InteractiveFly: GeneBrief

partner of inscuteable: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - partner of inscuteable

Synonyms - rapsynoid

Cytological map position - 98A--B

Function - interactor involved in asymmetric cell divisions

Keywords - asymmetric cell division, apical/basal polarity, regulation of spindle positioning; CNS

Symbol - pins

FlyBase ID: FBgn0040080

Genetic map position -

Classification - tetratrico-peptide repeats protein with GoLoco domains

Cellular location - cytoplasmic



NCBI link: EntrezGene

pins orthologs: Biolitmine

Recent literature
Tamada, M. and Zallen, J. A. (2015). Square cell packing in the Drosophila embryo through spatiotemporally regulated EGF receptor signaling. Dev Cell 35: 151-161. PubMed ID: 26506305
Summary:
Cells display dynamic and diverse morphologies during development, but the strategies by which differentiated tissues achieve precise shapes and patterns are not well understood. This study identified a developmental program that generates a highly ordered square cell grid in the Drosophila embryo through sequential and spatially regulated cell alignment, oriented cell division, and apicobasal cell elongation. The basic leucine zipper transcriptional regulator Cnc is necessary and sufficient to produce a square cell grid in the presence of a midline signal provided by the EGF receptor ligand Spitz. Spitz orients cell divisions through a Pins/LGN-dependent spindle-positioning mechanism and controls cell shape and alignment through a transcriptional pathway that requires the Pointed ETS domain protein. These results identify a strategy for producing ordered square cell packing configurations in epithelia and reveal a molecular mechanism by which organized tissue structure is generated through spatiotemporally regulated responses to EGF receptor activation.

Luchtenborg, A. M., Purvanov, V., Melnik, B. S., Becker, S. and Katanaev, V. L. (2015). Mode of interaction of the Gαo subunit of heterotrimeric G proteins with the GoLoco1 motif of Drosophila Pins is determined by guanine nucleotides. Biosci Rep. 35(6): pii: e0027. PubMed ID: 26487707
Summary:
Drosophila GoLoco motif-containing protein Pins is unusual in its highly efficient interaction with both GDP- and the GTP-loaded forms of the α-subunit of the heterotrimeric Go protein. This study analyzed the interactions of Gαo in its two nucleotide forms with GoLoco1 - the first of the three GoLoco domains of Pins - and the possible structures of the resulting complexes, through combination of conventional fluorescence and Forster resonance energy transfer measurements as well as through molecular modeling. The data suggest that the orientation of the GoLoco1 motif on Gαo significantly differs between the two nucleotide states of the latter. In other words, a rotation of the GoLoco1 peptide in respect with Gαo must accompany the nucleotide exchange in Gαo. The sterical hindrance requiring such a rotation likely contributes to the guanine nucleotide exchange inhibitor activity of GoLoco1 and Pins as a whole. These data have important implications for the mechanisms of Pins regulation in the process of asymmetric cell divisions.

Bergstralh, D.T., Lovegrove, H.E., Kujawiak, I., Dawney, N.S., Zhu, J., Cooper, S., Zhang, R. and St Johnston, D. (2016). Pins is not required for spindle orientation in the Drosophila wing disc. Development [Epub ahead of print]. PubMed ID: 27287805
Summary:
In animal cells, mitotic spindles are oriented by the dynein/dynactin motor complex, which exerts a pulling force on astral microtubules. Dynein/dynactin localization depends on Mud/NUMA, which is typically recruited to the cortex by Pins/LGN. In Drosophila neuroblasts, the Inscuteable/Baz/Par-6/aPKC complex recruits Pins apically to induce vertical spindle orientation, whereas in epithelial cells, Dlg recruits Pins laterally to orient the spindle horizontally. This study investigated division orientation in the Drosophila imaginal wing disc epithelium. Live imaging reveals that spindle angles vary widely during prometaphase and metaphase, and therefore do not reliably predict division orientation. Next, the mutants that have been reported to disrupt division orientation in this tissue were re-examined. Loss of Mud/NUMA misorients divisions, but Inscuteable expression and aPKC, dlg and pins mutants have no effect. Furthermore, Mud localizes to the apical-lateral cortex of the wing epithelium independently of both Pins and cell cycle stage. Thus, Pins is not required in the wing disc because there are parallel mechanisms for Mud localization and hence spindle orientation, making it a more robust system than other epithelia.
Schiller, E. A. and Bergstralh, D. T. (2021). Interaction between Discs large and Pins/LGN/GPSM2: A comparison across species. Biol Open. PubMed ID: 34596678
Summary:
The orientation of the mitotic spindle determines the direction of cell division, and therefore contributes to tissue shape and cell fate. Interaction between the multifunctional scaffolding protein Discs large (Dlg) and the canonical spindle orienting factor GPSM2 (called Pins in Drosophila and LGN in vertebrates) has been established in bilaterian models, but its function remains unclear. A phylogenetic approach was used to test whether the interaction is obligate in animals, and in particular whether Pins/LGN/GPSM2 evolved in multicellular organisms as a Dlg-binding protein. This study shows that Dlg diverged in C. elegans and the syncytial sponge O. minuta and proposes that this divergence may correspond to differences in spindle orientation requirements between these organisms and the canonical pathways described in bilaterians. It was also demonstrated that Pins/LGN/GPSM2 is present in basal animals, but the established Dlg-interaction site cannot be found in either Placozoa or Porifera. These results suggest that the interaction between Pins/LGN/GPSM2 and Dlg appeared in Cnidaria, and it is therefore speculated that it may have evolved to promote accurate division orientation in the nervous system. This work reveals the evolutionary history of the Pins/LGN/GPSM2-Dlg interaction and suggests new possibilities for its importance in spindle orientation during epithelial and neural tissue development.
Neville, K. E., Finegan, T. M., Lowe, N., Bellomio, P. M., Na, D. and Bergstralh, D. T. (2023). The Drosophila mitotic spindle orientation machinery requires activation, not just localization. EMBO Rep: e56074. PubMed ID: 36629398
Summary:
The orientation of the mitotic spindle at metaphase determines the placement of the daughter cells. Spindle orientation in animals typically relies on an evolutionarily conserved biological machine comprised of at least four proteins - called Pins, Gαi, Mud, and Dynein in flies - that exerts a pulling force on astral microtubules and reels the spindle into alignment. The canonical model for spindle orientation holds that the direction of pulling is determined by asymmetric placement of this machinery at the cell cortex. In most cell types, this placement is thought to be mediated by Pins, and a substantial body of literature is therefore devoted to identifying polarized cues that govern localized cortical enrichment of Pins. This study revisits the canonical model and finds that it is incomplete. Spindle orientation in the Drosophila follicular epithelium and embryonic ectoderm requires not only Pins localization but also direct interaction between Pins and the multifunctional protein Discs large. This requirement can be over-ridden by interaction with another Pins interacting protein, Inscuteable.
Parra, A. S., Moezzi, C. A., Johnston, C. A. (2023). Drosophila Adducin facilitates phase separation and function of a conserved spindle orientation complex. Frontiers in cell and developmental biology, 11:1220529 PubMed ID: 37655159
Summary:
Asymmetric cell division (ACD) allows stem cells to generate differentiating progeny while simultaneously maintaining their own pluripotent state. ACD involves coupling mitotic spindle orientation with cortical polarity cues to direct unequal segregation of cell fate determinants. In Drosophila neural stem cells (neuroblasts; NBs), spindles orient along an apical-basal polarity axis through a conserved complex of Partner of Inscuteable (Pins; human LGN) and Mushroom body defect (Mud; human NuMA). While many details of its function are well known, the molecular mechanics that drive assembly of the cortical Pins/Mud complex remain unclear, particularly with respect to the mutually exclusive Pins complex formed with the apical scaffold protein Inscuteable (Insc). This study identified Hu li tai shao (Hts; human Adducin) as a direct Mud-binding protein, using an aldolase fold within its head domain (Hts(HEAD)) to bind a short Mud coiled-coil domain (Mud(CC)) that is adjacent to the Pins-binding domain (Mud(PBD)). Hts is expressed throughout the larval central brain and apically polarizes in mitotic NBs where it is required for Mud-dependent spindle orientation. In vitro analyses reveal that Pins undergoes liquid-liquid phase separation with Mud, but not with Insc, suggesting a potential molecular basis for differential assembly mechanics between these two competing apical protein complexes. Furthermore, this study found that Hts binds an intact Pins/Mud complex, reduces the concentration threshold for its phase separation, and alters the liquid-like property of the resulting phase separated droplets. Domain mapping and mutational analyses implicate critical roles for both multivalent interactions (via Mud(CC) oligomerization) and protein disorder (via an intrinsically disordered region in Hts; Hts(IDR)) in phase separation of the Hts/Mud/Pins complex. This study identifies a new component of the spindle positioning machinery in NBs and suggests that phase separation of specific protein complexes might regulate ordered assembly within the apical domain to ensure proper signaling output.
BIOLOGICAL OVERVIEW

Rapsynoid/Partner of inscuteable was identified by two different research groups as an Inscuteable-binding protein. In one laboratory (Yu, 2000) Raps was identified using a yeast two hybrid screen. The second group (Schaefer, 2000) identified Raps by preparative immunoprecipitation and mass spectrometry. Raps is a new component of asymmetric divisions, required for the asymmetric localization of Inscuteable, the correct orientation of mitotic spindle, and resolution of distinct sibling cell fates. Raps is found to be complexed with heterotrimeric G-protein alpha subunit, implicating Raps in the activation of a heterotrimeric G-protein signaling cascade leading to the establishment of cell polarity.

Several proteins, Miranda, Staufen and Partner of Numb (Pon) (Lu, 1998) have been shown to act as a link between the apically localized Insc and the basally localized cell fate determinants. These adaptors act downstream of insc and are also asymmetrically localized, similar to the cell fate determinants they help to localize in an insc-dependent manner. Acting upstream of inscuteable is bazooka (baz), a Drosophila homolog of the nematode par3 gene, which encodes a mutiple PDZ domain protein that is required for the apical/basal polarity of the neuroepithelium. It is the only gene known to be required for asymmetric Insc localization. Baz is localized apically in the neuroepithelium as well as in dividing NBs and may act to link NB polarity to the apical/basal polarity of the epithelium by recruiting Insc to the apical cortex (Yu, 2000 and references therein).

partner of inscuteable encodes a novel protein with multiple repeats of the Tetratricopeptide (TPR) motif that complexes/interacts in vivo and in vitro with the Insc asymmetric localization domain. Raps colocalizes with Insc; the asymmetric cortical localization of both proteins is mutually dependent in dividing NBs and cells of mitotic domain 9. raps appears to be required for all aspects of insc function. Analyses of raps using both loss- and gain-of-function approaches suggest that the localization of Insc in neural progenitors involves at least two steps: (1) the initial localization of Insc to the apical cortex during delamination, while requiring baz, occurs independent of raps; (2) the maintenance of apical Insc (and Raps) later in interphase and during mitosis requires raps and insc (Yu, 2000 and Schaefer, 2000).

Baz is known to interact with Insc and to be required for Insc asymmetric localization. In the absence of baz function, Insc does not localize apically even in delaminating NBs and is cytoplasmic later in the cell cycle. In embryos lacking both maternal and zygotic baz, Raps distribution in mitotic NBs is mostly cortical, similar to its distribution in insc mutant NBs. Interestingly, Baz localization to the apical cortex of NBs is itself affected by raps and insc loss of function. In Raps- NBs, the apical cortical Baz crescents normally present in WT mitotic NBs cannot be detected from metaphase onward. However, occasional weak crescents can be found in mutant interphase/prophase NBs and these are always localized to the apical cortex. The Baz distribution in insc mutant NBs is similar to that seen in Raps- embryos. These observations suggest that the maintenance and/or stability of apical Baz in NBs requires both insc and raps (Yu, 2000).

Taken together these results indicate that the initial localization of Insc (e.g., to the apical stalk) requires baz but not raps; however, the maintenance of apical Baz/Raps/Insc later in the cell cycle (e.g., at metaphase) is mutually dependent, requiring all three components (Yu, 2000).

To further explore the relationship between raps and insc, attention was focussed on the epithelial cells that normally express but do not apically localize Raps and do not express Insc. insc is necessary for the apical localization of Raps in NBs and cells of mitotic domain 9. Would the ectopic expression of Insc in epithelial cells be sufficient to recruit Raps to the apical cortex? Ectopically expressed Insc, driven from a hsp70-insc transgene, localizes to the apical cortex in WT epithelial cells and, interestingly, causes Raps, which is normally localized to the lateral cortex, to also localize to the apical cortex. Conversely, apical localization of ectopically expressed Insc is dependent on raps. Insc ectopically expressed in Raps- epithelial cells does not localize as an apical crescent; rather it adopts a cytoplasmic distribution (primarily toward the apical side of the cell) during interphase and is undetectable during mitosis, presumably due to rapid degradation. This apparent instability of ectopically expressed Insc may be the reason why the 90° rotation in the mitotic spindles that occurs as a consequence of Insc ectopic expression in the WT epithelial cells no longer occurs when Insc is expressed in Raps- embryos. These results indicate that the ectopic expression of Insc is sufficient for Raps to be recruited to the apical cortex of WT epithelial cells; moreover, similar to NBs, the mutual dependence between Raps and ectopically expressed Insc is indicated by the apical localization of both proteins in these cells (Yu, 2000).

Where does Raps fit in the pathway that establishes and maintains cell asymmetry? Two proteins of approximately 70 kDa and 40 kDa are reproducibly coimmunoprecipitated with Inscuteable. The 70 kDa protein has been identified as Inscuteable. The sequences of two short peptide fragments of the 40 kDa protein could be determined. The sequences occur in both the Drosophila Galphai protein (Galpha65A, Swissprot accession number P20353) and Galphao protein (Galpha47A, Swissprot accession number P16377), but not in any other Drosophila protein or EST. It cannot currently be determine whether the 40 kDa band is Drosophila Galphao or Galphai. To test for a direct interaction between Inscuteable, Raps and Galphai/Galphao, in vitro binding assays were performed. In vitro translated Raps protein binds strongly to Inscuteable. Very weak binding is also detected between Insc and both Galphai and Galphao. In contrast, both Galphai and Galphao bind strongly to a Raps. These results suggest that the complex containing Inscuteable, Raps and Galphai/Galphao forms as a result of a direct protein interaction between Inscuteable and Raps, and between Raps and Galphai/Galphao, even though the weak interaction between Galphai/Galphao and Inscuteable may also contribute (Schaefer, 2000).

The fact that Raps contains three GoLoco domains, which are thought to be modulators of Galpha signaling, and that Raps exists in a complex with Galpha in vivo, offers the intriguing possibility that a heterotrimeric G-protein signaling cascade is involved in directing asymmetric cell divisions in Drosophila (Schaefer, 2000). No evidence exists that would suggest the involvement of extracellular signals (through G-protein coupled receptors) in orienting neuroblast divisions. Furthermore, asymmetric localization of Inscuteable during metaphase and asymmetric cell division can occur in cultured neuroblasts in the absence of any extracellular signal. Therefore, knowing whether and how G-proteins are involved in asymmetric cell division awaits identification of additional pathway elements.

Two proteins are known to be required for Insc asymmetric localization, Baz and Raps. They play apparently distinct roles in facilitating Insc localization in NBs. Baz is localized to the apical cortex of both neuroectodermal cells and NBs that delaminate from the neuroectoderm. Baz presumably acts as a link to allow NBs to retain the apical/basal polarity inherent to the neuroectodermal epithelium by facilitating the apical localization of Insc in interphase delaminating NBs before they lose contact with the neuroectoderm. Since Baz interacts with Insc in vivo and in vitro, it can in principle initiate Insc asymmetric localization by directly recruiting it to the apical cortex of delaminating NBs. Consistent with this view, in the absence of baz function, both the initiation and the maintenance of Insc asymmetric localization is defective (Schober, 1999; Wodarz, 1999; Yu, 2000).

In this context, it is interesting to note that the asymmetric localization of Baz, Insc, and Raps appears to follow a temporal order. Baz is the earliest apical localizing component. It is apical while the cells are still in the epithelium, preceding the apical localization of Insc in delaminating NBs. Although weak Raps signals can sometimes be detected in delaminating NBs, strong apical crescents are seen only in NBs following delamination. Therefore, and not surprisingly, raps is not required for the initiation of Insc apical localization. Following the baz-dependent localization of Insc to the apical stalk/cortex of interphase delaminating NBs, Raps is recruited to the apical cortex. In the absence of raps, the apical localization of both Insc and Baz fails to be maintained. It is therefore apparent that, as a delaminating NB progresses from interphase (G2) toward mitosis, the apical localization of Baz and Insc changes from being raps independent to being raps dependent. Since the (re)orientation of mitotic spindle and basal cortical localization of cell fate determinants occurs during mitosis and is insc dependent, it seems likely that the maintenance of an apical complex containing Insc, Raps, and Bazooka during mitosis would be essential for NB to divide asymmetrically. This appears to be the case because in Raps- NBs, where apical Insc/Baz/Raps fails to be maintained, all of these processes associated with the NB asymmetric cell divisions are defective, in effect giving phenotypes similar to those seen in insc mutants (Yu, 2000).

Interestingly, apical Baz and Raps also fail to be maintained in the absence of insc function; Baz and Raps apical crescents are absent by metaphase in mitotic NBs of insc embryos. Since Baz is also required for Insc asymmetric localization, the maintenance of apical Baz, Insc, and Raps appears to be dependent on all three components. This interdependence on multiple components for the asymmetric localization of a protein complex is reminiscent of the interaction exhibited by Par3, Par6, and Pkc-3, proteins involved in mediating the asymmetric blastomere divisions in the early nematode embryos (Yu, 2000 and references therein).

A direct interaction between Raps and Baz in yeast two hybrid and GST pull-down experiments could not be demonstrated. However, Baz complexes with Insc in vivo, and directly interacts with Insc in vitro (Schober, 1999; Wodarz, 1999). Since Raps interacts with Insc, these observations suggest that Insc may be acting to link Baz to Raps. Several observations support this view. (1) For NBs and cells of mitotic domain 9, Raps does not localize apically in the absence of insc function. (2) Also supportive is the apparent temporal order in which these genes are recruited to the apical cortex of NB: Baz (while part of the epithelia), followed by Insc (during delamination), followed by Raps (after delamination). (3) In epithelial cells that do not express Insc, Raps and Baz do not colocalize; Baz is found on the apical cortex and Raps shows lateral cortical distribution, yet the ectopic expression of Insc (which localizes apically) is sufficient to recruit Raps to the apical cortex of these cells. All of the available data are consistent with the model that the formation and maintenance of an apical protein complex that imparts apical/basal polarity in NBs comprises the following events: cells in the neuroepithelium destined to become NBs have apical/basal polarity as evidenced by the apical localization of Baz; as these interphase cells delaminate, Insc is recruited to the apical complex in a Baz-dependent manner; Raps is in turn recruited to this complex and this involves interaction with Insc. Some as yet undefined events must occur between delamination (interphase) and mitosis that change the nature of this complex such that its maintenance becomes codependent on these three molecules (Yu, 2000).

Both insc and raps are required for the execution of the more downstream processes associated with asymmetric cell divisions and the relative roles of the two genes are at present unclear. However, some interesting distinctions can be made between the two genes. insc was originally isolated on the basis of its expression in neural precursors. Insc expression is restricted, conforms to the prepattern-proneural-neurogenic-panneural cascade, and links general neuronal differentiation programs to lineage information; Raps shows a wider expression pattern and becomes involved in asymmetric cell divisions only when a signal (i.e., insc) is active. Raps and Insc also appear to follow different routes to reach the apical cortex -- Raps apparently transiting via the membrane but not Insc, which suggests that other interactors may be involved in linking Raps to the cortex. Finally, the only known direct links to downstream events associated with asymmetric cell divisions appear to be mediated through Insc (Yu, 2000).

Galphai generates multiple Pins activation states to link cortical polarity and spindle orientation in Drosophila neuroblasts

Drosophila neuroblasts divide asymmetrically by aligning their mitotic spindle with cortical cell polarity to generate distinct sibling cell types. Neuroblasts asymmetrically localize Gαi, Pins, and Mud proteins; Pins/Gαi direct cortical polarity, whereas Mud is required for spindle orientation. It is currently unknown how Gαi-Pins-Mud binding is regulated to link cortical polarity with spindle orientation. This study shows that Pins forms a "closed" state via intramolecular GoLoco-tetratricopeptide repeat (TPR) interactions, which regulate Mud binding. Biochemical, genetic, and live imaging experiments show that Gαi binds to the first of three Pins GoLoco motifs to recruit Pins to the apical cortex without "opening" Pins or recruiting Mud. However, Gαi and Mud bind cooperatively to the Pins GoLocos 2/3 and tetratricopeptide repeat domains, respectively, thereby restricting Pins-Mud interaction to the apical cortex and fixing spindle orientation. It is concluded that Pins has multiple activity states that generate cortical polarity and link it with mitotic spindle orientation (Nipper, 2007).

In complex, multicellular organisms, differentiated cell types are needed to perform diverse functions. One common mechanism for cellular differentiation is asymmetric cell division, in which the mitotic spindle is aligned with the cell polarity axis to generate molecularly distinct sibling cells. Asymmetric divisions have been proposed to regulate stem cell pool size during development, adult tissue homeostasis, and the uncontrolled proliferation observed in cancer. Thus, understanding how the mitotic spindle is coupled to the cell polarity axis is relevant to stem cell and cancer biology. This question was investigated in Drosophila neuroblasts, a model system for studying asymmetric cell division (Nipper, 2007).

Drosophila neuroblasts are stem cell-like progenitors that divide asymmetrically to produce a larger self-renewing neuroblast and a smaller ganglion mother cell (GMC) that differentiates into neurons or glia. Mitotic neuroblasts segregate factors that promote neuroblast self-renewal to their apical cortex and differentiation factors to their basal cortex. Precise alignment of the mitotic spindle with the neuroblast apical/basal polarity is required for asymmetric cell division and proper brain development: spindle misalignment leads to symmetric cell divisions that expand the neuroblast population and brain size (Nipper, 2007).

A key regulator of neuroblast cell polarity and spindle orientation is Partner of Inscuteable (Pins; LGN or mPins in mammals, GPR-1/2 in Caenorhabditis elegans). In metaphase neuroblasts, Pins is colocalized at the apical cortex with the heterotrimeric G protein subunit Gαi and the spindle-associated, coiled-coil Mushroom body defect protein (Mud; NuMA in mammals, Lin-5 in C. elegans). Pins and Gαi are interdependent for localization and for establishing cortical polarity. Pins also binds directly to Mud and recruits it to the apical cortex; Mud is specifically required to align the mitotic spindle with Gαi/Pins but has no apparent role in establishing cortical polarity (Nipper, 2007).

The mechanism underlying Pins regulation of cortical polarity and spindle-cortex coupling is unclear, and it is unknown how Gαi-Pins-Mud complex assembly is regulated. Pins has the potential to bind multiple Gαi·GDP molecules via three short GoLoco motifs, as do mammalian Pins homologs, but the role of these multiple binding sites is unknown. Moreover, via its tetratricopeptide repeats (TPRs), Pins can bind Mud, but the stoichiometry and regulation of this interaction has not been explored. Furthermore, like its mammalian homolog LGN, the regions of Pins containing the TPRs and GoLocos interact, raising the possibility of cooperative "opening" of Pins by Gαi and Mud ligands. This study tested the role of Pins intra- and inter-molecular interactions in coupling cortical polarity with spindle orientation. Biochemistry, genetics, and in vivo live imaging were used to test the role of Pins intramolecular interactions and whether Gαi and Mud bind Pins independently, cooperatively, or antagonistically. It is concluded that Pins has multiple functional states -- a form recruited by a single Gαi to the apical cortex that is unable to bind Mud but sufficient to induce cortical polarity, and a form saturated with Gαi that recruits Mud and links cortical polarity to the mitotic spindle. The multiple Pins states are due to cooperative binding of Mud and Gαi to Pins and result in a tight link between apical cortical polarity and mitotic spindle orientation (Nipper, 2007).

The NH2-terminal half of Pins contains seven TPRs, and the COOH-terminal half contains three GoLoco motifs, which is termed here the GoLoco region, or GLR. Each of the three GoLocos has the potential to bind GDP-bound Gαi, whereas the TPRs bind the Mud protein. Before testing whether the Pins intramolecular interaction regulates Pins-Gαi-Mud complex assembly, of the relevant individual domain interactions were tested: TPR-Mud, GLR-Gαi, and TPR-GLR. (1) The Pins TPRs bind Mud with a 1:1 stoichiometry as judged by the elution profile of the TPR-Mud complex on a calibrated gel-filtration column, indicating that Pins contains a single Mud binding site. (2) Each of the three Pins GoLoco domains binds Gαi·GDP (hereafter Gαi) equally well in a qualitative pull-down assay as well as in a more quantitative assay measuring Gαi binding by using the fluorescence anisotropy of tetramethylrhodamine attached to the COOH terminus of the Pins GLR. A binding isotherm describing three equivalent, independent sites with submicromolar Gαi affinities (Kd = 530 ± 80 nM) fits the data well and yields a linear Scatchard relationship. It is concluded that each GoLoco in the Pins GLR binds Gαi with a similar affinity and without cooperativity in the absence of the TPRs, similar to a three-GoLoco region of the protein AGS3. Finally, the interaction between the TPRs and GLR has an affinity of Kd = ~2 µM in trans, which may be enhanced in intact Pins because of the increase in effective concentration (Nipper, 2007).

To test whether the Pins intramolecular interaction regulates Pins-Gαi-Mud complex assembly, it was first determined whether Gαi or Mud binding disrupts TPR-GLR. Using a qualitative assay in which the TPRs and GLR are expressed as separate fragments, it was found that increasing concentrations of Gαi completely disrupt the trans TPR-GLR complex. The region of Mud that binds to Pins (Pins binding domain or PBD; contained within Mud residues 1825-1997) also disrupts the TPR-GLR complex, although not as efficiently as Gαi. Thus, Pins contains an intramolecular interaction that competes against both Gαi and Mud binding (Nipper, 2007).

Because Gαi and Mud are both coupled to the Pins intramolecular interaction, whether the two proteins bind cooperatively to Pins was tested by determining whether Gαi could enhance the affinity of Pins for Mud. 1 µM Pins binds weakly to a GST fusion of the Mud PBD. However, addition of Gαi induces a large increase in Pins binding and the formation of a Mud-Pins-Gαi ternary complex. It is concluded that Gαi increases the affinity of Pins for Mud (i.e., Gαi and Mud bind cooperatively to Pins) (Nipper, 2007).

Because Pins contains three GoLoco motifs and the Pins intramolecular interaction competes against Gαi binding, whether these Gαi binding sites are repressed equally in intact Pins was tested. Gel-filtration chromatography of full-length Pins and Gαi were used to determine how Gαi-GoLoco binding is affected by the intramolecular interaction. Pins elutes as a single peak with an elution volume consistent with the molecular weight for a monomer. Addition of low Gαi concentrations leads to formation of a 1:1 Gαi:Pins complex peak. Higher Gαi concentrations lead to the formation of a 3:1 Gαi:Pins complex with a very broad peak, suggestive of a lower affinity interaction. It is concluded that full-length Pins contains a single high-affinity Gαi-binding GoLoco and two low-affinity GoLocos (Nipper, 2007).

Because the three GoLocos are intrinsically equivalent, independent Gαi binding sites, the distinct Gαi binding behavior in full-length Pins suggests that Pins contains one GoLoco domain that is unregulated or only partially regulated by the intramolecular interaction and two GoLoco domains that are cooperatively repressed. To further explore this model, one or more GoLocos was inactivated by mutating a single critical arginine residue to phenylalanine in the context of full-length Pins. These mutations do not inhibit the ability of the TPRs and GoLocos to interact. Inactivation of GoLoco1 (Pins δGL1; R486F) specifically abolishes the high-affinity 1:1 complex, whereas inactivation of either GoLoco 2 or 3 has no effect on the high-affinity complex. Therefore GoLoco1 is classified as a high-affinity GoLoco in the context of full-length Pins. Disruption of GoLocos 2 and 3 (Pins δGL2/3; R570F, R631F) leads to the formation of a 1:1 complex at low concentrations of Gαi, further confirming that GoLoco1 is not repressed by the TPRs. It is concluded that the three GoLoco motifs are differentially regulated by the Pins intramolecular interaction: Gαi shows unregulated high-affinity binding to GoLoco1 and low-affinity, cooperative binding to GoLocos 2 and 3 (Nipper, 2007).

It was next asked how Gαi binding to the different Pins GoLoco domains affects cooperative Gαi-Pins-Mud complex assembly. When GoLoco1 is inactivated (Pins δGL1), Gαi can still enhance Mud binding, in a manner similar to the WT Pins. The activation is more efficient, however, presumably because of the lack of Gαi "buffering" by GoLoco1. In contrast, in the Pins δGL2/3 mutant, Gαi does not enhance Mud binding even though it binds GoLoco1 with high affinity. Thus, Pins differentially regulates the ability of Gαi to promote Pins-Mud binding: Gαi binding to GoLoco1 has no effect on Pins-Mud binding, whereas Gαi binding to GoLocos 2 and 3 strongly enhances Pins-Mud association (Nipper, 2007).

These results suggest that Gαi binding to GoLocos 2 and 3 "opens" Pins to allow Mud binding to the TPRs. To directly monitor the Pins conformational transition between "closed" and "open" states, a Pins fluorescence resonance energy transfer (FRET) sensor was constructed with YFP and CFP at the NH2 and COOH termini, respectively. This type of sensor has been used successfully to monitor the conformational transition of a mammalian Pins homolog, LGN. Surprisingly, addition of Gαi or Mud alone did not cause a significant change in the YFP-Pins-CFP FRET signal, even at high concentrations, suggesting that Gαi or Mud alone is insufficient to "open" Pins. The addition of both ligands together, however, leads to a large change in the FRET signal (nearly complete loss of energy transfer), indicating that Mud and Gαi are both required to induce the "open" Pins conformation. To test the model that Gαi binding to GoLoco1 cannot open Pins, a Pins δGL2/3 FRET sensor was analyzed. Mud and Gαi fail to induce the conformational change seen with the WT FRET sensor, consistent with Gαi binding at GoLoco1 not being coupled to the intramolecular interaction (Nipper, 2007).

Because Mud or Gαi alone are not able to "open" Pins, a simple model in which Mud and Gαi directly compete in a mutually exclusive fashion (e.g., sterically) with the intramolecular interaction can exclude be excluded. Although disruption of the Pins TPR-GLR interaction was observed in trans, this is likely to result from effective concentration effects in which the interaction is weaker when the two domains are not in the same polypeptide. It is concluded that Mud and Gαi allosterically modulate the TPRs and GoLocos, respectively, in a manner that leaves the intramolecular interaction intact but in a weakened state, poised to open upon binding of the second ligand. Thus, Pins can exist in a "closed" state (no Gαi or Mud bound), a "potentiated" closed state (with Gαi or Mud bound), and an "open" state (with both Gαi and Mud bound) (Nipper, 2007).

Based on the network of interactions present in Pins, Gαi binding to GoLoco1 should recruit Pins to the neuroblast apical cortex but not lead to Mud recruitment. To test this model, either HA:Pins WT or HA:Pins δGL2/3 was expressed in pins mutant neuroblasts and both Pins and Mud localization were examined. In third-instar larval central brain neuroblasts, both WT and δGL2/3 Pins localized to the apical cortex at metaphase. However, Mud was correctly recruited to the apical cortex in neuroblasts expressing WT Pins, and Mud recruitment in δGL2/3 neuroblasts was significantly reduced. Thus, Gαi binding to GoLoco1 is sufficient for Pins localization but not for efficient Mud targeting (Nipper, 2007).

To understand how cortically localized and Mud-recruiting Pins states are populated as Gαi accumulates at the apical cortex, Pins-Gαi binding was simulated based on the parameters described earlier. At low Gαi concentration, Pins with Gαi bound to GoLoco1 predominates because of its higher affinity relative to the other two GoLocos (which are repressed by the TPRs). Although this Pins form does not bind to Mud with high affinity, it was hypothesized that it is sufficient to induce aspects of cortical polarity (e.g., Insc polarization). At higher Gαi concentrations, GoLoco1 becomes saturated and binding can occur at GoLocos 2 and 3, allowing for Mud recruitment to the apical cortex. Thus, it is predictd that as Gαi accumulates at the apical cortex, it first recruits Pins in a form that is competent for cortical polarization but not spindle positioning. As Gαi levels further increase, however, GoLocos 2 and 3 become populated, weakening the intramolecular interaction and freeing the TPRs to recruit Mud to the apical cortex (Nipper, 2007).

The model that the population of Pins activation states is very sensitive to Gαi concentration was tested by examining Pins localization, Mud localization, and spindle orientation in larval neuroblasts with different levels of Gαi protein. The model strongly predicts that normal Gαi and Mud levels should "open" Pins to form a ternary complex at the apical cortex that is functional for spindle alignment, low Gαi levels would bind Pins GoLoco1 and recruit Pins to the apical cortex without allowing Mud binding or spindle orientation, and no Gαi protein would result in a failure to recruit Pins or Mud to the cortex. To test this model, larval neuroblasts were examined with normal, low, or no Gαi protein (WT zygotic mutants and maternal zygotic mutants, respectively). As expected, neuroblasts with WT levels of Gαi invariably colocalize Gαi, Pins, and Mud to an apical cortical crescent that is tightly coupled with the mitotic spindle, consistent with the activity of both Gαi and Mud "opening" Pins to form a ternary complex that is functional for spindle orientation. In contrast, neuroblasts with reduced Gαi levels formed robust Pins and Insc crescents but typically failed to localize Mud to the apical cortex and showed defects in spindle orientation. Neuroblasts lacking all Gαi protein fail to recruit Pins to the cortex and have spindle orientation defects. These results strongly support the model: low Gαi levels can recruit "closed" Pins to the cortex without recruiting Mud or promoting spindle orientation, whereas higher Gαi levels function together with Mud to "open" Pins and promote spindle orientation (Nipper, 2007).

To further test the model, time-lapse video microscopy was used to examine the dynamics of spindle behavior using a GFP-tagged microtubule-associated protein. In WT neuroblasts, the apical centrosome/spindle pole is anchored at the center of the Gαi/Pins/Mud crescent from prometaphase through telophase, although slight spindle rocking can be observed. In neuroblasts with reduced Gαi levels, where Gαi/Pins but not Mud are present at the apical cortex, it was found that the centrosome/spindle pole is not stably attached to the apical cortex and often shows excessive rotation. These data provide further support for the model that low levels of Gαi are sufficient to recruit Pins to the cortex via GoLoco1 binding but are insufficient to allow Pins to bind Mud and capture the apical spindle pole (Nipper, 2007).

Through interactions with Gαi and Mud, Pins regulates two fundamental aspects of asymmetric cell division: cortical polarity and alignment of the spindle with the resulting polarity axis. This study has investigated the mechanism by which Gαi regulates Pins interactions with the spindle orientation protein Mud. It was found that, although the three Pins GoLocos are intrinsically equivalent, independent Gαi binding sites, an intramolecular interaction with the Pins TPRs leads to differential Gαi binding. Gαi binding to GoLoco1 is not coupled to the Pins intramolecular interaction and therefore does not influence Mud binding but is sufficient to localize Pins to the cortex for Mud-independent functions (e.g., recruitment of Insc to the apical cortex). Gαi binding to GoLocos 2 and 3 destabilizes the Pins intramolecular interaction leading to cooperative Mud binding, and together the ligands induce an "open" Pins conformational state. This leads to a model in which Gαi induces multiple Pins activation states: one that localizes cortically but is not competent for Mud binding, and one that binds Mud linking localized Gαi to the mitotic spindle (Nipper, 2007).

Intramolecular interactions are common features of signaling proteins that typically act through "autoinhibition" of an enzymatic or ligand binding activity. Such interactions allow for coupling of regulatory molecule binding to an increase or decrease in downstream function, a critical aspect of information flow in signaling pathways. Pins is involved in the regulation of multiple downstream functions, and the results support the notion that the multiple Gαi binding sites present in Pins allow for the signal to branch into two pathways, one controlling cortical polarity and the other spindle positioning. A notable exception to the multiple GoLocos present in Pins-like proteins is the C. elegans Pins homologue GPR-1/2, which contains a single GoLoco domain. The lack of multiple GoLocos in GPR-1/2 may be consistent with their more limited role in C. elegans asymmetric cell division, where they regulate spindle positioning but not cortical polarity (Nipper, 2007).

In the model presented in this study, the Pins intramolecular interaction serves to regulate Mud binding. This may occur for several reasons. (1) Localization of Mud activity to the apical cortex appears to be important for aligning the spindle with the axis of cortical polarity. In this context, the Pins intramolecular interaction may be important for restricting Mud activity to the apical cortex. Mutant pins or Gαi neuroblasts may have low ectopic Mud activity at the basal or lateral cortex that leads to the observed misdirected spindle rotation seen in live neuroblast imaging. This observation is consistent with previous observations that too little Mud (in mud mutant neuroblasts) results in spindle position defects without any rotation. (2) Mud activity may be affected by its interaction with Pins. For example, LGN binds to a region of NuMA near its microtubule binding site such that LGN binding to NuMA competes with microtubule binding (Nipper, 2007).

A unique feature of the Pins intramolecular interaction is that autoinhibition is incomplete. Binding of GoLocos 2 and 3 to Gαi is repressed by the TPRs, but binding to GoLoco1 is not. This has two important consequences. (1) Whereas the three GoLocos are intrinsically equivalent and independent Gαi binding sites, TPR repression of GoLocos 2 and 3 significantly lowers the affinity of these GoLocos relative to GoLoco1. This leads to preferential population of GoLoco1, which may be important for temporal regulation of asymmetric cell division by ensuring that cortical polarity is established before the spindle is positioned. (2) The TPRs appear to repress GoLocos 2 and 3 cooperatively (Gαi binding to 2 or 3 increases the affinity at the other site). Cooperativity is a common property of signaling pathways that is used generate complex input-output profiles. Pins exhibits both homotropic (Gαi) and heterotropic (Gαi and Mud) binding cooperativity. In both cases, cooperativity is not an inherent property of the binding sites but is generated through the competition that results from the intramolecular interaction between the TPRs and GoLocos. Such "cooperative repression" of inherently equivalent binding sites through intramolecular interactions may be a general mechanism for generating cooperativity in signaling proteins (Nipper, 2007).

A NudE/14-3-3 pathway coordinates dynein and the kinesin Khc73 to position the mitotic spindle

Mitotic spindle position is controlled by interactions of cortical molecular motors with astral microtubules. In animal cells, Partner of Inscuteable (Pins) acts at the cortex to coordinate the activity of Dynein and Kinesin-73 (Khc73; KIF13B in mammals) to orient the spindle. Though the two motors move in opposite directions, their synergistic activity is required for robust Pins-mediated spindle orientation. This study identified a physical connection between Dynein and Khc73 that mediates cooperative spindle positioning. Khc73's motor and MBS domains link Pins to microtubule plus ends, while its stalk domain is necessary for Dynein activation and precise positioning of the spindle. A motif in the stalk domain binds, in a phospho-dependent manner, 14-3-3ζ, which dimerizes with 14-3-3ε. The 14-3-3ζ/ε heterodimer binds the Dynein adaptor NudE to complete the Dynein connection. The Khc73 stalk/14-3-3/NudE pathway defines a physical connection that coordinates the activities of multiple motor proteins to precisely position the spindle (Lu, 2013).

Mitotic spindle orientation requires the coordination of several pathways that act on astral microtubules. These pathways may establish cortical-microtubule connections and generate the forces necessary for movement of this large cellular structure with metaphase spindle lengths varying from 2 mm in yeast to 60 mm of a Xenopus single-cell stage. The spindle-orientation protein Pins has a domain that has been thought to capture microtubules (Pinslinker), and another that generates force (PinsTPR). This study attempted to understand how these two pathways function together by taking advantage of an induced polarity system in cultured S2 cells in which the two pathways can be selectively activated. This system allowed for the identification of the Khc73 stalk domain as a critical element that links PinsTPR and Pinslinker pathways. This observation was used as a platform for establishing a complete physical connection between the two pathways. This study has also clarified the role of 14-3-3 proteins in spindle orientation, establishing that their interaction with Pins is likely to be indirect (through Dlg and Khc73) (Lu, 2013).

Khc73 performs two functions in Pins-mediated spindle positioning. First, it functions in the Pinslinker pathway to mediate cortical microtubule capture through its MBS and motor domains, respectively. The N-terminal portion of Khc73 is sufficient for linker activity, which is likely occurring through a DlgGK/Khc73MBS interaction at the cortex and a microtubule/ Khc73motor interaction at the spindle. This suggests that Khc73's motor domain could function at the cortex by itself, however, Ed:Khc73motor did not have spindle positioning activity, indicating that other factors could be required or the motor domain is not functional in this context (e.g., as a monomer with the coiled-coil stalk). Khc73 must therefore rely on Dlg as an adaptor to target it to the cortex, which is where it can potentially function to facilitate the initial contact of astral microtubules (Lu, 2013).

Although Khc73's MBS domain directly interacts with Dlg, Khc73 is not seen to colocalize with cortical Pins, even though Dlg robustly localizes to Pins crescents. Instead, the motor protein is seen distinctly at the ends of microtubule, suggesting that Khc73 moves to the plus ends where it may be poised for capture by the cortical Pinslinker/Dlg complex. Thus, Khc73's N-terminal domains are likely to facilitate cortical microtubule capture by linking microtubule plus ends to cortical Dlg (Lu, 2013).

In addition to facilitating cortical microtubule capture, this study found that Khc73 also forms a physical connection to the PinsTPR/Mud/Dynein pathway with its stalk region, which is essential for the synergistic function of the two pathways. Khc73 may activate Dynein by delivering NudE to the cortex, where Dynein is presumably localized by PinsTPR/Mud. Although it is not possible to observe the localization of Dynein in S2 cells for technical reasons, there is good evidence that it is cortically localized by way of PinsTPR/Mud. In HeLa cells, Dynein localizes to the cortex with the mammalian homolog of Mud, NuMA, along with mPins, during mitosis (Lu, 2013).

It is proposed that a 14-3-3 motif in Khc73's stalk region activates an 'idling' cortically localized Dynein by cargoing NudE. Interestingly, although the Khc73 14-3-3 motif mutant Khc73S1374A has a distribution of spindle-orientation angles that isn't random, the distribution is bimodal such that the spindle angles are either fully aligned or orthogonal to the polarity axis. The bimodal phenotype is distinct from the Khc73motor+MBS fragment, which has a canonical intermediate distribution of spindle angles, suggesting that there may be additional regions or domains in the stalk that are contributing to the bimodal phenotype. It is hypothesized that an element within Khc73's stalk region is required for the proper application of the forces generated from by two motor proteins to properly orient the mitotic spindle. Nevertheless, biochemical and genetic studies demonstrate that the 14-3-3 binding motif is, at the very least, required for proper Pins-mediated spindle positioning and required for Khc73's interaction with the 14-3-3 proteins and NudE (Lu, 2013).

Pins mediates spindle positioning by coordinating two motor proteins that, as a pair, facilitate the cortical capture of microtubules and also provide pulling forces to robustly orient the mitotic spindle. A model is proposed in which orientation occurs through an ordered series of events, beginning with the initial polarization of Pins, followed by recruitment of Mud through its PinsTPR domain and Dlg through Pinslinker region. Cortical Mud then recruits cytoplasmic Dynein, which is not yet active and will remain inert, but poised at the cortex. Khc73 localizes to the plus ends of microtubules, where it establishes cortical-microtubule contacts through direct binding to Dlg and also delivers NudE to cortical Dynein, thereby activating it. As astral microtubules enter the proximity of the Dynein complex, Dynein can generate specifically timed cortical pulling forces necessary for robust spindle positioning. Future work will be directed at dissecting the precise timing of these synergistic events that underlie differentiation and tissue architecture (Lu, 2013).


REGULATION

Ric-8 controls Drosophila neural progenitor asymmetric division by regulating heterotrimeric G proteins; Ric-8 complexes with Pins through their mutual interactions with Galpha

Asymmetric division of Drosophila neuroblasts (NBs) and the C. elegans zygote uses polarity cues provided by the Par proteins, as well as heterotrimeric G-protein-signalling that is activated by a receptor-independent mechanism mediated by GoLoco/GPR motif proteins. Another key component of this non-canonical G-protein activation mechanism is a non-receptor guanine nucleotide-exchange factor (GEF) for Galpha, RIC-8, which has recently been characterized in C. elegans and in mammals. The Drosophila Ric-8 homologue is required for asymmetric division of both NBs and pI cells. Ric-8 is necessary for membrane targeting of Galphai, Pins and Gbeta13F, presumably by regulating multiple Galpha subunit(s). Ric-8 forms an in vivo complex with Galphai and interacts preferentially with GDP-Galphai, which is consistent with Ric-8 acting as a GEF for Galphai. Ric-8 complexes with Pins through their mutual interactions with Galpha. Comparisons of the phenotypes of Galphai, Ric-8, Gbeta13F single and Ric-8;Gbeta13F double loss-of-function mutants indicate that, in NBs, Ric-8 positively regulates Galphai activity. In addition, Gbetagamma acts to restrict Galphai (and GoLoco proteins) to the apical cortex, where Galphai (and Pins) can mediate asymmetric spindle geometry (Wang, 2005).

In neuroblasts (NBs), two apically localized protein cassettes - (Bazooka, Par3-DmPar6-DaPKC0 and (Galpha-Partner of Inscuteable [Pins, a GDP dissociation inhibitor (GDI) of Galpha)], which are linked by Inscuteable (Insc) -- mediate all aspects of NB asymmetric division. These two conserved protein cassettes are spatially separated in pI cells of the sensory organ precursor (SOP) lineage: Pins-Galpha localizes to the anterior, whereas Baz-Par-6-DaPKC localizes to the posterior cortex. In both Drosophila and C. elegans asymmetry models, the activation of heterotrimeric G-protein signalling apparently occurs via a receptor-independent mechanism that is mediated by proteins containing GoLoco/GPR (G-protein regulatory) motifs with GDI activity (for example, Drosophila Pins and nematode GPR1/2), which can compete with Gbetagamma for GDP-Galpha. With respect to the spindle geometry of Drosophila NBs, Gbeta13FGgamma1 seems to have a more crucial role than Galpha and Pins in this process. By contrast, Galpha subunits, GOA-1 and GPA-16, and the GoLoco proteins GPR1/2 are essential in C. elegans, for the generation of a net posterior force that is necessary for asymmetric spindle positioning. Gbetagamma, in contrast, does not play a positive role in this process. More recently, RIC-8, a novel non-receptor guanine nucleotide-exchange factor (GEF) for Galpha, has been shown to be required for asymmetric spindle positioning in the C. elegans zygote (Afshar, 2004; Couwenbergs, 2004; Tall, 2003; Hess, 2004). This study characterizes the role of the Drosophila Ric-8 homologue in neural progenitor asymmetric division (Wang, 2005).

Database searches of rat Ric-8A identified a putative Drosophila homologue, Ric-8 (CG15797, at cytological position 8D10 of the X chromosome), which shares ~31% amino-acid identity with rat Ric-8A. Ric-8 RNA is ubiquitously expressed with an abundant maternal component. In glutathione S-transferase (GST) pull-down assays, GST-Ric-8 interacts directly with Galpha in vitro. In co-immunoprecipitation experiments using embryonic extracts, Ric-8, similarly to Pins and Gbeta13F, interacts strongly with Galpha when GDP has been added in excess, but interacts poorly with Galpha in the presence of excess GTP-gammaS. This indicates that Ric-8 preferentially interacts with GDP-Galpha. These interactions are consistent with Ric-8 acting as a GEF for Galpha, similarly to its mammalian and nematode homologues (Wang, 2005).

To ascertain that the in vitro binding of Ric-8 with Galpha reflects an in vivo association, co-immunoprecipitation experiments were performed using embryonic extracts. Ric-8 was detected in immunocomplexes when precipitation was performed with anti-Galpha but not with the pre-immune control, indicating that Ric-8 complexes with Galpha in vivo. To further substantiate this interaction using a different approach, protein extracts from wild-type embryos were incubated with agarose beads coupled to bacterially expressed MBP-Galpha or MBP protein. Ric-8 was detected in the bound complex with MBP-Galpha but not MBP (Wang, 2005).

In Drosophila NBs, Galpha is present in at least two mutually exclusive complexes: a heterotrimeric complex with Gbeta13F, or with a GoLoco-containing protein, Pins, which acts as a GDI for, and can directly interact with Galphai. Conventional G-protein-coupled receptors (GPCRs) promote nucleotide exchange on the Galphai-Gbetagamma heterotrimeric complex, whereas the mammalian non-receptor GEF RIC-8A cannot act on the heterotrimer. To explore the molecular context in which Ric-8 might act on Galpha, whether Ric-8 can complex with Pins or Gbeta13F was examined in Drosophila using co-immunoprecipitation experiments with embryonic extracts. When precipitations were performed using anti-Ric-8, Pins was specifically detected in the immunocomplex; in precipitations using anti-Pins, Ric-8 was also specifically detected. No direct interaction was observed with Ric-8 and Pins in the in vitro binding assays, indicating that Ric-8 complexes with Pins through their mutual interactions with Galpha. To confirm these findings using a different approach, wild-type embryonic extracts were incubated with agarose beads coupled to bacterially expressed MBP-Ric-8 fusion protein. Pins but not Gbeta13F was found in the bound complex with MBP-Ric-8. Thus, Ric-8 preferentially binds to the GDP-Galpha-Pins complex, a similar finding to that seen in C. elegans embryos. This is in contrast to conventional GPCRs, which act on the heterotrimeric complex (Wang, 2005).

To determine the effects of ric-8 loss of function, several mutant alleles were isolated by imprecise excision of a P-element, EY05996. ric-8P587 removes the entire coding region (-953 bp to +1853 bp; ric-8 transcriptional start is +1), whereas ric-8P340 contains a larger deletion with unsequenced breakpoints. Both maternal and zygotic components were removed in the ric-8P340 and ric-8P587 germline clones (GLCs). These mutant embryos showed indistinguishable phenotypes, indicating that both are null alleles. Experiments were performed using embryos that were derived from ric-8P587 GLCs (Wang, 2005).

Galpha shows punctated, cytosolic distribution in dividing and non-dividing NBs of ric-8 GLCs, in contrast to the apical cortical crescents seen in wild-type NBs. Pins also seemed to be cytosolic, which is consistent with findings that Galpha is required for the recruitment of Pins to the cortex. The issue of whether Gbeta13F is also required for membrane targeting of Galpha was examined using a newly generated anti-Galpha antibody, as it was unclear whether the reported inability to detect Galpha in Gbeta13F mutant NBs by immunofluorescence was due to low sensitivity of the previously available antibody. The specificity of this new antibody was demonstrated by the absence of immunoreactivity in Galphai mutant embryos or nota in both immunofluorescence and Western experiment. It was found that Galpha was uniformly localized on the cortex of Gbeta13F GLC NBs, with clearly reduced intensity compared with the wild type. Pins was also uniformly cortical in Gbeta13F GLC NBs, which indicates that the residual Galpha on the cortex is sufficient to recruit Pins. The localization of Galpha and Pins in blastoderm embryos that were derived from ric-8 and Gbeta13F GLCs lends further support to these findings. Strikingly, Galpha and Pins localized as punctated, cytosolic 'spots' in ric-8 GLC embryos, whereas in both wild-type and Gbeta13F GLC embryos, Galpha was membrane associated. Therefore, ric-8, but not Gbeta13F, is crucial for the membrane targeting of Galpha in NBs and other cell types (Wang, 2005).

In ric-8 GLC NBs, Insc was cytosolic. Baz and aPKC localized non-uniformly/asymmetrically on the cortex, but with reduced intensity and often as broader crescents, indicating that residual polarity cues remained. Mira crescents were often mislocalized in metaphase ric-8 NBs; mitotic domain 9 cells failed to re-orient their spindle by 90°, indicating that ric-8 is required for spindle re-orientation in cells of mitotic domain 9. These defects are similar to those seen in Galphai mutant NBs. Ric-8 is also required for the asymmetric division of pI cells. In ric-8 mutant metaphase pI cells, Galpha and Pins did not form the anterior cortical crescents. Similarly, in Galphai metaphase pI cells, the anterior crescent of Pins did not form. In both ric-8 and Galpha mutants, the Pon crescent was undetectable or significantly reduced. Nevertheless, Pon localized at the anterior cortex in anaphase pI cells of both mutants (Wang, 2005).

Antibodies specific for Ric-8 were generated against the amino-terminal (aa 1-150) or carboxy-terminal (aa 425-573) region of Ric-8. Ric-8 was localized to the cytoplasm of NBs throughout the cell cycle, even though Galpha was seen as an apical crescent in mitotic NBs. However, interestingly, Ric-8 was also observed as 'spot'-like structures at the apical cortex of metaphase NBs, partially colocalizing with the Galpha, indicating that their interaction might occur on the cytosolic face of the plasma membrane or in the cytoplasm. Similarly, in pI cells, Ric-8 was also cytosolic throughout the cell cycle (Wang, 2005).

ric-8 GLCs also exhibit abnormal gastrulation, in addition to defects in asymmetric divisions. Since gastrulation defects were also seen in Gbeta13F and Ggamma1 GLC embryos but not in Galphai embryos, the relationship was examined between ric-8 and Gbeta13F. During cellular blastoderm formation, Gbeta13F is delocalized from the cortex and is largely cytosolic in ric-8 GLC embryos, indicating that ric-8 is required for cortical localization of Gbeta13F during these early stages. Consistently, Gbeta13F is also largely cytosolic in NBs throughout the various stages of the cell cycle in stage-10 embryos derived from ric-8 GLCs. Given that Galphai loss of function alone does not disturb Gbeta13F localization and Gbeta13F does not complex with Ric-8, it was hypothesized that Ric-8 mediates the cortical localization of Gbeta13F through its regulation of another Galpha subunit. To further explore this possibility, it was asked whether Ric-8 can complex with Pins in embryos devoid of maternal and zygotic Galphai. If there was another Galpha subunit involved, it might allow Ric-8 to complex with Pins by interacting with both, even in the absence of Galpha. Indeed, Ric-8 complexes with Pins in the absence of Galpha. Given that Ric-8 does not display a direct interaction with Pins, these data indicate that an, as yet unidentified, Galpha subunit that is also regulated by Ric-8 may act (possibly in conjunction with Galpha) to mediate Gbeta13F cortical localization (Wang, 2005).

Gbeta13F protein levels in ric-8 GLCs are significantly reduced compared with wild-type embryos; Galpha and Pins levels remain unaffected. By contrast, Galpha protein levels in Gbeta13F GLCs are reduced, whereas Ric-8 levels do not change in Galpha or Gbeta13F GLCs. Gbetagamma might normally be in excess; therefore, despite the reduction in Gbetagamma levels in ric-8 mutants, sufficient cytosolic levels may remain to stabilize normal levels of Galpha. These data indicate that Ric-8 is required only for membrane targeting of Galpha but not its stability; Gbeta13F is required for the stability of Galpha but not for its membrane targeting. In addition, Ric-8 is involved in both membrane association and the stability of Gbeta13F, possibly by acting through another Galpha subunit (Wang, 2005).

The requirement of Ric-8 for cortical localization and stability of Gbeta13F prompted an examination of whether NB spindle geometry and difference in daughter-cell size are severely disrupted in ric-8 mutants, as shown for Gbeta13F GLCs. In telophase NBs of wild-type stage-10 embryos, the ratio of ganglion mother cell (GMC) and NB (GMC/NB) diameter never exceeded 0.8 (average ratio = 0.42. By contrast, a hallmark of Gbeta13F or Ggamma1 loss is the high frequency of divisions that generate daughters of approximately equal size. These cells are telophase NBs in which the GMC diameter/NB diameter ratio was 0.8 or more (for Gbeta13F NBs, 64% of divisions were similar sized with an average GMC/NB ratio of 0.82. The residual size asymmetry which remained was shown to be due to the reduced levels of asymmetrically localized Par proteins. However, a surprising observation was that, although cortical Gbeta13F localization was disrupted in ric-8 mutant NBs, only 16% of telophase NBs divided into two similar-sized daughter cells, similar to those observed in Galphai mutant NBs. Thus, ric-8 GLC NBs did not display a phenotype similar to that of Gbeta13F loss-of-function mutants. Further removal of Baz (by RNA interference) in ric-8 GLCs resulted in similar-sized division in 94% of NBs, indicating that partially localized Baz (Par proteins) can provide some asymmetry cues in ric-8 mutant NBs. Therefore, Ric-8 probably acts in the same pathway as Galpha to redundantly regulate the difference in daughter-cell size in the Baz pathway. It was shown previously that in Gbeta13F mutants, the number of abdominal Even-skipped positive lateral (EL) neurons in stage-15 embryos was severely decreased, presumably because a high frequency of similar-sized divisions rapidly reduces the cell volume of daughter NBs, resulting in early cessation of divisions. It was found that wild-type embryos produced an average of 9.0 EL neurons per abdominal hemisegment at stage 15; both ric-8 GLCs and Galphai mutants showed a similar reduction of EL neurons. By contrast, Gbeta13F GLC embryos showed a more marked reduction in the numbers of EL neurons. These data indicate that, with respect to both numbers of EL neurons and NB daughter-cell size asymmetry, ric-8 and Galpha mutants exhibit similar phenotypes that are less severe than those seen in Gbeta13F mutants (Wang, 2005).

Two alternative explanations are envisioned for why ric-8 and Gbeta13F mutants have different effects on the asymmetric size of the daughter cells. (1) The generation of functional Gbetagamma may occur even in the absence of ric-8 function, despite the majority of the molecules being cytosolic. (2) Alternatively, the severe phenotypes seen in Gbeta13F or G gamma1 mutant NBs may be an indirect consequence caused by the uniform cortical distribution of Galpha (and Pins); the failure of ric-8 GLC NBs to exhibit a marked decrease in asymmetric daughter size would be because Galpha and Pins are both cytosolic in ric-8 mutants and presumably inactive. To distinguish between these possibilities, ric-8, Gbeta13F double mutant GLC embryos were made in which both ric-8 and Gbeta13F would be completely removed. Interestingly, the double mutant GLC NBs exhibited phenotypes similar to those of ric-8 GLC NBs rather than Gbeta13F GLC NBs. In double GLC NBs, Galpha and Pins are cytosolic, whereas Baz localized non-uniformly/asymmetrically on the cortex. Only 24% of NBs divided into two similar-sized daughter cells. These observations indicate that the cytoplasmic Gbetagamma in ric-8 GLC NBs is non-functional and further suggests that the marked decrease in the difference in daughter-cell size of Gbeta13F GLC NBs is an indirect consequence of the uniform cortical localization of Galpha (and Pins) (Wang, 2005).

These data indicate that ric-8 mutants mediate asymmetric division of NBs and SOPs by regulating heterotrimeric G-protein localization. ric-8 acts at the top of a hierarchy for the sequential membrane/cortical localization of the apical proteins Galphai-Pins-Insc. The role of Ric-8 in membrane targeting of Galpha is novel. Interestingly, Ric-8 also promotes cortical localization of Gbeta13F in Drosophila. These data raise the possibility that this may be mediated indirectly by additional substrate(s) of Ric-8, which are presumably additional Galpha subunit(s). Rat Ric-8A interacts with multiple brain membrane Galpha subunits, including Galpha13, Galphao, Galphaq and Galpha1,2. It is therefore speculated that Ric-8 may control the localization and stability of Gbeta13F by regulating multiple Galpha subunits. Precedence for a role of Galpha in Gbetagamma membrane localization has been reported in mammalian cells (Wang, 2005).

This analyses of ric-8, Galphai, Gbeta and ric-8;Gbeta mutants support the view that, in NBs, cortically localized Galpha mediates asymmetric spindle geometry and asymmetric daughter-cell size, which is positively regulated by Ric-8, and that an important role of Gbetagamma is to restrict Galpha from the basal cortex. In the absence of Gbetagamma, the GoLoco/Galpha complex expands from its normal apical localization, becomes uniformly cortical and can largely override the residual polarity cues that are provided by the asymmetrically localized, but drastically reduced levels of, Par proteins to greatly reduce spindle asymmetry and the difference in daughter size. The residual asymmetry that is present in the absence of Gbeta13F is lost following further removal of Par function. The negative regulation of Galphai by Gbeta13F in Drosophila NBs is similar to that in the C. elegans zygote, in which excess Galpha activity was observed following loss of function of Gbeta or Ggamma. The findings that ric-8 mutants are genetically epistatic to Gbeta mutants, both with respect to Galphai-Pins localization and to spindle geometry, are different from those reported in C. elegans embryos, in which inactivation of Gbetagamma alleviates the requirement for RIC-8 in asymmetric division. This indicates that different mechanisms of heterotrimeric G-protein regulation are present in the asymmetric division of nematode embryos and Drosophila NBs. These findings are consistent with a model in which Ric-8 has a crucial role in Galpha activity by localizing the GoLoco/Galpha complex onto the cortex and/or generating GTP-Galpha as a GEF to mediate spindle geometry. Ric-8 also regulates the cortical localization and activity of Gbeta, possibly through its regulation of multiple Galpha subunits; Gbeta acts to restrict Galpha localization only to the apical cortex. Galpha subunits that are asymmetrically localized at the apical cortex, in conjunction with Par proteins, mediate asymmetric spindle geometry and differences in daughter-cell size (Wang, 2005).

Robust spindle alignment in Drosophila neuroblasts by ultrasensitive activation of pins

Cellular signaling pathways exhibit complex response profiles with features such as thresholds and steep activation (i.e., ultrasensitivity). In a reconstituted mitotic spindle orientation pathway, activation of Drosophila Pins (LGN in mammals) by Gαi is ultrasensitive (apparent Hill coefficient of 3.1), such that Pins recruitment of the microtubule binding protein Mud (NuMA) occurs over a very narrow Gαi concentration range. Ultrasensitivity is required for Pins function in neuroblasts as a nonultrasensitive Pins mutant fails to robustly couple spindle position to cell polarity. Pins contains three Gαi binding GoLoco domains (GLs); Gαi binding to GL3 activates Pins, whereas GLs 1 and 2 shape the response profile. Although cooperative binding is one mechanism for generating ultrasensitivity, it was found GLs 1 and 2 act as 'decoys' that compete against activation at GL3. Many signaling proteins contain multiple protein interaction domains, and the decoy mechanism may be a common method for generating ultrasensitivity in regulatory pathways (Smith, 2011).

Complex input/output relationships generated by cell signaling networks allow for a multitude of cellular decision-making behaviors, such as bistability or hysteresis, which are necessary to implement diverse physiological processes. Ultrasensitivity is a building block for these types of behaviors, yet its molecular origins are poorly understood. While cooperativity is a well-described mechanism to generate ultrasensitivity, this study has uncovered a cellular regulatory system that uses another mechanism for obtaining sigmoidal responses with high apparent Hill coefficients (Smith, 2011).

It was found that activation of the mitotic spindle orientation protein Pins by Gαi is highly ultrasensitive, and this ultrasensitivity arises from a decoy mechanism as binding sites GLs 1 and 2 compete with the activating GL3 for the Gαi input. Cooperativity is commonly thought to be the source for ultrasensitivity in protein-protein interaction networks and protein-DNA interactions. However, the current observations of Pins activation are inconsistent with a cooperative mechanism for three reasons. First, activation of δGL1,2 occurs at a lower Gαi concentration than WT. Second, the sigmoidal response can be largely recapitulated through Gαi binding to GLs 1 and 2 in trans. Lastly, thresholding behavior is entirely dependent on the concentration of Pins present. These findings are supported by mathematical modeling and suggest that ultrasensitive responses can be generated without cooperativity from binary protein-protein interactions through a simple competition mechanism, similar to the competition that occurs in kinase signaling cascade (Smith, 2011).

Although competition and cooperativity are both potential origins of ultrasensitive responses, there are inherent differences between curves created by each of these mechanisms. Cooperativity- based ultrasensitivity can dramatically reduce the amount of input necessary to reach maximal output. For example, initial binding events of O2 are of low affinity and, without cooperativity, would require a large change in O2 concentration for saturation. The competition mechanism described in this study and in kinase cascades generates ultrasensitive responses from a threshold, as activation would occur in a graded fashion at low input concentrations without competition. Therefore, while yielding sigmoidal responses with high apparent Hill coefficients this mechanism may be more important for thresholding than the observed apparent steepness. Modeling studies have shown that multisite phosphorylation builds a good threshold, not necessarily a more switch-like response (although the Hill coefficient is often used as a measure of steepness, this single parameter is also influenced by the threshold). However, multisite phosphorylation is required for the bistable signaling nature of Xenopus oocyte maturation and cell cycle progression (Smith, 2011).

Expressing the nonultrasensitive δGL1,2 Pins failed to fully rescue the spindle positioning defect of the pinsP62 null allele relative to WT Pins, suggesting that ultrasensitive regulation of Pins is important for proper molecular function. The reduced spindle-orienting activity of the graded Pins mutant is caused by decreased pathway output because less apical Mud recruitment and spindle pole dynamics was seen relative to NBs expressing WT Pins. The δGL1,2 Pins spindle phenotype is similar to loss of Lis1 function, an adaptor protein that physically links the Gαi-Pins-Mud complex at the apical cortex to the Dynein motor protein, generating pulling forces on the spindle. Although ultrasensitivity is important for the robust spindle positioning observed in WT NBs, loss of ultrasensitivity had only a minor effect on spindle orientation, as all spindle angles measured in δGL1,2 Pins NBs were within 30° of the apical Pins crescent. This is likely because of redundant spindle-orienting cues in vivo as the mitotic spindle is not completely random in pinsP62 null NBs (Smith, 2011).

Why might ultrasensitive regulation of Pins be required for robust spindle-orienting function? In WT NBs, thresholding limits Pins output to the apical cortex where the Gαi input concentration is high. Thus, Pins is not activated at cortical sites where input concentration is low. In δGL1,2 Pins NBs, thresholding is absent such that Pins output can potentially occur both at the apical cortex and distal cortical regions. Loss of steepness results in only a slight difference in total Pins output between WT and δGL1,2 Pins in vitro (100% versus 85%), but transient activation of δGL1,2 Pins at cortical sites with low Gαi concentration could magnify this difference in vivo by titrating away Mud from the apical cortex. Thus, ultrasensitivity may be an important feature of the Gαi-Pins-Mud spindle orientation pathway, as it allows for generating maximal pathway output through spatial restriction of Pins activity. In this way, competition-based ultrasensitivity allows for increased pathway output by setting concentration thresholds to restrict signaling protein activity and may be a common theme in other regulatory pathways (Smith, 2011).

The modular architecture of signaling proteins is thought to be a means of coupling different inputs with new output functions, allowing for rapid evolution of new signaling functions. This feature is also important for creating signaling proteins that integrate multiple inputs to trigger a specific output. Protein modularity also can create new input/output relationships such as ultrasensitive responses through cooperative interactions between input domains. This analysis of Pins supports this idea but adds that modularity can shape pathway responses without cooperativity, simply by including multiple input domains. In this system it was shown that decoys can build either ultrasensitivity or thresholding depending on the affinities of the decoys relative to the activating site for the input. A high-affinity decoy sets a strong threshold, but lowering the decoy affinity can change thresholding into a more sigmoidal shaped curve, simply by blending the inflection point between thresholding and activation. This type of ultrasensitivity may be a fairly common component of cell signaling pathways, because autoinhibition and domain repeats are common features of cell signaling proteins. Thus, incorporating more domain repeats through genetic recombination events can modulate the response profile. The relative affinities of these sites could then be 'tuned' through point mutations to build thresholding behavior and/or apparent steepness into the signaling pathway (Smith, 2011).

Troponin I and Tropomyosin regulate chromosomal stability and cell polarity

The Troponin-Tropomyosin (Tn-Tm) complex regulates muscle contraction through a series of Ca(2+)-dependent conformational changes that control actin-myosin interactions (see video). Pre-cellular embryos of Troponin I, Tm1 and Tm2 mutants exhibit abnormal nuclear divisions with frequent loss of chromosome fragments. During cellularization, apico-basal polarity is also disrupted as revealed by the defective location of Discs large (Dlg) and its ligand Rapsynoid (Raps; also known as Partner of Inscuteable, Pins). In agreement with these phenotypes in early development, on the basis of RT-PCR assays of unfertilized eggs and germ line mosaics of TnI mutants, it was also shown that TnI is part of the maternal deposit during oogenesis. In cultures of the S2 cell line, native TnI is immunodetected within the nucleus and immunoprecipitated from nuclear extracts. SUMOylation at an identified site (see SUMO) is required for the nuclear translocation. These data illustrate, for the first time, a role for TnI in the nucleus and/or the cytoskeleton of non-muscle cells. It is proposed that the Tn-Tm complex plays a novel function as regulator of motor systems required to maintain nuclear integrity and apico-basal polarity during early Drosophila embryogenesis (Sahota, 2009).

Troponin I (TnI) and Tropomyosin (Tm) are actin-binding proteins that regulate muscle sarcomere contraction. The Tn-Tm complex contains three different Troponin polypeptides, C, T and I, and it regulates acto-myosin interactions in response to the rise of free calcium. Mammals have three genes expressing TnI known as slow twitch (TNNI1), fast twitch (TNNI2) and cardiac (TNNI3). In humans, mutations in TNNI2 and TNNI3 cause distal arthrogryposis type 2B and familial hypertrophic cardiomyopathy, respectively. In Drosophila, viable mutations in the single gene expressing TnI, wings up A (wupA) [also known as held up (hdp)], result in hypercontraction and degeneration of the indirect flight muscles of the thorax due to recessive hypomorphic point mutations. However, studies on lack of function mutations for this gene have been hampered by the fact that null alleles are dominant lethals. Mammals contain four tropomyosin genes, TPM1-4, while Drosophila has two, Tm1 and Tm2. In humans, mutant TPM1 is thought to be responsible for type 3 familial hypertrophic cardiomyopathy, whereas TPM2 is involved in nemaline myopathy and TPM3 has been linked to dominant nemaline myopathy. TPM1 has also been identified as a suppressor of malignant transformation as it is downregulated in mammalian transformed cells, and its expression is abolished in human breast tumors. Indeed, it is widely accepted that actin regulation plays a crucial role in cell motility, which is a key feature in metastatic cancers (Sahota, 2009 and references therein).

Although some of these pathological phenotypes appear unrelated to muscle biology, several lines of evidence indicate that these muscle-specific proteins could have a role in other cell types and processes. For instance, Tm1 is part of the maternal deposit during Drosophila oogenesis, it is required to localize the oskar mRNA at the posterior pole of the oocyte, and later in development it localizes to various cell types including the gut, brain and epidermis. Also, this study demonstrates that TnI RNA is detected in mature unfertilized eggs, which suggests a role in early embryogenesis. Thus, this study set out to analyze early development phenotypes and their mechanisms in TnI and Tm mutants (Sahota, 2009).

This study shows a novel function for the Tn-Tm complex in regulating nuclear divisions during early embryogenesis in Drosophila. Evidence is provided that TnI is required for maintaining stable chromosomal integrity, which was also show for Tm1 and Tm2. Importantly, the three genes seem required for correct epithelial apico-basal polarity; mutant phenotypes include cellularization defects that mislocalize the polarity markers Discs large (Dlg) and its ligand Rapsynoid (Raps) [also known as Partner of Inscuteable (Pins)]. Consistent with the function of these genes in cellularization and spindle integrity, defects in mitosis and chromosome segregation are observed. In a stable cell line, S2, TnI can be detected within the nucleus. Furthermore, the translocation of TnI to the nucleus is dependent upon a mechanism involving SUMOylation. Taken together, these data implicate the Tn-Tm complex in regulating nuclear functions. Moreover, the results suggest that the Tn-Tm complex is required to maintain correct segregation of chromosomes, as disruption of this complex leads to aberrations including chromosome fragment losses. This is the first evidence that the Tn-Tm complex can regulate both nuclear divisions and cell polarity in Drosophila. This is likely to have important implications in cancer progression since chromosomal instability and the generation of aneuploidies are characteristic hallmarks of many cancers (Sahota, 2009).

This study has immunolocalized TnI to the nucleus and shown nuclear phenotypes in the mutants. It should be noted, however, that the nuclear localization, either in the syncitial embryo or the regular S2 cells, seems dependent on the physiological state of the cell and nucleus. Also, with the techniques used in this study, it cannot be determined whether TnI is bound directly to the chromosomes or through intervening proteins. Because the repertoire of HeLa metaphase chromosome-associated proteins does not include TnI, nor other muscle proteins, the observed effects on chromosome integrity might be produced through indirect links. Nevertheless, one should realize that the referred repertoire is also subject to the technical constrains of the purification methods used in the study of HeLa cells (Sahota, 2009).

This study has also shown that the required nuclear translocation is achieved by SUMOylation, at least in the case of TnI. The putative SUMOylation sequence in exon 10 is required for nuclear import. This site, VKEE, is found in the C-termini of all TnI isoforms because it can be incorporated into the protein sequence, either from exon 9 or exon 10. Thus, all TnI isoforms could be tagged for their function. Other putative SUMOylation sites, if actually used for SUMOylation, could provide further functional diversity for TnI. This mechanism for tagging TnI in Drosophila is likely to be conserved in mammals since the VKEE motif is present in the three TnI gene types (slow twitch, fast twitch and cardiac). Although not addressed in this study, it is possible that a similar mechanism might be used to import Tm1 and Tm2 into the nucleus since they contain suitable motifs in the three isoforms of Tm2 and in one of the two isoforms of Tm1 (Sahota, 2009).

This work on the Tn-Tm complex provides an insight into how DNA aberrations and cellularization defects can be linked, and how this complex is crucially required for both DNA and cellular stability. Given that the Tn-Tm complex is also involved in muscle contraction, it appears likely that there may be other processes where disruption of this complex may be detrimental to the development of the organism. In support of this, it has been shown that mutant TnI allele 23437 displays severe defects in axon guidance and fasciculation and that the TnI L9/wupRA isoform rescues these defects. Considering the role of the Tn-Tm complex in sarcomere contraction and the range of phenotypes described in this study, it seems reasonable to propose that TnI, Tm1 and Tm2 are components of a force-generating complex within the nucleus and in the cytoplasm. However, this remains to be determined since the TnI-associated partners have not being investigated in this study (Sahota, 2009).

Being an actin-binding protein, TnI should perform its nuclear functions in association with actin. This protein is known to help RNA polymerase to move during gene transcription. It is currently a matter of debate whether this function requires actin in a globular or a filament structure. However, a recent study reports the interaction of vertebrate fast skeletal TnI with the estrogen receptor during transcription. By analogy to the role that TnI plays in the sarcomere, where the Tn-Tm complex interacts with the actin filaments, it seems likely that during transcription actin has a filament structure, as in the sarcomere thin filament. Actin is also important for morphogenesis of cells and organs in the early embryo, ranging from nuclear divisions and chromosomal segregation in conjunction with myosin, to the regulation of cell shape and movements. All these processes are also relevant to the formation and progression of tumors. In addition, chromosomal instability, mitotic defects and cell polarity defects are characteristic features of many cancers. The fact that TnI, Tm1 and Tm2 all regulate actin strengthens the argument that they execute this regulation as a complex. Defects in all three genes give rise to similar DNA defects, and also to similar defects in apico-basal cell polarity. These common features provide the basis for a mechanism leading to aneuploidy and aberrant cell signaling. That is, molecules that ensure proper actin function during nuclear divisions also ensure that actin correctly regulates cell polarity, which, in turn, is important in proliferation and growth. The tubulin spindle was also affected in the three mutants, indicating that the integrity of the cytoskeletal network may be compromised when any of these molecules are depleted (Sahota, 2009).

In addition to the cytoskeletal network, the localization of Dlg and Pins were also shown to be disrupted in TnI-Tm mutants. Dlg has been described as a neoplastic tumor suppressor and disruption of polarity is a hallmark of cancer progression. The Pins protein is involved in orientation of asymmetric cell divisions, which is important for specifying cell fate. Consistent with the altered Pins expression, spindle orientation defects are observed in the three mutants. Also, spindle orientation is particularly important for specifying neuronal identity in Drosophila neuroblasts. The recycling of molecules for distinct processes is a recurrent theme in development. Indeed, many actin-binding proteins were first identified for their effects on axon guidance and growth, and were subsequently shown to play important roles during cellularization. Also, Dlg was associated with synaptogenesis before its role in cellularization was determined. The novel function for the Tn-Tm complex uncovered in this study might have opened the way to reveal requirements in other actin-associated events. It was observed that TnI, as well as Tm1 and Tm2, are crucial for the correct development of the central nervous system. Further studies on the role of the Tn-Tm complex during nuclear divisions seem appropriate towards understanding how these proteins affect cell proliferation, and might provide novel targets for controlling cell divisions (Sahota, 2009).

Sgt1 acts via an LKB1/AMPK pathway to establish cortical polarity in larval neuroblasts

Drosophila neuroblasts are a model system for studying stem cell self-renewal and the establishment of cortical polarity. Larval neuroblasts generate a large apical self-renewing neuroblast, and a small basal cell that differentiates. A genetic screen was performed to identify regulators of neuroblast self-renewal, and a mutation was identified in sgt1 (suppressor-of-G2-allele-of-skp1) that had fewer neuroblasts. sgt1 neuroblasts have two polarity phenotypes: failure to establish apical cortical polarity at prophase, and lack of cortical Scribble localization throughout the cell cycle. Apical cortical polarity was partially restored at metaphase by a microtubule-induced cortical polarity pathway. Double mutants lacking Sgt1 and Pins (a microtubule-induced polarity pathway component) resulted in neuroblasts without detectable cortical polarity and formation of 'neuroblast tumors.' Mutants in hsp83 (encoding the predicted Sgt1-binding protein Hsp90), LKB1 (PAR-4), or AMPKα all show similar prophase apical cortical polarity defects (but no Scribble phenotype), and activated AMPKα rescued the sgt1 mutant phenotype. It is proposed that an Sgt1/Hsp90-LKB1-AMPK pathway acts redundantly with a microtubule-induced polarity pathway to generate neuroblast cortical polarity, and the absence of neuroblast cortical polarity can produce neuroblast tumors (Anderson, 2012).

This study presents evidence that the evolutionary-conserved protein Sgt1 acts with Hsp90, LKB1 and AMPK to promote apical localization of the Par and Pins complexes in prophase neuroblasts. It is proposed that Sgt1/Hsp90 proteins function together based on multiple lines of evidence: (1) they show conserved binding from plants to humans; (2) the sgt1s2383 mutant results in a five amino acid deletion within the CS domain, which is the Hsp90 binding domain; (3) sgt1 and hsp83 have similar cell cycle phenotypes; and (4) sgt1 and hsp83 have similar neuroblast polarity phenotypes. The Sgt1/Hsp90 complex either stabilizes or activates client proteins (Zuehlke, 2010); it is suggested that Sgt1 activates LKB1, rather than stabilizing it, because it was not possible to rescue the sgt1 mutant phenotype by simply overexpressing wild type LKB1 protein. No tests were performed for direct interactions between Sgt1 and LKB1 proteins, and thus the mechanism by which Sgt1 activates LKB1 remains unknown (Anderson, 2012).

LKB1 is a 'master kinase' that activates at least 13 kinases in the AMPK family. It is suggested that LKB1 activates AMPK to promote neuroblast polarity because overexpression of phosphomimetic, activated AMPKα can rescue the lkb1 and sgt1 mutant phenotype. It remains unclear how AMPK activity promotes apical protein localization. An antibody to activated AMPKα (anti-phosphoT385-AMPKα shows spindle and cytoplasmic staining that is absent in ampkα mutants, and centrosomal staining that persists in AMPKα null mutants, but no sign of asymmetric localization in neuroblasts. AMPK activity is thought to directly or indirectly activate myosin regulatory light chain to promote epithelial polarity. AMPK is activated by a rise in AMP/ATP levels that occur under energy stress or high metabolism; AMP binds to the γ regulatory subunit of the heterotrimeric complex and results in allosteric activation of the α subunit. ampkα mutants grown under energy stress have defects in apical/basal epithelial cell polarity in follicle cells within the ovary. In contrast, AMPKα mutants grown on nutrient rich food still show defects in embryonic epithelial polarity, neuroblast apical polarity, and visceral muscle contractio. Larval neuroblasts, embryonic ectoderm, and visceral muscle may have a high metabolic rate, require low basal AMPK activity, or use a different mechanism to activate AMPK than epithelial cells. What are the targets of AMPK signaling for establishing apical cortical polarity in larval neuroblasts? AMPK could directly phosphorylate Baz to destabilize the entire pool of apical proteins, but currently there is no evidence supporting such a direct model. AMPK may act via regulating cortical myosin activity: clear defects have been seen in cortical motility, ectopic patchy activated myosin at the cortex, and failure of cytokinesis in sgt1, lkb1, and ampkα mutants. This strongly suggests defects in the regulation of myosin activity, but how or if gain/loss/mispositioning of myosin activity leads to failure to establish apical cortical polarity remains unknown. Lastly, the defects in apical cell polarity seen at prophase could be due to the prometaphase cell cycle delays (Anderson, 2012).

What activates the Sgt1-LKB1-AMPK pathway to promote cell polarity during prophase? In budding yeast, Sgt1 requires phosphorylation on Serine 361 (which is conserved in Drosophila Sgt1) for dimerization and function (Bansal, 2009); this residue is conserved in Drosophila Sgt1 but its functional significance is unknown (Anderson, 2012).

Sgt1/Hsp90/LKB1/AMPK are all required for apical Par/Pins complex localization, but Sgt1 must act via a different pathway to promote Dlg/Scrib cortical localization, because only the sgt1 mutant affects Dlg/Scrib localization, and overexpression of activated AMPKα is unable to restore cortical Scrib in sgt1 mutants. The mechanism by which Sgt1 promotes Dlg/Scrib cortical localization is unknown (Anderson, 2012).

This study has shown that sgt1 mutants lack Par/Pins apical polarity in prophase neuroblasts, but these proteins are fairly well polarized in metaphase neuroblasts. The rescue of cortical polarity is microtubule dependent, probably occurring via the previously described microtubule-dependent cortical polarity pathway containing Pins, Dlg and Khc-73. The weak polarity defects still observed in sgt1 metaphase neuroblasts may be due to the poor spindle morphology. The lack of microtubule-induced polarity at prophase, despite a robust microtubule array in prophase neuroblasts, suggests that the microtubule-induced cortical polarity pathway is activated at metaphase. Activation of the pathway could be via expression of the microtubule-binding protein Khc-73; via phosphorylation of Pins, Dlg or Khc-73 by a mitotic kinase like Aurora A; or via a yet unknown pathway (Anderson, 2012). e

Prefoldin and Pins synergistically regulate asymmetric division and suppress dedifferentiation

Prefoldin is a molecular chaperone complex that regulates tubulin function in mitosis. This study shows that Prefoldin depletion results in disruption of neuroblast polarity, leading to neuroblast overgrowth in Drosophila larval brains. Interestingly, co-depletion of Prefoldin and Partner of Inscuteable (Pins) leads to the formation of gigantic brains with severe neuroblast overgrowth, despite that Pins depletion alone results in smaller brains with partially disrupted neuroblast polarity. This study shows that Prefoldin acts synergistically with Pins to regulate asymmetric division of both neuroblasts and Intermediate Neural Progenitors (INPs). Surprisingly, co-depletion of Prefoldin and Pins also induces dedifferentiation of INPs back into neuroblasts, while depletion either Prefoldin or Pins alone is insufficient to do so. Furthermore, knocking down either α-tubulin or β-tubulin in pins- mutant background results in INP dedifferentiation back into neuroblasts, leading to the formation of ectopic neuroblasts. Overexpression of α-tubulin suppresses neuroblast overgrowth observed in prefoldin pins double mutant brains. These data elucidate an unexpected function of Prefoldin and Pins in synergistically suppressing dedifferentiation of INPs back into neural stem cells (Zhang, 2016).

Control of tissue homeostasis is a central issue during development. The neural stem cells, or neuroblasts, of the Drosophila larval brain is an excellent model for studying stem cell homeostasis. Asymmetric division of neuroblasts generates a self-renewing neuroblast and a different daughter cell that undergoes differentiation pathway to produce neurons or glia. Following each asymmetric division, apical proteins such as aPKC are segregated into the neuroblast daughter and function as 'proliferation factor', while basal proteins are segregated into a smaller daughter cell to act as 'differentiation factors'. At the onset of mitosis, the Partitioning defective (Par) protein complex that is composed of Bazooka (Baz)/Par3, Par6 and atypical protein kinase C (aPKC) is asymmetrically localized at the apical cortex of the neuroblast. Other apical proteins including Partner of Inscuteable (Pins), the heterotrimeric G protein Gαi, and Mushroom body defect (Mud) also accumulate at the apical cortex through an interaction of Inscuteable (Insc) with Par protein complex. Apical proteins control basal localization of cell fate determinants Numb, Prospero (Pros), Brain tumor (Brat) and their adaptor proteins Miranda (Mira) and Partner of Numb (Pon) that are segregated into the ganglion mother cell (GMC) following divisions. Apical proteins and their regulators also control mitotic spindle orientation to ensure correct asymmetric protein segregation at telophase. Several centrosomal proteins, Aurora A, Polo and Centrosomin, regulate mitotic spindle orientation (Zhang, 2016).

There are at least two different types of neuroblasts that undergo asymmetric division in the larval central brain. Perturbation of asymmetric division in either type of neuroblast can trigger neuroblast overproliferation and/or the induction of brain tumors. The majority of neuroblasts are type I neuroblasts that generate a neuroblast and a GMC in each division, while type II neuroblasts generate a neuroblast and an intermediate neural progenitor (INP), which undergoes three to five rounds of asymmetric division to produce GMCs. Ets transcription factor Pointed (PntP1 isoform), exclusively expressed in type II neuroblast lineages, promotes the formation of INPs. Failure to restrict the self-renewal potential of INPs can lead to dedifferentiation, allowing INPs to revert back into 'ectopic neuroblasts'. Notch antagonist Numb and Brat function cooperatively to promote the INP fate. Loss of brat or numb leads to 'ectopic type II neuroblasts' originating from uncommitted immature INPs that failed to undergo maturation. A zinc-finger transcription factor Earmuff functions after Brat and Numb in immature INPs to prevent their dedifferentiation. Earmuff also associates with Brahma and HDAC3, which are involved in chromatin remodeling, to prevent INP dedifferentiation. However, the underlying mechanism by which INPs possess limited developmental potential is largely unknown (Zhang, 2016).

Prefoldin (Pfdn) was first identified as a hetero-hexameric chaperone consisting of two α-like (PFDN3 and 5) and four β-like (PFDN 1, 2, 4 and 6) subunits, based on its ability to capture unfolded actin (Vainberg, 1998). Prefoldin promotes folding of proteins such as tubulin and actin by binding specifically to cytosolic chaperonin containing TCP-1 (CCT) and by directing target proteins to it. The yeast homologs of Prefoldin 2–6, named GIM1-5 (genes involved in microtubule biogenesis) are present in a complex that facilitates proper folding of α-tubulin and γ-tubulin. All Prefoldin subunits are phylogenetically conserved from Archaea to Eukarya. Structural study of the Prefoldin hexamer from the archaeum M. thermoautotrophicum showed that Prefoldin forms a jellyfish-like shape consisting of a double β barrel assembly with six long tentacle-like coiled coils that participate in substrate binding. The function of Prefoldin as a chaperone has also been illustrated in lower eukaryotes like C. elegans, in which loss of prefoldin resulted in defects in cell division due to reduced microtubule growth rate. Depletion of PFDN1 in mice displayed cytoskeleton-related defects, including neuronal loss and lymphocyte development defects. The only Prefoldin subunit in Drosophila that has been characterized to date, Merry-go-round (Mgr), the Pfdn3 subunit, cooperates with the tumor suppressor Von Hippel Lindau (VHL) to regulate tubulin stability (Delgehyr, 2012). However, the functions of Prefoldin in the nervous system remain elusive (Zhang, 2016).

This study describes the critical role of evolutionarily-conserved Prefoldin complex in regulating neuroblast and INP asymmetric division and suppressing INP dedifferentiation. Mutants for two Prefoldin subunits, Mgr and Pfdn2, displayed neuroblast overgrowth with defects in cortical polarity of Par proteins and microtubule-related abnormalities. Interestingly, co-depletion of Pins in mgr or pfdn2 mutants led to massive neuroblast overgrowth. Prefoldin and Pins synergistically regulate asymmetric division of both neuroblasts and INPs. Surprisingly, they also synergistically suppress dedifferentiation of INPs back into neuroblasts. Knocking down tubulins in pins mutant background resulted in severe neuroblasts overgrowth, mimicking that caused by co-depletion of Prefoldin and Pins. These data provide a new mechanism by which Prefoldin and Pins regulates neural stem cell homeostasis through regulating tubulin stability in both neuroblasts and INPs (Zhang, 2016).

pfdn2/CG6302, encoding a Prefoldin β-like subunit, was identified from a RNA interference (RNAi) screen in larval brains. Ectopic neuroblasts labeled by a neuroblast marker, Deadpan (Dpn), were formed upon knocking down pfdn2 under a neuroblast driver insc-Gal4. Only one neuroblast was observed in control type I neuroblast lineages using insc-Gal4 and type II neuroblast lineages using worniu-Gal4 with asense (ase)-Gal80. In contrast, upon pfdn2 RNAi excess neuroblasts were observed in both type I neuroblast lineages and type II neuroblast lineages, respectively. To verify the function of Pfdn2 in neuroblasts, a putative hypomorphic allele of pfdn2, pfdn201239, was analyzed that has a P element inserted at the 5′ untranslated region (UTR) of pfdn2. Hemizygous larval brains of pfdn201239 over Df(3L)BSC457 (referred to as pfdn2 thereafter) displayed 235.3 ± 31.7 neuroblasts per brain hemisphere, suggesting that Pfdn2 inhibits the formation of ectopic neuroblasts in larval brains. Consistently, an increase of EdU (5-ethynyl-2′-deoxyuridine)-incorporation was also observed in pfdn2 mutants compared to the control. To generate pfdn2 null alleles, a P element, EY06124, was mobilized. Its imprecise excision yielded two loss-of-function alleles, pfdn2Δ10 and pfdn2Δ17, both deleting the entire opening reading frame (ORF) of pfdn2. pfdn2Δ10 and pfdn2Δ17 mutants survive to pupal stage and display strong phenotypes with ectopic neuroblasts labeled by Dpn. These phenotypes in pfdn2Δ10 and pfdn2Δ17 mutant brains can be fully rescued by overexpression of wild-type pfdn2 or pfdn2-Venus transgene. Pfdn2 is abundantly expressed in neuroblasts, INPs and their immediate neural progeny- GMCs, detected by a specific antibody generated against Pfdn2 full length and a transgenic Pfdn2 with a Venus tag at the C-terminus. In addition, Pfdn2 expression under the tubulin-Gal4 fully rescued the lethality of both pfdn2Δ10 and pfdn2Δ17 mutants. Pfdn2 protein was undetectable in pfdn2Δ10 zygotic mutants, further supporting that it is a null allele. Both type I and type II MARCM (Mosaic Analysis with Repressible Cell Marker) clones of pfdn2Δ10 generated excess neuroblasts. These phenotypes were slightly weaker than pfdn2Δ10 zygotic mutants, likely due to residual Pfdn2 protein in the clones. These data indicate that Pfdn2 is required in both type I and type II neuroblast lineages to prevent the formation of ectopic neuroblasts (Zhang, 2016).

This study has identified an unexpected synergism between Prefoldin and Pins in suppressing neuroblasts overgrowth. Barious subunits of Prefoldin complex are implicated in asymmetric division of neuroblasts, especially during asymmetric protein segregation at telophase. It is known that depletion of Pins results in the formation of smaller larval brains, despite partial loss of neuroblasts polarity. Interestingly, co-depletion of Pfdn2 and Pins results in severe neuroblasts overgrowth, while Pfdn2 depletion alone only causes mild brain overgrowth. This phenotype is contributed by a combination of loss of neuroblast polarity, defects of asymmetric division of INPs, as well as INP dedifferentiation. Knocking down tubulins in pins mutant background mimics the co-depletion of Prefoldin and Pins, suggesting that tubulin stability appears to be critical for the suppression of neuroblast overgrowth in the absence of Pins function. The data also suggest that Prefoldin function and tubulin stability in INPs are important to suppress their dedifferentiation back into neuroblasts (Zhang, 2016).

How microtubules induce cortical polarity is poorly understood in Drosophila neuroblasts. Previously, one report showed that kinesin Khc-73, which localized at the plus end of astral microtubules, and Discs large (Dlg) induced cortical polarization of Pins/Gαi in neuroblasts. However, microtubules are considered not essential for neuroblast polarity. This study shows that Drosophila Prefoldin regulates asymmetric division of both neuroblasts and INPs through tubulins, suggesting an important role of microtubules in neuroblast polarity. The essential role of microtubules directly regulating cell polarity is found in various systems. During C. elegans meiosis, a microtubule-organizing center is necessary and sufficient for the establishment of the anterior-posterior polarity. In the fission yeast Schizosaccharomyces pombe, interphase microtubules directly regulate cell polarity through proteins such as tea1p. In mammalian airway cilia, microtubules are required for asymmetric localization of planer cell polarity proteins (Zhang, 2016).

This study shows that the role of Drosophila Prefoldin complex in regulating asymmetric division is very likely dependent on microtubules. This is consistent with the known essential role of Prefoldin for maintaining tubulin levels in various organisms such as yeast, C. elegans, plants and mammals. In yeast, Gim (Prefoldin) null mutants become super-sensitive to the microtubule-depolymerizing drug benomyl as a result of a reduced level of α-tubulin. In the absence of Prefoldin, the function of the chaperone pathway is damaged and unable to fold sufficient amount of tubulins for normal yeast growth. In C. elegans, reducing Prefoldin function causes defects in cell division presumably due to the reduction of tubulin levels and microtubule growth rate. Genetic analysis of mammalian Prefoldin also suggests that cytoskeletal proteins like actin and tubulin make up the major substrate of Prefoldin in mammals. These studies in different organisms together suggest that Prefoldin complex plays a conserved central role in tubulin folding (Zhang, 2016).

'Telophase rescue', a term refers to the phenomenon that protein mis-localization at metaphase is completely restored at telophase, is observed in many mutants that affect neuroblast asymmetric division. However, both apical and basal proteins are still mis-segregated in pfdn2 and mgr mutants, suggesting that 'telophase rescue' is defective in these mutants. Telophase rescue is regulated by TNF receptor-associated factor (DTRAF1), which binds to Baz and acts downstream of Egr/TNF. Telophase rescue also depends on Worniu/Escargot/Snail family proteins and a microtubule-dependent Khc-73/Dlg pathway. Pins did not form a protein complex with Mgr, α-tubulin or β-tubulin in co-immunoprecipitation assay. Given that Dlg is a Pins-interacting protein, Prefoldin appears to function in a different pathway with Dlg or Khc-73 during asymmetric division (Zhang, 2016).

Recently, merry-go-round (mgr), encoding Prefoldin 3 (Pfdn3)/VBP1/Gim2 subunit, was reported to regulate spindle assembly. Loss of mgr led to formation of monopolar mitotic spindles and loss of centrosomes because of improper folding and destabilization of tubulins. The current analysis on Pfdn2 indicates that pfdn2 mutants displayed similar spindle and centrosome abnormalities. In addition, the incorrectly folded tubulin due to loss of mgr may be eliminated by Drosophila von Hippel Lindau protein (Vhl), an E3 ubiquitin-protein ligase. Interestingly, the data suggest that Prefoldin has a tumor-suppressor like function in preventing neuroblast overgrowth. However, Drosophila Vhl is not important for brain tumor suppression, as its loss-of-function neither affects number of neuroblasts nor suppresses overgrowth observed in pfdn2 RNAi or mgr RNAi (Zhang, 2016).

This study shows a novel synergism between Prefoldin and Pins in suppressing dedifferentiation of INPs back into neuroblasts. Prefoldin and Pins apparently suppress dedifferentiation through regulating tubulin levels. It is likely that appropriate tubulin levels in INPs are important for their differentiation, while reducing tubulin levels can increase the risk of INP dedifferentiation. Currently, several cell fate determinants such as Brat, Numb and the SWI/SNF chromatin remodeling complex with its cofactors Erm and Hdac3 are critical to suppress INP dedifferentiation back into neuroblast. It is currently unknown whether or how Prefoldin/Pins are linked to these known suppressors of dedifferentiation. It is possible that symmetric division of INPs causes reduced levels of Brat and Numb in these abnormal INP daughters, leading to their dedifferentiation. Alternatively, Prefoldin might regulate transcription of genes within INPs to suppress dedifferentiation. It was reported that the human homolog of Pfdn5, MM-1, has a role in transcriptional regulation by binding to the E-box domain of c-Myc and represses E-box-dependent transcriptional activity. Interestingly, Prefoldin Subunit 5 gene is deleted in Canine mammary tumors, suggesting that it may be a tumor suppressor gene. This study has revealed a novel mechanism by which Prefoldin and Pins function through tubulin stability to suppress stem cell overgrowth. It is expected to contribute to the understanding of mammalian/human Prefoldin function in tumorigenesis (Zhang, 2016).

Protein Interactions

Interaction of rapsynoid/partner of inscuteable with inscuteable

If the yeast two-hybrid interaction reflects an interaction in vivo, it should be possible to identify an embryonic complex containing both Rapsynoid/Partner of Inscuteable and Inscuteable. To show the existence of this complex, a transgenic fly strain was used that is induced by heat shock to ubiquitously express a FLAG-tagged version of Insc. This engineered version of Insc is fully functional and its expression can rescue the defects associated with insc loss of function. Protein extracts were prepared from heat-shocked transgenic embryos and extracts prepared from non-heat-shocked transgenic embryos processed in parallel were used as controls. An anti-FLAG immunoaffinity column was used for both control and experimental extracts. Raps, which runs as an ~75 kDa band that is absent in extracts from raps minus embryos, specifically copurifies with the extract prepared from the heat-shocked transgenic embryos containing the FLAG-tagged Insc and not with the control extract. These results indicate that Insc and Raps/Pins interact either directly or indirectly in vivo (Yu, 2000a).

To further characterize the Raps/Insc interaction and to ascertain whether it might be direct, in vitro-translated 35S-labeled full-length Raps was incubated with sepharose beads coupled to GST and various Insc-GST fusion proteins. Raps is able to bind to all Insc-GST fusion proteins containing the asymmetric localization domain. To further characterize this interaction, 35S-labeled full-length Raps/Pins (FL-Pins), as well as the N-terminal portion (N-Pins, containing the seven TPR repeats, aa 1-378) and the C-terminal portion (C-Pins, aa 364-658) of Pins, were produced by in vitro translation. The translation products were incubated with sepharose beads coupled to a full-length Insc-GST fusion protein. Full-length and N-terminal Pins can bind Insc-GST, whereas C-terminal Pins can not bind to Insc-GST. These results are consistent with the data from the two yeast two-hybrid assays; together, they indicate that the TPR repeat-containing the N-terminal region of Raps/Pins is necessary and sufficient for direct interaction with the Insc asymmetric localization domain in vitro (Yu, 2000a).

As a first step toward understanding the role raps might play with respect to the genes that are known to be involved in asymmetric cell divisions, Pins/Raps distribution was examined in embryos homozygous for loss-of-function alleles of miranda, prospero, partner of numb, numb, and insc. With the exception of insc mutants, Raps expression is wild type (WT) in these mutants. In insc null embryos, Raps distribution is no longer asymmetric in mitotic NBs as well as dividing cells of mitotic domain 9; Raps distribution is primarily cortical and the intensity of anti-Raps staining is also strongly reduced. Hence asymmetric localization of Raps requires insc (Yu, 2000a).

The apical localization of Insc involves and a maintenance step that requires Bazooka and Partner of Inscuteable

The asymmetry of neuroblast cell divisions might arise from neuroblast-specific expression of the proteins required for asymmetric division. Alternatively, both neuroblasts and neuroepithelial cells could be capable of dividing asymmetrically, but in neuroepithelial cells other polarity cues might prevent asymmetric division. By disrupting adherens junctions the symmetric epithelial division of epidermal cells can be changed into asymmetric division. The adenomatous polyposis coli (APC) tumor suppressor protein is recruited to adherens junctions, and both APC and microtubule-associated EB1 homologs are required for the symmetric epithelial division along the planar axis. These results indicate that neuroepithelial cells have all the necessary components to execute asymmetric division, but that this pathway is normally overridden by the planar polarity cue provided by adherens junctions (Lu, 2001).

Drosophila neuroblasts delaminate from a polarized epithelial layer in the ventral neuroectoderm and divide asymmetrically along the apical-basal axis to produce larger apical neuroblasts and smaller basal ganglion mother cells. Inscuteable (Insc) as a central protein in organizing neuroblast division. Insc provides positional information that couples mitotic spindle orientation with the basal localization of cell-fate determinants such as Numb and Prospero together with their respective adaptor proteins Partner of Numb (Pon) and Miranda (Lu, 2001 and references therein).

The apical localization of Insc involves both a Baz-dependent initiation step and a maintenance step that requires Baz and Partner of Inscuteable (Pins). The expression of Baz and Pins in both neuroblasts and neuroepithelial cells suggests that these cells share certain apical-basal polarity information. Consistent with this notion is the observation that, when Pon is expressed ectopically in epithelial cells it is localized to the basal cortex, as in neuroblasts. Unlike neuroblasts, however, epithelial cells divide symmetrically along the planar axis and segregate ectopic Pon equally between the two daughter cells. These observations raise further questions: do epithelial cells have the ability to couple spindle orientation with protein localization, and segregate proteins asymmetrically between two unequally sized daughter cells? If so, what prevents them from executing this asymmetric division (Lu, 2001)?

To characterize epithelial division by monitoring it in live embryos, transgenic embryos expressing Pon and tau proteins fused with green fluorescent protein (GFP) were used. During epithelial cell cycle, tau-GFP-labelled mitotic spindle is formed along the planar axis of the embryo, and Pon-GFP is initially uniformly associated with the cortex and then localized to a basal crescent. The mitotic spindle remains oriented along the planar axis throughout mitosis. After cytokinesis, the Pon-GFP crescent is bisected by the cleavage furrow and is equally distributed between two equally sized daughter cells. This in vivo analysis shows that the machinery for basal protein localization is intact in epithelial cells, but it is uncoupled from spindle orientation (Lu, 2001).

Double-stranded (ds) CRB RNA was injected into transgenic embryos expressing Pon-GFP and tau-GFP. In about 70% (n = 200) of crb(RNAi) embryos, the organization of the ectodermal epithelium is disrupted, with epithelial cells losing their columnar shape, adopting rounded morphology, and becoming separated from each other. Live imaging of epithelial divisions in these embryos reveals that nearly all the epithelial cells show a tight coupling between the positioning of Pon-GFP crescents and the orientation of the mitotic spindle. Pon-GFP crescents were found at basal and lateral positions and less frequently at apical positions on the cell cortex, and one of the spindle poles was positioned underneath the Pon-GFP crescent (Lu, 2001).

After cytokinesis, Pon-GFP was segregated to one of the two similarly sized daughter cells. Asymmetric segregation of Pon-GFP to one of two similarly sized daughter cells was also observed in crb zygotic mutant embryos. Immunostaining of crb(RNAi) embryos with antibodies against Asense, Prospero and Insc indicates that epithelial cells do not express these neuronal markers, suggesting that the ability of these cells to undergo asymmetric division is not a result of cell-fate change (Lu, 2001).

Overexpression of the membrane-bound cytoplasmic tail of Crb (Crb-intra) causes similar disorganization of the epithelium as seen in crb mutants. The effect of overexpressing Crb-intra on epithelial division was examined. As observed in crb(RNAi) embryos, epithelial cells overexpressing Crb-intra show coupling of the mitotic spindle with the Pon-GFP crescent and asymmetric segregation of Pon-GFP to one of the daughter cells. Thus, when the formation of the adherens junction is disrupted, epithelial cells switch from a symmetric to an asymmetric division pattern (Lu, 2001).

In addition to its function in localizing Insc and regulating division axis in the neuroblasts, Baz is also required for the formation of adherens junction and the maintenance of epithelial polarity. The function of Baz in epithelial division was examined. The baz(RNAi) embryos showed overall disruption of epithelium organization similar to that observed in crb(RNAi) embryos. Unlike in crb(RNAi) embryos, however, epithelial cells in baz(RNAi) embryos divide in a symmetric fashion, with Pon-GFP distributed uniformly around the cell cortex throughout mitosis and the mitotic spindle orients in random directions. After cytokinesis, two equally sized daughter cells are produced and Pon-GFP is equally distributed between them (Lu, 2001).

Daughter cell size asymmetry in neuroblast division is largely unaffected in baz(RNAi) embryos. In crb(RNAi) epithelial cells Baz can still be localized into a crescent but the crescent is mispositioned and Pon-GFP is always localized to the opposite side of the Baz crescent. This suggests that, although mispositioned, Baz is still functional in directing Pon-GFP localization in crb(RNAi) embryos. To test whether the coupling of Pon-GFP localization with spindle orientation observed in crb(RNAi) embryos is Baz dependent, double RNAi was performed by co-injecting a mixture of baz and crb dsRNAs. Epithelial divisions in the co-injected embryos appeared similar to baz single-injected embryos, with Pon-GFP segregated equally between two equally sized daughter cells. It is therefore concluded that epithelial cells depend on Baz to couple spindle orientation with protein localization when the adherens junction is disrupted (Lu, 2001).

To investigate the molecular mechanism underlying the planar positioning of spindles by the adherens junction, the function of proteins associated with the adherens junction was examined. A ubiquitously expressed, epithelial-cell-enriched APC (E-APC) is localized to the adherens junction, and, in shotgun and crb mutants, this adherens junction localization of E-APC is disrupted. The human APC protein interacts with a microtubule-associated EB1 protein, and the yeast homolog of EB1 (Bim1), together with the cortical marker Kar9, has been implicated in a search-and-capture mechanism of spindle positioning. Therefore, the function of E-APC in epithelial cell division was tested (Lu, 2001).

In about 60% of E-APC(RNAi) embryos, the positioning of Pon-GFP crescent and orientation of mitotic spindle became tightly coupled during epithelial division. At cytokinesis, epithelial cells divided asymmetrically to produce two unequally sized daughter cells, and Pon-GFP was always segregated to the smaller daughter cell. The asymmetric segregation of Pon-GFP and the ability to undergo unequal cytokinesis all depend on Baz, because in baz and E-APC double RNAi embryos, Pon-GFP is equally segregated to two similarly sized daughter cells. Therefore, in the absence of E-APC, epithelial cells divide asymmetrically in a Baz-dependent fashion. This suggests that adherens-junction-associated E-APC promotes spindle positioning along the planar axis and prevents the coupling of spindle positioning with asymmetric basal protein localization (Lu, 2001).

To test whether E-APC functions with EB1 to orient the mitotic spindle, RNAi was performed on a closely related fly homolog of EB1 (dEB1). In dEB1(RNAi) embryos, the epithelial divisions are also asymmetric, producing two unequally sized daughter cells, with Pon-GFP segregated to the smaller cell. The penetrance of dEB1(RNAi) phenotype (20%) is lower than that of E-APC(RNAi). Since there is strong maternal contribution of dEB1, the low penetrance might be due to a perdurance of maternal dEB1 protein. Alternatively, it might be due to functional compensation by two other distantly related EB1 homologs in the fly genome. It has been noted that E-APC lacks the carboxy-terminal domain, which is required for interaction with EB1, and no direct interaction between E-APC and EB1 could be detected in in vitro binding assays. It therefore remains to be determined whether the two are functionally linked together in vivo through some cofactor(s), or whether E-APC functions mainly to maintain adherens junction integrity and EB1 interacts with other unidentified molecules to orient spindles (Lu, 2001).

These results indicate that two sets of polarity cues exist for spindle positioning in epithelial cells: a planar polarity cue mediated by the adherens junction and an apical-basal polarity cue regulated by Baz. The division pattern of wild-type epithelial cells suggests that the planar polarity cue is normally dominant over the apical-basal polarity cue. Epithelial cells within the procephalic neurogenic region (PNR) that express endogenous Insc or epithelial cells outside of the PNR that express ectopic Insc are known to orient their mitotic spindle along the apical-basal axis during division. This suggests that the dominance of planar polarity over apical-basal polarity can be overcome by the expression of Insc. The normal appearance of the adherens junction in epithelial cells in the PNR, together with the observation that these cells divide along the planar axis and maintain their normal monolayer organization in an insc mutant, suggests that Insc functions by strengthening the apical-basal polarity instead of weakening the planar polarity through changing the behavior of the adherens junction (Lu, 2001).

When neuroblasts delaminate from the epithelium layer, they undergo morphological changes from columnar to round shape, lose their contacts with the surrounding cells and thus the adherens junction structures. This situation may be reminiscent of epithelial cells in adherens-junction mutants in which the planar polarity cue is lost. In both cases, the Baz-mediated polarity pathway takes over. That one polarity cue can dominate over another cue in orienting axis division may have its precedent in other organisms. Budding yeast can divide in either an axial or a bipolar pattern. Mutations in genes such as AXL1, BUD3, BUD4 and BUD10/AXL2 result in loss of polarity cue for axial bud formation and the cells divide in a bipolar fashion. This suggests that axial and bipolar cues coexist and that the axial cue is normally dominant over the bipolar cue. During mammalian cortical neurogenesis, neural progenitors switch from early symmetric divisions to later asymmetric divisions. It will be interesting to determine whether similar mechanisms and molecules are used to control this division symmetry switch in mammals. These results on E-APC highlight the importance of tumor suppressors in regulating not only cell growth but also polarity and asymmetric division (Lu, 2001).

Partner of Inscuteable interacts directly with Discs-large in the establishment planar polarity during asymmetric cell division in Drosophila

In the dorsal thorax (notum) of the Drosophila pupa, the pI cell divides unequally with its spindle axis aligned with the anterior-posterior (a-p) axis of the fly body. It produces two different daughter cells, pIIa and pIIb. During this division, Numb and its adaptor protein Partner of Numb (Pon) form an anterior crescent and segregate unequally into the anterior pIIb cell. In fz mutant pupae, the division of the pI cell is oriented randomly relative to the a-p axis and the Numb crescent does form, but at a random position. Thus, Fz is not required to establish planar asymmetry per se, but is necessary to orient the axis of the asymmetric cell division. This indicates that additional genes may be required for establishing, rather than orienting, planar asymmetry in the pI cell (Bellaïche, 2001 and references therein).

Fz organizes the actin cytoskeleton at the site of hair formation. Planar polarity in the pI cell is established by a mechanism that involves a remodeling of the previously established apical-basal polarity. During the pI cell division, Baz and DaPKC relocalize from the apical cortex to the posterior lateral cortex, while Discs large (Dlg) and Pins accumulate asymmetrically at the anterior lateral cortex. This redistribution along the a-p axis leads to the formation of two complementary planar domains at the cell cortex. This mechanism of polarity establishment is distinct from the one described in Drosophila neuroblasts. In these cells, Pins is recruited via Insc by Baz to the apical cortex, and acts in a Dlg-independent manner to maintain the Baz/DmPAR-6/DaPKC/Insc complex at the apical cortex. Dlg interacts directly with Pins and regulates the localization of Pins and Baz. Pins acts with Fz to localize Baz posteriorly, but Baz is not required to localize Pins anteriorly. Finally, Baz and the Dlg/Pins complex are required for the asymmetric localization of Numb. Thus, the Dlg/Pins complex responds to Fz signaling to establish planar asymmetry in the pI cell (Bellaïche, 2001).

In the dividing pI cell, Numb and Pon colocalize at the anterior pole of the lateral cortex, marked with Fasciclin3 (Fas3), below the adherins junction (AJ), marked with DE-Cadherin (Shotgun). In epithelial cells in interphase, Baz colocalizes with Shotgun at the AJ around the apical cortex. In the pI cell, Baz accumulates at the posterior cortex during mitosis. Prior to chromosome condensation, this accumulation is seen at the level of the AJ. Then, during prophase and metaphase, Baz forms a posterior crescent below AJ and opposite to Numb. At telophase, the pIIa cell inherits a higher level of Baz than its sister cell. DaPKC shows a similar distribution to Baz in the pI cell (Bellaïche, 2001).

In neuroblasts, a key function of the Baz/DaPKC/DmPAR-6 complex is to recruit the Insc and the Pins proteins. However, in the pI cell, Insc is not expressed and Pins does not colocalize with Baz at the posterior cortex. Rather, it localizes to the anterior pole in early prophase and colocalizes with Numb at the anterior lateral cortex at metaphase (Bellaïche, 2001).

Because DaPKC and Baz have a dual function in epithelial polarity and asymmetric neuroblast division, it was hypothesized that genes required for epithelial polarity might also regulate planar polarity in the pI cell. To test this hypothesis, the planar distribution of various proteins known to be distributed asymmetrically along the apical-basal axis of epithelial cells was examined. Of these, only Dlg was identified as a protein localizing asymmetrically along the planar axis in the pI cell. Dlg overlaps with Fas3 below the AJ in interphase cells. In dividing pI cells, Dlg redistributes in part along the planar a-p axis. From late prophase onward, Dlg becomes enriched at the anterior cortex, where it colocalizes with Numb and Pins. During this time, Dlg does remain detectable at the posterior lateral cortex. At telophase, a higher level of Dlg segregates into the pIIb cell. Thus, the accumulation of Dlg/Pins and Baz at opposite poles of the cell defines two complementary cortical domains oriented along the a-p planar axis of the pI cell. The position of the mitotic spindle at metaphase correlates with the localization of these two cortical domains. The posterior spindle pole is positioned near the accumulation of Baz, and the anterior spindle pole lies near the accumulation of Dlg. In both pI and epidermal cell, the mitotic spindle poles are found below the AJ, which appear to remain functional since they retain their ability to recruit Arm (Bellaïche, 2001).

To determine the possible function of Baz in the planar polarization of the pI cell, clones of baz mutant cells were studied in the notum. Loss of baz activity does not affect the localization of Shotgun and Dlg, indicating that apical-basal polarity in the notal epithelium is maintained in the absence of Baz. In the dividing pI cell, Numb either does not localize asymmetrically or forms a weak crescent at the anterior cortex at prometaphase. In contrast, Pins localizes asymmetrically at the cortex of the pI cell during division. Moreover, baz mutant pI cells divide within the plane of the epithelium with a normal a-p orientation with Pins localizing at the anterior cortex. This shows that baz is required for the asymmetric localization of Numb but is not essential to establish asymmetry nor to orient polarity along the a-p axis (Bellaïche, 2001).

The function of Pins during the asymmetric division of the pI cell was analyzed using a viable null allele of pinsDelta1-50 that does not affect epithelial cell polarity. To study the function of Dlg, two hypomorphic alleles, dlgSW and dlg1P20 were used that were predicted to encode truncated proteins lacking the C-terminal 14 and 43 amino acids, respectively and which do not perturb apical-basal polarity. The GUK domain of Dlg is partly deleted in the mutant Dlg1P20 protein, but should be unaffected in the mutant DlgSW protein. In the pI cell, the DlgSW protein accumulates normally at the anterior cortex, whereas the mutant Dlg1P20 protein is cortical, but fails to accumulate anteriorly (Bellaïche, 2001).

The possible role of Dlg and Pins in regulating the position of the mitotic spindle was investigated. Spindle movements were analyzed in living pupae using Tau-GFP. It was found that the a-p orientation of the pI division does not depend on the activity of pins and is not affected in the dlg1P20mutant. In wild-type and pins mutant pI cells, the spindle lines up with the planar polarity axis 3-4 min prior to the metaphase-anaphase transition. In contrast, the spindle often rotates throughout metaphase in dlg mutant pI cells. It is concluded that Dlg regulates the localization or the activity of factors responsible for spindle rotation (Bellaïche, 2001).

The roles of Dlg and Pins in the asymmetric localization of Numb and Pon were examined. The interphase localization of Numb at the cortex and of Pon around the nucleus does not depend on the function of the dlg or pins genes. At metaphase, however, the anterior localization of both proteins requires the activity of both dlg and pins. Thus, in pins mutant cells at prometaphase, the crescent of Numb and Pon is either not detected or weak. Nevertheless, both proteins segregate into the anterior cell at anaphase and telophase. In dlg1P20 mutant pI cells, Numb does not accumulate at the anterior cortex and Pon remains cytoplasmic at metaphase. At telophase, Numb and Pon segregate equally into both daughter cells. These results show that Dlg and Pins are required to localize Numb and Pon at the anterior cortex in the pI cell. Consistently, nonsensory cells are transformed into neurons leading to a bristle loss phenotype in adult flies. Furthermore, the genetic interaction seen between dlgsw and pins suggests that dlg and pins act in the same process to specify the fate of the pI daughter cells (Bellaïche, 2001).

Pins colocalizes with the anterior accumulation of Dlg and dlg and pins mutations genetically interact. This raises the possibility that the two proteins interact directly. Indeed, in a yeast two-hybrid screen using full-length Dlg as bait, one Pins clone (encoding amino-acid residues 235 to 658) was isolated. To further test for a direct interaction between Dlg and Pins and to identify the Pins interaction domain of Dlg, blot overlay experiments were performed using GST-fusion proteins. A biotinylated Pins protein has been found to interact with the SH3 domain but not with the PDZ1, PDZ2, PDZ3, HOOK, or GUK domains of Dlg. The Dlg-Pins complex is also detected in brains and imaginal discs by coimmunoprecipitation experiments. This interaction is abolished by a single amino-acid substitution (L556P) in the SH3 domain, which does not noticeably affect Dlg stability in dlgm30 mutant larvae but does result in disc overgrowth and late larval lethality (Bellaïche, 2001).

Consistent with this direct interaction, Pins and Dlg are mutually dependent for their accumulation at the anterior cortex. A very weak crescent of Pins is seen at the anterior cortex in dlg1P20 mutant pI cells at metaphase, suggesting that the GUK domain might facilitate the interaction between the SH3 domain of Dlg and Pins. Conversely, Dlg does not become enriched at the anterior cortex of pins mutant pI cells at metaphase. It is concluded that Dlg directly interacts with Pins via its SH3 domain, and that this interaction is important for the anterior accumulation of both Dlg and Pins (Bellaïche, 2001).

The role of Pins and Dlg in localizing Baz asymmetrically was examined. In pins mutant pI cells, Baz accumulates at the posterior cortex at metaphase, but the asymmetry is less pronounced than in wild-type cells. This raises the possibility that Pins participates in the asymmetric localization of Baz. In dlg1P20 mutant pupae, Baz is correctly localized to the apical posterior cortex prior to chromosome condensation, but does not form a cortical crescent below the AJ during late prophase and prometaphase. Instead, Baz accumulates in the cytoplasm and remains cortical only at the level of the AJ. Thus, the initial posterior localization of Baz at the level of the AJ does not depend on the activity of the GUK domain of Dlg, but its cortical localization below the AJ does require dlg activity. It is concluded that planar polarization of the pI cell cannot be maintained without Dlg activity (Bellaïche, 2001).

To test whether the initial Dlg-independent localization of Baz at the posterior cortex depends on Fz signaling, the distribution of Baz was studied in fz mutant pupae. In wild-type pupae, a clear accumulation of Baz is seen at the level of the AJ in 61% of the interphase pI cells. By contrast, an asymmetric distribution of Baz at the apical cortex is detected in only 19% of the interphase pI cells in fz mutant pupae. In the remaining 81% of the cells, the asymmetric accumulation of Baz is either weak or similar to that seen in the surrounding epithelial cells. This indicates that Fz signaling regulates the initiation of the asymmetric localization of Baz at the posterior cortex. At metaphase, however, Baz and Pins form misoriented crescents relative to the a-p axis that localize at opposite poles in fz mutant pI cells. It is concluded that the formation of the two opposite Baz and Pins domains does not depend on fz activity, and that planar asymmetry can be established in the absence of Fz signaling. However, as previously seen for pins, the asymmetric distribution of Baz is less pronounced in fz mutant pI cells than in wild-type cells. Moreover, Dlg is distributed around the entire cell cortex, indicating that Fz signaling is required for the anterior accumulation of Dlg (Bellaïche, 2001).

Since Pins localizes asymmetrically in a Fz-independent manner, it was asked whether Pins is necessary to localize Baz at one pole of the pI cell in the absence of Fz. It was found that Baz localizes uniformly around the basal-lateral cortex in 82% of the fz;pins double mutant pI cells at metaphase. Moreover, although Numb forms a crescent at anaphase in pI cells mutant for pins or fz, no Numb crescent is seen at either metaphase or anaphase in fz;pins double mutant pI cells. Consistently, loss of fz activity enhances the pins bristle loss phenotype. These data show that Pins and Fz act in a redundant manner to exclude Baz from the anterior cortex and to establish planar asymmetry in the pI cell (Bellaïche, 2001).

These results show that Pins localizes to the anterior cortex in a Baz-independent manner, in an orientation opposite that of Baz, as does Numb. Pins cooperates with Fz to exclude Baz from the anterior cortex of the pI cell. In contrast, in neuroblasts, Pins localizes in a Baz-dependent manner to the apical pole, opposite Numb, and stabilizes the Insc/Baz/DmPAR-6/DaPKC complex. Nevertheless, Pins promotes the localization of Numb in both cell types (Bellaïche, 2001).

One important difference between pI cells and neuroblasts is the lack of insc expression in pI cells. To test the functional significance of this lack of Insc, Insc was expressed in the pI cell. Under these circumstances, Insc and Pins localize at the anterior cortex. Insc triggers the anterior relocalization of Baz, while Numb forms a posterior crescent at anaphase. The pI cell division remains planar. This contrasts with the effect of Insc in epithelial cells. In these cells, Insc localizes apically and orients the spindle along the apical-basal axis. This further indicates that the apical-basal polarity is remodeled in the pI cell. It is concluded that the ectopic expression of Insc is sufficient to reverse the planar polarity axis of the pI cell and to modify the activity of Pins relative to Baz. In the absence of Insc, the Dlg/Pins complex excludes Baz, while expression of Insc leads to the formation of a Pins/Insc/Baz complex. In both cases, Numb localization is opposite that of Baz (Bellaïche, 2001).

Pins interacts with Galphai to direct asymmetric cell division

In Drosophila, distinct mechanisms orient asymmetric cell division along the apical-basal axis in neuroblasts and along the anterior-posterior axis in sensory organ precursor (SOP) cells. Heterotrimeric G proteins are essential for asymmetric cell division in both cell types. The G protein subunit Galphai (FlyBase designation G-oalpha65A) localizes apically in neuroblasts and anteriorly in SOP cells before and during mitosis. Interfering with G protein function by Galphai overexpression or depletion of heterotrimeric G protein complexes causes defects in spindle orientation and asymmetric localization of determinants. Galphai is colocalized and associated with Pins, a protein that induces the release of the ßgamma subunit and might act as a receptor-independent G protein activator. Thus, asymmetric activation of heterotrimeric G proteins by a receptor-independent mechanism may orient asymmetric cell divisions in different cell types (Schaefer, 2001).

While a significant amount of Galphai coimmunoprecipitates with Insc and Pins, no Galphao can be detected in the immunoprecipitate. It is concluded that Galphai but not Galphao is part of the Insc/Pins complex in vivo. To determine the subcellular localization of Galphai, Drosophila embryos were stained for Galphai, DNA, and Insc or Bazooka. Before stage 12 of embryogenesis, Galphai is expressed in all cells and localizes to the cell cortex. Costaining for the apical marker Bazooka reveals that Galphai is concentrated basolaterally in epithelial cells. Upon neuroblast delamination, when the expression of Insc starts, Galphai concentrates in an apical stalk that extends into the epithelial cell layer and then colocalizes with Insc in a crescent along the apical cell cortex during interphase, prophase, and metaphase until anaphase, when Insc disappears and Galphai becomes delocalized. Galphai but not the associated ß subunit is asymmetrically localized in neuroblasts, suggesting that Gß13F is also bound to other Galpha subunits, possibly Galphao (Schaefer, 2001).

To test whether Insc is required for asymmetric Galphai localization in neuroblasts, inscP72 mutant embryos were stained for Galphai and DNA. During neuroblast delamination, Galphai fails to localize apically in insc mutants and in 87% of insc mutant metaphase neuroblasts, the protein is distributed around the whole cell cortex. To test whether ectopic expression of Insc is sufficient for the apical localization of Galphai, insc was ubiquitously expressed from a heat-inducible transgene. While Galphai is localized basolaterally in epidermal cells of heat-shocked control embryos, heat-shock-induced ectopic expression of insc in these cells results in apical concentration of Galphai. Thus, expression of insc is both required and sufficient for apical recruitment of Galphai (Schaefer, 2001).

Since Galphai directly binds to Pins, the subcellular localization of Galphai was tested in pins mutants. No apical localization of Galphai was observed in 100% of the pins mutant metaphase neuroblasts. This might be an indirect consequence of the defect in Insc localization in pins mutant metaphase neuroblasts. However, initiation of Galphai localization also fails in 88% of pins mutant delaminating neuroblasts. Insc is normally localized in pins mutants at this stage and so it is concluded that both Insc and Pins are required for the apical localization of Galphai in neuroblasts (Schaefer, 2001).

Genetic analysis of Galphai is complicated by the presence of another gene within the first intron and the lack of identified P-element insertions near the gene. However, a P-element inserted into the 5' untranslated region of the Gß13F gene was identified and this was used to generate mutants by imprecise excision. Two lethal imprecise excisions were isolated, one of which (Gß13FDelta1-96A) removes the entire coding region, can be rescued to viability by a transgene containing the Gß13F genomic region, and was used in all experiments (Schaefer, 2001).

Since Gß13F has a strong maternal contribution, all experiments were performed in embryos from Gß13F mutant germline clones (here called Gß13F mutants). Gß13F mutants have characteristic morphological defects during gastrulation that lead to the formation of anterior and posterior holes in the cuticle. The defects and cuticle phenotypes are similar to embryos mutant for concertina (cta). Cta is a heterotrimeric G protein alpha subunit and the phenotypic similarity suggests that Cta signals through Gß13F. Galphai protein levels and localization are unaffected in cta mutants. In Gß13F mutants, however, Galphai disappears during gastrulation and is undetectable by immunofluorescence in all cell types during stage 10 of embryogenesis when neuroblasts undergo their first round of asymmetric cell division. Thus, both Galphai and Gß13F are absent from neuroblasts of Gß13F mutant embryos (Schaefer, 2001).

Staining for the neuronal marker Asense has shown that neuroblasts are correctly specified, delaminate, and enter mitosis shortly after delamination both in cta and Gß13F mutants. Furthermore, staining for DmPar-6 reveals no defects in epithelial polarity. However, while 86% of the asymmetric cell divisions in cta mutant neuroblasts are oriented along the apical-basal axis, only 26% of the divisions in Gß13F mutant neuroblasts have this orientation, whereas the others are misoriented by more than 30 degrees. Miranda localizes into a basal cortical crescent in 100% of the cta mutant metaphase neuroblasts, but only in 6% of the Gß13F mutant neuroblasts. In 29% of the Gß13F mutant neuroblasts, crescents are misoriented, whereas in 65%, Miranda is largely cytoplasmic. Defects in asymmetric localization are also observed for Numb. Thus, Gß13F mutants have defects in asymmetric cell division similar to or stronger than those observed in insc mutants, and therefore Insc distribution was analyzed in these mutants. When neuroblasts delaminate from the neuroectoderm, Insc begins to accumulate in a stalk that extends into the epithelium, and this initial localization is unchanged in Gß13F mutants. In Gß13F mutants, cortical localization of the protein is progressively lost after delamination. Weak cortical Insc crescents were found in 11% of the metaphase neuroblasts, but in 25%, the protein was partially, and in 64% completely, localized into the cytoplasm. Thus, heterotrimeric G proteins are required for maintaining Insc localization and for directing spindle orientation and asymmetric protein localization during neuroblast division (Schaefer, 2001).

Heterotrimeric G proteins can interact with their downstream targets either via the Gßgamma subunit or the GTP-bound Galpha subunit. Overexpression of wild-type Galphai and GalphaiQ205L, a GTPase-deficient mutant form, should distinguish between these possibilities. Wild-type Galphai should bind and deplete free Gßgamma and inhibit its downstream interactions. GalphaiQ205L, in contrast, should be in the GTP-bound form that does not bind Gßgamma and should not interfere with Gßgamma signaling. Signaling via the alpha subunit, however, should be enhanced by GalphaiQ205L, but not be affected by the wild-type form (Schaefer, 2001).

Asymmetric cell division was therefore analyzed in control embryos or embryos overexpressing wild-type Galphai from a ubiquitous maternal promoter. While both Pins and Galphai localize apically in control metaphase neuroblasts, they are uniformly distributed around the cortex of neuroblasts overexpressing Galphai. The intensity of cortical Pins staining is higher in Galphai-overexpressing embryos, indicating that Pins is recruited from the cytoplasm to the cell cortex. Miranda localizes into a cortical crescent in control metaphase neuroblasts but in only 20% of the Galphai-overexpressing neuroblasts. Instead, the protein is uniformly cortical (6%) or localizes partially or completely into the cytoplasm. Defects in asymmetric localization are also observed for Numb, even though Numb does not relocalize to the cytoplasm. Mitotic spindles (visualized by gamma-Tubulin staining) are oriented along the apical-basal axis in controls, but are misoriented in 74% of the Galphai-overexpressing neuroblasts. Insc localization is initiated during neuroblast delamination both in control and in Galphai-overexpressing neuroblasts. In metaphase neuroblasts, however, Insc forms an apical crescent in the controls, but localizes partially (40%) or completely (60%) to the cytoplasm upon Galphai overexpression (Schaefer, 2001).

If the defects observed upon Galphai overexpression are due to depletion of free Gßgamma, overexpression of GalphaiQ205L should be without effect. Asymmetric cell division was therefore analyzed in neuroblasts after overexpression of GalphaiQ205L under the same ubiquitous maternal promoter. Like wild-type Galphai, GalphaiQ205L localizes around the cell cortex when overexpressed in neuroblasts. However, the mutant form fails to recruit Pins to the cell cortex and has no effect on Pins localization. No defects in spindle orientation or basal localization of Numb and Miranda were observed and Insc was still localized into an apical crescent in 91% of the metaphase neuroblasts. This suggests that the phenotypes caused by Galphai overexpression might be a consequence of Gßgamma depletion (Schaefer, 2001).

However, several observations indicate that the defects observed after Galphai overexpression are not caused only by depletion of Gß13F. In addition to the defects described above, Galphai overexpression also causes phenotypes that are not observed in Gß13F mutants. While the size difference between daughter cells is unaffected in most Gß13F mutant neuroblasts, staining of the cell cortex by anti-alpha-spectrin reveals that 80% of the Galphai-overexpressing neuroblasts produce two equal sized daughter cells. While in Gß13F mutants, Miranda localization fails during metaphase but is largely normal during late stages of mitosis (similar to insc mutants), Galphai overexpression causes defects throughout mitosis and incorrectly positioned Miranda crescents are often bisected by the cleavage furrow. Thus, even though some of the Galphai overexpression phenotypes may be caused by depletion of Gß13F, other mechanisms like depletion of another Gß subunit or signaling via the GDP-bound form of Galphai may contribute to these phenotypes (Schaefer, 2001).

The strong overexpression phenotypes caused by wild-type Galphai but not by GalphaiQ205L suggest that the GDP-bound form of Galphai may have a function in asymmetric cell division. To test whether Pins interacts preferentially with the GTP- or the GDP-bound form, Galphai was immunoprecipitated in the presence or absence of the slowly hydrolyzable GTP-analog GTPgammaS. While Pins can be readily coimmunoprecipitated with Galphai in the presence of GDP, only trace amounts of Pins can be coimmunoprecipitated in the presence of GTPgammaS, suggesting that Pins preferentially interacts with the GDP-bound form of Galphai (Schaefer, 2001).

The GDP-bound form of Galpha is thought to be inactive and tightly associated with its ßgamma subunit. To test whether Galphai in the Insc/Pins complex is bound to the ß subunit, the Insc/Pins/Galphai complex was immunoprecipitated using a ß-Gal-tagged version of the functional domain of Insc. No Gß13F can be found in the complex, even though a significant amount of Gß13F can be detected in a control experiment where equal amounts of Galphai are precipitated by anti-Galphai. Thus, Galphai is bound to Gß13F in vivo but is free of the ß subunit in the complex with Insc and Pins. To test whether Pins is responsible for the release of the ß subunit, Galphai was immunoprecipitated in the presence of recombinant Pins protein. A significant amount of Gß13F is bound to Galphai in control experiments, but addition of an MBP (maltose binding protein)-fusion of full-length Pins (MBP-Pins) or the Pins GoLoco domains (MBP-GoLoco) during the immunoprecipitation causes the release of the ß subunit. The same effect can be achieved by addition of a 38 aa peptide corresponding to the last GoLoco domain of the Pins protein, but not with a peptide in which a conserved phenylalanine had been mutated to arginine. Thus, the Pins GoLoco domains cause the dissociation of Gß13F from Galphai (Schaefer, 2001).

These results suggest that Galphai exists in an unusual form in Drosophila neuroblasts that is bound to GDP but free of the ß subunit. Furthermore, the observation that recombinant Pins triggers the release of the ß subunit from Galphai is consistent with the hypothesis that Pins activates heterotrimeric G proteins without nucleotide exchange on the alpha subunit in the absence of an extracellular ligand (Schaefer, 2001).

To test whether G proteins also function in Insc-independent asymmetric cell division, the distribution of Galphai was analyzed in SOP cells during pupal development. In interphase, when Numb is homogeneously distributed around the cell cortex, Galphai is asymmetrically localized to the anterior cell cortex in SOP cells. During metaphase, both Numb and Galphai are found at the anterior cell cortex and in telophase, they segregate into the same daughter cell. Similar results were obtained for Pins. Thus, Pins and Galphai localize asymmetrically in SOP cells but in contrast to neuroblasts, they are at the same side as Numb (Schaefer, 2001).

Asymmetric cell divisions in SOP cells are oriented along the anterior-posterior axis of epithelial planar polarity. To test whether planar polarity is required for Galphai localization, Galphai localization was analyzed in frizzled mutants where planar polarity is disrupted. As in wild-type, Galphai localizes asymmetrically in frizzled mutant interphase SOP cells and localizes to the same side as Numb in mitosis. However, both the Numb and the Galphai crescents are misoriented in these mutants, suggesting that planar polarity determines the position of Galphai accumulation but is not required for its asymmetric localization per se (Schaefer, 2001).

To determine whether G proteins are required for asymmetric cell division in SOP cells, Gß13F mutant clones generated by mitotic recombination in eye imaginal discs were analyzed. No Galphai protein could be detected on the cell cortex of Gß13F mutant cells but it is not possible to distinguish between delocalization and degradation of the protein. While Numb localizes asymmetrically and Gß13F is uniformly cortical in mitotic SOP cells outside the clones, no asymmetric localization of Numb is seen within the clone where Gß13F cannot be detected. To directly test a requirement of Galphai in SOP cells, heritable RNAi was used to disrupt Galphai function. Expression of double-stranded Galphai RNA significantly reduces Galphai protein levels in all SOP cells. Eleven percent of the SOP cells no longer stained for Galphai and in these cells, Pins no longer localizes to the cell cortex. Numb is distributed around the cell cortex in metaphase, leading to cell fate transformations in the bristle lineage. Mitotic spindles are misoriented in the SOP cells that have lost Galphai, but their low frequency makes a quantitative analysis of the spindle orientation phenotype difficult. Similar defects are observed in SOP cells in mitotic clones mutant for the strong allele pins83. Neither Galphai nor Numb are asymmetrically localized in these cells, indicating that Pins and Galphai are codependent for their asymmetric localization in SOP cells. It is concluded that Galphai and Pins are also required for Insc-independent asymmetric cell divisions in SOP cells (Schaefer, 2001).

In neuroblasts, Galphai function does not seem to involve the GTP-bound form of Galphai. To test whether this is also the case in SOP cells, wild-type Galphai and GalphaiQ205L were overexpressed in SOP cells. Upon overexpression, Galphai is no longer asymmetrically localized and Numb is uniformly distributed around the cell cortex. Thirty-eight percent of the Galphai overexpressing SOP cells (n = 149) but only 9% of the controls divided at an angle that deviated more than 45° from the anterior-posterior axis. However, unlike in neuroblasts, in this case similar defects can be generated by overexpression of the activated GalphaiQ205L mutant form. The different effects of GalphaiQ205L overexpression in neuroblasts and SOP cells suggest that distinct pathways might function downstream of G proteins in the two cell types (Schaefer, 2001).

In neuroblasts, the Insc protein is critical for the asymmetric localization of Galphai and its binding partner Pins. Neuroblasts arise from epithelial cells in which Insc is not expressed and Galphai is localized basolaterally. When neuroblasts delaminate, Insc expression starts and the protein functions as an adaptor that links the Pins/Galphai complex to the Bazooka/DmPar-6/DaPKC complex that is inherited from the apical cortex of the epithelial cells. Neither Pins nor Galphai are required for Insc localization during this stage. In delaminated neuroblasts, however, Insc, Pins, and Galphai become codependent for their apical localization. At this point, their subcellular localization in various mutants can no longer be explained simply by protein-protein interactions of the known components. When Galphai is overexpressed, for example, Pins is recruited to the cell cortex whereas Insc relocalizes into the cytoplasm, suggesting that the two proteins no longer interact. Thus, events that happen downstream of Galphai seem to be involved in maintaining the colocalization of the more upstream components. The simplest model is that G proteins establish a positional cue at the apical cell cortex during neuroblast delamination -- this cue is needed for maintaining apical protein localization in delaminated neuroblasts and ultimately, for orienting asymmetric cell division. In Drosophila, this downstream activity remains to be identified, but a similar feedback loop for asymmetric protein localization is found in yeast and here its molecular components are well understood. Local activation of a heterotrimeric G protein in response to the pheromone alpha-factor recruits Cdc24 to the site of G protein activation. Cdc24 is an exchange factor that locally activates the small G protein Cdc42 and activated Cdc42, in turn, is needed to maintain Cdc24 localization. Thus, the initiation of an autoregulatory feedback loop at a particular position may be a common theme in cell polarity (Schaefer, 2001).

The function of heterotrimeric G proteins in directing cell polarity and asymmetric cell division is not restricted to Drosophila. In C. elegans, a Gßgamma subunit is required for correct orientation of mitotic spindles during early development and two Galpha subunits function redundantly in asymmetric spindle positioning and generation of different daughter cell sizes. Since the role of the Bazooka/DmPAR-6/DaPKC complex is also conserved from C. elegans to Drosophila, a homologous machinery may direct asymmetric cell division in the two organisms. RNAi experiments so far have failed to reveal a function for the C. elegans Pins homolog, but recently, two other proteins containing a GoLoco domain have been found to be required for asymmetric cell division in a chromosome-wide RNAi screen. G proteins are not asymmetrically localized and not required for the asymmetric segregation of determinants in C. elegans, but it is possible that asymmetric activation of G proteins by GoLoco domain proteins is a conserved mechanism to orient mitotic spindles in the two organisms (Schaefer, 2001).

Distinct roles of Galphai and Gß13F subunits of the heterotrimeric G protein complex in the mediation of Drosophila neuroblast asymmetric divisions

The asymmetric division of Drosophila neuroblasts involves the basal localization of cell fate determinants and the generation of an asymmetric, apicobasally oriented mitotic spindle that leads to the formation of two daughter cells of unequal size. These features are thought to be controlled by an apically localized protein complex comprised of two signaling pathways: Bazooka/Drosophila atypical PKC/Inscuteable/DmPar6 and Partner of inscuteable (Pins)/Galphai. In addition, Gß13F is also required, however, the role of Galphai and the hierarchical relationship between the G protein subunits and apical components are not well defined. This study describes the isolation of Galphai mutants and shows that Galphai and Gß13F play distinct roles. Galphai is required for Pins to localize to the cortex, and the effects of loss of Galphai or pins are highly similar, supporting the idea that Pins/Galphai act together to mediate various aspects of neuroblast asymmetric division. In contrast, Gß13F appears to regulate the asymmetric localization/stability of all apical components, and GßF loss of function exhibits phenotypes resembling those seen when both apical pathways have been compromised, suggesting that it acts upstream of the apical pathways. Importantly, these results have also revealed a novel aspect of apical complex function, that is, the two apical pathways act redundantly to suppress the formation of basal astral microtubules in neuroblasts (Yu, 2003b).

This study reports the isolation and analysis of loss of function mutations in Galpha and show that the loss of Galpha and Gß13F have distinct effects on NB asymmetric cell divisions. Galphai is required for Pins cortical association and asymmetric localization; loss of Galphai causes Pins to localize to the cytosol, and mutant NBs exhibit phenotypes that are highly similar to those seen in pins mutants. Analyses of double mutant combinations confirm Galphai RNAi results showing that Pins/Galphai and Baz/DaPKC/Insc act in an redundant fashion to mediate the formations of an asymmetric mitotic spindle and the generation of NB daughters of unequal size. Importantly, these analyses also revealed a new aspect of apical complex function: that the two apical pathways also act redundantly to suppress the formation of astral microtubules from the basal centrosome of NBs. In contrast, Gß13F appears to act upstream of the apical components and is required for their asymmetric localization/stability. The defects associated with NBs lacking G圩F function are highly similar to those seen when the function of both apical pathways have been compromised. In addition, it was shown that high level overexpression of two different Galpha subunits, which can bind/complex to Gß13F, results in similar phenotypes seen in G圩F mutant NBs, suggesting that it is the depletion of free Gß13F, which is responsible for the mutant phenotypes (Yu, 2003b).

Pins and Galphai apical localization are mutually dependent. In pins NBs, Galphai is evenly distributed to the NB cortex, and in Galpha mutant NBs, Pins localizes to the cytosol. Pins asymmetric localization to the apical cortex of the NBs is a two-step process: Pins needs to be targeted to the cortex first: this requires the COOH-terminal Goloco motifs that can bind Galphai before Galphai can be recruited to the apical cortex in a process which requires the Galphai NH2-terminal TPR that can interact with Insc. The current results therefore suggest that Pins cortical targeting is most likely mediated by Galphai, which not only binds Pins, but also is able to localize to the plasma membrane through lipid modifications (Yu, 2003b).

However, in G圩F mutant NBs, although the levels of Pins are drastically reduced, the residual Pins is localized both to the cytosol and to the cell cortex. This poses a problem since in the G圩F mutant NBs not only is Gß13F absent but Galphai also is undetectable with an anti-Galphai antibody. One possible explanation is that although Galphai is undetectable, there is still some Galphai remaining in the G圩F NBs: this may account for the low level residual uniform cortical distribution of Pins. Alternatively, the possibility cannot be ruled out that the cortical Pins in G圩F NBs is due to some unknown molecule that can recruit Pins to cortex in the absence of both Galphai and Gß13F (Yu, 2003b).

The analysis of G圩F function is complicated by the fact that in the G圩F mutant NBs, Galphai levels are also down-regulated presumably due to the instability of the protein in the absence of Gß13F. Although loss of either Galpha or G圩F causes aberrations in localization of the basal components and orientation of the mitotic spindle, it is clear that at least some of the defects associated with the loss of G圩F cannot be attributable solely to the depletion of Galphai. In the great majority of Galphai mutant NBs, DaPKC and Baz still localize asymmetrically to a subset of the cell cortex. And consistent with the proposal that spindle geometry and the size asymmetry of the NB daughters are mediated by two redundant apical pathways, Pins/Galphai and Baz/DaPKC, the great majority (79%) of the Galpha mutant NBs generate an asymmetric mitotic spindle and divide to produce unequal size daughters. In contrast, in G圩F NBs not only do Pins/Galphai always fail to become asymmetrically localized but the majority of mutant NBs (71%) also fail to asymmetrically localize Baz/DaPKC; consequently ~65% of NBs fail to generate an asymmetric mitotic spindle and divide to produce equal size daughters. Therefore, at least formally, Gß13F acts upstream of the two apical pathways (Yu, 2003b).

It is believed that the major reason for the phenotypes associated with loss of Gß13F function is due to the disruption of Gßgamma signaling. Overexpression of Galphai will cause a high frequency of equal size divisions. In addition, overexpression of Galphao, a Galpha subunit that interacts with Gß13F but is not itself required for asymmetric divisions in wt NBs, will also mimic the Gß13F loss of function phenotype. For both overexpression of Galphai and Galphao, the frequency of equal size divisions is significantly higher than that seen in Gß13F loss of function. This difference may be due to the existence of other Gß subunits which might also function in NB asymmetric divisions. Three Gß genes have been identified by the Drosophila genome project, and although one of these genes, concertina, appears not to be involved in the process, it is possible that overexpression of Galpha molecules may deplete not only Gß13F but also Gß76C. This possibility could be addressed by the analysis of double mutants of Gß genes. Nevertheless, these observations are consistent with the view that the depletion of free Gßgamma, and not Galphai, is the major cause for the symmetric divisions seen in G圩F mutant NBs. Hence, although previous analysis of G圩F loss of function did not report any effects on NB daughter size, the current data are consistent with the notion that G圩F plays a major role in mediating the distinct size of NB daughter cells (Yu, 2003b).

The apical centrosome associates with prominent astral microtubules, whereas the basal centrosome connects to few if any astral microtubules in wt NBs and in mutants in which one of the two apical pathways is compromised. In contrast, in NBs that lack both apical pathways a symmetric mitotic apparatus is established that features extensive arrays of astral microtubules at both centrosomes. Therefore, either of the two apical pathways appears sufficient to prevent formation of basal astral microtubules. It is not clear how this might be accomplished at a mechanistic level. However, one might speculate that there exists an asymmetrically localized molecule, which can act to promote the formation of astral microtubules. When either of the apical pathways is functional, this molecule is asymmetrically localized and promotes the formation of astral microtubules only over the centrosome it overlies. However, when both apical pathways are mutated, or when G圩F is mutated or when all apical components become uniformly cortical, e.g., when Galphai is overexpressed, then the hypothetical molecule becomes uniformly cortical and can promote the formation of astral microtubules over both centrosomes. This type of model can readily explain why either loss or uniform cortical localization of both apical pathways leads to symmetric astral microtubule formation over both centrosomes (Yu, 2003b).

In summary, the results demonstrate that for NB asymmetric divisions Galphai and Gß13F play distinct roles. Galphai and Pins are members of one of the two apical pathways and Baz/DaPKC/Insc forms the other. Loss of Galphai function results in defects in NB asymmetry that are essentially indistinguishable from those seen in pins mutants. Gß13F (Gßgamma) functions upstream of both Pins/Galphai and Baz/DaPKC/Insc pathways to mediate their stability and/or asymmetric localization (and function). Without Gß13F, the function of both apical pathways are attenuated; Galphai levels are dramatically reduced and Pins/Galphai pathway is defective; in addition, the asymmetric localization of members of the Baz/DaPKC/Insc pathway is often defective. Consequently, loss of Gß13F function yields phenotypes that are similar to those seen when both apical pathways are disrupted by mutations (Yu, 2003b).

A mutation in Gβ13F sheds light on the involvement of heterotrimeric G proteins in regulating daughter cell size asymmetry in Drosophila neuroblast divisions

Cell division often generates unequally sized daughter cells by off-center cleavages, which are due to either displacement of mitotic spindles or their asymmetry. Drosophila neuroblasts predominantly use the latter mechanism to divide into a large apical neuroblast and a small basal ganglion mother cell (GMC), where the neural fate determinants segregate. Apically localized components regulate both the spindle asymmetry and the localization of the determinants. Asymmetric spindle formation depends on signaling mediated by the Gβ subunit of heterotrimeric G proteins. Gβ13F distributes throughout the neuroblast cortex. Its lack induces a large symmetric spindle and causes division into nearly equal-sized cells with normal segregation of the determinants. In contrast, elevated Gβ13F activity generates a small spindle, suggesting that this factor suppresses spindle development. Depletion of the apical components also results in the formation of a small symmetric spindle at metaphase. Therefore, the apical components and Gβ13F affect the mitotic spindle shape oppositely. It is proposed that differential activation of Gβ signaling biases spindle development within neuroblasts and thereby causes asymmetric spindles. Furthermore, the multiple equal cleavages of Gβ mutant neuroblasts accompany neural defects: this finding suggests indispensable roles of eccentric division in assuring the stem cell properties of neuroblasts (Fuse, 2003).

During mitosis, neuroblasts localize the cell fate determinants Prospero and Numb to the basal cortex and orient the mitotic spindle along the apical-basal axis to segregate the determinants into GMCs. These processes are regulated by the apical protein complex that includes Inscuteable, Bazooka, atypical protein kinase C (DaPKC), the G protein subunit Gαi, and Partner of Inscuteable (Pins). The depletion of any single apical component does not severely affect the cell size difference between the neuroblast daughters. However, a recent study shows that the two signaling pathways, Bazooka/DaPKC and Pins/Gαi, within the apical complex control in parallel the production of unequal-sized daughters (Fuse, 2003).

During a mutational screen with Miranda, the adaptor protein of Prospero, the f261 mutant, which is defective in unequal-sized neuroblast divisions, was obtained. In germline clone embryos that are both maternally and zygotically mutant for f261 (f261 mutant), the neuroblasts produce nearly equal-sized daughters, although the GMC is still slightly smaller than the sibling neuroblast after the initial divisions. Nevertheless, after a slight delay in crescent formation, Miranda localizes normally in f261 neuroblasts and segregates to the GMC. Consequently, Prospero is inherited by the GMCs. The abnormal division in f261 causes neuroblasts to be smaller and smaller after each succeeding division. The f261 mutant turned out to be a protein null mutant of the Gβ13F gene that encodes a β subunit of heterotrimeric G proteins. In wild-type neuroblasts, this protein distributes uniformly at the cell cortex. A deletion mutant of Gβ13F has been reported (Schaefer, 2001) to show delayed localization of Miranda and randomized orientation of neuroblast division, as well as gastrulation defects, all of which occur in f261 embryos, but cell size defects have not been described. Deletion mutants lacking the entire Gβ13F coding sequence have been created. Such a mutant, Gβ13FΔ15, as well as the deletion mutant reported previously (Schaefer, 2001) indeed show the same neuroblast phenotypes as those of f261 embryos. Therefore, the loss of Gβ13F activity affects cell size asymmetry but essentially does not affect the segregation of the cell fate determinants. The neuroblast phenotypes observed in the Gβ13F mutants are not consequences of morphological defects before neuroblast formations because the neuroblast-specific expression of Gβ13F rescues the phenotype of cell size asymmetry (Fuse, 2003).

Cell cleavage occurs at the plane crossing the midzone of the central spindle, where the two spindle halves overlap after chromosomal segregation. In Drosophila neuroblasts, both the basal displacement and asymmetric shape of the spindle allocate the spindle midzone basally, resulting in a basal shift of the cleavage site. In wild-type neuroblasts, microtubules are asymmetrically organized from metaphase, with larger apical and smaller basal halves of the spindle and asters; then, the apical half continues to grow, whereas the basal half stops growing or even shortens from anaphase onward. In contrast, in f261 neuroblasts, spindle and astral microtubules develop well from the two centrosomes as though both spindle halves were apical, and the entire microtubule structure remains symmetrical throughout mitosis (Fuse, 2003).

Measuring centrosome positions along the division axis also indicates that spindle asymmetry is abolished in the f261 neuroblasts. However, the spindle position shifts toward the side where Miranda localizes, indicating that the property of spindle displacement still resides in the f261 neuroblasts. This effect confers some residual asymmetry of daughter cell size. Possible contributions of the apical components to this asymmetry were examined in f261 neuroblasts; however, the tested apical components no longer localize normally in the absence of Gβ13F. This remaining asymmetry was found to be due to bazooka activity. The depletion of bazooka activity in f261 results in the complete loss of asymmetry, with uniform distribution of Miranda and disruption of both spindle asymmetry and displacement. Neuroblasts consequently produce two indistinguishable daughters in this Gβ13F-bazooka double mutant, whereas neuroblasts mutant only for bazooka show no gross defect in cell size asymmetry. Therefore, spindle displacement involves both bazooka and Gβ13F, but the asymmetry in the mitotic spindle depends largely on Gβ13F function and determines the difference in daughter cell size (Fuse, 2003).

In canonical heterotrimeric G protein signaling, the Gβ and Gγ complex (Gβγ) associates with the GDP form of Gα, but the conversion of GDP to GTP releases Gβγ from Gα; both Gβγ and Gα can then signal downstream. In Drosophila neuroblasts, it is unlikely that GTP-Gαi acts as a signal. Instead, it has been suggested that the GDP form of Gαi binds to Pins to release the Gβγ subunit (Schaefer, 2001). According to this model, Pins-dependent activation of Gβ signaling occurs at the apical cortex, where Pins and Gαi are colocalized. Unlike the Gβ13F mutants, defects in unequal-sized divisions are observed only in a small fraction of pins mutant neuroblasts, probably because of the bazooka/DaPKC activity that functions in parallel to form asymmetric spindles. Therefore the effects of pins and Gβ13F on microtubule development were compared under conditions in which bazooka activity is simultaneously depleted. In the absence of both and bazooka, metaphase neuroblasts form a large symmetric spindle resembling that seen in f261. In contrast, the simultaneous loss of pins and bazooka activities results in the formation of a small symmetric spindle at metaphase, which is rather similar to the basal half of the wild-type spindle. Therefore, Gβ and Pins exert opposite effects on spindle formation during metaphase in the absence of bazooka. This reciprocal effect of Pins and Gβ on spindle development is not straightforwardly deduced from the model that shows that Pins induces the free and active Gβγ. These states of the mitotic spindle in the double mutants appear to persist throughout mitosis because the midbody, the bundled central spindle at telophase, is notably narrower in the pins-bazooka double mutant than in the Gβ-bazooka mutant. In comparison, astral microtubules develop to a similar extent from anaphase onward under those two mutant conditions. The asters in these double mutants develop more than the basal half of wild-type but less than that seen in f261 and appear at an intermediate level. The differential influence of the mutations on the mitotic spindle (or central spindle) and asters may originate from different mechanisms that regulate these microtubule structures. This possibility has been suggested by the existence of asterless mutants, in which asters are apparently absent, whereas the mitotic spindle appears to develop normally. The role of astral microtubules in cell size asymmetry is controversial because asterless mutant neuroblasts still bud off small GMCs by forming an asymmetric central spindle (Fuse, 2003).

To clarify the functions of G protein subunits in neuroblasts, the subunits were overexpressed and their effects on microtubule development were examined. Whereas overexpression of Gβ13F alone has no effect on division, the simultaneous overexpression of Gγ1, which is expressed endogenously in neuroblasts, and Gβ13F drastically reduces microtubule organization. At metaphase, Gγ1- plus Gβ13F-expressing neuroblasts (22 of 30) form a small symmetric or disorganized spindle, as though both spindle halves were basal. As a result, some telophase neuroblasts undergo equal cleavage (6 of 90), but others also frequently show defective cytokinesis (59 of 90). Overexpressed Gαi, which should be largely in the GDP form, has a uniformly cortical distribution in neuroblasts and often causes equal divisions (Schaefer, 2001). In these cells, a large symmetric spindle and asters emerge, as in f261. Because GDP-Gα sequesters free Gβγ, the symmetry of division in Gαi-overexpressing cells may be due to the Gαi-mediated repression of Gβ activity. Therefore, the gain of Gβγ activity and its loss by the f261 mutation (or Gα overexpression) exert opposite effects on microtubules even though equal division occurs under both conditions. This effect suggests that Gβ signaling directly or indirectly prevents microtubule development. This idea is supported by the observation that the mitotic spindle becomes shrunken in cultured S2 cells that simultaneously overexpress Gβ13F and Gγ1 (15 of 30); however, as with the embryos, expression of Gβ13F alone has no effect on the cultured cells. Therefore, the observations obtained with cultured cells and mutant embryos are consistent with the idea that, in mitotic neuroblasts, Gβ13F inhibits microtubule development on the basal side to define its small spindle half (Fuse, 2003).

The Gβγ complex is anchored to the cell membrane via the C-terminal lipidation of Gγ; this finding suggests that Gβ13F acts cortically to regulate microtubules. Consistent with this idea, the effect of Gβ13F overexpression on the spindles requires Gγ coexpression. This effect of Gγ can be replaced by the fusion of Gβ13F with a domain of Miranda that is sufficient for its localization to the basal cortex. The fusion protein redistributes throughout the cortex and causes microtubule shrinkage during metaphase (27 of 31 compared to 0 of 37 in the wild-type) like that seen with Gβγ overexpression. Furthermore, a mutant exhibits the same defects as Gβ13F mutants. Therefore, Gβ signaling that regulates microtubule development likely operates at the cell cortex (Fuse, 2003).

Finally, the roles played by eccentric neuroblast divisions in neural development were investigated by taking advantage of the Gβ13F mutant that shows nearly equal-sized cleavages despite the normal segregation of the determinants. In the wild-type, neuroblasts repeatedly bud off small GMCs with a constant volume throughout neurogenesis and thereby gradually reduce the volume of neuroblasts. In contrast, in the Gβ13F mutants, consecutive equal divisions cause both the neuroblasts and the sibling GMCs, which reach the same size as ordinary GMCs by stage 14, to rapidly reduce their cell volume. Although wild-type neuroblasts continue their asymmetric division after stage 14, the f261 neuroblasts exhibit several defects around this stage. (1) The numbers of cells expressing Asense and Deadpan (Dpn), which mark neuroblasts, rapidly decrease by stage 14 in f261 embryos. For example, at stage 14, the f261 mutants have 38.8 ± 7.6 Dpn+ cells/segment compared with 60.8 ± 6.7 cells/segment in the wild-type (n = 14). (2) By stage 14, fewer neuroblasts divide in f261 embryos than in wild-type embryos; this finding suggests that the mutant neuroblasts are experiencing cell cycle retardation or early cessation of division. Observations of the production of neurons that express Even-skipped (Eve) in f261 embryos support the latter possibility. The f261 embryos generate early-born Eve+ neurons, such as the RP2 neurons derived from the first division of neuroblast 4-2. However, although neuroblast 3-3, which normally generates ten Eve+ neurons called EL neurons, produces the first five EL neurons in the f261 embryos, the five later-born neurons are not generated. These defects in neural development are rescued by paternal supply of the wild-type Gβ13F gene, which in contrast does not rescue the gastrulation defects in f261 embryos. In addition, concertina and folded gastrulation mutants, which have essentially the same gastrulation defects as f261 but do not show equal-sized neuroblast divisions, do not exhibit the neural defects observed in f261. Therefore, it is unlikely that the neural phenotypes in the f261 mutant are indirect consequences of the gastrulation defects of this mutant. These data together indicate that neuroblasts rapidly lose their normal properties in the absence of Gβ13F and that this loss probably is due to the smaller cell sizes that result from the equal cleavages. Unequal-sized division may serve to maintain the stem cell properties of neuroblasts by minimizing the reduction in neuroblast cell volume (Fuse, 2003).

In the first division of C. elegans eggs, eccentric cleavage occurs due to the displacement of the symmetric spindle, which is pulled asymmetrically by astral microtubules. In contrast, the unequal-sized divisions of Drosophila neuroblasts are predominantly promoted by the asymmetric organization of the mitotic spindle, which requires biased microtubule development along the apical-basal axis. Gβ13F plays an essential role in forming asymmetric spindles in neuroblasts. The elimination of Gβ13F activity enhances spindle development, but its elevation inhibits spindle growth. These findings suggest that Gβ signaling acts to suppress microtubule development. In comparison, simultaneous disruption of the two apical pathways appears to reduce the size of mitotic spindles; this finding suggests that these signals normally act to enhance spindle development. Therefore, Gβ and the two apical signals likely exert opposite effects on microtubule development. These observations led to a simple model in which Gβ signaling is active on the basal cortex to suppress spindle growth but is inhibited by the apical signals on the apical side. In an alternative model, the apical signals enhance spindle growth, and Gβ13F acts to exclude this activity of the apical complex from the basal side. Currently, both models equally explain the data obtained in this study and suggest that Gβ signaling confers the basal character to the cell cortex. This differential Gβ signaling ultimately induces biased spindle development, which results in the asymmetric spindle. For better understanding of the mechanisms that regulate spindle asymmetry, it would be necessary to assess where Gβ13F is active in neuroblasts and to elucidate how it relates to the apical signals (Fuse, 2003).

Locomotion defects, together with Pins, regulates heterotrimeric G-protein signaling during Drosophila neuroblast asymmetric divisions

Heterotrimeric G proteins mediate asymmetric division of Drosophila neuroblasts. Free Gßgamma appears to be crucial for the generation of an asymmetric mitotic spindle and consequently daughter cells of distinct size. However, how Gßgamma is released from the inactive heterotrimer remains unclear. This study shows that Locomotion defects (Loco) interacts and colocalizes with Galphai and, through its GoLoco motif, acts as a guanine nucleotide dissociation inhibitor (GDI) for Galphai. Simultaneous removal of the two GoLoco motif proteins, Loco and Pins, results in defects that are essentially indistinguishable from those observed in Gß13F or Ggamma1 mutants, suggesting that Loco and Pins act synergistically to release free Gßgamma in neuroblasts. Furthermore, the RGS domain of Loco can also accelerate the GTPase activity of Galphai to regulate the equilibrium between the GDP- and the GTP-bound forms of Galphai. Thus, Loco can potentially regulate heterotrimeric G-protein signaling via two distinct modes of action during Drosophila neuroblast asymmetric divisions (Yu, 2005).

Heterotrimeric G proteins have been shown to be involved in controlling distinct microtubule-dependent processes in one-cell embryos of C. elegans. Gßgamma is important for correct centrosome migration around the nucleus and spindle orientation, while Galpha subunits, GOA-1 and GPA-16, are required for asymmetric spindle positioning. Recent studies have shown that the GoLoco-motif-containing proteins, GPR1/2, act as GDIs for GOA-1 and GPA-16 to translate polarity cues, mediated by the asymmetrically localized Par proteins, into asymmetric spindle positioning in the C. elegans zygote (Colombo, 2003; Gotta, 2003; Srinivasan, 2003). In Drosophila NBs, heterotrimeric G proteins Gß13F and Ggamma1 are required for the asymmetric localization/stability of the apical components and, hence, the formation of an asymmetric spindle (Yu, 2003b). This is likely to be achieved through the generation of free Gßgamma since depletion of Gßgamma function by overexpression of wild-type Galphai/Galphao or loss of Gß13F or Ggamma1 function can lead to the generation of a symmetric and centrally placed mitotic spindle, and NBs frequently divide to produce daughter cells of similar size (henceforth referred to as 'similarsized divisions,'). Thus, generation of free Gßgamma is crucial for NB asymmetric divisions. However, it is not clear whether Gßgamma mediates spindle geometry independently of the Galpha subunit(s) or alternatively by controlling the localization of Galpha subunit(s) and/or the GoLoco proteins. Pins has previously been shown to act as a GDI to facilitate the dissociation of Gßgamma from heterotrimers by binding to and stabilizing the GDP-bound form of Galphai (GDP-Galphai). However, paradoxically, loss of pins function does not produce the severe spindle defects seen in the Gß13F or Ggamma1 mutant NBs, suggesting that the absence of the Pins GDI activity does not prevent the generation of free Gßgamma. Similarly, loss of Galphai, while causing defects in spindle orientation and the localization of the basal proteins up to metaphase, like pins loss of function, also does not cause the severe spindle asymmetry defects seen in Gß13F or Ggamma1 mutant NBs; however, it remains possible that additional Galpha subunits may be involved in this process (Yu, 2005 and references therein).

This study shows that locomotion defects (loco), a gene previously shown to be required for glial cell differentiation and dorsal-ventral patterning, encodes a novel component of the NB apical complex that exhibits both guanine nucleotide dissociation inhibitor (GDI) and GTPase-activating protein (GAP) activities for Galphai. Loco interacts with GDP-Galphai through its GoLoco motif and forms a complex with Galphai in vivo. Loco colocalizes with Galphai and Pins at the apical cortex of NBs throughout mitosis and is required for the asymmetric localization/stabilization of Pins/Galphai. Analyses of various double-mutant NBs suggest that Loco, like Pins and Galphai, functions redundantly with the Baz/DaPKC pathway in regulating spindle geometry. Interestingly, loss of both loco and pins functions leads to similar-sized divisions in the majority of NBs, similar to that seen in either Gß13F or Ggamma1 mutants, suggesting that activation of Gßgamma is mediated in a redundant manner by both Loco and Pins. These data therefore provide functional support for the idea that the activation of heterotrimeric G-protein signaling through the generation of free Gßgamma, crucial for NB asymmetric divisions, can occur via a receptor-independent mechanism by using multiple GDIs that functionally overlap. Moreover, Loco can, through its RGS domain, also function as a GAP to regulate the balance between GDP-Galphai and GTP-Galphai. Hence, both the GDI and GAP functions of Loco are important for NBs to regulate the activities of Galphai and Gßgamma (Yu, 2005).

Previous studies have shown that heterotrimeric G-protein components play important roles in NB asymmetric divisions. This study considers the issues of how heterotrimeric G-protein activation might be mediated during NB asymmetric divisions and the roles that Gßgamma, GTP-Galphai, and GDP-Galphai play in this process. Loco is shown to be a novel asymmetrically localized component of the NB asymmetric division machinery that possesses both GDI and GAP activities for Galphai. Evidence is provided that indicates that the redundant GDI activities of Pins and Loco lead to the generation of free Gßgamma, which plays a crucial role for the formation of an asymmetric mitotic spindle and daughter cells of distinct size. Based on loss-of-function phenotype, Galphai appears to play a less important role than Gßgamma in this process; however, the proper balance between the levels of GTP- and GDP-bound forms of Galphai, which may be mediated, at least in part, by the GAP activity of Loco, is crucial for the asymmetric localization of Pins and Insc. It is important to note that there may exist additional Galpha subunit(s) that might functionally overlap with Galphai in the generation of an asymmetric spindle. Therefore the possibility that Gßgamma might mediate asymmetric spindle geometry by regulating the localization Galpha subunit(s) (and GoLoco proteins) cannot be excluded at this point (Yu, 2005).

Heterotrimeric G proteins are classically known to transmit extracellular signals to targets within the cell through seven transmembrane, G-protein coupled receptors (GPCRs). Upon ligand binding, GPCR acts as a GEF to stimulate release of GDP from the Galpha subunit, which, in turn, is converted to the GTP-bound form. GTP-Galpha and Gßgamma dissociate and activate their respective effectors to initiate downstream signaling. G-protein signaling is attenuated through the hydrolysis of GTP to GDP by the GTPase activity of Galpha, which is accelerated by GAPs, which often contain an RGS domain. GDP-Galpha can reassociate with and inactivate Gßgamma (Yu, 2005).

Analyses of loss of function of Gß13F and Ggamma1 as well as gain of function of Galphai in NBs have provided compelling support for the view that free Gßgamma is required for the asymmetric localization/stability of both apical pathway components as well as the generation of asymmetric spindle and daughter cell size. Galphai is required primarily for the asymmetric localization of Pins and makes only a minor contribution in regulating spindle geometry and asymmetric daughter cell size. The mechanism by which heterotrimeric G-protein activation (generation of free Gßgamma) is mediated in NBs has been unclear. The fact that no G-protein-coupled receptors (GPCRs) have been implicated in NB asymmetric divisions, the apparent intrinsic polarity exhibited by cultured NBs, as well as the observed GDI activity associated with Pins have raised the possibility that heterotrimeric G-protein activation may occur via a receptor-independent mechanism since GoLoco-containing molecules like Pins should be able to generate free Gßgamma from the heterotrimeric complex by competing for binding to GDP-Galphai. However, loss of pins does not cause the majority of NBs to produce daughters of similar size and is therefore inconsistent with a failure to activate G-protein signaling (Yu, 2005).

This apparent contradiction is resolved by observations that indicate that receptor-independent activation of heterotrimeric G-protein signaling may be mediated through the GDI activities of both Pins and Loco. Like Pins, Loco can interact with GDP-Galphai through its GoLoco motif and form an in vivo complex with Galphai. In NBs, Loco colocalizes with Galphai and Pins at the apical cortex throughout mitosis. Removal of maternal and zygotic loco leads to delocalization of Pins/Galphai. Analysis of double mutants indicates that Loco functions redundantly with the Baz/DaPKC pathway with respect to the generation of differential daughter size. Simultaneous loss of both loco and pins results in phenotypic defects essentially indistinguishable from those seen in Gß13F or Ggamma1 loss-of-function NBs. These observations indicate that receptor-independent activation of heterotrimeric G proteins during Drosophila NB asymmetric division may be achieved through the actions of the two functionally redundant GDI activities of Pins and Loco (Yu, 2005).

In addition to its GDI activity, Loco also possesses an RGS domain that exhibits GAP activity for Galphai in vitro, suggesting that Loco can regulate Galphai via two distinct modes of action, both as a GDI and as a GAP. These studies suggest that Gßgamma, activated by the GDI activity of Pins and Loco, is crucial for NBs to produce daughters of unequal size, while the equilibrium between GDP-Galphai and GTP-Galphai, regulated, at least in part, by the GAP activity of Loco, is required for the localization of Insc/Pins/Loco at the apical cortex in NBs. When the equilibrium is shifted toward GTP-Galphai, that is, when GalphaiQ205L (the constitutively GTP-bound form) is expressed in the absence of endogenous wild-type Galphai, Pins becomes delocalized/destabilized because it requires binding to GDP-Galphai to localize to the cell cortex; however, the ability to generate an asymmetric spindle and unequal-size daughters is not compromised since Gßgamma function should not be compromised. Conversely, when the equilibrium is shifted toward GDP-Galphai, through the ectopic expression of GalphaiG204A (the constitutively GDP-bound form) in the absence of endogenous wild-type Galphai, free Gßgamma fails to be generated and defects similar to those seen in Gß13F or Ggamma1 loss of function result (Yu, 2005).

While the Loco-associated GAP activity can facilitate the conversion of GTP-Galphai to GDP-Galphai in NBs, how might the reverse reaction be catalyzed without invoking the involvement of a GPCR-associated GEF activity? A possible nonreceptor GEF that can fulfill this role may be the Drosophila homolog of the mammalian Ric-8A (Synembrin). Mammalian Ric-8A has been shown to act as a nonreceptor GEF for Galphao, Gq, and Galphai1 subunits. Ric-8A is evolutionarily conserved from worm to mammals. More recent reports on C. elegans RIC-8 suggest that it functions as a GEF to regulate asymmetric divisions in the zygote for the Galpha subunits (GOA-1 and GPA-16). The fly homolog, DmRic-8, is indeed able to associate with Galphai and is involved in NB asymmetric divisions (Yu, 2005).

While receptor-independent activation of heterotrimeric G-protein signaling appears to be a mechanism conserved between fly and nematode, there are clear differences between the two systems. In the nematode zygote, previous studies have suggested that the Galpha subunits, GOA-1 and GPA-16, are required for generation of a net pulling force from the posterior cortex that leads to the displacement of the mitotic spindle toward the posterior cortex. Either (possibly both) of the GoLoco/GPR motif proteins, GPR1/2, which are enriched at the posterior pole of the zygote (Colombo, 2003; Gotta, 2003), can act as GDIs to asymmetrically activate heterotrimeric G-protein signaling. The Galpha subunits and GPR1/2 both appear to act downstream of the PAR proteins and their inactivation using RNAi results in identical spindle phenotypes that resemble those seen in par-2 mutants for which a reduction in cortical spindle forces have been directly demonstrated (Colombo, 2003; Gotta, 2003). More recently, it has been reported that loss of ric-8 function also disrupts the movement of the posterior centrosome, suggesting that RIC-8 acts in the same pathway as GPR-1/2 to establish Galpha-dependent force generation, whereas loss of function of rgs-7, encoding a GAP protein for GOA-1, leads to overly vigorous posterior spindle rocking and more exaggerated size difference between two daughter cells, indicating that Galpha passes through the GTP-bound state during its activity cycle to regulate the force in one-cell-stage nematode embryos. In contrast, Gßgamma does not appear to regulate spindle displacement in the worm zygote (Yu, 2005).

For Drosophila NBs, spindle geometry and displacement appear to be regulated to a large extent through Gßgamma activation by the GoLoco proteins Loco and Pins. The spindle defects associated with loco/pins double loss-of-function NBs resemble those seen in the Gß13F and Ggamma1 mutants. However, it is clear that in Gß13F and Ggamma1 mutants there is a small degree of residual asymmetry in the size of the NB daughters; this residual size difference can be removed by the additional loss of baz function. There is no evidence implicating a major role for Galphai in spindle asymmetry since loss of Gi has relatively mild effects. However, the possibility that multiple Galpha subunits redundantly regulate NB spindle geometry cannot be ruled out (Yu, 2005).

Furthermore, in contrast to the C. elegans zygote where heterotrimeric G-protein signaling acts downstream of the PAR polarity cues, the precise hierarchical relationship between the heterotrimeric G proteins and the PAR proteins in Drosophila NBs is more complex. Some observations can be interpreted, at least formally, to suggest that free Gßgamma acts upstream of the apical components, since mutations in Gß13F and Ggamma1 cause delocalization of Pins/Loco/Galphai and affect the stability (intensity) of the Baz and DaPKC apical crescents. However, reduced levels of Baz and DaPKC can nevertheless asymmetrically localize and maintain residual levels of asymmetry despite the loss of free Gßgamma, suggesting that some aspects of NB asymmetry and PAR polarity cues act in parallel or upstream of heterotrimeric G proteins. This study provides evidence that in Drosophila NBs, both Loco and Pins contribute toward the generation of free Gßgamma and the asymmetric localization of Pins/Loco/Galphai depends not only on Gßgamma but also the right balance of GDP-Galphai and GTP-Galphai. It remains to be seen whether in NBs Gßgamma mediates the formation of an asymmetric spindle by regulating Galpha subunits (Yu, 2005).

Pins functions in the asymmetric localization of Neuralized

In Drosophila, Notch signaling regulates binary fate decisions at each asymmetric division in sensory organ lineages. Following division of the sensory organ precursor cell (pI), Notch is activated in one daughter cell (pIIa) and inhibited in the other (pIIb). The E3 ubiquitin ligase Neuralized localizes asymmetrically in the dividing pI cell and unequally segregates into the pIIb cell, like the Notch inhibitor Numb. Furthermore, Neuralized upregulates endocytosis of the Notch ligand Delta in the pIIb cell and acts in the pIIb cell to promote activation of Notch in the pIIa cell. Thus, Neuralized is a conserved regulator of Notch signaling that acts as a cell fate determinant. Polarization of the pI cell directs the unequal segregation of both Neuralized and Numb. It is proposed that coordinated upregulation of ligand activity by Neuralized and inhibition of receptor activity by Numb results in a robust bias in Notch signaling (Le Borgne, 2003).

The mechanisms by which Neur localized at the anterior cortex of the dividing pI cell were investigated. The role of the cytoskeleton was studied by applying drugs to cultured nota. Colcemid, a microtubule-depolymerizing agent, was found to have no significant effect. In contrast, both Latrunculin A, an agent that depolymerizes actin microfilaments, and the myosin motor inhibitor butanedione-2-monoxime (BDM) strongly impaired or completely inhibited the asymmetric localization of Neur. Thus, both myosin motor activity and an intact actin cytoskeleton are required for the formation and/or maintenance of the Neur crescent at the anterior cortex of the dividing pI cell. These requirements for Neur localization are similar to the ones seen earlier for Numb and Pon. Neur also behaves in a manner similar to Numb and Pon in that localization of Neur at the anterior cortex of the pI cell depends on planar polarity genes and on the polarity genes discs-large and pins. Moreover, mispartitioning of Neur in dlg and pins mutant cells correlates with a loss in asymmetric internalization of Dl. These data indicate that Neur and Numb share part of the same molecular machinery to localize asymmetrically in the pI cell (Le Borgne, 2003).

Strabismus promotes Pins anterior localization during asymmetric division of sensory organ precursor cells

Cell fate diversity is generated in part by the unequal segregation of cell-fate determinants during asymmetric cell division. In the Drosophila bristle lineage, the sensory organ precursor (pI) cell is polarized along the anteroposterior (AP) axis by Frizzled (Fz) receptor signaling. Fz localizes at the posterior apical cortex of the pI cell prior to mitosis, whereas Strabismus (Stbm) and Prickle (Pk), which are also required for AP polarization of the pI cell, co-localize at the anterior apical cortex. Thus, asymmetric localization of Fz, Stbm and Pk define two opposite cortical domains prior to mitosis of the pI cell. At mitosis, Stbm forms an anterior crescent that overlaps with the distribution of Partner of Inscuteable (Pins) and Discs-large (Dlg), two components of the anterior Dlg-Pins-Galphai complex that regulates the localization of cell-fate determinants. At prophase, Stbm promotes the anterior localization of Pins. By contrast, Dishevelled (Dsh) acts antagonistically to Stbm by excluding Pins from the posterior cortex. It is proposed that the Stbm-dependent recruitment of Pins at the anterior cortex of the pI cell is a novel read-out of planar cell polarity (Bellaïch, 2004).

Planar polarization of the pI cell occurs prior to division and is required, upon entry into mitosis, to direct the Dlg-Pins-Galphai and Baz-Par6-aPKC complexes at the anterior and posterior cortex, respectively. The localization of Pins at the anterior cortex is regulated positively by the Stbm-Pk complex and negatively by Dsh. (1) Loss of stbm activity results in a delay in the cortical localization of Pins during prophase; (2) concomitant expression of Stbm and Pk leads to a broadening of the cortical crescent of Pins at prophase; (3) loss of dsh PCP activity similarly results in an extended Pins crescent at prophase. Moreover, analysis of the defective partitioning of Pon::GFP suggests that the Stbm-Pk complex acts antagonistically to Dsh to localize at the anterior cortex a centrosome-attracting activity. It is proposed that the Stbm-Pk complex organizes the anterior cortex and recruits the Dlg-Pins-Galphai complex as well as molecules regulating spindle positioning (Bellaïch, 2004).

Cortical localization of Pins is a novel read-out of PCP signaling in the pI cell that is distinct from the ones previously identified in wing and eye cells. In wing epidermal cells, Fz promotes the formation of a polarized actin cytoskeleton via a pathway that possibly involves a direct interaction between Dsh and a Daam1-Rho complex and a Rho Kinase-dependent phosphorylation of cytoplasmic myosin. Whether Dsh also regulates microfilament assembly in pI cells remains to be studied. In photoreceptor cells, the read-out for PCP signaling is the transcriptional regulation of the Delta gene in R3. Thus, the conserved core of PCP signaling molecules have different, cell-type specific read-outs (Bellaïch, 2004).

How does Stbm direct the localization of Pins to the anterior cortex? One hypothesis is that Stbm directs the anterior localization of Pins via the regulated assembly of a Stbm-Dlg-Pins complex. The anterior accumulation of Pins depends on its interaction with Dlg. Evidence is provided that Stbm may bind Dlg. (1) In vitro binding studies indicate that Stbm interacts with Dlg. It is noted, however, that PDZ-containing proteins other than Dlg may also bind Stbm in this assay. (2) The localization of Stbm overlaps with the distribution of Pins and Dlg in dividing pI cells. (3) The PBM motif of Stbm appears to regulate the re-localization of Stbm in pI cells. The data are therefore consistent with a model in which, upon mitosis, the binding of Stbm to Dlg in turn promotes the binding of Pins to Dlg and, hence, localization of Pins at the anterior cortex where Stbm and Dlg accumulations overlap. This model predicts that the PBM of Stbm should be required for the anterior localization of Pins. It was found, however, that StbmDeltaPBM is fully functional and that Pins is properly recruited at the anterior cortex in stbm6c mutant pI cells expressing StbmDeltaPBM. One interpretation of this result is that Stbm regulates the localization of Pins not only via the PBM-dependent assembly of the Dlg-Pins complex but also via a second PBM-independent mechanism. Since Dsh acts redundantly with Baz to localize Pins asymmetrically, it is suggested that this second mechanism may involve Dsh. Accordingly, in stbm6c mutant pI cells, uniformly distributed Dsh activity would prevent Pins cortical localization. By contrast, since the PCP function of stbm does not depend on its PBM, the activity of Dsh should be restricted to the posterior cortex in stbm6c mutant pI cells expressing StbmDeltaPBM. Dsh should therefore restrict Pins localization to the anterior cortex in this mutant background. Another interpretation of the correct localization of Pins in stbm6c mutant pI cells expressing StbmDeltaPBM is that Stbm recruits Pins via a mechanism that does not involve an interaction with Dlg (or any other PDZ-containing proteins). Future studies will address how the Stbm-Pk complex regulates the localization of Pins in the pI cell (Bellaïch, 2004).

Different mechanisms appear to cooperate to maintain Pins asymmetric localization. baz is required for the asymmetric localization of Pins in the absence of dsh PCP activity. This indicates that Baz can regulate the maintenance of Pins asymmetric localization at prometaphase. The loss of asymmetric localization of Pins in dsh baz mutant pI cells suggests that Dsh may also contribute to maintain Pins asymmetric localization at prometaphase. Dsh does not merely act by excluding Stbm, a positive regulator of Pins localization in prophase, because Pins localizes asymmetrically in baz stbm double mutant pI cells. The mechanisms by which Baz and Dsh regulates Pins localization are not known. However, because Pins regulates its own localization via a Gß13F-dependent positive feedback loop, one hypothesis is that Baz and/or Dsh negatively regulates Gß13F signaling activity (Bellaïch, 2004).

One of the best examples of PCP in mammals is the stereotyped planar orientation of the stereociliary bundles that are located at the apical cortex of each mechanosensory hair cell within the cochlea. In these cells, the first sign of polarization is the stereotyped movement, at the luminal surface of the cell and along the neural-abneural axis, of the kinocilium, the single tubulin-based cilium, from the center towards the abneural pole of the cell. Recently, a mutation in a stbm homolog, Vangl2, has been shown to result in the defective orientation of the stereociliary bundles. This planar cell polarity defect appears to result from the randomly oriented center-to-periphery movement of the kinocilium. Because LGN, a mammalian homolog of Pins, is known to regulate microtubule stability, it is tempting to speculate that Vangl2 may regulate via LGN a microtubule-dependent process regulating kinocilium movement along the neural-abneural axis. Future studies will reveal whether the regulation of Pins/LGN cortical localization is a conserved read-out of PCP (Bellaïch, 2004).

Differential functions of G protein and Baz-aPKC signaling pathways in Drosophila neuroblast asymmetric division - a predominant role of Partner of Inscuteable-Gi in spindle orientation

Drosophila neuroblasts (NBs) undergo asymmetric divisions during which cell-fate determinants localize asymmetrically, mitotic spindles orient along the apical-basal axis, and unequal-sized daughter cells appear. This study identified a Drosophila mutant in the Ggamma1 subunit of heterotrimeric G protein, which produces Ggamma1 lacking its membrane anchor site and exhibits phenotypes identical to those of Gß13F, including abnormal spindle asymmetry and spindle orientation in NB divisions. This mutant fails to bind Gß13F to the membrane, indicating an essential role of cortical G1-Gß13F signaling in asymmetric divisions. In Ggamma1 and Gß13F mutant NBs, Pins-Galphai, which normally localize in the apical cortex, no longer distribute asymmetrically. However, the other apical components, Bazooka-atypical PKC-Par6-Inscuteable, still remain polarized and responsible for asymmetric Miranda localization, suggesting their dominant role in localizing cell-fate determinants. Further analysis of Gßgamma and other mutants indicates a predominant role of Partner of Inscuteable-Gi in spindle orientation. It is thus suggested that the two apical signaling pathways have overlapping but different roles in asymmetric NB division (Izumi, 2004).

Because the Gß13F-Ggamma1 complex, which distributes uniformly in the cortex, functions in asymmetric organization of the spindle, differential activation or inactivation of Gßgamma signaling must occur in the apical-basal direction. Two apical signaling pathways are implicated in the apical-basal difference in spindle development in a redundant fashion. What is the relationship between the apical signals and the Gßgamma signal? Spindle size is reduced by an increase in the amount of Gßgamma, but a lack of Gßgamma results in formation of a large, symmetric spindle. These findings raise the possibility that spindle development is suppressed by the Gßgamma signal, which is repressed by the presence of an apical complex on the apical side in the wild-type cells, resulting in a large apical and small basal spindle. This model suggests that the apical complex acts upstream of the Gßgamma signal. In contrast, elimination of Gß13F affects the localization of the apical components: Pins becomes uniformly distributed and Galphai becomes undetectable. In addition, Ggamma1N159 and G圩F mutations appear to destabilize the localization of the components in the Baz-DaPKC pathway, as judged by the reduced staining by their antibodies (although this may be an indirect consequence of the mislocalization of Pins-Galphai). The Gßgamma signal is thus required for normal distribution of the components of both apical pathways, consistent with the idea that the apical pathways acts downstream of the Gßgamma signal in regulating spindle asymmetry. Tests for epistasis between the apical pathways and the Gßgamma signal are needed to clarify their relationship in the regulation of spindle organization (Izumi, 2004).

The effects of Ggamma1N159 and G圩F mutations on cell-size asymmetry are remarkable but different from those in double mutants in which both apical pathways are disrupted simultaneously, where daughter cell sizes are completely equal. The cell-size ratio of GMCs to their sibling NBs shows a broad distribution: from 0.6 to 1 in the Ggamma1 (and G圩F) mutants. This residual asymmetry in daughter cell size is due to Baz-DaPKC activity. The components of this pathway indeed distribute asymmetrically in Ggamma1 (and G圩F) mutant NBs in which Pins-Galphai activity is no longer asymmetric (Pins is uniformly distributed and Galphai is absent) (Izumi, 2004).

Why does this polarized Baz-DaPKC activity cause less asymmetry in daughter cell size in spite of the redundant function of the Baz-DaPKC pathway and Pins-Galphai? Antibody staining for Baz, DaPKC, and DmPar-6 suggests that their levels and their polarized distribution are weakened in Ggamma1 (and G圩F) mutants. A possible explanation is that low levels of polarized Baz-DaPKC activity confer only low levels of asymmetry to the daughter cell size in the absence of polarized Pins-Galphai. Thus, the degree of cell-size asymmetry resulting from NB divisions may depend on the dosage of the components of one apical pathway when the other is absent or uniformly distributed. In contrast, Miranda localization does not appear to be severely impaired in Ggamma1N159 and G圩F mutants until late embryonic stages, indicating that the polarized Baz-DaPKC activity in these mutants is sufficient to localize Miranda. Therefore, full asymmetry in daughter cell size may require relatively higher levels of Baz-DaPKC activity than does polarized distribution of cell-fate determinants does (Izumi, 2004).

In Ggamma1N159 and G圩F mutants, Insc has a distribution different from the other components of the Baz-DaPKC pathway. In most of these mutant NBs, Insc distributes broadly to both the cytoplasm and the cortex in a slightly asymmetric way, but Baz, DaPKC, and DmPar-6 localize asymmetrically in the cortex. The cytoplasmic distribution of Insc is also slightly asymmetric in pins mutant NBs. It is not known whether cytoplasmic Insc is functional. Interestingly, Insc distribution often appears to correlate better with the asymmetry in daughter cell size than do the other components of the Baz-DaPKC pathway in Ggamma1N159 and G圩F mutants: in most telophase NBs that are cleaving into two equal daughters, DaPKC and DmPar-6 are excluded from the daughter GMC, but Insc tends to distribute evenly to both daughter cells. This occurs also in pins mutants, in which ~15% of NBs divide equally but most NBs divide unequally. In pins NBs cleaving equally, Insc is found equally in the cytoplasm of both daughter cells, but DaPKC and DmPar-6 remain in the newly forming NB; in unequally dividing NBs, all three components are found preferably on the NB side. These observations raise the intriguing possibility that Insc has more important roles in the generation of spindle asymmetry than do the other components of the Baz-DaPKC pathway. Because the absence of Baz results in mislocalization of Insc and vice versa, it is technically difficult to discriminate Insc-specific from Baz-specific functions. It may be Insc or some unknown Insc-associating effectors, rather than Baz, that functions in parallel with Pins-Galphai in the establishment of cell-size asymmetry (Izumi, 2004).

The question of whether the two apical pathways have redundant functions in aspects of NB division other than cell-size asymmetry has been elusive. In this paper, examination of Ggamma1N159 and G圩F mutant NBs, as well as those overexpressing baz, suggests that the asymmetric localization of Miranda depends solely on polarized Baz activity and not on Pins-Galphai function. Miranda always distributes on the cortical side, opposite the distribution of Baz in these mutants and in the wild-type. This also occurs for sensory precursor cells in the peripheral nervous system: in sensory precursor cell division Insc is not expressed, and Pins and Baz distribute on cortical sides opposite to each other, unlike in NBs; however, both Miranda and Numb localize to the cortex opposite Baz, as seen in NBs (Izumi, 2004).

Phosphorylation of the Lethal (2) giant larvae protein by DaPKC directs the localization of cell-fate determinants to the basal cell cortex. When baz is overexpressed in NBs, ectopically distributed Baz excludes Miranda from the Baz region and DaPKC colocalizes with the ectopic Baz. In contrast, a decrease in Baz activity in the wild-type results in cytoplasmic localization of DaPKC and uniform cortical distribution of Miranda. All these findings suggest that the Baz-directed localization of DaPKC excludes Miranda from the apical cortex via Lethal (2) giant larvae phosphorylation. In the absence of Baz, Miranda is eventually concentrated to the budding GMC during telophase by unknown mechanisms, a phenomenon called 'telophase rescue'. This phenomenon did not occur by depleting both baz activity and Gßgamma signaling, suggesting that telophase rescue involves Gßgamma signaling or asymmetric Pins-Galphai localization (Izumi, 2004).

The absence of any single component of the apical complex has the same effect on spindle orientation during NB division, which is normally perpendicular to the apical-basal axis. Thus, proper orientation of the spindle has been thought to require all the apical components. However, observations on epithelial cells and mitotic domain 9 cells indicated that the spindle always points to the location of Pins when Pins is localized in the cell. This alignment of the spindle toward Pins occurs irrespective of the localization of the Baz-pathway components. For instance, wild-type epithelial cells divide parallel (Pins direction) but not perpendicular to the apical-basal axis (Baz direction); so do most epithelial cells and mitotic domain 9 cells in G圩F and Ggamma1 mutants. Therefore, the Pins-Galphai pathway, rather than the Baz-DaPKC complex, is likely to play a dominant role in controlling spindle orientation (Izumi, 2004).

In most NBs in pins, G圩F, and Ggamma1 mutants, the spindle is oriented in the direction of Baz localization and therefore follows the localization of the cell-fate determinants. This coincidence results in the determinants' virtually normal segregation to one daughter cell despite the random orientation of division. Thus, only when Pins-Galphai are absent or uniformly distributed in NBs, polar Baz activity appears to be capable of directing spindle orientation. Alternatively, the mitotic spindle may position the Baz-DaPKC complex over one spindle pole (Izumi, 2004).

In the NB in which the Baz-DaPKC pathway is depleted, Pins-Galphai can still localize asymmetrically and orient the spindle. Interestingly, the Pins crescent forms in random orientations in this situation, leading to random spindle orientation. This fact suggests that the Baz-DaPKC complex or its combination with Pins-Galphai is necessary to orient the Pins-Galphai crescent in the apical direction of the NB, raising an intriguing possibility that there are unknown mechanisms by which formation of the apical complex occurs on the apical side. This postulated mechanism may involve interactions with neighboring epithelial cells (Izumi, 2004).

What is the molecular mechanism by which Pins-Galphai orient the spindle? It is interesting to assume that Pins has the ability to attract the spindle pole. This idea is consistent with previous evidence; although epithelial cells do not normally express Insc, its ectopic expression in these cells recruits Pins-Galphai to the apical cortex and reorients the mitotic spindle in the apical-basal direction. The C. elegans homologues of Pins, GPR-1/GPR-2, interact with Galphai/Galphao and a coiled-coil protein, LIN-5, which is required for GPR-1/GPR-2 localization. All these molecules are indeed involved in the regulation of forces attracting spindles during early cleavages. Although Lin-5 has no obvious homologue in other species, functional homologues may regulate Pins localization and/or the connection between the spindle pole and Pins in Drosophila. Furthermore, the C. elegans gene ric-8, which interacts genetically with a Galphao gene, is also required for embryonic spindle positioning. Its homologue in mammals acts as a guanine nucleotide exchange factor for Galphao, Galphaq, and Galphai. An analysis of the Drosophila RIC-8 homologue may give insight into the mechanisms by which Pins-Galphai regulate spindle orientation (Izumi, 2004).

Microtubule-induced Pins/Gαi cortical polarity in Drosophila neuroblasts

Cortical polarity regulates cell division, migration, and differentiation. Microtubules induce cortical polarity in yeast, but few examples are known in metazoans. Astral microtubules, kinesin Khc-73, and Discs large (Dlg) induce cortical polarization of Pins/Gαi in Drosophila neuroblasts; this cortical domain is functional for generating spindle asymmetry, daughter-cell-size asymmetry, and distinct sibling fates. Khc-73 localizes to astral microtubule plus ends, and Dlg/Khc-73 and Dlg/Pins coimmunoprecipitate, suggesting that microtubules induce Pins/Gαi cortical polarity through Dlg/Khc-73 interactions. The microtubule/Khc-73/Dlg pathway acts in parallel to the well-characterized Inscuteable/Par pathway, but each provides unique spatial and temporal information: The Inscuteable/Par pathway initiates at prophase to coordinate neuroblast cortical polarity with CNS tissue polarity, whereas the microtubule/Khc-73/Dlg pathway functions at metaphase to coordinate neuroblast cortical polarity with the mitotic spindle axis. These results identify a role for microtubules in polarizing the neuroblast cortex, a fundamental step for generating cell diversity through asymmetric cell division (Siegrist, 2005).

A current model for the establishment of neuroblast cortical polarity is that an unknown cue recruits Baz, aPKC, Par-6, and Insc to the apical cortex of the neuroblast just prior to prophase, which is closely followed by the apical recruitment of Pins/Gαi proteins, presumably via Insc-Pins direct interactions. This is termed the cortical “Insc/Par pathway” of Pins/Gαi localization to distinguish it from the Insc-independent “microtubule-based pathway” of Pins/Gαi localization that is the focus of this paper (Siegrist, 2005).

insc22 null mutant embryos (insc mutants) lack apical localization of the Insc/Par complex proteins (Insc, Baz, aPKC, and Par-6), but interestingly that Pins, Gαi, and Dlg still form robust crescents in the majority of insc mutant metaphase neuroblasts. Similar results were observed in mitotic neuroblasts from embryos homozygous for the TE35 deficiency in which insc is not transcribed. Although Pins/Gαi/Dlg crescents form in insc mutants, the timing and position of crescent formation differed from wild-type. First, in wild-type neuroblasts Pins/Gαi/Dlg crescents always formed at the apical surface adjacent to the overlying ectoderm, whereas in insc mutant neuroblasts Pins/Gαi/Dlg crescents were found at all positions around the cortex. Second, in wild-type neuroblasts Pins/Gαi crescents formed by early prophase (94%), whereas in insc mutants Pins/Gαi crescents were not detected at prophase but only at metaphase (78%). These results suggest that there is an Insc/Par-independent pathway that is active at metaphase to induce formation of Pins/Gαi/Dlg cortical crescents (Siegrist, 2005).

A clue to the identity of the Insc/Par-independent pathway was the observation that Pins/Gαi/Dlg crescents were always colocalized over one spindle pole, which can be mispositioned relative to the overlying ectoderm in insc mutants. This observation suggested that either spindle microtubules induced cortical polarity, or cortical polarity formed spontaneously at a nonapical position and induced spindle alignment. To distinguish between these mechanisms, microtubules were depolymerized in insc mutant neuroblasts with Colcemid, and Pins/Gαi/Dlg cortical crescents were scored. Colcemid treatment of insc mutant neuroblasts resulted in a nearly complete loss of Pins/Gαi/Dlg crescents: Pins is mostly cytoplasmic and Gαi/Dlg are uniform cortical. In contrast, Colcemid treatment of wild-type neuroblasts had no effect on Pins/Gαi/Dlg crescent formation, likely due to the association of Pins/Gαi/Dlg with the apical Insc/Par complex. In fact, the Insc/Par pathway of Pins/Gαi/Dlg localization requires only Insc and Baz proteins, because aPKC mutants that lack aPKC/Par-6 protein localization but retain Baz/Insc localization still formed Pins/Gαi/Dlg crescents in the absence of microtubules. It is concluded that spindle microtubules have the ability to induce Pins/Gαi/Dlg cortical crescents over one spindle pole in the absence of an Insc/Par pathway (Siegrist, 2005).

Recent work has shown that microtubules can directly regulate cortical polarity in yeast during C. elegans meiosis and in migrating cells. An important question is the extent to which microtubules regulate cortical cell polarity in other contexts. This study identifies a microtubule/kinesin pathway for inducing cortical polarity in Drosophila neuroblasts. This pathway is sufficient to induce cortical polarization of the evolutionarily conserved Dlg, Pins, and Gαi proteins and is necessary for reliable spindle orientation relative to apical Insc/Par cortical proteins (Siegrist, 2005).

A model is presented for the microtubule/Khc-73/Dlg pathway, in the absence of the Insc/Par function.

Asymmetric localization of Pins/Gαi proteins can be induced by two distinct pathways in embryonic neuroblasts: a well-characterized cortical pathway involving the Insc/Par proteins and a microtubule-dependent Khc-73/Dlg pathway. Each pathway is regulated differently and has unique features that provide different temporal and spatial information for generating cortical polarity (Siegrist, 2005).

First, each pathway is initiated by a different mechanism and provides unique information for the timing of Pins/Gαi polarization. The Insc/Par pathway is initiated at late interphase in response to an unknown extrinsic cue and is required for the early prophase cortical polarization of Pins/Gαi. In contrast, the Khc-73/Dlg pathway is initiated later at prometaphase/metaphase by astral microtubules and is required for cortical polarization of Pins/Gαi only in the absence of Insc/Par complex proteins. Consistent with this timeline, asymmetric enrichment of Dlg normally occurs well after polarization of Insc/Par/Pins/Gαi during the prometaphase/metaphase transition, and this temporal progression of Dlg cortical enrichment is not affected in insc mutants. The temporal polarization of Dlg coincides precisely with the onset of Pins/Gαi cortical polarity at prometaphase/metaphase that occurs in the absence of the Insc/Par pathway (Siegrist, 2005).

Next, each pathway provides different spatial information for the cortical polarization of Pins/Gαi. The Insc/Par pathway recruits Pins/Gαi to the apical cortex of the neuroblast at a position just below the overlaying epithelium, thus coordinating neuroblast cortical polarity with the neuroblast environment. In the absence of this pathway (e.g., insc mutant neuroblasts), cortical polarity can be generated but is not linked to tissue polarity, resulting in mispositioning of neuroblast progeny. In contrast, the microtubule/Khc-73/Dlg pathway induces Pins/Gαi crescent formation over one spindle pole, thus coordinating the neuroblast cortical polarity with the spindle axis. In the absence of this pathway (e.g., dlg mutant or Khc-73 RNAi neuroblasts), Insc/Baz can still recruit Pins/Gαi to the apical cortex, yet the spindle is not always properly aligned with this cortical polarity. Together these two pathways ensure the correct temporal and spatial positioning of apical complex proteins relative to extrinsic and intrinsic landmarks (Siegrist, 2005).

Drosophila sense organ precursors (SOPs) divide asymmetrically to generate an anterior pIIb cell and a posterior pIIa cell. During this division, Pins, Gαi, Dlg, and Numb form cortical crescents over the anterior spindle pole, and Baz localizes over the posterior spindle pole. Cell division orientation is fixed along the anterior/posterior axis by planar polarity cues mediated by the seven pass transmembrane receptor Frizzled. However, Frizzled signaling is required only for the position of Dlg/Pins crescents, not for their formation. When both frizzled and microtubules were remove together, it was found about 10% of the mitotic SOPs lack Pins crescents. This mild phenotype suggests that while astral microtubules may contribute to Dlg/Pins polarization in SOPs, there must be an additional mechanism involved. The best candidates for this third mechanism are the Par proteins because Par crescents still form in frizzled mutant SOPs at metaphase (Siegrist, 2005).

There are many similarities between asymmetric division of fly neuroblasts and the C. elegans zygote, but there are also striking differences. One of the most noteworthy differences is that C. elegans par mutants undergo symmetrically sized embryonic cell divisions, whereas in Drosophila, par or insc mutants maintain sibling cell size asymmetry. This work provides an explanation for this discrepancy. It is shown that astral microtubules are capable of generating Pins/Gαi cortical polarity in the absence of localized Par proteins and that this microtubule-induced Pins/Gαi cortical polarity is fully functional for generating an asymmetric spindle, cell size, and unique daughter cell fates. It is likely that C. elegans lacks this “microtubule-based pathway” for inducing GPR1/2 (Pins) and Gα cortical polarity, at least during the first embryonic cell division, because posterior cortical localization of GPR1/2 is absent in par mutants and the daughter cells are equal in size. Interestingly, an increase is observed in symmetrically dividing neuroblasts in neuroblasts lacking both Insc/Par and microtubule pathways, compared to loss of single pathways alone. It appears that either the Insc/Par or microtubule/Khc-73/Dlg pathway is sufficient to induce Pins/Gαi cortical polarity, which generates daughter cells of different sizes and fates (Siegrist, 2005).

The microtubule/kinesin-induced Dlg clustering pathway described in this study may be evolutionarily conserved. In mammals, hDlg and the Khc-73 ortholog GAKIN are coexpressed in T cells and coimmunoprecipitate, and T cell activation leads to recruitment of hDlg to the immunological synapse (Hanada, 2000). Interestingly, GAKIN targets hDlg into ectopic cellular projections in MDCK cells, and this targeting depends on microtubules (Asaba, 2003). This has lead to the hypothesis that GAKIN may use a microtubule-based mechanism to target hDlg to the T cell immune synapse, similar to the microtubule/Khc-73 pathway described in this paper (Siegrist, 2005).

The Drosophila NuMA homolog Mud regulates spindle orientation in asymmetric cell division

During asymmetric cell division, the mitotic spindle must be properly oriented to ensure the asymmetric segregation of cell fate determinants into only one of the two daughter cells. In Drosophila neuroblasts, spindle orientation requires heterotrimeric G proteins and the Gα binding partner Pins, but how the Pins-Gαi complex interacts with the mitotic spindle is unclear. This study shows that Pins binds directly to the microtubule binding protein Mud, the Drosophila homolog of Nuclear Mitotic Apparatus (NuMA) protein. Like NuMA, Mud can bind to microtubules and enhance microtubule polymerization. mud mutants form functional spindles and the neuroblasts are correctly polarized. Consistent with this, Brat and Numb form crescents in mud mutant neuroblasts, but the spindle is not aligned with them. Mitotic spindles in neuroblasts fail to align with the polarity axis. Therefore, the spindle orientation defect is a direct consequence of Mud loss of function. mud mutation can lead to symmetric segregation of the cell fate determinants Brat and Prospero, resulting in the misspecification of daughter cell fates and tumor-like overproliferation in the Drosophila nervous system. The data suggest a model in which asymmetrically localized Pins-Gαi complexes regulate spindle orientation by directly binding to Mud (Bowman, 2006; Izumi, 2006; Siller, 2006).

The role of heterotrimeric G proteins in asymmetric cell division is well studied in Drosophila. In embryonic neuroblasts, G proteins make three major contributions: (1) maintenance of the apical localization of Inscuteable and the Par complex, (2) regulation of spindle orientation at metaphase, and (3) generation of spindle asymmetry at anaphase. It is thought that both free Gβγ and Pins-Gαi, as well as Par complex members Baz and aPKC, have a role to play in the control of spindle asymmetry. Whether G proteins can directly regulate spindle orientation is less clear because of the complexity of G protein phenotypes. Misregulation of G proteins can cause Insc and Par complex delocalization as well as spindle orientation defects. As a result, it is difficult to determine whether it is actually G proteins that are responsible for spindle misorientation, or whether the orientation defect is a secondary consequence of a general loss of polarity. mud mutants, however, show spindle misorientation without Insc or Par delocalization. Since Mud binds to Pins and localizes asymmetrically in neuroblasts, this suggests that Pins-Gαi regulates spindle orientation through its interaction with Mud (Bowman, 2006).

In vertebrates, the Pins-Gαi complex is proposed to control the attachment of astral microtubules to the cortex through its interaction with NuMA. This model of spindle positioning is supported by an experiment in which overexpressed Pins causes spindle rocking movements that can be inhibited by coexpressing a short fragment of NuMA or disrupting astral microtubules with low concentrations of nocodazole. In Drosophila, astral microtubules are also important for spindle positioning. Mutations in centrosomin and asterless prevent the formation of centrosomes and astral microtubules, and neuroblasts in these mutant backgrounds often fail to coordinate the mitotic spindle with the crescent of cell fate determinants at metaphase. Abolishing astral microtubules pharmacologically produces similar results. It is proposed that Mud forms a complex with Pins and Gαi that regulates the attachment of astral microtubules to the cortex, and that this regulation is necessary for the mitotic spindle to assume the correct orientation in asymmetric cell division. In mud mutants, faulty microtubule-cortical attachment results in a failure to coordinate the mitotic spindle with the axis of polarity. Accordingly, the spindle assumes orientations that do not align with the crescents of Insc and Miranda, and regulators of cell size as well as cell fate determinants can be inherited symmetrically (Bowman, 2006).

The identification of Mud and LIN-5 as NuMA homologs indicates that three different model organisms use NuMA-like proteins to regulate spindle movements. During the first division of the C. elegans zygote, the mitotic spindle is set up along the A/P axis in the center of the cell. In anaphase, the spindle rocks vigorously as the posterior centrosome is displaced toward the posterior cortex. Following this division, mitosis begins in the daughter cells, which initially align their centrosomes transverse to the A/P axis. However, the spindle in the posterior cell eventually rotates 90° and orients along the A/P axis. These spindle rocking and displacement movements require the NuMA-like protein LIN-5. Because LIN-5 is found in a complex with the Pins-like GoLoco motif proteins GPR-1 and GPR-2, and because the phenotype of GPR-1/-2 loss of function is nearly identical to that of LIN-5, it is thought that LIN-5 and GPR-1/-2 act together to generate the forces required for spindle rocking and spindle orientation in mitosis (Bowman, 2006).

In rodents, NuMA, mammalian Inscuteable (mInsc), and G proteins regulate spindle orientation in the asymmetric division of self-renewing stem cells. Epidermal stem cells localize mInsc, NuMA, and Pins to an apical crescent and align the spindle parallel to the apical-basal axis. If apical localization of Pins and NuMA is disrupted, spindle orientation becomes randomized. In the developing neocortex, neural progenitors divide with their spindles orthogonal to the apical-basal axis for symmetric divisions and parallel to this axis for asymmetric divisions. Reliable coordination of the spindle with the apical-basal axis during asymmetric division requires mInsc, free Gβγ, and the Pins-like protein AGS3. If the function of any of these proteins is compromised, asymmetric divisions fail because of misoriented spindles. Furthermore, NuMA and Pins can create spindle-rocking movements during mitotis. This work shows that the NuMA-like protein Mud forms a complex with Pins and Gαi and is required for spindle orientation in asymmetrically dividing Drosophila neuroblasts. Taken together, these studies strongly suggest that asymmetric cell divisions in C. elegans, Drosophila, and vertebrates all use NuMA-Pins-Gαi complexes to regulate spindle orientation (Bowman, 2006).

In mud mutants, failure of asymmetric division leads to an expansion of the neuroblast pool. This places mud with lgl and brat in a class of genes in which zygotic loss of function produces ectopic neuroblasts. Because of the interaction of Pins with Mud, pins mutants could also be expected to have defective spindle orientation and symmetric divisions that produce two neuroblasts. Surprisingly, pins mutant neuroblasts do not overproliferate. In fact, they exhibit a mild underproliferation phenotype (Bowman, 2006).

How can the difference in the proliferative behavior of mud and pins mutant neuroblasts be explained? First, the possibility that in addition to regulating spindle orientation, Mud directly inhibits proliferation by an unknown mechanism cannot be excluded. Since the overproliferation in mud mutants is mild compared to that in lgl or brat mutants, this seems unlikely. Second, Pins could be acting redundantly with Loco to regulate spindle orientation, so a potential pins mutant overproliferation is masked by the presence of Loco. Since Mud-C does not bind to Loco under the same conditions with which it binds to Pins, the notion that Loco substitutes for Pins by interacting with Mud is questionable. Alternatively, the proliferative differences could be explained by the localization of aPKC. A recent study in larval neuroblasts suggests that inheritance of cortical aPKC can confer the ability to self-renew. Since work in embryos has shown that Pins is required to maintain the apical localization of the Par complex, it follows that in pins mutant brains, aPKC localizes weakly to the cortex and cytoplasm of metaphase neuroblasts. By contrast, aPKC forms a cortical crescent in mud mutants. In this model, pins mutant daughter cells inheriting cytoplasmic aPKC are more likely to exit the cell cycle, while, in mud mutants, the daughter cells inheriting cortical aPKC continue to proliferate as neuroblasts. The data neither prove nor disprove this hypothesis (Bowman, 2006).

Drosophila Pins-binding protein Mud regulates spindle-polarity coupling and centrosome organization

The orientation of the mitotic spindle relative to the cell axis determines whether polarized cells undergo symmetric or asymmetric divisions. Drosophila epithelial cells and neuroblasts provide an ideal pair of cells to study the regulatory mechanisms involved. Epithelial cells divide symmetrically, perpendicular to the apical-basal axis. In the asymmetric divisions of neuroblasts, by contrast, the spindle reorients parallel to that axis, leading to the unequal distribution of cell-fate determinants to one daughter cell. Receptor-independent G-protein signalling involving the GoLoco protein Pins is essential for spindle orientation in both cell types. This study identifies Mud as a downstream effector in this pathway. Mud directly associates and colocalizes with Pins at the cell cortex overlying the spindle pole(s) in both neuroblasts and epithelial cells. The cortical Mud protein is essential for proper spindle orientation in the two different division modes. Moreover, Mud localizes to centrosomes during mitosis independently of Pins to regulate centrosomal organization. It is proposed that Drosophila Mud, vertebrate NuMA4 and Caenorhabditis elegans Lin-5 have conserved roles in the mechanism by which G-proteins regulate the mitotic spindle (Izumi, 2006).

Drosophila neuroblasts delaminate from the epithelial cell layer and undergo asymmetric divisions to produce a chain of smaller ganglion mother cells (GMCs) on the basal side. These divisions are accomplished by localizing cell-fate determinants such as Numb, Prospero and its adaptor Miranda asymmetrically to the basal cortex, and rotating the spindle 90° to ensure unequal partitioning of the determinants. The atypical protein kinase C (aPKC)-Par complex (including Bazooka/Par-3, aPKC and Par-6) acts to create cell polarity in both epithelial cells and neuroblasts. The oriented division of those cells also requires heterotrimeric G-protein signalling, which involves the heterotrimeric G-protein subunit Galphai and guanine nucleotide dissociation inhibitors (GDIs) with the GoLoco motif (Pins and Loco), and can activate the Galpha and Gßgamma subunits independently of receptor signalling. Whereas in epithelial cells, Galphai and Pins localize to the lateral cortex, Inscuteable is expressed in neuroblasts. Inscuteable then recruits Pins-Galphai to the apical cortex by interacting with both Baz and Pins (Izumi, 2006).

Although a growing body of evidence indicates that the Pins-Galphai pathway is involved in the regulation of spindle orientation and spindle configuration in Drosophila, Caenorhabditis elegans and mammals, the underlying mechanisms are poorly understood. To address this question, molecules were sought that mediate the interactions of the Pins-Galphai complex with astral microtubules in Drosophila by coimmunoprecipitation with Pins. FLAG-tagged variants of Pins were overexpressed in embryos, and their extracts were subjected to immunoprecipitation with anti-FLAG antibody. A protein with a relative molecular mass of more than 200,000 was specifically coimmunoprecipitated with the amino-terminal region of Pins, PinsDelta5-FLAG. Mass spectrometry revealed this protein to be Mushroom body defect (Mud). The mud gene, which was previously identified from mutations affecting adult brain morphology, encodes several large coiled-coil proteins. Of the three characterized Mud isoforms, the longest isoform (2,501 amino acids) is mainly expressed in embryos. When wild-type embryos were subjected to immunoprecipitation with the anti-Pins antibody, the endogenous Mud protein coimmunoprecipitated with Pins. In in vitro binding assays, the Pins amino-terminal region directly interacts with a domain in the longest Mud isoform, which was found in the Mud fragment that was sufficient for the asymmetric distribution to the apical cortex. These results strongly indicate that Mud directly associates with Pins in vivo (Izumi, 2006).

Mud and Pins were compared in terms of their subcellular localization by generating several antibodies specific to different parts of Mud. In neuroblasts, Mud was detected at the apical cortex throughout the cell cycle, whereas it is barely detectable in the basal cortex. In addition, Mud emerged in both the apical and basal centrosomal regions during mitosis. Mud staining was stronger for the apical centrosome, reflecting the differential sizes of the two centrosomes. Mud and Pins colocalized at the apical cortex, although Pins was absent in the centrosomal regions. In epithelial cells, Mud and Pins colocalize along the lateral cortex throughout the cell cycle, whereas Mud (but not Pins) is also detected in the two centrosomal regions during mitosis. Both apical and centrosomal distributions of Mud were observed in dividing cells in mitotic domain 9 of the procephalic neuroepithelium, where cells divide perpendicular to the embryo's surface. These results indicate that the cortical domains where the two proteins colocalize are tightly correlated with spindle orientation in those three mitotic cell types (Izumi, 2006).

Next, how the subcellular localization of Mud is determined was examined. In both pins and Galphai mutant neuroblasts, Mud remains in the two centrosomal regions but fails to localize to the apical cortex during mitosis. Since the absence of Mud does not affect the asymmetric localization of Pins, this finding indicates that Pins recruits Mud to the apical cortex in mitosis via a direct molecular interaction. By contrast, during interphase, Mud localization is not affected in pins mutant cells, indicating that a secondary mechanism functions for Mud apical localization in interphase. By contrast, microtubules are required for the centrosomal, but not the cortical, localization of Mud; it distributes along microtubules near centrosomes during mitosis. When wild-type embryos were treated with colcemid to depolymerize the microtubules, Mud remained at the apical cortex in neuroblasts, but not in the centrosomes. Given this set of findings, it is concluded that Mud distributes in mitotic cells in two mutually independent ways: a Pins-Galphai-dependent mechanism for cortical localization, and a Pins-independent, microtubule-dependent mechanism for centrosomal accumulation (Izumi, 2006).

To investigate the role of Mud in spindle orientation, embryos homozygous for strong or null mud alleles were examined, since germline clone embryos that are both maternally and zygotically homozygous for any available mud mutation do not develop. From embryonic stage 11-12 onwards, Mud immunoreactivity becomes virtually undetectable, indicating that such embryos are in strongly hypomorphic states. During mitosis, these mutant neuroblasts localize Pins (and aPKC) and Miranda (and Prospero) in opposite cortical crescents. In wild-type neuroblasts, the asymmetric localization of these components represents cortical polarity that is perpendicular to the overlying epithelium. The orientation of the Miranda crescent in wild-type and mud neuroblasts is essentially indistinguishable during metaphase, indicating that mud neuroblasts retain cortical polarity with normal orientation. However, spindle orientation is severely affected in the mutant neuroblasts. In wild-type neuroblasts, the mitotic spindle orients along the apical-basal axis, tightly aligning with the polar distribution of Miranda (and Pins) from metaphase onwards. The spindle in mud neuroblasts, however, frequently fails to orient in the apical-basal direction, which results in its poor coordination with the basal Miranda crescent. 'Spindle coupling' is defined as how the spindle axis aligns in respect to the Miranda crescent or cortical polarity. The spindle uncoupling that is observed in mud neuroblasts continues until the completion of cytokinesis. It is concluded that Mud is required for the coupling of spindle orientation to cortical polarity (Izumi, 2006).

A similar failure in spindle coupling has been observed in pins-mutant neuroblasts; pinsp62 germline clone embryos were used to examine pins-null phenotypes, which are designated as 'pins-' or 'pins mutant' hereafter). However, pins mutants differ from mud mutants in two aspects: first, during metaphase and anaphase, both the Miranda crescent and the spindle misorient from the apical-basal axis in pins neuroblasts, whereas Miranda is oriented normally in mud metaphase neuroblasts. This indicates that Mud acts with Pins in spindle coupling with cortical polarity, but not in a separate role of Pins in maintaining the orientation of cortical polarity. Second, spindle coupling in pins mutants is, nevertheless, recovered to a large extent during telophase, a phenomenon that is termed 'telophase rescue'. Telophase rescue does not occur in mud mutants, indicating that Mud has a Pins-independent role at telophase (Izumi, 2006).

The size difference between two daughter cells is also affected in mud neuroblast divisions: the more tilted the spindle orientation, the less different the two daughter cells tend to be in stage-11 embryos. The aPKC-Par complex and Galphai-Pins function redundantly to make the two daughter cells unequal in size, presumably by regulating spindle organization. In mud-mutant neuroblasts, these components normally localize as apical crescents, generating normal cortical polarity. It is speculated that spindles that are oblique to the apical-basal axis would decrease differential effects of the apical signals on their two poles (or asters), which in turn reduces asymmetric spindle organization. When neuroblasts divide perpendicular to the apical-basal axis, spindles are indeed nearly symmetric. In these extreme cases, neuroblasts undergo centric divisions into two equal-sized daughters, both of which inherit Prospero. Although it is unclear how those daughter cells retain the properties of the neuroblast or the GMC, neuronal fate defects and/or loss of progeny neurons may occur. As expected, aberrant neuronal progeny were observed in mud-mutant embryos (Izumi, 2006).

In addition to the spindle uncoupling phenotype, supernumerary centrosomes are often observed in mud-mutant mitotic cells. These extra centrosomes are not accompanied by the formation of multipolar spindles, although infrequently a faint microtubule array emanates from an extra centrosome. Instead, a virtually normal bipolar spindle with astral microtubules is formed from a pair of centrosomes in those neuroblasts. Centrosome amplification may arise from abnormal assembly of centrosomes or cytokinesis defects. The absence of observable multinuclear figures or polyploidy in mud mutants indicates that cytokinesis occurs normally. Thus, in centrosomes, Mud seems to function in centrosome assembly or maintenance (Izumi, 2006).

Spindle uncoupling in mud mutants may be due to the loss of cortical Mud or, alternatively, result from the selection of two abnormally positioned centrosomes from supernumerary centrosomes to form the spindle. To distinguish between these two possibilities, spindle orientation was compared relative to the Miranda crescent in metaphase neuroblasts with two centrosomes and those having three or more centrosomes. The spindle orientation relative to the Miranda crescent was indistinguishable in the two neuroblast populations, indicating that spindle uncoupling occurs independently of the centrosome number in mud mutants. It is inferred from these results that cortical Mud is required for spindle coupling with cortical polarity. Mud in the centrosome may also contribute to a Pins-independent role of Mud in spindle coupling, which is suggested by the absence of telophase rescue in mud mutants, although other possibilities have not been ruled out (Izumi, 2006).

Unlike neuroblasts, proliferating epithelial cells orient the mitotic spindle parallel to the epithelial plane; this spindle alignment requires laterally distributing Pins. In mud-mutant epithelia, the spindle occasionally fails to orient in this direction, as has been described for pins mutants. The Mud-Pins complex therefore acts in spindle orientation in both neuroblasts and epithelial cells, such that the spindle orients towards the cortical domain where this complex resides (Izumi, 2006).

To further investigate the functional relationship between Mud and Pins, the effect of ectopic expression of Inscuteable was examined in epithelial cells, which do not normally express this protein. Ectopic Inscuteable relocalizes Pins from the lateral cortex to the apical cortex due to the ability of Inscuteable to bind both Bazooka and Pins, and rotates the division axis 90° to reorient along the apical-basal axis. The misexpression of Inscuteable also results in the relocalization of Mud to the apical cortex throughout the cell cycle, indicating that the Pins-Inscuteable complex can, in a dominant fashion, recruit Mud to the cortical region where the complex distributes. When Inscuteable is expressed in pins-mutant epithelial cells, the spindle does not rotate 90° due to the failure to localize Insc cortically, and frequently orients randomly, as observed in pins-mutant epithelial cells. The apical localization of Mud is never observed in these cells. These observations and the requirement of Pins for Mud localization in neuroblasts indicate that Pins is both necessary and sufficient to determine Mud cortical localization during mitosis (not interphase), and that the cortical position of the Mud-Pins (and Galphai) complex determines spindle orientation. Thus, cortical Mud probably acts downstream of Pins-Galphai to couple the spindle with cortical polarity (Izumi, 2006).

This study has shown that Mud forms a cortical complex with Pins-Galphai to act in spindle orientation during both the symmetric division of epithelial cells and the asymmetric division of neuroblasts. In vertebrates, NuMA associates with a Pins homologue LGN, which in turn binds Galphai/Galphao through its GoLoco motif and regulates spindle movement. C. elegans Lin-5 forms a complex with GoLoco proteins (GPR-1/2) and Galpha (GOA-1/GPA-16) to generate the pulling force for astral microtubules in one-cell zygotes. Therefore, NuMA, Lin-5 and Mud seem to have similar roles in the cortical processes that regulate spindle positioning. A short sequence shared by Mud, NuMA and Lin-5 is found within the respective GoLoco GDI-binding regions. This shared sequence may be important for the association with their GoLoco GDI partners (Izumi, 2006).

How then does Mud regulate spindle orientation at the cell cortex? The cortical Mud-Pins-Galphai complex probably interacts with the plus end of astral microtubules, either directly with tubulin or with microtubule plus-end-binding proteins (+TIPs). NuMA, indeed, interacts with the Dynein-Dynactin complex, which localizes to the plus-end of microtubules. Mud, therefore, links astral microtubules with Pins-Galphai, which is in turn connected with the aPKC-Par complex by Inscuteable. This sequential association between the apical components seems to achieve coupling of the spindle with cortical polarity in dividing neuroblasts. A recent study indicates that Pins apical localization is dictated by astral microtubules via +TIP Kinesin Khc-73 and the cortical Discs large protein. Mud may interact with this kinesin to affect spindle coupling (Izumi, 2006).

Another feature that is common to Mud, NuMA and Lin-5 is their localization to the centrosomal regions. The centrosomal localization of Mud is Pins-independent, and centrosome assembly is abnormal in mud mutants, but not in pins mutants. Mud function in the centrosome therefore seems to be independent of G-protein signalling. The Dynein/Dynactin complex cooperates with NuMA in the coalescence of spindle poles, as in the cell cortex. Drosophila mutants for Lis1 and glued (components of the Dynein-Dynactin complex) show defects in assembling microtubule minus ends at the pole. These observations raise the possibility that Mud may also function with the Dynein-Dynactin-Lis1 complex in centrosomal organization. Interestingly, a genome-wide two-hybrid analysis indicated that Mud binds Centrosomin, a protein that is necessary for centrosomal organization. Centrosomin may bind Mud at the centrosome to localize Mud (Izumi, 2006).

These findings suggest that the cortical complex of Galpha, GoLoco proteins and a coiled-coil protein (Mud, NuMA or Lin-5) functions in an evolutionarily conserved, receptor-independent mechanism that regulates spindle orientation. The 'search and capture' mechanism that is driven by molecules such as APC and EB1 has been proposed as another general mechanism for orienting the spindle. This mechanism is also thought to involve the Dynein-Dynactin complex. How the receptor-independent G-protein pathway and the search and capture system are related is an open question (Izumi, 2006).

The NuMA-related Mud protein binds Pins and regulates spindle orientation in Drosophila neuroblasts

Asymmetric cell division generates cell diversity during development1, 2 and regulates stem-cell self-renewal in Drosophila and mammals. In Drosophila, neuroblasts align their spindle with a cortical Partner of Inscuteable (Pins)-Galphai crescent to divide asymmetrically, but the link between cortical polarity and the mitotic spindle is poorly understood. This study shows that Pins directly binds, and coimmunoprecipitates with, the NuMA-related Mushroom body defect (Mud) protein. Pins recruits Mud to the neuroblast apical cortex, and Mud is also strongly localized to centrosome/spindle poles, in a similar way to NuMA. In mud mutants, cortical polarity is normal, but the metaphase spindle frequently fails to align with the cortical polarity axis. When spindle orientation is orthogonal to cell polarity, symmetric division occurs. It is proposed that Mud is a functional orthologue of mammalian NuMA and Caenorhabditis elegans Lin-5, and that Mud coordinates spindle orientation with cortical polarity to promote asymmetric cell division (Siller, 2006).

The Mud-Pins interaction was confirmed by showing that a short C-terminal portion of Mud containing the NLM domain and 142 amino acids of the amino-terminal sequence (amino acids 1825-1997) directly interacts with Pins in vitro. Further analysis revealed that Mud binds the amino-terminal Pins tetratricopeptide (TPR)1-7 domain, but not the C-terminal GoLoco domain. Although Insc binds TPR1-4, no Mud binding was observed to any region of Pins that was smaller than TPR1-7, indicating that all seven TPRs are required for proper presentation of the Mud-binding epitope. Consistent with the Mud-Pins direct interaction, Mud and Pins can be coimmunoprecipitated from embryonic lysates. The Mud-Pins interaction is likely to be evolutionarily conserved, since homologous domains in NuMA and Mud mediate their interaction with LGN and Pins, respectively. It is concluded that the Mud C-terminus can bind the Pins TPRs, and both proteins are part of a common protein complex in vivo (Siller, 2006).

In embryonic neuroblasts, Mud and Pins were both enriched at the cortex over the apical centrosome/spindle pole from late interphase and up to the end of metaphase. By late anaphase-telophase, Mud showed bipolar apical and basal cortical crescents over both spindle poles; this can be seen most clearly in neuroblasts that are cultured in vitro, where there are fewer surrounding cells. In addition, Mud shows strong spindle-pole/centrosome localization and weaker spindle and astral microtubule localization in all neuroblasts. In larval neuroblasts, Mud is always present at the apical cortex with Pins: it either forms cortical crescents over both spindle poles or is uniformly cortical. In mud null mutants, larval neuroblasts have virtually no detectable Mud protein, confirming the antibody specificity. It is concluded that Mud and Pins form apical cortical crescents during late interphase and prophase (this is the time when spindle orientation is established in larval neuroblasts), and that Mud is also detected at the basal cortex later in mitosis, as well as on spindle poles and microtubules. The minor differences in Mud localization between embryonic and larval neuroblasts may be due to differences in fixation/visualization or in cell types. Thus, Drosophila Mud, C. elegans Lin-5 and mammalian NuMA all share a common localization profile of cell cortex, spindle poles and spindle microtubules (Siller, 2006).

Whether Mud and Pins rely on each other for apical cortical localization was tested. In mud mutant larval neuroblasts, normal apical-basal localization of Pins-Galphai and all other tested cortical polarity proteins was found. By contrast, pins or Galphai maternal-zygotic null-mutant neuroblasts always lacked apical enrichment of Mud: it was either cytoplasmic or cytoplasmic with residual uniform cortical localization, although centrosome/spindle-pole localization was unaffected. In addition, the C-terminal truncated Mud protein that is encoded by the mud allele fails to localize to the cortex or spindle poles in larval neuroblasts. It is concluded that Pins recruits Mud to the neuroblast apical cortex, probably via interaction with the Mud C-terminal domain (Siller, 2006).

The function of Mud in spindle orientation was investigated. Because Mud is maternally provided and required during meiosis, spindle orientation was analyzed in larval neuroblasts. Wild-type larval neuroblasts invariably aligned their metaphase spindle within 15° of the centre of the Pins apical crescent or the Mira basal crescent. By contrast, mud mutant neuroblasts showed significant defects in metaphase spindle alignment with the apical Pins crescent. Also, formation of bent spindles were observed in 29%-40% of all mud mutant neuroblasts, but these are not correlated with spindle-orientation defects and arise after spindle orientation is fixed. It is concluded that Mud is required for metaphase spindle orientation. Despite severe defects in metaphase spindle orientation, it was found that the mitotic spindle and cortical polarity markers were nearly always re-aligned by telophase in mudmutant neuroblasts. In the rare neuroblasts in which 'telophase rescue' of spindle-cortex alignment failed to occur, and the spindle axis remained nearly perpendicular to the cell polarity axis, it was found that the neuroblast division was invariably symmetric with regards to cortical polarity and sibling cell size. Thus, Mud specifically regulates spindle orientation, but spindle orientation defects can affect the asymmetry of cell division. It is concluded that: (1) Mud is required to align the mitotic spindle with Pins cortical polarity at metaphase; (2) a Mud-independent mechanism can rescue spindle-cortex alignment at telophase, and (3) proper spindle-cortex alignment is necessary to promote asymmetric cell division of larval neuroblasts (Siller, 2006).

Time-lapse imaging of larval neuroblasts was used to address two important questions: when do the spindle orientation defects arise in mud mutants, and how are the spindle orientation defects 'rescued' at telophase? Mitotic larval neuroblasts were imaged in whole brain explants expressing a spindle marker labelled with green fluorescent protein (transgenic line G147) and/or an enhanced yellow fluorescent protein (EYFP)-Baz apical cortical marker. In wild-type neuroblasts, it was found that the two fully separated centrosomes were always aligned along the future apical-basal axis by the end of prophase. Thus, in contrast to embryonic neuroblasts in which spindle rotation is reported to occur at metaphase, larval neuroblasts fix spindle-pole/centrosome alignment at prophase and maintain spindle orientation up to the end of telophase. Analysis of the EYFP-Baz apical cortical marker revealed that cortical polarity was always established prior to fixation of centrosome position and accurately predicted the final axis of spindle orientation. This is consistent with the tight alignment of centrosomes and cortical polarity axes that were observed from the end of prophase to telophase in fixed preparations. It is concluded that wild-type neuroblasts establish cortical polarity by prophase, establish centrosome position by the end of prophase and maintain tight spindle-cortex alignment during telophase (Siller, 2006).

In mud mutant neuroblasts, it was found that spindle orientation was also established at prophase, with little or no spindle movement through telophase. However, significant defects were found in the alignment of the mitotic spindle with the EYFP-Baz cortical crescent, including neuroblasts in which the spindle and cortical polarity axes were nearly perpendicular. When examined mud mutant neuroblasts were examined during anaphase/telophase, movement of the mitotic spindle to bring it into alignment with the EYFP-Baz cortical polarity axis was never observed, despite data from fixed preparations showing that the majority of metaphase spindle orientation defects are corrected by telophase. Finally, it was observed that mud mutants could divide asymmetrically or symmetrically. Symmetric divisions occurred only when the spindle was nearly orthogonal with the cortical polarity axis and it was inferred that these neuroblasts correspond to the equally dividing neuroblasts that were seen in fixed preparations. Three conclusions were drawn from the live imaging experiments. First, the mud spindle-orientation defects are due to a failure in centrosome/spindle-pole positioning at prophase, prior to the formation of the metaphase spindle. This further supports the conclusion that metaphase spindle morphology defects are not the source of the spindle-orientation defects. Second, mud mutants do not rotate their spindle towards the cortical polarity axis at anaphase-telophase, indicating that the observed 'telophase rescue' of spindle-cortical polarity occurs by modification of cortical protein distribution to match the spindle axis. Third, Mud does not directly promote asymmetric cell division, but it does regulate spindle orientation relative to cortical polarity, and only when the spindle is orthogonal to the cortical polarity axis does the cell division become symmetric. Thus, spindle orientation dictates whether the cell division is symmetric or asymmetric (Siller, 2006).

This study has shown that Mud has the properties of a functional orthologue of the vertebrate NuMA and C. elegans Lin-5 proteins. All three proteins contain coiled-coil regions and an adjacent NLM domain (found only in NuMA-related proteins), and all three proteins directly interact with similar Galpha-binding proteins (Pins, LGN, GPR1/2). In addition, all three proteins are localized to the cell cortex, spindle poles and spindle microtubules, and at least Mud and Lin-5 have some role in spindle orientation and generating unequal daughter cell size. However, there are differences. NuMA and Lin-5 cortical association depends on LGN and GPR1/2, respectively, whereas Mud can localize to the cortex (albeit uniformly) in the absence of cortical Galphai and Pins. Pins-independent Mud cortical localization may be mediated by the Mud C-terminal putative transmembrane domains, which are absent in NuMA and Lin-5 proteins. Conversely, NuMA and Lin-5 facilitate cortical localization of LGN and GPR1/2, respectively, whereas Mud is not required for Pins localization. Finally, it is unknown how Mud interacts with the mitotic spindle. NuMA directly binds tubulin through a domain containing the NLM motif, raising the possibility that the Mud NLM domain mediates microtubule association. Alternatively, Mud may associate with the spindle via dynein/dynactin, as has been shown for NuMA (Siller, 2006).

Pins and Galphai regulate cortical polarity, spindle orientation, spindle asymmetry and the establishment of sibling cell size differences. Previously, all Drosophila mutants in cortical polarity proteins either severely disrupted cortical polarity, thereby precluding analysis of cortical-spindle alignment, or had no effect on spindle orientation. Reduction in Mud or Khc-73 levels affects spindle orientation without altering cortical polarity; each has a partially penetrant phenotype, so they may function redundantly. mud mutants affect only spindle orientation without directly regulating any other known Pins-Galphai-dependent functions, such as regulation of cortical polarity or sibling cell size. Only when the spindle is aligned orthogonally to the Pins-Galphai crescent are there defects in sibling cell size, presumably due to the equalized activity of Pins-Galphai in both siblings. Whether each of the many essential Pins-Galphai functions has a unique effector protein, similar to the role of Mud in regulation of intrinsic spindle orientation, will be an interesting question for the future (Siller, 2006).

Dual roles for the trimeric G protein Go in asymmetric cell division in Drosophila; Go interacts with Pins

During asymmetric division, a cell polarizes and differentially distributes components to its opposite ends. The subsequent division differentially segregates the two component pools to the daughters, which thereby inherit different developmental directives. In Drosophila sensory organ precursor cells, the localization of Numb protein to the cell's anterior cortex is a key patterning event and is achieved by the combined action of many proteins, including Pins, which itself is localized anteriorly. This study describes a role for the trimeric G protein Go in the anterior localization of Numb and daughter cell fate specification. Go is shown to interact with Pins. In addition to a role in recruiting Numb to an asymmetric location in the cell's cortex, Go transduces a signal from the Frizzled receptor that directs the position in which the complex forms. Thus, Go likely integrates the signaling that directs the formation of the complex with the signaling that directs where the complex forms (Katanaev, 2006 see full text of article).

Because Fz appears to act as the exchange factor for Go in the Wnt and PCP pathways (Katanaev, 1995), The effects of GoWT and GoGTP on wing margin bristles were examined when Fz levels were modulated. The effects of overexpression of GoWT fell to zero in fz–/– wings, but the GoGTP overexpression phenotypes were not reduced; rather, they were enhanced. Why the aberrations increased is not clear, but this result shows that GoGTP is a potent disturber of asymmetric division in the absence of Fz, whereas WT Go requires it. This finding suggests that Go requires Fz to convert it into the 'active' GTP-bound state and predicts that overexpression of Fz should enhance the potency of Go. Indeed, co-overexpression of Fz and GoWT enhances the asymmetric division defects. Overexpression of Fz alone produced orientation defects but no asymmetric division aberrations (Katanaev, 2006).

In Drosophila, Wnt-1 (Wingless, Wg) is transduced by the Go-dependent receptors Fz and Dfz2. Therefore whether co-overexpression of Dfz2 could also enhance the effects of overexpression of Go was tested. Overexpression of Dfz2 alone characteristically induced ectopic margin bristles (activation of the Wg pathway) that showed no asymmetric division defects. But when Dfz2 and GoWT were co-overexpressed, they mutually enhanced their respective phenotypes, suggesting that Go enhanced the ability of Dfz2 to ectopically activate Wg signaling, and Dfz2 potentiated the ability of Go to disturb the asymmetric divisions. Dfz2 is usually down-regulated in the SOP region of the wing margin and likely does not normally influence Go activity there, but its forced expression shows an ability to potentiate the effects of Go (by inference catalyzing it into the GTP-activated form). These results provide the first example of the ability of Dfz2 to activate signaling in a pathway other than 'canonical' Wnt cascade (Katanaev, 2006).

Gβ13F and Gγ1 likely represent the β- and γ-subunits of the Go trimeric complex. Receptor-catalyzed exchange of GDP for GTP occurs on Gα-subunits complexed with βγ. Thus, βγ-subunits should be required for the effects of GoWT overexpression. Indeed, GoWT overexpression effects were attenuated when one gene copy of Gγ1 was removed, arguing that these effects were not due to sequestration of βγ moieties from another α-subunit such as Gi. Ablation of Gβ13F or Gγ1 genes was reported to affect neuroblast divisions. It was also found that loss or overexpression of Gγ1 and Gβ13F (but not Gβ5) resulted in adult bristle defects similar to those of loss or overexpression of Go. Taken together, these observations suggest that Gβ13F and Gγ1 represent the β- and γ-subunits of the Go trimeric complex (Katanaev, 2006).

Various roles for trimeric G proteins have been reported for asymmetric cell divisions; for example, Caenorhabditis elegans Gα-subunits GOA-1 and GPA-16 redundantly regulate posterior displacement of the mitotic spindle required for the asymmetric division of the zygote, and β- and γ-subunits are involved in orientating the mitotic spindle. In Drosophila, evidence for trimeric G protein function in both the formation of the asymmetric spindle and the correct localization of various cell fate determinants have come from manipulation of βγ-subunits in the neuroblasts. Additionally, Gi is known to be involved in asymmetric divisions and to interact with Pins; cell fate determinant localizations are aberrant during metaphase but are restored by telophase (Katanaev, 2006).

In this report, strong and pervasive roles have been documented for Go in Drosophila asymmetric divisions. Five major points are made: (1) In SOP asymmetric divisions, there are two patterning mechanisms: the establishment of the asymmetric complexes and the orientation of the asymmetry. Go appears to act in both functions and is therefore a likely molecular integrator of the two. (2) Go appears to function in both the neuroblast-type and SOP divisions and is therefore likely used in all asymmetric divisions in Drosophila. (3) Go binds to and genetically interacts with Pins. One function of Go, then, is likely mediated by a direct interaction with Pins. (4) Hitherto, Gi was considered the major Gα-subunit functioning in asymmetric cell divisions. Go shows significantly stronger phenotypes, suggesting a greater role, but genetic interaction between the two suggests a degree of functional redundancy. (5) Both Fz and Dfz2 appear able to act as exchange factors for Go in the SOP divisions. The role for Fz is supported by many different results, but whether Dfz2 normally functions here remains unclear (Katanaev, 2006).

Go appears to play parallel bifunctional roles in the establishment of asymmetries in both SOPs and PCP, as evidenced by the following: (1) polarized structures form in both; in PCP, it is the focal organizer of hair outgrowth, and in SOPs, it is the Numb crescent; (2) in both processes, Fz signaling organizes the polarized distribution of 'core group' PCP proteins. For example, Fz itself becomes localized to the distal and posterior ends of PCP cells and SOPs, respectively, whereas Van Gogh/Strabismus is found proximal and anterior in PCP cells and SOPs, respectively. (3) In both processes, these Fz-dependent localizations do not critically contribute to the final polarized structures, because loss of Fz (or other core group proteins) only leads to randomization in the positioning of the (usually) single-hair focus or Numb complex. Thus, there appear to be two semiindependent mechanisms: (1) the polarization of the core group PCP proteins, which instructs (2) the position of the self-assembling complexes (Katanaev, 2006).

Go appears to work in both these mechanisms. Mildly Go-compromised cells lose correct orientation of hairs or Numb complexes, consistent with an orientation function. Cells with strongly disturbed Go function lose the ability to polarize; in the SOP, Numb becomes diffuse or forms a number of small foci; and in PCP, many hair initiation sites are produced. Phenotypes of fz or other core group mutants occasionally result in two hairs per cell, but Go mutants frequently induce cells with five or six hairs (Katanaev, 2006).

The question now arises as to whether Go functions in the same way in both processes. In terms of the Fz-mediated orientation step, it is likely that Go performs the same role; in both, Fz is directed to one end of the cell (distal or posterior), and Go itself becomes preferentially distributed to the other end (proximal or anterior). This local enrichment of Go possibly serves as the point of integration with the internal asymmetry formation step. In the SOP case, anterior Go may recruit Pins and seed the formation of the anterior Numb crescent. In the PCP case, Go localizes opposite to the site of hair growth, suggesting that the highest depletion of Go specifies the site of hair growth. In the absence of the Fz orienting information, it may be a stochastic increase of Go localization (or activity) that establishes the initial asymmetric bias. Alternatively, the asymmetric distribution of Go may only be a manifestation of the Fz-mediated orientation, being essentially irrelevant to the subsequent step. In this case, the activity of Go (rather than its site of accumulation) would be required for the formation of the Numb crescent or the hair initiation point (Katanaev, 2006).

Identification of an Aurora-A/PinsLINKER/ Dlg spindle orientation pathway using induced cell polarity in S2 cells

Asymmetric cell division is intensely studied because it can generate cellular diversity as well as maintain stem cell populations. Asymmetric cell division requires mitotic spindle alignment with intrinsic or extrinsic polarity cues, but mechanistic detail of this process is lacking. A method has been developed to construct cortical polarity in a normally unpolarized cell line and this method was used to characterize Partner of Inscuteable (Pins; LGN/AGS3 in mammals) -dependent spindle orientation. A previously unrecognized evolutionarily conserved Pins domain (PinsLINKER) was identified that requires Aurora-A phosphorylation to recruit Discs large (Dlg; PSD-95/hDlg in mammals) and promote partial spindle orientation. The well-characterized PinsTPR domain has no function alone, but placing the PinsTPR in cis to the PinsLINKER gives dynein-dependent precise spindle orientation. This 'induced cortical polarity' assay is suitable for rapid identification of the proteins, domains, and amino acids regulating spindle orientation or cell polarity (Johnston, 2009).

A surprising result of these studies is the importance of the PinsLINKER domain for spindle orientation in the S2 assay and within neuroblasts in vivo. Only this domain is sufficient for spindle orientation, and a single point mutation in the linker domain (S436A) results in spindle orientation defects in larval neuroblasts that closely mimic the pins null mutant phenotype. On the basis of domain mapping and epistasis analysis, a linear pathway has been identified from cortical PinsLINKER to the plus ends of astral microtubules: (1) Aurora-A phosphorylates PinsLINKER on a single amino acid, serine 436, (2) the phosphorylated PinsLINKER binds and recruits Dlg, (3) the kinesin Khc-73 moves to astral microtubule plus ends using its motor domain and may be anchored at the plus ends by its Cap-Gly domain (Siegrist, 2005), and (4) the Khc-73MBS domain binds the cortical DlgGK domain, thereby linking Khc-73+ astral microtubule plus ends to the Dlg cortical domain. Interestingly, this pathway is active in both directions during mitosis. Cortical Pins acts through Dlg and Khc-73 to regulate spindle orientation, and spindle-associated Khc-73 acts through Dlg and Pins to induce Pins/Galphai functional cortical polarity in neuroblasts (Johnston, 2009).

Why does the PinsLINKER pathway provide only partial spindle orientation function? Live imaging rules out several possible explanations, such as PinsLINKER-induced spindle rocking variability, or that PinsLINKER functions during only a narrow window during mitosis. Live imaging shows that in PinsLINKER cells, the spindle drifts until it is immobilized at the edge of the crescent. This is consistent with the fact that Khc-73 is a plus end-directed motor protein, and thus unable to generate pulling forces to bring the centrosome closer to the cell cortex; at best, it would provide a static link between astral microtubules and the cell cortex (Johnston, 2009).

The PinsTPR domain can improve the PinsLINKER spindle orientation to a level matching wild-type neuroblasts. It is proposed that the PinsTPR domain directly binds Mud and that Mud interacts with the dynein/dynactin/Lis1 complex to enhance PinsLINKER spindle orientation. This model is based on five observations. First, the PinsTPR domain binds Mud in vitro and the two proteins coimmunoprecipitate from in vivo lysates; this interaction is conserved in the related C. elegans and mammalian proteins. Second, the PinsTPR and PinsTPR+LINKER but not the PinsLINKER can recruit Mud to the cortex of S2 cells. Third, PinsTPR+LINKER-mediated spindle orientation requires the dynein complex proteins Dlc and Lis1. Fourth, PinsTPR+LINKER-mediated spindle orientation exhibits rapid, directional spindle movement toward the center of the Pins cortical crescent, similar to dynein-dependent spindle orientation in Drosophila neuroblasts. Fifth, PinsTPR+LINKER-mediated spindle orientation leads to dynein-dependent movement of the spindle pole close to the cell cortex, consistent with dynein minus end-directed pulling of astral microtubules, as observed in other cell types (Johnston, 2009).

If PinsTPR recruits Mud, and Mud recruits the dynein complex, then why doesn't PinsTPR have spindle-orienting function on its own? The simplest model is that PinsTPR/Mud alone is unable to recruit or activate the dynein complex. Alternatively, the PinsLINKER pathway could be required for 'presenting' microtubule plus ends to an active PinsTPR/Mud/Dynein complex, which fits with the requirement for PinsTPR and PinsLINKER acting in cis. In summary, these data show that the PinsTPR and PinsLINKER domains provide distinct functions, both of which are required for optimal spindle orientation. Interestingly, spindle orientation in S2 cells does not show 'telophase rescue'—a phenomenon whereby spindles that are partially oriented in metaphase/anaphase neuroblasts become aligned with the cell polarity axis by telophase -- consistent with the absence of redundant spindle orientation pathways in this assay (Johnston, 2009).

The PinsTPR pathway is regulated by Galphai binding to the GoLoco domain, relieving intramolecular TPR-GoLoco interactions, and making the TPR domain accessible for intermolecular interactions. In addition, Galphai is required to recruit Pins to the cell cortex, where it can interact with regulator and effector proteins. In the S2 spindle orientation assay, a requirement for Galphai can be bypassed by simply deleting the GoLoco domain (thereby freeing the TPR for intermolecular interactions) and tethering the PinsTPR+LINKER protein to the cortex by fusion with the Ed transmembrane protein. Thus, Galphai is important to activate and localize full-length Pins, but not as an effector of Pins-mediated spindle orientation (Johnston, 2009).

In contrast, the PinsLINKER pathway is not regulated by Galphai, because full-length Pins in the absence of Galphai provides equal spindle orientation to PinsLINKER, suggesting that the PinsLINKER is active when Pins is in the 'closed' form. The Khc-73 mammalian ortholog GAKIN transports hDlg to the cell cortex, but there are several reasons to think that this mechanism does not activate the PinsLINKER pathway. First, cortically tethered DlgGK domain requires Khc-73 for spindle orientation, which rules out a role for Khc-73 in merely transporting Dlg to the cortex; second, khc-73 RNAi does not block the ability of PinsLINKER to recruit Dlg to the cortex (Johnston, 2009).

This study has shown that Aurora-A kinase activates the PinsLINKER spindle orientation pathway by phosphorylating S436 in the linker domain and that this pathway is required for accurate spindle orientation in vivo for larval neuroblast asymmetric cell division. Neuroblasts expressing the nonphosphorylatable form of Pins (S436A) have a weaker spindle orientation phenotype than aurora-A null mutants, as expected because of Aurora-A regulation of multiple Pins-independent processes required for spindle orientation, such as centrosome maturation, cell-cycle progression, and cell polarity in flies. However, this study shows that a Pins phosphomimetic mutant (S436D) allows spindle orientation even after RNAi depletion of Aurora-A levels, suggesting that Aurora-A phosphorylation of PinsS436 is essential for Pins-dependent spindle orientation in the S2 cell assay. Furthermore, the finding that the PinsS436A protein has no spindle orientation activity in pins mutant larval neuroblasts, and has dominant-negative activity in the presence of endogenous Pins, shows that the Aurora-A/PinsLINKER pathway is required for spindle orientation in larval neuroblasts as well (Johnston, 2009).

The Pins spindle orientation pathway is cell-cycle regulated: interphase S2 cells that have polarized PinsTPR+LINKER do not capture centriole/centrosomal microtubules. There are at least two reasons for the lack of Pins interphase activity. First, the level of the Aurora-A kinase is low during interphase, and Aurora-A phosphorylation of Pins S436 has been shown to be is essential for Pins-mediated spindle orientation. Second, interphase centrosomes are immature, lacking Cnn and nucleating few microtubules. Expression of the Pins S436D protein, which is fully functional during mitosis even after Aurora-A depletion, still has no ability to capture centrioles during interphase. Thus, both centrosome maturation and Aurora-A activation are required for Pins-mediated spindle orientation in S2 cells (Johnston, 2009).

Cell polarity and spindle orientation has been induced in a cultured cell line in this study. This system was used to identify two pathways regulating spindle orientation, to establish molecular epistasis within each pathway, and to identify the target of the mitotic kinase Aurora-A that coordinates cell-cycle progression with spindle orientation. In the future, this system should be useful for characterizing spindle orientation pathways from other Drosophila cell types or from other organisms, identifying the mechanisms that control centrosome or spindle asymmetry, and characterizing the establishment and maintenance of cortical polarity. In each of these cases, the induced polarity system should be useful for rapid protein structure/function studies and high-throughput drug or RNAi loss-of-function studies (Johnston, 2009).

Drosophila GoLoco-protein Pins is a target of Galpha(o)-mediated G protein-coupled receptor signaling

G protein-coupled receptors (GPCRs) transduce their signals through trimeric G proteins, inducing guanine nucleotide exchange on their Gα-subunits; the resulting Gα-GTP transmits the signal further inside the cell. GoLoco domains present in many proteins play important roles in multiple trimeric G protein-dependent activities, physically binding Gα-subunits of the Gαi/o class. In most cases GoLoco binds exclusively to the GDP-loaded form of the Gα-subunits. This study demonstrates that the poly-GoLoco-containing protein Pins of Drosophila can bind to both GDP- and GTP-forms of Drosophilao. Pins GoLoco domain 1 is identified as necessary and sufficient for this unusual interaction with Gαo-GTP. A lysine residue located centrally in this domain is pinpointed as necessary for the interaction. These studies thus identify Drosophila Pins as a target of Gαo-mediated GPCR receptor signaling, e.g., in the context of the nervous system development, where Gαo acts downstream from Frizzled and redundantly with Gαi to control the asymmetry of cell divisions (Kopein, 2009).

These observations expand a previous report that Pins could interact with Gαo in the context of the asymmetric cell divisions during formation of Drosophila adult sensory bristles. In that work, a genetic interaction was demonstated, as well as an ability of both GDP- and GTPγS-loaded forms of recombinant Gαo to pulldown endogenous Pins from Drosophila extracts. However, when the interaction between purified recombinant Gαo and Pins proteins was tested, only the GDP-loaded Gαo revealed the binding to Pins. This discrepancy is interpreted by proposing that certain Drosophila proteins could enhance the interaction between the GTP-loaded Gαo and Pins, while the interaction between the purified proteins was 'canonical' and only happened in the presence of GDP (Kopein, 2009).

Although the existence of helper proteins enhancing the in vivo interactions between GTP-loaded Gαo and Pins is still a possibility, this study found that the nontagged or (His)6-tagged Gαo-GTPγS efficiently binds purified Pins in multiple experimental setups, while Gαo used in previous experiments was GST-tagged. It was also found that the point Q205L mutation on Gαo, rendering it unable to hydrolyze GTP and thus constitutively GTP-bound, allows highly efficient Pins binding comparable to that of the Gαo[GDP]. Although it cannot be fully explained why the GST-tagged Gαo-GTPγS is unable to bind purified Pins, it is noted that the bulky GST tag reduces the GTP-binding activity of Gαo 3-5 times. Thus, it is concluded that the active, GTP-loaded Gαo binds Pins both in vivo and in vitro (Kopein, 2009).

This unusual interaction of the GTP-loaded Gαo and Pins is confined to the GoLoco1 domain of Pins. Lys15 of the GoLoco1 domain is necessary for the efficient binding to GTP-loaded Gαo. Substitution of Lys15 of GoLoco1 domain with Gly located in the identical position of GoLoco3 domain uncouples the interaction with GTP-loaded Gαo but only moderately affects the binding to GDP-loaded Gαo, and thus recapitulates the GoLoco3 domain-binding pattern. It is thus proposed that Lys15 of the GoLoco1 domain might stabilize the γ-phosphate of GTP during interaction with GTP-loaded Gαo (Kopein, 2009).

This work provides the second clear demonstration of the interaction of a GoLoco domain-containing protein with the GTP-loaded form of a Gα-subunit. The only other clearly confirmed case of a similar interaction is the binding of the activated rat Gαz to Rap1GAP. It is interesting to note that Lys15 of the GoLoco1 domain of Pins is absent from the equivalent position of the Rap1GAP' GoLoco domain. It thus might be proposed that multiple mechanisms stabilizing the GoLoco domain interaction with GTP-loaded Gα may exist. Additional evidence is provided by the current experiments with homologues of Gαo and Pins. Gαi, being 69% identical to Gαo, binds Pins or its domains exclusively in the GDP-conformation. This biochemical result is paralleled with in vivo experiments where only Gαi[GDP] but not Gαi[GTP] could affect asymmetric divisions in Drosophila. Furthermore, rat Gαo, 81% identical to Drosophilao, shows no ability to interact with Drosophila Pins in the GTPγS-loaded form, but interacts efficiently in the GDP-form. Additionally, both Drosophila and rat Gαo-GTPγS fail to bind the GoLoco region of mammalian Pins homologues AGS3 and LGN, despite the presence of Lys15 in the GoLoco4 domain of AGS3 and LGN. It is still possible that other Gαo/Pins homologues may reveal an interaction in the GTP state. For example, efficient binding of C. elegans AGS3 (which has Lys15 in GoLoco1 domain and Arg15 in GoLoco2 domain to GAO-1[GDP] and GAO-1[GTP] was demonstrated in the yeast two-hybrid assay, but the biochemical confirmation of this interaction is missing. The detailed information this study provides on the specificity of GoLoco binding to the GTP-loaded Gαo (Gαo, but not Gαi; Drosophila, but not rat Gαo; Drosophila Pins, but not its mammalian homologues; GoLoco1 domain of Pins, but not other Drosophila GoLoco domains) will help elucidate the structural mechanism of this interaction (Kopein, 2009).

Pins and its homologues have the conserved activity in the regulation of the asymmetric cell divisions. In Drosophila sensory organ formation, the process of the asymmetric cell divisions appears under the redundant control of Gαo and Gαi. Down-regulation of Gαi alone, either by genetic ablation or by targeted RNAi expression, does not result in any defects in the structure of the adult sensory bristles, unlike same manipulations of Pins. In contrast, loss-of-function or overactivation of Gαo result in aberrations in the process of asymmetric cell divisions and visible defects in the adult bristle structure. However, this study shows that no apparent defects are induced by targeted expression of pertussis toxin, which uncouples Gαo (and not any other Gα-protein in Drosophila) from its cognate GPCRs such as Frizzled. This observation is not unexpected, as loss of Frizzled itself leads only to the randomization of the axis of the asymmetric cell divisions, but not to the loss of asymmetry or defects in the adult bristle structure. However, the redundancy between Gαo and Gαi is revealed by a concomitant expression of the Gαi-RNAi and pertussis toxin, as this now phenocopies Pins loss-of-function. The same phenotype is produced by the concomitant down-regulation of Frizzled (acting upstream from Gαo) and Gαi. These data suggest that Gαo and Gαi act coordinately in the process of the asymmetric cell division of the sensory precursor cells, perhaps similarly to what has been demonstrated for the asymmetric division of the C. elegans zygote. The three individual GoLoco domains of Pins bind Gαi identically; furthermore, multiple Gαi molecules can simultaneously bind a single Pins scaffold. Similarly, this study shows that Gαo and Gαi can simultaneously bind Pins most likely occupying different GoLoco domains. This study also shows that this trimeric complex exists when the two G proteins are bound to different nucleotides: Gαo to GTP and Gαi to GDP. Such a multiprotein complex might allow a more effective regulation of the process of the asymmetric cell division (Kopein, 2009).

The results on the in vivo function of Frizzled, Gαo, Gαi, and Pins in the Drosophila sensory organ lineage further support the idea that Pins acts as a target and not as an activator of G protein signaling in this physiological process. Indeed, similarity of the Frizzled-RNAi + Gαi-RNAi phenotypes on one hand, and the pertussis toxin + Gαi-RNAi phenotypes on the other hand clearly shows the redundancy of the Frizzled→Gαo module with the Gαi function for the process of asymmetric cell divisions. This redundancy implies that both Gαo and Gαi act upstream from Pins. While generation of active Gαo from the trimeric Go complexes can be achieved by Frizzled receptors, it is not clear how Gαi is released from the trimeric Gi complexes. Ric-8 (a non-GPCR guanine nucleotide exchange factor) might be implicated in activation of Gαi. Downstream from Pins, a known regulator of the asymmetry of cell divisions is NuMA (known as Mud in flies) that anchors the mitotic spindle at the correct location within the plasma membrane (Kopein, 2009).

While Pins and its homologues have the conserved activity in the regulation of the asymmetric cell divisions, additional functions of these proteins exist. The Pins homologues AGS3 and LGN are strongly expressed in the brain as is Gαo, where AGS3 is involved e.g., in drug sensitization and seeking behavior. At the molecular level Pins homologues regulate plasma membrane localization and activity of several transmembrane receptors and channels. Drosophila Pins is also expressed in the larval and adult brain. Additionally, Pins affects motor axon guidance and synaptogenesis in Drosophila. Thus a variety of GPCRs are likely to engage Pins and potentially other GoLoco domain-containing proteins through liberation of Gαo-subunits from the trimeric Go protein complexes. In addition, some non-GPCR guanine nucleotide exchange factors such as Ric-8 might be involved in the generation of the Pins-interacting Gαo-GTP. Although clear data demonstrate that Pins and its homologues can modulate activities of Gαi, the capacity of the activated Gαo to bind Pins demonstrated in this study highlights the possible important function of Pins as a general transducer of GPCR signaling. Yeast two-hybrid screens have identified multiple interaction partners of Pins. The multidomain structure of Pins may suggest that this protein serves as a scaffold to organize signal transduction downstream from various GPCRs (Kopein, 2009).

Canoe binds RanGTP to promote PinsTPR/Mud-mediated spindle orientation

Regulated spindle orientation maintains epithelial tissue integrity and stem cell asymmetric cell division. In Drosophila neural stem cells (neuroblasts), the scaffolding protein Canoe (Afadin/Af-6 in mammals) regulates spindle orientation, but its protein interaction partners and mechanism of action are unknown. This paper uses a recently developed induced cell polarity system to dissect the molecular mechanism of Canoe-mediated spindle orientation. A previously uncharacterized portion of Canoe was shown to directly bind the Partner of Inscuteable (Pins) tetratricopeptide repeat (TPR) domain. The Canoe-PinsTPR interaction recruits Canoe to the cell cortex and is required for activation of the Pins(TPR)-Mud (nuclear mitotic apparatus in mammals) spindle orientation pathway. The Canoe Ras-association (RA) domains directly bind RanGTP, and both the CanoeRA domains and RanGTP are required to recruit Mud to the cortex and activate the Pins/Mud/dynein spindle orientation pathway (Wee, 2011).

Spindle orientation is essential to maintain epithelial integrity; planar spindle orientation results in both daughter cells maintaining apical junctions and remaining part of the epithelium, whereas apical/basal spindle orientation can lead to the loss of the basal daughter cell from the epithelium. Spindle orientation is also important during asymmetric cell division of stem, progenitor, and embryonic cells; when the spindle orients along an axis of intrinsic or extrinsic polarity, it will generate two different daughter cells, but, when the spindle aligns perpendicular to the axis of polarity, it will generate two identical daughter cells. Proper spindle orientation may even be necessary to prevent tumorigenesis. Thus, it is essential to understand the molecular mechanisms that regulate spindle orientation, particularly those that use evolutionarily conserved proteins and pathways, to help direct stem cell lineages and potentially treat pathological conditions caused by aberrant spindle orientation (Wee, 2011).

Drosophila neuroblasts provide an excellent system for studying spindle orientation during asymmetric cell division. Neuroblasts have an apical/basal polarity and orient their mitotic spindle along this cortical polarity axis to generate distinct apical and basal daughter cells. The apical neuroblast inherits fate determinants responsible for neuroblast self-renewal, whereas the basal daughter cell inherits fate determinants responsible for neuronal/glial differentiation. Genetic studies have identified proteins that regulate spindle orientation during asymmetric cell division, including the apically localized proteins Inscuteable, Partner of Inscuteable (Pins; LGN/AGS-3 in mammals), Mushroom body defect (Mud; nuclear mitotic apparatus [NuMA] in mammals), Discs large (Dlg), and Gai. In addition, many proteins that are not asymmetrically localized are required for spindle orientation, including the dynein complex and the Aurora A and Polo kinases (Wee, 2011).

An induced cell polarity/spindle orientation system has been developed using the normally apolar S2 cell line to biochemically dissect Drosophila and vertebrate spindle orientation (Johnston, 2009; Ségalen, 2010). Using this system to characterize Drosophila spindle orientation, it was shown that cortical Pins nucleates two spindle orientation pathways: (1) the PinsLINKER domain is phosphorylated by Aurora A, which allows recruitment of Dlg, which interacts with the kinesin Khc-73 to promote partial spindle orientation; and (2) the Pins tetratricopeptide repeat (TPR) domain (PinsTPR) binds Mud, which promotes dynein-dynactin complex-mediated spindle orientation (Johnston, 2009). This induced cell polarity system was used to characterize Dishevelled-mediated spindle orientation in the zebrafish embryo and in Drosophila sensory organ precursor cells, identifying a Dishevelled domain that is necessary and sufficient to bind Mud and regulate spindle orientation in both cell types (Wee, 2011).

The scaffolding protein Canoe has been shown to regulate spindle orientation and cell polarity in Drosophila neuroblasts (Speicher, 2008), although the mechanisms involved remain unknown. Canoe contains two Ras-association (RA) domains, a Forkhead domain, a myosin-like Dilute domain, and a PSD-95, Dlg, and ZO-1 (PDZ) domain. In addition to regulating neuroblast cell polarity and spindle orientation, it integrates Notch, Ras, and Wnt pathways during Drosophila muscle progenitor specification and serves as a Rap1 effector within the Jun N-terminal kinase pathway during dorsal closure of the Drosophila embryo, and the mammalian orthologue Afadin links cadherins to the actin cytoskeleton at adherens junctions. This study mapped direct Pins/Canoe and Canoe/RanGTP-binding domains and used the induced cell polarity/spindle orientation system to show that Canoe/RanGTP is required for Pins to recruit Mud and activate the Pins/Mud/dynein spindle orientation pathway (Wee, 2011).

How might Canoe/RanGTP promote Mud recruitment to the Pins cortical domain? One model is that Ran sequesters importin-a/β away from the Mud NLS, thereby allowing Mud to interact with Pins. This model is based on the observation that RanGTP inhibits binding of importin-β to the NLS of NuMA (the mammalian orthologue of Mud), increasing the pool of NuMA available to promote spindle formation. The model predicts that Mud can bind importin-a/β and that this binding prevents Mud/Pins association. Consistent with the model, importin-β/Mud were coimmunoprecipitated from S2 cell lysates, and a GST:Mud fragment containing the adjacent Mud TPR-interacting peptide (TIP)-NLS domains (GST:MudTIP-NLS) could bind purified importin-β in the presence of importin-a. However, it was found that increasing the concentration of purified importin-a/β did not effect the amount of Pins pulled down with GST:MudTIP-NLS, which does not support a model in which Ran must sequester importin-a/β to allow Pins/Mud binding. Furthermore, a GFP-tagged MudTIP-NLS fragment localized to Ed:PinsTPR+LINKER crescents independently of the Canoe/Ran pathway, showing that the Mud NLS is not involved in the Canoe/Ran-regulated localization mechanism. Interestingly, Canoe/RanGTP regulation is required for recruitment of full-length endogenous Mud but not for the recruitment of the smaller MudTIP-NLS fragment; this indicates that Canoe/RanGTP normally functions by blocking an unknown inhibitor of the Mud-PinsTPR interaction (Wee, 2011).

In conclusion, this study has characterized the molecular mechanism by which Canoe regulates spindle orientation. A region of Canoe (amino acids 1,755-1,950) was identified that directly interacts with the PinsTPR domain, and it was showm that these domains are necessary and sufficient for Canoe-Pins association. It was shown that the Canoe RA domains bind directly to RanGTP, that both the Canoe RA domains and Ran are necessary for the PinsTPR/Mud spindle orientation pathway, and that Canoe/RanGTP acts by promoting Mud recruitment to the cortical Pins domain. All of the proteins in the Pins/Canoe/Ran/Mud pathway are conserved from flies to mammals, suggesting that this pathway could be widely used to regulate spindle orientation (Wee, 2011).

LGN/mInsc and LGN/NuMA complex structures suggest distinct functions in asymmetric cell division for the Par3/mInsc/LGN and Galphai/LGN/NuMA pathways

Coupling of spindle orientation to cellular polarity is a prerequisite for epithelial asymmetric cell divisions. The current view posits that the adaptor Inscuteable (Insc) bridges between Par3 and the spindle tethering machinery assembled on NuMA-LGNGαiGDP, thus triggering apico-basal spindle orientation. The crystal structure of the Drosophila ortholog of LGN (known as Pins) in complex with Insc reveals a modular interface contributed by evolutionary conserved residues. The structure also identifies a positively charged patch of LGN binding to an invariant EPE-motif present on both Insc and NuMA (Mushroom body defect or Mud). In vitro competition assays indicate that Insc competes with NuMA for LGN binding, displaying a higher affinity, and that it is capable of opening the LGN conformational switch. The finding that Insc and NuMA are mutually exclusive interactors of LGN challenges the established model of force generators assembly, which this study revises on the basis of the newly discovered biochemical properties of the intervening components (Culurgioni, 2011).

This study reports the characterization of the PinsTPR dInscPEPT complex and provides a molecular explanation for the mutual exclusive interaction of Insc and NuMA to LGN. While this manuscript was in preparation, Zhu and coworkers arrived to similar conclusions analyzing the structure of the LGN-NuMA complex (Zhu, 2011).

A 38-residue fragment of Drosophila Insc encompasses the PinsTPR binding region. This fragment of Insc shares a high sequence similarity to functional homologues recently discovered in mammals, fully supporting the notion that the basic mechanism responsible for the recruitment of force generators at polarity sites is evolutionary conserved. With the exception of a short N-terminal α-helix, the InscPEPT is intrinsically unstructured, and lines on the scaffold provided by the superhelical TPR arrangement of Pins with an extended conformation. The interaction surface is organized around a core module involving the EPE motif of InscPEPT and the central TPR5-6 of Pins, whose specificity is primarily dictated by charge complementarity. The binding is further stabilized by polar and hydrophobic interactions contributed by the αA helix of InscPEPT. Not surprisingly, the large interaction surface characterizing the topology of the PinsTPR;InscPEPT heterotypic dimer accounts for an elevate;d binding affinity (of about 5 nM for the fly proteins and 13 nM for the human counterparts). The structure of mouse LGN191–350, corresponding to what is named TPR5-8, with Insc19–40 suggests that vertebrate proteins assemble with organizational principles similar to the fly ones. However, the short mouse constructs only depict the interaction of LGNTPR with the αA helix of InscPEPT, up to the first Glu of the EPE motif. Intriguingly, the mouse LGN;Insc interaction seems to be characterized by lower affinity compared to human and fly ones (with KD of 63 nM for LGNTPR5–8;Insc19–40, and of 47 nM for LGNTPR1–8;Insc20–57) (Zhu, 2011).

The evidence that NuMA forms a complex with the same LGNTPR domain associating to Insc raised the question of whether it binds in a similar manner. Indeed, comparison of the primary sequence of InscPEPT with the known LGN-binding portion of NuMA revealed the presence of an EPE triplet that turned out to be essential for the LGN recognition, with a similar molecular signature of the EPE motif of the InscPEPT. Notably, the NuMA ortholog in fly (Mud) codes for two consecutive EDE-EGE motifs in the Pins-binding region, whose interplay remains to be clarified. The structure of LGN in complex with NuMAPEPT fully supports the notion that the EPE-interaction module represents a common region required for docking unstructured ligands on the LGNTPR scaffold. In the case of NuMA, the interface is further contributed by a helical fragment forming a bundle with helices αA2 and αA3 of LGNTPR. The consequence of the partial overlap between the Insc and NuMA binding sites is that their concomitant loading on LGN is excluded (Zhu, 2011).

A key step during the assembly of the force generators is the opening of the LGN conformational switch that keeps the molecule in an inactive state. Binding of NuMA to LGNTPR induces the release of the intramolecular interactions holding the molecule in a closed form. In agreement with the similarity in the binding modes, it was demonstrated that also Insc disengages the LGN GoLoco motifs from the TPR domain. Together these findings imply that the GoLoco motifs contact the TPR repeats in the same region occupied by Insc and NuMA. Primary sequence inspection revealed that the GoLocos of both Pins and LGN do not contain EPE triplets, suggesting that either the head-to-tail interaction involves alternative TPR patches sterically occluded by the presence of Insc and NuMA, or that less conserved negatively charged triplets are accommodated on the same TPR5-6 of LGN (Zhu, 2011).

The well established model for force generators recruitment at polarity sites rests on the assumption that Insc and NuMA can be part of the same apically localized multisubunit complexes containing Par proteins. This model stems from colocalization experiments showing that in asymmetric mitoses Par3, Insc, LGN, and NuMA cluster together in apical crescents, complemented by coimmunoprecipitation assays in which LGN;Gαi were found in association with Par3;Insc and NuMA. The finding that Insc and NuMA are mutually exclusive partners of LGN is both unexpected and puzzling. In particular, the higher affinity characterizing the Insc binding to LGN shifts the balance of the unmodified proteins towards the Insc;LGN complex formation, which is instrumental in recruiting LGN with Par proteins at the onset of mitosis but cannot account for microtubule-pulling forces. What is the possible mechanism for transferring LGN from Insc to NuMA? The architecture of the InscPEPT;PinsTPR structure whereby an extended ligand is accommodated on a large domain allows a high degree of regulation of the interaction strength. Posttranslational modifications on either side of the dimer might locally alter the contacts without affecting the rest of the interface, as it has been demonstrated for the similarly organized complex between the cytoplasmic domain of E-cadherin and β-catenin. Such modulating modifications can in principle occur on Insc, NuMA, or on LGN. To date, no experimental information is available regarding putative Insc or NuMA modifications. More controversial is the literature relative to LGN phosphorylations. In mitotic Drosophila neuroblasts, Pins has been found phosphorylated by Aurora-A on Ser436 at about half of the linker connecting the TPR domain with the GoLoco motifs. Using an “induced polarity” assay in S2 cells, phospho-Ser436Pins was shown to trigger a redundant NuMA-independent spindle orientation pathway engaging the membrane associated Dlg protein. It is to date unclear if such pathway is conserved in vertebrates. Notably, during oriented symmetric cell divisions of MDCK cells, phosphorylation on a similarly positioned Ser401 of LGN functions in excluding force generators from the apical cortex in order to prevent apico-basal spindle orientation. In this context, phospho-Ser401LGN would selectively prevent binding of LGN to apical Gαi. Based on structural and biochemical results, it is difficult to provide a molecular explanation as to whether these LGN phosphorylations could also impact on the Insc and NuMA binding. Recent observations support the notion that the pool of NuMA;LGN;Gαi colocalizing with Par3;Insc in embryonic mouse skin progenitors is tightly regulated to set the balance between symmetric and asymmetric divisions, though no mechanism for this has been put forward. In summary, more has to be learned to understand what brings LGN from Insc to NuMA (Zhu, 2011).

An additional question relates to the mechanism maintaining effective NuMA;LGN;GαiGDP species at the correct cortical sites in the absence of Insc. Based on the knowledge acquired in this study, a step-wise model is proposed that can be schematized as follows (see Both NuMA and Insc open the LGN conformational switch): (1) in the early phases of mitosis LGN is brought to the apical membrane in conjunction with Par proteins by the high-affinity interaction with the preformed Par3;Insc complex. Binding of LGN to Insc triggers the conformational switch transition enabling the relocation of GαiGDP moieties previously distributed all around the plasma-membrane with Gβγ; (2) upon mitotic progression, when LGN is already at the correct sites, a yet unidentified molecular event alters the relative affinities of Insc and NuMA for LGN to shift the balance between the Insc-bound and the NuMA-bound LGN pools. It is hypothesized that the four Gαi subunits present on LGN at this stage are sufficient to transiently hold cortical NuMA;LGN;GαiGDP in proximity of Par proteins to allow directional microtubule pulling. It is speculated that NuMA;LGN;GαiGDP is a short-lived complex and disassemble, possibly assisted by a specialized GEF for Gαi such as as Ric-8A, releasing apo-LGN in the cytoplasm to start a new cycle. Such a dynamical interaction network would allow for a continuous regulation of the force exerted on astral microtubules throughout mitosis. Future attempts to validate the model in vivo will greatly benefit from the biochemical tools presented in this study (Zhu, 2011).

An ana2/ctp/mud complex regulates spindle orientation in Drosophila neuroblasts

Drosophila neural stem cells, larval brain neuroblasts (NBs), align their mitotic spindles along the apical/basal axis during asymmetric cell division (ACD) to maintain the balance of self-renewal and differentiation. This study identified a protein complex composed of the tumor suppressor anastral spindle 2 (Ana2), a dynein light-chain protein Cut up (Ctp), and Mushroom body defect (Mud), which regulates mitotic spindle orientation. Two ana2 alleles were isolated that displayed spindle misorientation and NB overgrowth phenotypes in larval brains. The centriolar protein Ana2 anchors Ctp to centrioles during ACD. The centriolar localization of Ctp is important for spindle orientation. Ana2 and Ctp localize Mud to the centrosomes and cell cortex and facilitate/maintain the association of Mud with Pins at the apical cortex. These findings reveal that the centrosomal proteins Ana2 and Ctp regulate Mud function to orient the mitotic spindle during NB asymmetric division (Wang, 2011).

The Drosophila larval brain neural stem cell, or neuroblast (NB), has recently emerged as a new model for studying stem cell self-renewal and tumorigenesis. NBs divide asymmetrically to generate a self-renewing daughter NB and a ganglion mother cell (GMC) that is committed to differentiation. Asymmetric localization/segregation machinery ensures the polarized distribution of 'proliferation factors,' including atypical protein kinase C (aPKC), and 'differentiation factors,' including basal proteins such as Numb, Miranda (Mira), Brain tumor (Brat), and Prospero, to the daughter NB and GMC, respectively. The failure of asymmetric division of NBs can result in their hyperproliferation and the induction of tumors (Wang, 2011).

To ensure the correct asymmetric segregation of cell fate determinants, the mitotic spindle has to be properly oriented with respect to the polarized proteins on the cell cortex. Inscuteable (Insc) and the heterotrimeric G proteins Gαi and Gβγ and their regulators Partner of Insc (Pins) and Ric-8 control mitotic spindle orientation (Wang, 2011).

Recent work has also implicated centrosome-associated proteins in the regulation of spindle orientation and tumorigenesis. Centrosomes function as major microtubule-organizing centers in most animal cells. A centrosome is composed of a pair of centrioles surrounded by an amorphous matrix of pericentriolar material (PCM). Centriole duplication is regulated by centriolar components, such as Asterless (Asl), Sas6, Sas4, and anastral spindle 2 (Ana2). ana2 was identified from genome-wide RNA interference (RNAi) screens, where ana2 RNAi-treated S2 cells exhibited an anastral spindle phenotype. The Ana2 overexpression phenotype and its interaction with Sas6 have suggested a role for Ana2 in centriole duplication (Stevens, 2010). However, no ana2 mutants were previously available for further functional studies (Wang, 2011).

This study has isolated two ana2 alleles that are defective in apical/basal spindle orientation during NB asymmetric division. Ana2 is demonstrated to be a tumor suppressor that suppresses NB overproliferation. The centriolar protein Ana2 directly interacts with Ctp, a dynein light chain that also localizes to the centrioles, and Mud, leading to their localization to the centrosomes. This finding suggests that the tumor suppressor Ana2 ultimately regulates Mud function to direct asymmetric division and prevent tumor formation (Wang, 2011).

This study investigated the role of Drosophila Ana2 during NB asymmetric cell division, focusing on mitotic spindle orientation. Two ana2 alleles were isolated from a genetic screen that produced supernumerary NBs in larval brains and failed to properly align the mitotic spindle with asymmetrically localized proteins. It was demonstrated that Drosophila Ana2 functioned as a tumor suppressor in a transplantation experiment. Using ana2 mutants, it was shown that Ana2 is important for centriole function. Ana2 interacts with Sas-6 through the C-terminal region of Ana2 (201-420 aa), which contains the conserved STAN motif and coiled-coil domain (Stevens, 2010). The data suggest that the N terminus of Ana2 (1-274 aa), which interacted with Ctp, a Ddlc1 (Drosophila Dynein light chain), is sufficient for its function in centriole assembly and spindle orientation. This is not in direct contradiction with the interaction between Ana2 and Sas6 because the C-terminal region of Ana2 (201-420 aa), which interacts with Sas-6, partially overlaps with the Ana2 N1 (1-274 aa). However, this result suggests surprisingly that the STAN motif may be dispensable for Ana2's function during centriole formation. The mammalian Ana2-related protein STIL, which also contains the STAN motif, has been implicated in primary microcephaly, a neurodevelopmental disorder characterized by a reduced brain size. The apparently disparate phenotypes reported for mammalian STIL and fly Ana2 during brain development are likely due to different developmental contexts (Wang, 2011).

The reason that NB overproliferation occurs in ana2 mutants, but not in asterless or sas4 mutants with spindle or centriole defects, may be due to the different behaviors of these mutants in 'telophase rescue,' a phenomenon whereby proteins delocalized from the cortex during early mitosis are restored at anaphase/telophase by a poorly understood compensatory mechanism. The spindle misorientation phenotype in ana2 mutants is much more severe than sas4 or asterless mutants. Likely as a consequence of a relatively weak spindle misorientation phenotype, 'telophase rescue' still occurred in 100% of the asterless and sas4 mutant telophase NBs, and all asymmetrically localized proteins were correctly segregated into different daughter cells. In contrast, in ana2 mutants or mud mutants, which have NB overgrowth in larval brains, asymmetrically localized proteins sometimes mis-segregate into different daughters at telophase (Wang, 2011).

The RNAi screen identified Ctp as an important player in mitotic spindle orientation because ctp mutants displayed spindle misorientation during NB asymmetric division. ctp null mutants display spindle misorientation in NBs similar to that seen in ctp RNAi. It is noted that Ctp localizes to centrioles in Drosophila. Ana2 directly binds and anchors Ctp to the centrioles during NB division. The centriole localization is important for Ctp function during spindle orientation because membrane-targeted CtpCAAX fails to rescue the spindle misorientation phenotype in the ctp null mutant. The interaction between Ctp and Ana2 on the centrioles may be critical for dynein to organize astral microtubules and move its cargo proteins along the microtubules (Wang, 2011).

A dynein component, Ctp, can also bind directly to Mud, a protein downstream of heterotrimeric G protein signaling, that regulates spindle orientation. This interaction is conserved in vertebrates; Xenopus NuMA, a Mud-related protein, also forms a complex with dynein. Ana2 and Ctp are important for spindle pole localization of Mud during spindle orientation in NBs, whereas Mud is not required for centriolar localization of Ana2 or Ctp. Ana2 also directly interacts with Mud. These data suggested that Mud may be an important downstream target of Ana2 and Ctp during spindle orientation. Ana2, Ctp, and Mud are also found in the same protein complex in vivo and in vitro. Mud is involved in spindle pole/centrosome engagement, which has not been reported in previous analyses of Mud function. Ana2 and Ctp also played a similar role during spindle pole/centrosome attachment. Together, these data indicate that the Ana2, Ctp, and Mud complex functioned to regulate spindle pole assembly and spindle orientation during asymmetric division of NBs (Wang, 2011).

Apical/basal spindle orientation is controlled by a two-step mechanism: an early, centrosome-dependent alignment and a later spindle-cortex interaction. The data indicate that Ana2 is not only critical for the early, centrosome-dependent step, but also for the later spindle-cortex interaction. Although the loss of Ana2 or Ctp function does not affect Pins asymmetric localization in NBs, Ana2 and Ctp appear to be important for the interaction between Pins and Mud in larval brains because the Pins-Mud interaction is diminished in ana2 or insc-CtpCAAX, ctp mutant larval brains. These findings suggested that the Dynein-Dynactin complex cooperate with the centriolar protein Ana2 to mediate the spindle-cortex interaction. The spindle-cortex interaction may require the 'search and capture' mechanism, driven by the plus-end microtubule-binding protein EB1 and Dynein-Dynactin complex). It is speculated that Ana2 and Ctp may be involved in such a 'search and capture' mechanism during apicobasal spindle orientation (Wang, 2011).

These data suggested that a multiprotein complex composed of Ana2, Ctp, and Mud is critical during the regulation of spindle orientation. Ana2 and Ctp regulated Mud localization on centrosome/spindle poles as well as on the cell cortex, whereas the heterotrimeric G protein pathway is only important for cortical Mud localization. Thus, the centrosomal Ana2/Ctp/Mud complex converges with the heterotrimeric G protein pathway during spindle orientation. Very little is known about the molecular mechanisms by which centrosomal proteins regulate spindle orientation. Aur-A, a PCM protein, has been shown to phosphorylate Pins on S436 of the Pins Linker domain, which is required for accurate spindle orientation. The current findings suggest important functional links among the centriolar protein Ana2, the dynein complex, and Mud during asymmetric division of NBs. This raises the possibility that a similar mechanism whereby centrosomal proteins interact with dynein complexes to mediate cortical protein localization may exist during asymmetric division and stem cell self-renewal in mammals (Wang, 2011).

Inscuteable regulates the Pins-Mud spindle orientation pathway

During asymmetric cell division, alignment of the mitotic spindle with the cell polarity axis ensures that the cleavage furrow separates fate determinants into distinct daughter cells. The protein Inscuteable (Insc) is thought to link cell polarity and spindle positioning in diverse systems by binding the polarity protein Bazooka (Baz; aka Par-3) and the spindle orienting protein Partner of Inscuteable (Pins; mPins or LGN in mammals). This study investigated the mechanism of spindle orientation by the Insc-Pins complex. Previously, two Pins spindle orientation pathways were defined: a complex with Mushroom body defect (Mud; NuMA in mammals) is required for full activity, whereas binding to Discs large (Dlg) is sufficient for partial activity. The current study examined the role of Inscuteable in mediating downstream Pins-mediated spindle orientation pathways. It was found that the Insc-Pins complex requires Galphai for partial activity and that the complex specifically recruits Dlg but not Mud. In vitro competition experiments revealed that Insc and Mud compete for binding to the Pins TPR motifs, while Dlg can form a ternary complex with Insc-Pins. These results suggest that Insc does not passively couple polarity and spindle orientation but preferentially inhibits the Mud pathway, while allowing the Dlg pathway to remain active. Insc-regulated complex assembly may ensure that the spindle is attached to the cortex (via Dlg) before activation of spindle pulling forces by Dynein/Dynactin (via Mud) (Mauser, 2012).

Spindle positioning is important in many physiological contexts. At a fundamental level, spindle orientation determines the placement of the resulting daughter cells in the developing tissue, which is important for correct morphogenesis and tissue organization. In other contexts, such as asymmetric cell division, spindle position ensures proper segregation of fate determinants and subsequent differentiation of daughter cells. This study examined the function of a protein thought to provide a 'passive' mark on the cortex for subsequent recruitment of the spindle orientation machinery. During neuroblast asymmetric cell division, Insc has been thought to mark the cortex based on the location of the Par polarity complex (Mauser, 2012).

Ectopic expression of Insc in cells that normally do not express the protein has revealed that it is sufficient to induce cell divisions oriented perpendicular to the tissue layer, reminiscent of neuroblast divisions. Expression of the mammalian ortholog of Inscuteable, mInsc, in epidermal progenitors has shown that this phenotype is not completely penetrant over time. Expression of mInsc leads to a transient re-orientation of mitotic spindles, in which mInsc and NuMA initially co-localize at the apical cortex. After prolonged expression, however, the epidermal progenitors return to dividing along the tissue polarity axis, a scheme in which mInsc and NuMA no longer co-localize. These results indicate that Insc and Mud can be decoupled from one another (Mauser, 2012).

This study examined the effect of Insc-Pins complex formation both in an induced polarity spindle orientation assay and in in vitro binding assays. The results indicate that Insc plays a more active role in spindle positioning than previously appreciated. Rather than passively coupling polarity and spindle positioning systems, Insc acts to regulate the activity of downstream Pins pathways. The Dlg pathway is unaffected by Inscuteable expression while the Mud pathway is inhibited by Insc binding (Mauser, 2012).

Recent work on the mammalian versions of these proteins explains the structural mechanism for competition between the Insc-Pins and Pins-Mud complexes. The binding sites on Pins for these two proteins overlap making binding mutually exclusive because of steric considerations. The observation of Insc dissociation of the Pins-Mud complex in Drosophila (this work) and mammalian proteins (LGN-NuMA) suggests that Insc regulation of Mud-binding is a highly conserved behavior (Mauser, 2012).

This competition between Mud and Insc for Pins binding is consistent with previous work done with a chimeric version of Inscuteable/Pins (Yu, 2000b). This protein, in which the Pins TPR domain was replaced with the Inscuteable Ankyrin-repeat domain, bypasses the Insc-Pins recruitment step of apical complex formation. In these cells, the chimeric Insc-Pins protein was able to rescue apical/basal polarity and spindle orientation in metaphase pins mutant neuroblasts. As this protein lacks the Mud-binding TPR domain, Mud binding to Pins is not absolutely necessary for spindle alignment. Importantly, the PinsLINKER domain is still intact in the Insc-Pins fusion, implying that Dlg, not Mud, function is sufficient for partial activity, as observed in the S2 system (Mauser, 2012).

The Mud and Dlg pathways may play distinct roles in spindle positioning. The Dlg pathway, through the activity of the plus-end directed motor Khc73, may function to attach the cortex to the spindle through contacts with astral microtubules. In contrast, the Mud pathway, through the minus-end directed Dynein/Dynactin generates force to draw the centrosome towards the center of the cortical crescent. Fusion of the Pins TPR motifs, which recruit Mud, to Echinoid does not lead to spindle alignment, indicating that the Mud pathway is not sufficient for spindle alignment. The PinsLINKER domain does have partial activity on its own, however, and when placed in cis with the TPRs leads to full alignment. In this framework, the function of Insc may be temporal control, ensuring that microtubule attachment by the Dlg pathway occurs before the force generation pathway is activated (Mauser, 2012).

In the temporal model of Insc function, what might cause the transition from the Insc-Pins-Dlg complex, which mediates astral microtubule attachment, to the Mud-Pins-Dlg complex, which generates spindle pulling forces? By early prophase, Inscuteable recruits Pins and Gαi to the apical cortex. During this phase of the cell cycle, Mud is localized to the nucleus in high concentration. Apically-localized Pins binds Dlg, creating an apical target for astral microtubules. During early phases of mitosis, Inscuteable would serve to inhibit binding of low concentrations of cytoplasmic Mud to the Pins TPRs to prevent spurious activation of microtubule shortening pathways. After nuclear envelope breakdown, Mud enters the cytoplasm in greater concentrations and could then act to compete with Insc for binding to Pins, allowing Pins output to be directed into microtubule-shortening pathways (see Proposed model for Inscuteable regulation of spindle orientation). Future work will be directed towards testing additional aspects of this model (Mauser, 2012).

Discs Large Links Spindle Orientation to Apical-Basal Polarity in Drosophila Epithelia

Mitotic spindles in epithelial cells are oriented in the plane of the epithelium so that both daughter cells remain within the monolayer, and defects in spindle orientation have been proposed to promote tumorigenesis by causing epithelial disorganization and hyperplasia. Previous work has implicated the apical polarity factor aPKC, the junctional protein APC2, and basal integrins in epithelial spindle orientation, but the underlying mechanisms remain unclear. This study shows that these factors are not required for spindle orientation in the Drosophila follicular epithelium. Furthermore, aPKC and other apical polarity factors disappear from the apical membrane in mitosis. Instead, spindle orientation requires the lateral factor Discs large (Dlg), a function that is separable from its role in epithelial polarity. In neuroblasts, Pins recruits Dlg and Mud to form an apical complex that orients spindles along the apical-basal axis. Pins and Mud are also necessary for spindle orientation in follicle cells, as is the interaction between Dlg and Pins. Dlg localizes independently of Pins, however, suggesting that its lateral localization determines the planar orientation of the spindle in epithelial cells. Thus, different mechanisms recruit the conserved Dlg/Pins/Mud complex to orient the spindle in opposite directions in distinct cell types (Bergstralh, 2013).

Dlg is recruited by Pins to the cortex of asymmetrically dividing cells, such as neuroblasts and SOPs, and is required to orient the spindle toward the Pins crescent. Since Dlg colocalizes with Pins and Mud at the lateral cortex of the follicle cells, whether it is also necessary for spindle orientation in this epithelium was investigated. Dlg is essential for apical-basal polarity in epithelia, however. This complicates the analysis of its role in spindle orientation, because cells homozygous mutant for a strong loss-of-function allele, dlg14 (also called dlgm52), round up and lose their epithelial organization. The analysis was therefore restricted to those dlg14 mutant clones in which the cells remained in a monolayer, and it was observed that the spindles were randomly oriented (Bergstralh, 2013).

Dlg interacts with Pins through its C-terminal guanylate kinase (GUK) domain, which is disrupted in cells homozygous for the mutant allele dlg18, a premature stop mutation that removes the last 43 amino acids of the protein. Importantly, dlg18 does not disrupt the lateral localization of Dlg, and apical-basal polarity is unaffected in early-stage mutant clones, which form a normal epithelial monolayer. Despite this wild-type epithelial organization, dlg18 randomizes the orientation of the mitotic spindles (Bergstralh, 2013).

Spindles are oriented normally in dlgsw, which removes the last 14 amino acids of Dlg, leaving the GUK domain intact. Thus, Dlg is required for spindle orientation in the follicle cells, and this function is separable from its role in epithelial polarity. The role of Dlg in spindle orientation depends on the presence of an intact GUK domain and therefore presumably requires its interaction with Pins, strongly suggesting that the Dlg/Pins/Mud complex orients the spindle in epithelia, as it does in asymmetrically dividing cells (Bergstralh, 2013).

In neuroblasts, Pins is required for the apical localization of Dlg during mitosis, whereas Dlg reinforces the apical localization of Pins through a pathway that depends on astral microtubules. The situation in epithelia appears to be different, however, as Dlg localizes normally along the lateral cortex in clones of the pins null mutant, pinsp62. Since Dlg localizes laterally throughout the cell cycle, it is presumably localized by the same polarity-related mechanisms in interphase and mitotic cells. Whether Dlg is required for the localization of Pins was examined and it was observed that Pins still localizes around the cortex during mitosis in the absence of Dlg (dlg14) but is not enriched laterally The lateral enrichment of Pins also appears reduced in cells homozygous for the GUK domain mutant dlg18, suggesting that its interaction with Dlg contributes to its recruitment to the lateral cortex, although this phenotype is more variable than in the null (Figure 4F) (Bergstralh, 2013).

It has previously been proposed that the aPKC excludes Pins from the apical domain during mitosis in MDCK cells and the Drosophila wing imaginal disc, although not in chick neuroepithelial cells. In agreement with the latter finding, Pins-YFP shows a wild-type lateral localization during mitosis in apkcts/apkck06403 transheterozygous flies maintained at 18o. Thus, the lateral enrichment of Pins in mitotic follicle cells is independent of aPKC (Bergstralh, 2013).

In conclusion, this study has demonstrated that the planar orientation of the mitotic spindle in the follicular epithelium is independent of apical, junctional, or basal cues and depends instead on Dlg, Pins, and Mud. It therefore seems likely that the spindle is aligned within the plane of the epithelium by the same mechanisms that orient the spindle along the apical-basal axis in neuroblasts and that the key determinant of spindle orientation in both cell types is the location of the Dlg/Pins/Mud complex. The restriction of this complex to the lateral cortex in epithelial cells depends on Dlg, and its dual role in apical-basal polarity and spindle positioning therefore provides a mechanism to couple spindle orientation with the overall polarity of the tissue (Bergstralh, 2013).

Aurora kinases phosphorylate Lgl to induce mitotic spindle orientation in Drosophila epithelia

The Lethal giant larvae (Lgl) protein was discovered in Drosophila as a tumor suppressor in both neural stem cells (neuroblasts) and epithelia. In neuroblasts, Lgl relocalizes to the cytoplasm at mitosis, an event attributed to phosphorylation by mitotically activated aPKC kinase and thought to promote asymmetric cell division. This study shows that Lgl also relocalizes to the cytoplasm at mitosis in epithelial cells, which divide symmetrically. The Aurora A and Aurora B kinases directly phosphorylate Lgl to promote its mitotic relocalization, whereas aPKC kinase activity is required only for polarization of Lgl. A form of Lgl that is a substrate for aPKC, but not Aurora kinases, can restore cell polarity in lgl mutants but reveals defects in mitotic spindle orientation in epithelia. It is proposed that removal of Lgl from the plasma membrane at mitosis allows Pins/LGN to bind Dlg and thus orient the spindle in the plane of the epithelium. These findings suggest a revised model for Lgl regulation and function in both symmetric and asymmetric cell divisions (Bell, 2014).

Cell polarity regulates biased myosin activity and dynamics during asymmetric cell division via Drosophila Rho kinase and Protein kinase N

Cell and tissue morphogenesis depends on the correct regulation of non-muscle Myosin II, but how this motor protein is spatiotemporally controlled is incompletely understood. This study shows that in asymmetrically dividing Drosophila neural stem cells, cell intrinsic polarity cues provide spatial and temporal information to regulate biased Myosin activity. Using live cell imaging and a genetically encoded Myosin activity sensor, Drosophila Rho kinase (Rok) was found to enrich for activated Myosin on the neuroblast cortex prior to nuclear envelope breakdown (NEB). After NEB, the conserved polarity protein Partner of Inscuteable (Pins) sequentially enriches Ro and Protein Kinase N (Pkn) on the apical neuroblast cortex. These data suggest that apical Rok first increases phospho-Myosin, followed by Pkn-mediated Myosin downregulation, possibly through Rok inhibition. It is proposed that polarity-induced spatiotemporal control of Rok and Pkn is important for unequal cortical expansion, ensuring correct cleavage furrow positioning and the establishment of physical asymmetry (Tsankova, 2017).

In asymmetrically dividing fly neural stem cells the protein kinases Rok and Pkn respond to cell polarity cues to regulate Myosin activity and dynamics in a stereotypical, spatiotemporal manner. It is proposed that the sequential regulation mediated by these two kinases is necessary to control Myosin activity and actomyosin dynamics, triggering stereotypic cell shape changes at various steps in the neuroblast cell cycle; first to induce cell rounding as neuroblasts enter mitosis, to permit cell elongation and unequal cortical expansion during anaphase, and finally to complete cytokinesis and the establishment of physical asymmetry (Tsankova, 2017).

Myosin recruitment before NEB is mediated by Rok. This kinase, implicated in Myosin phosphorylation, is already localized at the neuroblast cortex before NEB and in rok mutants, Myosin remains cytoplasmic. At NEB both Rok and Myosin enrich on the apical neuroblast cortex. This apical enrichment, but not cortical localization, depends on the polarity protein Pins since only apical Rok and Myosin enrichment is lost if Pins localization is compromised. Based on these data it is proposed that Rok responds to cell cycle cues, presumably through the small GTPase Rho1, to phosphorylate Myosin's regulatory subunit, enabling activated Myosin to engage with F-actin at the cell cortex prior to NEB. Subsequently, polarity cues enhance Rok on the apical cortex, resulting in the elevation of phosphorylated and, thus, activated Myosin on the apical neuroblast cortex at NEB (Tsankova, 2017).

With Pkn a second kinase has been identified, responding to polarity cues since its apical localization, starting at NEB and peaking by the end of metaphase, is dependent on Pins. Pkn is not absolutely necessary for cortical Myosin enrichment; pkn mutant neuroblasts still retain apical Myosin, although elsewhere on the cortex its localization is dramatically reduced. However, Pkn is required for Myosin's timely relocalization from the apical cortex. Wild-type neuroblasts clear Myosin from the apical cortex in early anaphase, creating an asymmetric distribution that is necessary for the unequal cortical expansion. In pkn mutants, however, both Rok and Myosin dynamics are changed, retaining both on the apical neuroblast cortex, causing aberrant cortical constrictions and concomitantly inverted polar expansion (Tsankova, 2017).

Based on these results, the following model is proposed. (1) Rok triggers cortical Myosin accumulation before NEB. (2) At NEB, apically localized Pins enriches Rok on the apical neuroblast cortex and concomitantly increases phospho-Myosin apically. (3) Pins also induces the apical enrichment of Pkn, which is necessary for the timely relocalization of Myosin from the apical neuroblast during metaphase. It is further proposed that Pkn is downregulating Myosin activity through inhibiting or downregulating apical Rok activity. Whether Pkn downregulates Rok activity by direct phosphorylation remains an attractive hypothesis, since vertebrate Rock2 has recently been identified as a Pkn target. Alternatively, Rok activity could be regulated independently of phosphorylation but governed by the length of its coiled-coil tether, linking the kinase domain with the membrane binding domain (Tsankova, 2017).

This sequential regulation of Myosin dynamics seems to be a key regulatory mechanism underlying physical asymmetric cell divisions. For instance, apical Myosin relocalization always precedes basal Myosin clearing in wild-type neuroblasts. Similarly, biasing the localization of activated Myosin affects cleavage furrow positioning and physical asymmetry (Tsankova, 2017).

It is hypothesized that polarity cues provide a cell-intrinsic timer, priming Myosin relocalization on the apical cortex, thereby ensuring the generation of physical asymmetry through unequal cortical extension. Polarity-induced enrichment of activated Myosin on the apical cortex could thus provide a symmetry breaking event, necessary for the subsequent induction of apical Myosin clearing. Consistent with this model is the finding that pins mutants, or uniform cortical localization of Pins, cause Myosin to clear from both poles at the same time and divide symmetrically by size (Tsankova, 2017).

Tissue and organ growth critically depends on the correct spatiotemporal regulation of cell division. This study provides a conceptual framework of how Rok and Pkn respond to both cell cycle and polarity cues. These cues, in conjunction with spindle-dependent signals, ensure correct physical asymmetric cell division that is necessary for stem cell homeostasis and cell differentiation. Spatial and temporal regulation of Myosin activity has also been shown to be important for pulsatile cell shape changes in the Drosophila embryo. Rok and Pkn play important roles during vertebrate development and morphogenesis , and it will be interesting to see how spatiotemporal cues, affecting local cell shape changes, are coordinated with overall tissue morphogenesis in flies and beyond (Tsankova, 2017).


DEVELOPMENTAL BIOLOGY

Embryonic

To determine the subcellular localization of Raps, antibodies were generated against a Raps fusion protein. In NBs, Raps is localized as a crescent to the apical cortex. Apical crescents can be detected in interphase NB following delamination. More intensely labeled Raps apical crescents can be seen during mitosis from prophase to anaphase. In telophase, Raps show a weak cortical distribution and disappears only after telophase. Double labelings with anti-Insc indicate that, with the exception of delaminating NBs, the two proteins are largely colocalized during the NB cell cycle. In delaminating NBs, high levels of Insc staining can be seen on the apical stalk, which extends from the NB toward the surface of the neuroectoderm. In comparison, high levels of apical Raps are detected only following NB delamination. These observations suggest that the initial localization of Insc to the apical stalk of NBs during delamination (interphase) may precede that of the Raps apical localization; however, during mitosis the two proteins are colocalized as apical crescents. Apical cortical crescents of Raps can also be found in the dividing cells of the procephalic mitotic domain 9 (Yu, 2000).

In situ hybridization using a full-length Raps probe reveals that the gene is ubiquitously expressed until stage 12 of embryonic development. Expression is slightly higher in neuroblasts and progressively restricted to the CNS starting with stage 13. To determine the subcellular localization of Raps, mouse antibodies against Raps were generated and used to stain stage 10 Drosophila embryos. In contrast to Inscuteable, Raps protein is present in epithelial cells, where the protein is concentrated at the cell cortex with no sign of asymmetric localization. In delaminating neuroblasts, Inscuteable protein is first detected in an apical stalk that extends into the epithelial cell layer. Raps protein is concentrated in the stalk during delamination and colocalizes with Inscuteable at the apical cell cortex in fully delaminated neuroblasts. This apical colocalization of Inscuteable and Raps is maintained through mitosis and, in anaphase, both proteins disappear and became delocalized and are hardly detectable in telophase. Thus, Inscuteable and Raps colocalize in neuroblasts from delamination to anaphase of the first cell cycle (Schaefer, 2000).

Bazooka colocalizes with Inscuteable in neuroblasts but, in contrast to Inscuteable, Bazooka is also apically localized in epithelial cells. To compare the subcellular localisation of Raps with Bazooka, stage 10 embryos were stained for Raps, Bazooka and DNA. Whereas Bazooka localizes to the apical cell cortex in epithelial cells, Raps is found around the cell cortex and no apical concentration is observed in wild-type embryos. In neuroblasts, however, Raps and Bazooka colocalize at the apical cell cortex. Asymmetric localisation of Raps is also observed in sensory organ precursor (SOP) cells and epithelial cells of the procephalic neurogenic region (PNR): all these cells express Inscuteable. Thus, Inscuteable, Bazooka and Raps colocalize in cells that express Inscuteable, such as neuroblasts, SOP cells and cells of the PNR, but Raps does not colocalize with Bazooka in epithelial cells, which do not express Inscuteable (Schaefer, 2000).

Larval and Pupal

To study the localization of Raps, an antibody was raised against a fusion protein containing Raps without the four first TPR repeats. A focus was placed on the expression of Raps during larval stages because some of the rapsynoid mutants are lethal at this stage. In the larval CNS, the neuroblasts are derived from quiescent embryonic neuroblasts. They are also polarized and produce small GMCs constantly at the same pole. However, there is no clear apical-basal orientation perpendicular to the surface of the brain, and different neuroblasts are polarized in different orientations. As in embryonic neuroblasts, Insc is asymmetrically localized and forms a crescent at the face opposite that of the future GMC during metaphase. Such a crescent is not visible during interphase, although this is reported in embryonic neuroblasts (Parmentier, 2000).

Raps is expressed in the neuroblasts in interphase. It is localized mainly cortically, and the staining is punctate, as opposed to the Discs-Large staining that is homogenously present all along the plasma membrane. Raps localization is different in dividing neuroblasts. There is a clear crescent of Raps protein during prophase, metaphase, and anaphase, which disappears during telophase. Double staining for Raps and Insc shows colocalization of the Raps and Insc crescents in all dividing neuroblasts observed (Parmentier, 2000).

Generation of cell-fate diversity in Metazoan depends in part on asymmetric cell divisions in which cell-fate determinants are asymmetrically distributed in the mother cell and unequally partitioned between daughter cells. The polarization of the mother cell is a prerequisite to the unequal segregation of cell-fate determinants. In the Drosophila bristle lineage, two distinct mechanisms are known to define the axis of polarity of the pI and pIIb cells. Frizzled (Fz) signaling regulates the planar orientation of the pI division, while Inscuteable (Insc) directs the apical-basal polarity of the pIIb cell. The orientation of the asymmetric division of the pIIa cell is identical to the orientation of its mother cell, the pI cell, but, in contrast, is regulated by an unknown Insc- and Fz-independent mechanism. Drosophila E-Cadherin-Catenin (Shotgun-Armadillo) complexes are shown to localize at the cell contact between the two cells born from the asymmetric division of the pI cell. The mitotic spindle of the dividing pIIa cell rotates to line up with asymmetrically localized Shotgun-Armadillo complexes. While a complete loss of Shotgun function disrupts the apical-basal polarity of the epithelium, both a partial loss of Shotgun function and expression of a dominant-negative form of Shotgun affect the orientation of the pIIa division. Furthermore, expression of dominant-negative Shotgun also affects the position of Partner of Inscuteable (Pins) and Bazooka, two asymmetrically localized proteins known to regulate cell polarity. These results show that asymmetrically distributed Shotgun regulates the orientation of asymmetric cell division (Le Borgne, 2002).

Three distinct mechanisms regulate the stereotyped orientation of the first three asymmetric cell divisions in the seemingly simple lineage that generates the sense organs on the Drosophila notum. (1) In the pI cell, Fz signaling orients the mitotic spindle along the AP axis of the body, regulates the formation of the Dlg/Pins and Baz complexes at the anterior and posterior poles, respectively, and thereby directs the asymmetric localization of the Numb crescent to the anterior cortex. (2) By analogy to the neuroblasts, an apical Baz/Insc/Pins complex is thought to direct the apical-basal orientation of the pIIb division. This analogy is supported by the observation that Pins, Baz, and Insc colocalize at the apical cortex of the dividing pIIb cell. (3) The pIIa cell divides with the same orientation as its mother cell in a Fz- and Insc-independent manner. In the pIIa cell, a specific cortical domain formed at the region of cell-cell contact between the pIIb/pIIIb and pIIa cells appears to regulate the precise orientation of this division. Five lines of evidence support this last conclusion: (1) Shotgun (Shg), Arm, and alpha-Catenin-GFP localize asymmetrically in a cortical patch at the anterior pole of the dividing pIIa cell; (2) the mitotic spindle of the pIIa cell rotates to specifically line up with this cortical domain; (3) expression of a dominant-negative form of Shg perturbs both the formation of this cortical domain, the orientation of the pIIa division, and the precise positioning of Pins at the anterior lateral cortex; (4) loss of Shg activity in clones leads to defects in the orientation of the pIIa division; (5) Pins localizes opposite of Baz in the pIIa cell along a polarity axis defined by the patch of Shg, and dominant-negative Shg affects the orientation of these two domains relative to this patch. Noticeably, a strong loss of Shg function does not randomize the orientation of the mitotic spindle or of the Pins/Baz domains. Thus, one function of Shg in the pIIa cell is to ensure precision in the orientation of the polarity axis. Although loss of Fz activity randomizes the orientation of the pI cell, Shg appears to play a role formally similar to Fz in defining the polarity axis in the pIIa cell. This is the first evidence of a regulatory role of E-Cadherin in the orientation of asymmetric cell divisions (Le Borgne, 2002).


EFFECTS OF MUTATION

To determine whether Raps has a role in asymmetric cell division, raps null mutants were generated by imprecise excision of a P element inserted near the raps gene. The raps null mutants are homozygous viable, suggesting that maternal Raps protein is sufficient for all gene functions until adulthood. The raps mutant phenotype was therefore analysed in embryos from homozygous mutant females that lack both maternal and zygotic gene function (these mutants are called raps mutant embryos). Even though raps mutant embryos never hatch, no obvious morphological defects are seen in these embryos. Epithelial polarity, analysed by staining for the apically localized proteins Armadillo and Bazooka, is unaffected up to stage 13 of embryonic development in these mutants, suggesting that Raps does not function in epithelial cells (Schaefer, 2000).

To analyse defects in asymmetric cell division, raps mutant embryos were stained for beta-tubulin and DNA, or for Miranda and DNA. Whereas in wild-type embryos 70% of mitotic spindles in neuroblasts are oriented along the apical-basal axis, only 33% of raps mutant neuroblasts show apical-basal spindle orientation and spindles are frequently misoriented. Miranda localizes in a basal cortical crescent in wild-type embryos. In raps mutants, Miranda localisation is abnormal: in many metaphase neuroblasts, Miranda fails to localize asymmetrically or the Miranda crescent forms at incorrect positions around the cell cortex. Similar results were obtained for Numb and Partner of numb (Pon). Thus, the absence of maternal and zygotic Raps leads to defects that are very similar to those observed in inscuteable mutants (Schaefer, 2000).

Inscuteable localisation in raps mutants was tested by double staining raps mutant embryos for Inscuteable and DNA. As in wild-type embryos, Inscuteable is concentrated in the stalk during neuroblast delamination in raps mutants. After delamination, however, Inscuteable fails to form an apical cortical crescent but relocalizes into the cytoplasm instead and, during mitosis, the protein is homogeneously distributed throughout the cytoplasm. Despite the localisation defect, however, Inscuteable becomes degraded during anaphase. It is concluded that Raps is required for orienting asymmetric cell divisions and that Raps and Inscuteable are interdependent for asymmetric apical localisation in neuroblasts (Schaefer, 2000).

In order to assess the function of raps, advantage was taken of a P(w+) transposon, EP3559, which was inserted at cytological location 98A-B, ~700 bp 5' to the raps cDNA. By mobilizing this element, several small deletions were generated which, as judged by Southern blots, removes all or part of the raps coding region. Analyses of embryos homozygous for four different antigen-negative raps alleles that removed either the entire coding region, pinsP120 and pinsP17, or part of the coding region, pinsP62 and pinsP89, indicate that the loss of the zygotic component of raps/pins shows no defects with respect to the localization of Insc, Miranda, Prospero, or Partner of numb; neither were neuronal cell fate changes evident; moreover, spindle orientation in NBs and mitotic domain 9 cells were normal. The lack of any phenotypes is not surprising since staining of the homozygous mutants with anti-Raps demonstrates that protein derived from a maternal component persists until stage 16 of embryonic development. Moreover, the zygotic component appears not to be absolutely essential since animals lacking the zygotic component can survive to adulthood, although at reduced frequencies, and females can lay fertilized eggs (Yu, 2000).

To assess the effects of removing both maternal and zygotic raps on Insc localization, genotypically mutant embryos were obtained, derived from mutant mothers either homozygous or transheterozygous for the mutants pinsP62 and pinsP89 (referred to as Raps- embryos). Insc localization is dramatically affected in these embryos. In mitotic NBs and interphase NBs that have completed delamination, as well as in dividing cells of mitotic domain 9, Insc is localized to the cytoplasm. In NBs this failure to asymmetrically localize appears to be a defect in maintenance, since the initial apical localization of Insc occurs normally. This is most convincingly seen in delaminating NBs that are known to have completed S phase and are at the G2 stage of the cell cycle. Delaminating NBs possess a membrane stalk that emanates from their apical surface, which retains contact with the epithelial surface; this is where apical cortical localization of Insc is initially seen. This initial localization of Insc to the apical stalk occurs normally in Raps- embryos; however, apical Insc localization cannot be maintained and later in interphase and during mitosis, Insc no longer associates with the cortex and adopts a cytoplasmic localization. Hence while the initial apical localization of Insc during delamination does not require raps, the maintenance of this asymmetric localization later in interphase and throughout mitosis is raps dependent (Yu, 2000).

The role of insc in orienting the mitotic spindle, localizing Pros/Mir and Pon/Numb in neural progenitors, mediating alternative cell fate, and effecting nuclear size asymmetry of specific sibling neurons has been previously demonstrated. In order to ascertain the role of raps in mediating these processes, the phenotype of Raps- embryos was analyzed with anti-beta-tubulin to assess spindle orientation in cells of mitotic domain 9. Anti-Mir, anti-Pros, anti-Pon, anti-Numb, and DNA stainings were used to examine protein localization in NBs. Anti-Eve staining was used to assess whether distinct cell fates and distinct nuclear cell sizes are specified for RP2/RP2sib, a pair of sibling neurons. Raps- embryos display phenotypes similar to those seen in insc mutants. Mitotic spindle orientation is defective. In the cells of mitotic domain 9, the phenotype is similar to that seen in insc mutants where the 90° reorientation, which normally occurs in WT and results in the orientation of the spindle along the apical/basal axis, fails to occur in the mutant. Mitotic spindle orientation of NBs in the segmented CNS, deduced from DNA staining, also often fails to adopt an apical/basal orientation. Mir/Pros and Pon/Numb normally localize as basal crescents in WT metaphase NBs. However, in Raps- metaphase NBs these proteins often show defective localization, in the form of mislocalized crescents and cortical localization, similar to that seen in insc mutants. Where misplaced Mir/Pon crescents (>45° deviation from basal) form, they can either overlie one of the mitotic spindle poles (termed 'coupled') or not ('uncoupled'). An interesting difference between the raps and insc phenotype is that the frequency of coupled protein crescents is higher in Raps- NBs than in insc NBs. These observations suggest that the coordination of mitotic spindle orientation with protein localization may be less disrupted in Raps- than in insc metaphase NBs (Yu, 2000).

Resolution of distinct fates for the sibling neurons RP2 and RP2sib also frequently fails to occur. In ~60% of the mutant hemisegments, duplicated RP2 neurons (Eve-expressing neurons at the RP2 position) are found at the expense of the RP2sib. Moreover, the two RP2 neurons appear to have indistinguishable nuclear size, a phenotype also seen in insc mutants. In ~15% (n = 350) of the hemisegments, no Eve-expressing RP2/RP2sib neurons are produced due to a failure to correctly specify the GMC that is the progenitor for RP2/RP2sib. This similarity in the raps and insc loss of function across a range of phenotypes indicates that the raps-mediated maintenance of Insc asymmetric localization is necessary for the correct execution of neural progenitor asymmetric cell divisions (Yu, 2000).

Rapsynoid/Partner of Inscuteable controls asymmetric division of larval neuroblasts in Drosophila

Asymmetric cell division generates daughter cells with different developmental fates. In Drosophila neuroblasts, asymmetric divisions are characterized by (1) a difference in size between the two daughter cells and (2) an asymmetric distribution of cell fate determinants, including Prospero and Numb, between the two daughter cells. In embryonic neuroblasts, the asymmetric localization of cell fate determinants is under the control of the protein Inscuteable (Insc), which is itself localized asymmetrically as an apical crescent. Rapsynoid (Raps), which interacts in a two-hybrid assay with the signal transduction protein Galphai, is localized asymmetrically in dividing larval neuroblasts and colocalizes with Insc. Moreover, in raps mutants, the asymmetric divisions of neuroblasts are altered: (1) Insc is no longer asymmetrically localized in the dividing neuroblast; and (2) the neuroblast division produces two daughter cells of similar sizes. However, the morphologically symmetrical divisions of raps neuroblasts still lead to daughter cells with different fates, as shown by differences in gene expression. The data show that Raps is a novel protein involved in the control of asymmetric divisions of neuroblasts (Parmentier, 2000).

Because Insc is involved in the control of asymmetric division of neuroblasts, the colocalization of Raps with Insc suggests a possible role of Raps in the same process. To analyze Raps function in neuroblasts, flies mutant for rapsynoid were obtained by imprecise excision of a P-element, l(3)S031807, adjacent to the rapsynoid gene. The P-element is situated in an intron of another gene proximal to rapsynoid, oriented in the opposite direction. Flies homozygous for any of the rapsynoid deletions die as young pupae and, based on their phenotype over Df(3R)IR16 that deletes the whole raps gene, behave genetically as strong hypomorphs for the phenotypes studied (Parmentier, 2000).

Because colocalization of Raps with Insc was observed in wild type, disruption of Insc localization was examined in raps mutants. The Insc crescent fails to form in the mutant metaphase neuroblasts, and only a punctate staining similar to that seen in interphase is visible. This phenotype is rescued in all raps mutants when a rapsynoid transgene is added under the control of the hsp70 promoter. The basal expression, at 25°C, of two copies of the transgene is sufficient for a complete rescue of Insc asymmetrical localization. It is thus concluded that Raps is necessary for the asymmetrical localization of Insc in neuroblasts (Parmentier, 2000).

The effect of a raps mutation was studied on the localization of Miranda, whose localization on the GMC side of the neuroblast during division is dependent on Insc function. As expected, Miranda is asymmetrically localized in wild-type mitotic neuroblasts, but is no longer asymmetrically localized in raps mutant neuroblasts (Parmentier, 2000).

An important aspect of neuroblast asymmetric divisions is their morphology. In wild-type third-instar larvae, at day 5 after egglaying, all neuroblast divisions are morphologically asymmetric, producing a GMC that is one-eighth the size of the neuroblast. This is not the case in raps mutants, where 28% of neuroblasts divisions are morphologically symmetrical. Correlatively, the neuroblasts of third-instar larvae are smaller than in wild type, and their size approaches that of GMCs. These phenotypes are rescued in the presence of a rapsynoid transgene (Parmentier, 2000).

Molecular markers have been used to determine whether the loss of morphological asymmetry in dividing raps neuroblasts reflects a defect in cell fate determination during these divisions. A neuroblast marker (deadpan-LacZ) was used that is expressed at a high level in neuroblasts and at a low level in GMCs, and a GMC marker, Prospero, was used which is expressed in the nuclei of GMCs but not in the nuclei of neuroblasts. In raps mutants, a neuroblast-like cell expresses the neuroblast marker and no Prospero (or expresses Prospero very weakly), whereas the other daughter cell expresses Prospero strongly. Although the division is morphologically symmetrical, and although Insc is not apically localized, there is still some asymmetry in the division to lead to the differentiation of two dissimilar daughter cells (Parmentier, 2000).

Effects of mutation: Apical complex genes control mitotic spindle geometry and relative size of daughter cells in Drosophila neuroblast and pI asymmetric divisions

Drosophila neuroblast asymmetric divisions generate two daughters of unequal size and fate. A complex of apically localized molecules mediates basal localization of cell fate determinants and apicobasal orientation of the mitotic spindle, but how daughter cell size is controlled has remained unclear. Mitotic spindle geometry and unequal daughter cell size were shown to be controlled by two parallel pathways (Bazooka/DaPKC and Pins/Galphai) within the apical complex. While the localized activity of either pathway alone is sufficient to mediate the generation of an asymmetric mitotic spindle and unequally sized neuroblast daughters, loss of both pathways results in symmetric divisions. In sensory organ precursors, Bazooka/DaPKC and Pins/Galphai localize to opposite sides of the cortex and function in opposition to generate a symmetric spindle (Cai, 2003).

Thus members of the NB apical protein complex control the generation of daughter cells of unequal size. There are two redundant pathways: (1) Baz/DaPKC/ (and presumably DmPar6) as well as (2) Pins/Gαi, either of which, when asymmetrically localized to the NB cortex, can lead to the formation of an asymmetric mitotic spindle through the preferential elongation of the proximal spindle arm and the displacement of the spindle toward the distal cell cortex, resulting in the production of unequal-sized daughter cells. In addition, in NBs, Insc is required for the function of the Baz/DaPKC/(DmPar6) pathway. When both pathways are inactivated/attenuated, spindle asymmetry and displacement fail to occur and equal-sized daughter cells are produced at high frequency. In the PNS progenitor, pI, where Baz/DaPKC are localized to the posterior cortex and Pins/Gαi are localized to the anterior cortex, the mitotic spindle is symmetric. Consistent with this hypothesis that both pathways can act to cause the preferential elongation of the proximal spindle arm relative to the distal spindle arm, removing posterior baz function without abolishing the localization and function of the anterior components results in the production of an asymmetric spindle with an anterior bias; removing anterior pins function without affecting the function of the posterior components results in a posteriorly biased asymmetric spindle; if components of both pathways are localized to the anterior cortex through the ectopic expression of Insc, an anteriorly biased asymmetric spindle results. These findings suggest that DaPKC and hetrotrimeric G protein signaling work in conjunction in the NB to produce an asymmetric spindle and in opposition in pI to produce a symmetric spindle (Cai, 2003).

Several lines of evidence suggest that localized signaling is essential to generate an asymmetric spindle and daughter cells of unequal size. (1) When both signaling pathways are abolished/attenuated (e.g., in insc/pins double mutant) or when signaling is uniform, which is assumed to be the case when Baz/DaPKC/Pins/Gαi are all uniformly localized throughout the cell cortex (e.g., in the case of Gαi overexpression in wt NBs), equal-sized daughters are generated. (2) When pins function is removed and DaPKC/Baz is asymmetrically localized (e.g., in pins mutant NB) or when Pins/Gαi are uniformly cortical but DaPKC/Baz are asymmetrically localized (e.g., in 69% [n = 51] of wt NBs overexpressing C-Pins), the site of the DaPKC/Baz localization coincides with the position where the larger daughter forms. (3) When Pins/Gαi is asymmetrically localized but baz/DaPKC function has been compromised (e.g., in insc mutant) or when Pins/Gαi is asymmetrically localized but Baz/DaPKC is uniformly cortical (in the case of NBs with basal Pins-C-Pon crescents), the site of localization coincides with the larger daughter and the extended spindle arm. These observations indicate that just one localized signal source, mediated presumably by either heterotrimeric G protein or DaPKC, is sufficient to cause proximal spindle arm elongation and the generation of unequal-sized daughters (Cai, 2003).

The situation is different in pI where Baz/DaPKC/(DmPar6) and Pins/Gαi act in opposition and where Insc is not required for the function of the Baz/DaPKC/(DmPar6) with respect to spindle elongation. Here, a distinction can be made between two possible models for explaining how spindle asymmetry/geometry is mediated. The first model is that the presence of either asymmetrically localized Baz/DaPKC/(DmPar6) or Pins/Gαi on one side of the cell is sufficient to cause elongation of the proximal spindle arm, regardless of what occurs on the other side of the cell. A second model would be that the signals from the opposite sides of the cortex are integrated and the bias in the spindle geometry depends on the relative magnitude of the two signals. The simplest prediction of the first model would be that the distance from the cleavage furrow to the spindle pole of wt telophase pI should be equivalent to the longer of the two spindle arms in telophase pI mutant for either baz or pins. This appears not to be the case. The average length of the longer spindle arm in telophase pI mutant for pins or baz is greater than that of a wt spindle arm and the length of the shorter of the spindle arms in mutant pI is less than that of a wt spindle arm . An equivalent analysis is difficult to do with NBs, since the size of the 30 or so NBs found in each hemisegment is more variable. Nevertheless, based on these observations the second type of model is favored (Cai, 2003).

Previous work has shown that Pins binds to the GDP bound form of Gαi and can cause Gαi to dissociate from Gβ13F; moreover, some phenotypes seen when Gαi is overexpressed in wt NBs (e.g., equal size divisions) are not seen when GαiQ205L, an activated form of Gαi lacking GTPase activity that should be in the GTP bound form, is overexpressed, or when Gβ13F function is abolished. These phenotypes therefore are unlikely to be induced by GTP bound Gαi or by depletion of Gβγ, suggesting that the GDP bound form of Gαi may be responsible for the equal size NB divisions seen when wt Gαi is overexpressed. These findings clearly support the view that the Pins/GDP-Gαi complex has a role for generating the signal associated with spindle asymmetry. (1) Equal size divisions seen when Gαi is overexpressed in wt NBs is drastically reduced when overexpression is performed in the absence of Pins. (2) Whenever unequal size division occurs when Baz/DaPKC function is compromised, Pins and Gαi are always colocalized to the side of the cell where the future larger daughter is formed (Cai, 2003).

Although in the nematode embryo generation of unequal-sized daughters involves only the posterior displacement of a symmetric spindle, there appears to be some parallels between the two model systems. In the wt nematode P0 division, the magnitude of the forces acting on the two spindle poles apparently depend on the character of the anterior and posterior cortex. In wt P0, PAR-3 and PAR-2 localize to the anterior and posterior cortex, respectively, and the mitotic spindle is displaced toward the posterior pole, correlating with a greater net posterior force acting on the posterior spindle pole relative to the net anterior force acting on the anterior spindle pole. In par-2 mutants, PAR-3 expands to occupy the whole of the cortex, imparting anterior character throughout, and the net force acting on both spindle poles has a magnitude equivalent to that of the wt force acting on the anterior spindle pole. Conversely in par-3 P0, PAR-2 becomes cortical, imparting posterior character to the entire cortex, and the magnitude of both forces acting on the spindle poles is equivalent to that of the wt posterior acting force. In both par-2 and par-3 mutants, the forces acting on the spindle poles are equalized, mitotic spindle is no longer displaced, and equal-sized daughters result (Cai, 2003).

In Drosophila NBs, although spindle displacement occurs, the generation of an apically biased mitotic spindle mediated by either asymmetrically localized Baz/DaPKC or Pins/Gαi makes the major contribution to the difference in daughter cell size. It is proposed that the asymmetric localization of components of either of these pathways can make the region of the cell cortex they occupy different from the cortical regions that they don't occupy through localized DaPKC or heterotrimeric G protein signaling mediated through Pins/Gαi. In wt NBs, the components of either pathway would impart apical character to the cell cortex where they are localized. One effect of the asymmetric signaling is to generate the preferential elongation of the spindle arm closest to the site of the localized signal. If signaling is symmetric, for example either when Baz/DaPKC and Pins/Gαi are all uniformly cortical, or when Baz/DaPKC and Pins/Gαi are localized to opposite sides of a dividing progenitor, as in pI, a symmetric spindle results. Hence, in both the nematode P0 and in Drosophila NBs the generation of unequal-sized daughters is regulated by asymmetrically localized cortical components. In the nematode there is compelling evidence that differential forces acting on the two spindle poles mediate spindle displacement and the generation of unequal daughters. However, NBs of Drosophila asterless mutants are apparently devoid of functional centrosomes and astral microtubules, yet they form functional asymmetric anastral mitotic spindles and undergo unequal cytokinesis to generate unequal size daughters. It remains to be seen how the localized properties of the NB cell cortex influences its spindle geometry (Cai, 2003).

Induction of tumor growth by altered stem-cell asymmetric division in Drosophila melanogaster

Loss of cell polarity and cancer are tightly correlated, but proof for a causative relationship has remained elusive. In stem cells, loss of polarity and impairment of asymmetric cell division could alter cell fates and thereby render daughter cells unable to respond to the mechanisms that control proliferation. To test this hypothesis, Drosophila larval neuroblasts were generated containing mutations in various genes that control asymmetric cell division and then their proliferative potential was assayed after transplantation into adult hosts. It was found that larval brain tissue carrying neuroblasts with mutations in raps (also called pins), mira, numb or pros grew to more than 100 times their initial size, invading other tissues and killing the hosts in 2 weeks. These tumors became immortal and can be retransplanted into new hosts for years. Six weeks after the first implantation, genome instability and centrosome alterations, two traits of malignant carcinomas, appeared in these tumors. Increasing evidence suggests that some tumors may be of stem cell origin. These results show that loss of function of any of several genes that control the fate of a stem cell's daughters may result in hyperproliferation, triggering a chain of events that subverts cell homeostasis in a general sense and leads to cancer (Caussinus, 2005).

Malignant transformation and loss of cell polarity are tightly correlated in human carcinomas. Likewise, Drosophila larval tissues with mutations in dlg1, l(2)gl or scrib have impaired apicobasal polarity and neoplastic growth in the imaginal epithelia and nervous system. There are several hypotheses to explain how loss of polarity contributes to neoplastic transformation. Most of them involve models in which changes in cellular architecture impinge directly on the cell cycle either by inhibiting signals that restrain cell proliferation or by enhancing mitogenic pathways. An alternative hypothesis is that loss of polarity in stem cells that divide asymmetrically impairs the mechanisms that specify the fate of the resulting daughter cells. If these daughter cells are unable to follow their normal developmental program, they may not respond to the mechanisms that control proliferation in the wild-type lineage (Caussinus, 2005).

Drosophila neuroblasts are stem cells whose asymmetric cell-division machinery is fairly well characterized and thus provide a good model to test this hypothesis. In the embryo, Insc integrates into the apical cortex of two neuroblast protein complexes, Baz-DmPar6-aPKC and Gialpha-Raps, by associating with Baz and Raps. These two complexes mediate the basal localization of Mira and Pon and their interacting proteins, Pros and Numb, whose segregation into the ganglion mother cell (GMC) is required for the unequal fate of the two neuroblast daughter cells. The basal localization of Mira and Pros, as well as the spindle orientation and asymmetry of daughter-cell sizes, require the functions provided by dlg1, l(2)gl and scrib. Larval neuroblasts originate from quiescent embryonic neuroblasts, and their asymmetric division seems to be controlled by the same molecular complexes, although minor differences have been reported (Caussinus, 2005).

To assess the effect of disrupted stem-cell asymmetric division on cell proliferation, larval neuroblasts were generated with mutations in aPKC, raps, mira, pros or numb and their proliferation potential was assayed after transplantation into adult hosts. No substantial growth of 101 pieces of wild-type larval brains were observed 2 weeks after transplantation. Similar results were observed for 109 implants that carried homozygous aPKCk06403 clones, none of which grew to any noticeable extent. In contrast, pieces of brains from rapsP89/raps P62 larvae or from larvae carrying homozygous numb03235, miraZZ176 or pros 17 clones grew to more than 100 times their original size, severely damaging and displacing the host's organs in the abdomen. Of the 103 flies studied in detail, 92% had one or more small tumor colonies derived from the implanted tissue but located at a long distance from the point of injection. The efficiency of tumor development ranged from 8% for numb03235 clones to 20% for rapsP89/rapsP62 tissue (Caussinus, 2005).

To assess further the growth potential of these tumors, they were cut into pieces and reimplanted into new hosts. More than 90% of these flies developed a tumor, even when they were implanted with numb 03235 tissue that had initially developed tumors in only 8% of implanted adults. This result suggests that the growing tumor mass adapts itself very rapidly to its new environment. Pieces of brain lobes from 9- to 12-d-old homozygous brat k06028 and l(3)mbt ts1 larvae, in which overgrowth was already apparent, developed tumors in 91% and 58%, respectively, of the implanted hosts (Caussinus, 2005).

All the tumors described here have been maintained in the laboratory, some for more than 2 years. This shows that the transformed cells became immortal and can proliferate without end, in contrast to cells of wild-type imaginal discs implanted into adult hosts, which remain alive after years of culture but very rarely proliferate. Among the established cell lines, substantial differences were observed in speed of growth, host lifespan or frequency or average number of additional tumor colonies, that could be attributed to the mutant background from which the tumors originated. Using the same criteria, these tumors were indistinguishable from dlg1, l(2)gl and scrib neuroblastomas (Caussinus, 2005).

Attempts were made to determine the kinds of cells that could be found in these tumors. Using green fluorescent protein as a clonal marker, it was observed that in tumors derived from tissue carrying numb 03235, miraZZ176 or pros17 clones induced by mitotic recombination, neither the wild-type twin nor the heterozygous background cells were able to proliferate upon implantation and were lost within 2 weeks. These cells accounted for most of the implanted mass, and so their inability to hyperproliferate provided a valuable internal control to substantiate the conclusion that tumor growth in this assay required the loss of the genes under study and was not just the result of dissection and transplantation into adult hosts. It also showed that the tumor growth induced by the loss of function of these genes was cell-autonomous (Caussinus, 2005).

Immunofluorescence staining for cell-specific markers identified the neuroblasts as relatively large cells, 8-12 microm in diameter, that expressed Mira. In miraZZ176 tumors, neuroblasts were identified by the expression of Wor. Ganglion cells were identified as small cells, 4-6 microm in diameter, that did not express Mira but did express Pros or, in pros 17-derived tumors, Numb. The intermediately sized cells that did not express Pros, some of which showed weak Mira staining, might be GMCs. Neuroblasts accounted for most of the mitotic activity observed in these tumors (86%). Daughter-cell size and Mira segregation during mitosis were symmetric in neuroblasts derived from rapsP89/rapsP62 tumors but asymmetric in those derived from numb03235 and pros 17 tumors. Daughter-cell size was also asymmetric in neuroblasts from miraZZ176 tumors (Caussinus, 2005).

Neither neuroblasts nor ganglion cells were markedly diluted or over-represented as the tumors aged from host to host. Therefore, like l(2)gl and dlg1 tumors, the tumors derived from numb03235, miraZZ176, pros17 and raps P89/rapsP62 were neuroblastomas that resulted from the uncontrolled division of neuroblast stem cells and were largely composed of the undifferentiated cell types that belong to this lineage. The mechanism by which these tumors grew is not understood, but it must account for the observed continuous expansion of both the neuroblast and the ganglion cell populations. One plausible mechanism could be a low frequency of neuroblast divisions that generate two neuroblast daughters. Real-time analysis of cell proliferation in these tumors may provide an answer to this issue (Caussinus, 2005).

In most solid human tumors, malignancy is very often correlated with genome instability, which is thought to contribute to multistage carcinogenesis. As in most animal cells, the frequency of natural cases of genome instability in wild-type Drosophila neuroblasts and GMCs is low (less than 10-3). This is also the case in numb03235, miraZZ176, pros 17 and rapsP89/raps P62 tumors shortly after transplantation. In 40-d-old tumors, however, 10%-15% of the cells presented different kinds of karyotype defects. Of the 340 karyotypes obtained from numb, mira, pros and raps tumors, 62% included segmental aneuploid; 9% were monosomic, trisomic or tetrasomic with respect to one or more chromosomes; 6% were triploid or tetraploid; and the remaining 23% included cells that could not be karyotyped owing to very high levels of ploidy, chromosome fragmentation or chromosome condensation (Caussinus, 2005).

The karyotypes obtained from cells in a single tumor were as different from one another as they were from the karyotypes of cells from other tumors, and none of the tumor lines that were established presented a distinct set of chromosome aberrations. Therefore, no substantial differences were observed attributable to the mutant condition that originated the tumor. In most tumor lines, the frequency of cells that contained abnormal karyotypes did not change noticeably over time, with one exception: 3 months after the first implantation, genome instability affected more than 95% of the cells in mirTF, a tumor line derived from miraZZ176. The absence or very low incidence of genome instability during the first round of implantation suggests that genome instability did not cause tumor formation in these tumor lines. But the onset of genome instability correlates well with a marked increase in the frequency of hosts that developed a tumor in later transplantations. Therefore, the possible contribution of genome instability to the evolution of these tumors remains to be assessed. Genome instability has also been reported in l(2)gl neuroblastomas (Caussinus, 2005).

In mammalian carcinomas, genome instability is tightly correlated with severe alterations of the centrosome cycle that affect the number of centrosomes per cell as well as centrosome size and shape. Supernumerary centrosomes can result in multipolar spindles and contribute to the generation of aneuploidy. Like the DNA cycle, the centrosome cycle is tightly controlled in wild-type neuroblasts, so that cells that have an abnormal number of centrosomes are exceptionally rare in wild-type tissue. This was not the case in numb03235, mira ZZ176, pros17 or raps P89/rapsP62 tumors: forty days after the first implantation, 15%-20% of those cells had more than two centrosomes. Some of these centrosomes were irregularly shaped, and their size range was much wider than that of control cells. A fraction of these could be centriole-less aggregates of pericentriolar material. The cells that had supernumerary centrosomes seemed to be hyperploid (Caussinus, 2005).

None of the mutant conditions from which these tumors originated has been reported to affect chromosome segregation or the centrosome cycle, which were both unaffected in early tumors. In addition, the cells of wild-type imaginal discs that have been kept for years in adult hosts maintain a stable genome and can differentiate into adult structures. Therefore, genome instability and impaired centrosome cycles in numb 03235, miraZZ176, pros17 and rapsP89/rapsP62 tumors cannot be considered a consequence of the mutant background or long-term exposure to the adult abdomen environment. Rather, the onset of genome instability and centrosome alterations suggests that once the mechanisms that control cell proliferation have been over-ridden, hyperproliferation triggers a chain of events that subverts cell homeostasis in a very general sense, including the DNA and centrosome cycles (Caussinus, 2005).

In summary, neoplastic transformation of Drosophila larval neuroblasts can be triggered by perturbing several of the functions that mediate asymmetric stem-cell division. In terms of growth rate, cell types, metastatic activity and extent of genome and centrosome instability, the resulting tumors are essentially indistinguishable from one another, regardless of the mutant from which they derive. The main conclusion that can be drawn from these data is that these tumors might have a common etiology: perturbation of neuroblast polarity and the resulting impairment of cell-fate determination. This argument is strengthened by the case of the homeobox-containing transcription factor Pros, which lies downstream of the other genes required for neuroblast asymmetric division (Caussinus, 2005).

The tumors in this study are practically indistinguishable from the neuroblastomas that arise in adults implanted with pieces of dlg1, l(2)gl or scrib mutant larval brains. Because these three neoplastic tumor suppressors are required for multiple aspects of neuroblast asymmetric cell division, including the basal localization of Mira, Numb and Pros, mislocalization of these proteins might explain, at least partially, the uncontrolled cell proliferation produced by loss of dlg1, l(2)gl or scrib function in larval neuroblasts (Caussinus, 2005).

The unequal segregation of cell-fate determinants resulting from asymmetric cell division is a fundamental mechanism for generating cellular diversity during development, organ homeostasis and repair. If impaired segregation of cell-fate determinants can cause the hyperproliferation of larval neuroblasts of Drosophila, it may similarly affect tissue stem cells in other species. At the moment, however, any parallel to stem-cell models of human cancer remains purely speculative. Consistent with this hypothesis, the inactivation of both Numb and Numb-like in the mouse dorsal forebrain leads to impaired neuronal differentiation, hyperproliferation of neural progenitors and delayed cell-cycle exit. In addition, loss of Lgl1 (also called Mlgl or Hugl), one of the two L(2)gl homologs in the mouse, results in a failure to asymmetrically localize Numb and leads to severe brain dysplasia (Caussinus, 2005).

In most human tumors, the identity of the first carcinogenic cell remains elusive. Indirect but growing evidence suggests that in some cases, the founders may be stem cells. Stem cells are self-renewing, have limitless replicative potential and produce differentiating cells, three features found in many cancers. Carcinomas occur in tissues that are maintained by a continuous supply of differentiating daughter cells originating from stem-cell division. Moreover, some of the signaling pathways that control stem-cell self-renewal, like the Notch, Wnt-ß-catenin and Hedgehog pathways, are known to have a role in carcinogenesis in these tissues. The results show that inactivation of any of several molecular mechanisms that control the asymmetry of the segregation of cell-fate determinants during stem-cell division may result in hyperproliferation of the stem-cell compartment and could contribute to cancer (Caussinus, 2005).

Drosophila Ric-8 regulates Galphai cortical localization to promote Galphai-dependent planar orientation of the mitotic spindle during asymmetric cell division

Localization and activation of heterotrimeric G proteins have a crucial role during asymmetric cell division. The asymmetric division of the Drosophila sensory precursor cell (pl) is polarized along the antero-posterior axis by Frizzled signalling and, during this division, activation of Galphai depends on Partner of Inscuteable (Pins). This study establish that Ric-8, which belongs to a family of guanine nucleotide-exchange factors for Galphai, regulates cortical localization of the subunits Gαi and Gβ13F. Ric-8, Gαi and Pins are not necessary for the control of the anteroposterior orientation of the mitotic spindle during pl cell division downstream of Frizzled signalling, but they are required for maintainance of the spindle within the plane of the epithelium. On the contrary, Frizzled signalling orients the spindle along the antero-posterior axis but also tilts it along the apico-basal axis. Thus, Frizzled and heterotrimeric G-protein signalling act in opposition to ensure that the spindle aligns both in the plane of the epithelium and along the tissue polarity axis (David, 2005).

In the dorsal thorax (notum) of the Drosophila pupa, approximately 100 sensory precursor (pI) cells each divide asymmetrically with an antero-posterior planar polarity to produce a posterior cell, pIIa, and an anterior cell, pIIb, which will further divide to give rise to a mechanosensory organ. The antero-posterior planar polarity of the pI cell division is dependent on Frizzled (Fz) activity. It is marked by the anterior asymmetric localization of the cell-fate determinants Numb and Neuralized. This anterior localization of Numb depends on Bazooka (Baz), which localizes at the posterior pI cell cortex, and on Pins and Gαi, which accumulate at the anterior cortex. Pins belongs to a family of guanine nucleotide dissociation inhibitors for Gα subunits and restricts the localization of Baz to the posterior cortex of the dividing pI cell. Baz, in turn, promotes the asymmetric localization of Numb. Analysis of Gαi-null dividing pI cells reveals that Gαi is required for localization of a functional Pins–YFP (yellow fluorescent protein) fusion protein and of Baz). Furthermore, the orientation of the mitotic spindle of the pI cell ensures that its division takes place along the antero-posterior axis and within the plane of the epithelium. The antero-posterior orientation of the spindle depends on Fz activity, and Pins and Gαi have been proposed to participate in this process. However, the mechanisms that ensure apico-basal orientation of the spindle have not been analysed (David, 2005).

Recently, Ric-8, a guanine nucleotide-exchange factor (GEF) for Gαi and Gαo, has been characterized in Caenorhabditis elegans and in mammals. This study analysed the role of a Drosophila ric-8 homologue in pI cell polarity and spindle orientation. In doing so, the first mechanism that ensures correct apico-basal orientation of the mitotic spindle during pI cell division has been identified (David, 2005).

Although putative ric-8a and ric-8b have been identified in the fly genome, ric-8b is likely to be a pseudogene. To study ric-8a function, the expression was used of a ric-8a double-stranded RNA (dsRNA), which strongly reduced the Ric-8a protein level as assessed by RNA interference (RNAi). A severe hypomorph or null P element allele of ric-8a, G0397 was used. The ric-8aG0397 insertion is lethal and this lethality was rescued by a Ric-8a–YFP protein, which was uniformly distributed in the cytoplasm of both epithelial and pI cells during interphase and mitosis (David, 2005).

In ric-8a-RNAi sensory organs, pIIa to pIIb cell-fate transformations were observed; Therefore these were analysed to see whether these fate transformations might arise from the role of Ric-8a in pI cell polarization by comparing the distribution of Numb, Baz and Pins in control and in ric-8a-RNAi pI cells (similar results were obtained in a smaller number of dividing pI cells by analysing ric-8a mutant somatic clones). Whereas Numb formed an anterior crescent in control dividing pI cells, it failed to localize asymmetrically in 46% of ric-8a-RNAi pI cells in prometaphase or metaphase. As observed for pins and Gαi, a telophase rescue mechanism operates in ric-8a-RNAi cells as Numb formed a weak anterior crescent in 96% of ric-8a-RNAi pI cells in telophase. The localization of Baz, which was restricted to the posterior half of the cortex in control cells at metaphase, was affected in 63% of dividing ric-8a-RNAi pI cells, leading to a circular localization in 75% of the affected pI cells. Similarly, the localization of Pins to the anterior cortex was lost in 88% of ric-8a-RNAi pI cells in prometaphase or metaphase. These data demonstrate that ric-8a is required to polarize dividing pI cells and may act upstream of, or in parallel to, baz and pins (David, 2005).

Consistent with the involvement of ric-8a in regulating the anterior accumulation of Pins, it was found that, in ric-8a-RNAi cells, the anterior accumulation of Gαi was weaker than in control cells and was even lost in 62% of cells. Strikingly, in such cells in which Gαi anterior accumulation was lost, Gαi was absent from the lateral cortex. This phenotype is not a consequence of defective cell polarity as Gαi is still present, although symmetric, at the cell cortex of dividing pins pI cells. Therefore, ric-8a not only affects pI cell polarity but also seems to be required for accumulation of Gαi at the cell cortex during division (David, 2005).

To test whether this unexpected function of ric-8a was specific to the pI cell, Gαi localization was analyzed in epithelial cells. Gαi was detectable at the baso-lateral cortex of epithelial control cells and its staining is augmented during mitosis. But, as in the ric-8a-RNAi pI cells, Gαi is lost from the cortex of both interphase and mitotic ric-8a-RNAi epithelial cells. ric-8a is therefore required for the accumulation of Gαi at the cortex of both pI and epithelial cells. As mouse Ric-8 is a GEF for both Gαi and Gαo (Tall, 2002), the localization of Gαo was examined in ric-8a-RNAi cells. Gαo appeared to be uniformly distributed at the cortex of dividing pI and epithelial control cells but, unlike Gαi, the cortical localization of Gαo was not affected in ric-8a-RNAi cells. Gβ13F was uniformly distributed at the cortex of control pI cells. Like the staining for Gαi, this staining was strongly reduced in ric-8a-RNAi cells. Again, this phenotype was not due to defective cell polarity, because Gβ13F was still cortical in both pins and Gαi mutant pI cells. Gβ13F was also detectable at the cell cortex of both interphase and mitotic epithelial cells, and this staining was equally lost in ric-8a-RNAi cells. These data demonstrate that ric-8a is required for the cortical accumulation of Gαi and Gβ13F (David, 2005).

The loss of cortical staining for Gαi and Gβ13F could result either from a reduction in the amount of these proteins or from their failure to localize at the plasma membrane. Therefore the fluorescent signal for Gαi and Gβ13F were first quantified and compared between control cells and neighbouring ric-8a mutant clones. Only a slight reduction was found in signal intensity, which could not account for the pronounced reduction in the cortical staining. Moreover, the levels of Gαi and Gβ13F proteins were compared in wild-type and ric-8a second instar larval brains and no significant differences was detected between the two strains. These results support the idea that Ric-8a is required for the cortical localization of Gαi and Gβ13F. To corroborate this idea, either Gαi or both Gβ13F and Gγ1 were overexpressed in ric-8a mutant cells. Although overexpressed Gαi or Gβ13F are cortical in wild-type cells, they are mainly cytoplasmic in ric-8a mutant cells. Furthermore, the overexpression of Gαi in ric-8a mutant epithelial cells could not rescue Gβ13F cortical localization and the overexpression of both Gβ13F and Gγ1 in ric-8a mutant epithelial cells did not lead to Gαi cortical localization. Altogether, these data demonstrate that Ric-8a mildly regulates Gαi and Gβ13F stability, but is mainly required for their localization at the plasma membrane (David, 2005).

Palmitoylation of Gα and its association with Gβγ are both required to allow the Gαβγ trimer to reach the plasma membrane. Drosophila Ric-8a may be required for Gαi palmitoylation or for its association with Gβγ. In mammals, Ric-8 does not interact with Gβ B12">12 , so the effect on Gβ13F is likely to be indirect. Gβ13F remained cytoplasmic in ric-8a,Gαi double-mutant epithelial cells, excluding the fact that Gβ13F was held in the cytoplasm by mislocalized Gαi in ric-8a mutant cells. It is therefore envisaged that Ric-8a affects other Gα subunits that are necessary for Gβ13F to reach its destination. This proposition is consistent with the demonstration that mammalian Ric-8 is a GEF for Gαi and Gαo but can also interact with Gαq and Gα13 (David, 2005).

The role of ric-8a was then analyzed in mitotic-spindle positioning. The microtubule-associated protein Tau–GFP (green fluorescent protein) was expressed under the control of a neuralized-GAL4 driver to follow the dynamics of the mitotic spindle in dividing pI cells. In wild-type cells, the spindle is oriented along the antero-posterior axis. This strict orientation is dependent on Fz signalling. In ric-8a mutant cells, the spindle is still oriented along the antero-posterior axis, and is not randomized as in fz mutant cells. This result led to a reanalysis of spindle orientation in Gαi and pins pI cells, and it was found that Gαi and Pins are not required downstream of Fz for the antero-posterior orientation of the mitotic spindle (David, 2005).

The orientation of the spindle is also strictly controlled along the apico-basal axis, so that division takes place in the plane of the epithelium. This was quantified by measuring αz, which represents the angle of the mitotic spindle relative to the plane of the epithelium. In wild-type cells, the spindle is almost, but not exactly, parallel to the plane of the epithelium, the posterior centrosome being always slightly more apical than the anterior one. In ric-8a mutant pI cells, the spindle appeared more tilted along the apico-basal axis, with 25% of the cells displaying a tilt of more than 30°, a situation that was never observed in wild-type cells. This apico-basal phenotype was stronger in pins and Gαi mutant pI cells, the posterior centrosome being always largely more apical than the anterior one. Therefore, Ric-8a, Pins and Gαi are required in pI cells to maintain the spindle in the plane of the epithelium. Furthermore, pins,Gαi double-mutant pI cells displayed a phenotype that was identical to the one observed in pins or Gαi single-mutant pI cells, demonstrating that Pins and Gαi act together in controlling apico-basal spindle orientation (referred to as Pins/Gαi signaling). Spindle orientation was also analyzed in Gγ1 mutant pI cells and it was found that the spindle is similarly tilted along the apico-basal axis. This could be a consequence of the absence of Gαi and Pins asymmetric localization in the Gγ1 mutant. However, two additional phenotypes were observed in the Gγ1 mutant: a mild effect on the antero-posterior orientation of the mitotic spindle and oscillatory movements of the mitotic spindle throughout division. This latter phenotype is reminiscent of that observed in C. elegans Gβ knockdown, which was interpreted to be a result of Gα hyperactivation (David, 2005).

Analysis of spindle orientation in epithelial cells revealed that division takes place in the plane of the epithelium in both wild-type and pins mutant cells. This demonstrates, first, that the requirement for Pins/Gαi to maintain the planar orientation of the spindle is specific to pI cells and, second, that a pI-specific activity tilts the spindle in the absence of Pins/Gαi signalling. Fz signalling was an obvious candidate for this pI-specific activity for two reasons. First, Fz signalling is still active in the pins and Gαi mutants as the spindle was correctly oriented along the antero-posterior axis in these mutants. Second, Fz accumulates at the posterior apical cortex of pI cells and this accumulation of Fz is maintained in Gαi pI cells. It is therefore envisaged that, although orienting the spindle along the antero-posterior axis, Fz signalling may also be responsible for tilting the spindle along the apico-basal axis in the absence of Pins/Gαi signalling. fz,pins double mutants were also analyzed to test this hypothesis. Strikingly, in the absence of both Fz and Pins, the spindle was parallel to the plane of the epithelium. Therefore, in the absence of Pins/Gαi signalling, the activity tilting the spindle along the apico-basal axis is Fz-dependent. Intriguingly, in fz,pins pI cells, the spindle was even less tilted than in wild-type cells, indicating that Fz may also tilt the spindle in wild-type cells along their apico-basal axis. To test this, spindle orientation was analyzed in the fz mutant. In the absence of Fz, division takes place within the plane of the epithelium, the spindle being less tilted than in wild-type cells. Together, these results demonstrate that in pI cells, a Fz-dependent activity tends to tilt the spindle along the apico-basal axis. This activity is counterbalanced by a Ric-8a/Pins/Gαi-dependent one that maintains the spindle in the plane of the epithelium. Orientation of the spindle in wild-type cells arises from this balance. Finally, the analysis of spindle orientation in baz mutant pI cells revealed that Fz exerts its activity on the spindle independently of Baz, and hence probably independently of the Par complex. The tight control of the spindle apico-basal orientation probably regulates the morphogenesis of the pIIb cell and of the differentiated sensory organs (David, 2005).

In C. elegans, ric-8 regulates spindle positioning in anaphase, downstream of the par genes and upstream or downstream of the GPR-Gαi complex, which is the homologue of the Pins-Gαi complex. The data demonstrate that, in the dividing pI cell, Ric-8a is required for asymmetric localization of Pins, Baz and Numb and for mitotic-spindle positioning. It is proposed that these activities of Ric-8a depend on an unexpected function of Ric-8a: localizing Gαi and Gβ13F at the plasma membrane. This study of ric-8a also revealed that, in the pI cell, ric-8a, pins, Gαi and Gγ1 are all required for orientation of the spindle within the plane of the epithelium. The milder apico-basal phenotype that was observed in ric-8a pI cells could be accounted for by some persistence of the Ric-8a protein in somatic clones. Alternatively, an intriguing possibility is that ric-8a may also affect Gαo activity, which has recently been proposed to act downstream of Fz signalling. ric-8a loss of function would thereby affect both the Fz- and Gαi-dependent activities exerted on the spindle, resulting in a milder apico-basal tilt (David, 2005).

Importantly, developmental processes ranging from gastrulation, neural-tube closure, neurogenesis and retina formation to asymmetric segregation of cell-fate determinants require that spindle orientation is controlled in two directions: along the polarity axis of the tissue (antero-posterior, animal-vegetal, central-peripheral, etc) and parallel to the plane of the epithelium. This study has shown that, in dividing pI cells, these two orientations are controlled by different and opposing activities. A Fz-dependent activity orients the spindle along the antero-posterior axis but tends to tilt it along the apico-basal axis, and a Gαi-dependent activity maintains the spindle parallel to the plane of the epithelium. The Fz- and Gαi-dependent activities are likely to act through forces pulling on astral microtubules. Fz and heterotrimeric G signalling are implicated in mitotic-spindle positioning during both symmetric and asymmetric cell division. The elucidation of the molecular mechanisms underlying these forces in the pI cell might therefore generally contribute to understanding of the mechanisms that control mitotic-spindle positioning (David, 2005).

Lgl, Pins and aPKC regulate neuroblast self-renewal versus differentiation

How a cell chooses to proliferate or to differentiate is an important issue in stem cell and cancer biology. Drosophila neuroblasts undergo self-renewal with every cell division, producing another neuroblast and a differentiating daughter cell, but the mechanisms controlling the self-renewal/differentiation decision are poorly understood. This study tested whether cell polarity genes, known to regulate embryonic neuroblast asymmetric cell division, also regulate neuroblast self-renewal. Clonal analysis in larval brains shows that pins mutant neuroblasts rapidly fail to self-renew, whereas lethal giant larvae (lgl) mutant neuroblasts generate multiple neuroblasts. Notably, lgl pins double mutant neuroblasts all divide symmetrically to self-renew, filling the brain with neuroblasts at the expense of neurons. The lgl pins neuroblasts show ectopic cortical localization of atypical protein kinase C (aPKC), and a decrease in aPKC expression reduces neuroblast numbers, suggesting that aPKC promotes neuroblast self-renewal. In support of this hypothesis, neuroblast-specific overexpression of membrane-targeted aPKC, but not a kinase-dead version, induces ectopic neuroblast self-renewal. It is concluded that cortical aPKC kinase activity is a potent inducer of neuroblast self-renewal (Lee, 2005).

Drosophila neuroblasts are an excellent model system in which to investigate the molecular control of self-renewal versus differentiation. Larval neuroblasts repeatedly divide asymmetrically to self-renew a neuroblast and to produce a smaller daughter cell, called a ganglion mother cell (GMC), that typically makes two postmitotic neurons; this process enables a single neuroblast to generate many hundreds of neurons. Self-renewal is defined as the capacity of a neuroblast to maintain all attributes of its cell type (molecular markers and proliferation potential). In this regard, a neuroblast is very similar to a germline stem cell: both maintain their stem cell identity while generating differentiating progeny. About 100 neuroblasts per brain lobe are formed during embryogenesis, where they proliferate briefly before entering quiescence. Brain neuroblasts re-enter the cell cycle between 10 and 72 h after larval hatching (ALH), and then a stable population of ~100 mitotic, self-renewing neuroblasts is maintained. This invariant neuroblast number was used to screen for mutants altering self-renewal versus differentiation: mutants in which a neuroblast makes two neuroblast progeny (ectopic self-renewal) will have >100 neuroblasts, whereas mutants in which a neuroblast makes two GMC progeny (failure in self-renewal) will have <100 neuroblasts. This assay was used to test known cell polarity mutants for a role in neuroblast self-renewal (Lee, 2005).

Two classes of cell polarity regulators were assayed for an effect on larval neuroblast self-renewal. lgl and discs large (dlg) zygotic mutants were examined, because these mutants form brain tumours and promote basal protein targeting in embryonic and larval neuroblasts. Lgl and Dlg have several protein interaction motifs and are localized around the neuroblast cortex. In addition, pins and Galphai zygotic mutants were examined; these genes regulate cell polarity in embryonic neuroblasts, but have not been well characterized in larval neuroblasts. Pins and Galphai are colocalized with Inscuteable and the evolutionarily conserved Bazooka-Par6- aPKC proteins at the apical cortex of mitotic neuroblasts, and all of these proteins are partitioned into the neuroblast during cytokinesis (Lee, 2005).

In wild-type larvae, a population of ~100 neuroblasts could be identified by the markers Worniu, Deadpan and Miranda, and by labelling with a pulse of 5-bromodeoxyuridine (BrdU); by contrast, the thousands of differentiating GMCs and neurons rapidly downregulate neuroblast markers and express nuclear Prospero and/or Elav. A clear increase in neuroblast number is observed in lgl and dlg mutants; there are supernumerary neuroblasts at all stages examined; all extra neuroblasts expressed Deadpan and Miranda and are proliferative on the basis of their ability to incorporate BrdU. Galphai zygotic mutants have a complex phenotype that will be described in a later publication; however, pins zygotic mutants show a marked decrease in neuroblast number. Notably, this phenotype is not due to a subset of neuroblasts remaining quiescent, because neuroblast numbers peak and then decline over time, and it is not due to neuroblast cell death. The relatively late onset of the pins phenotype is probably due to the gradual depletion of maternal pins gene product in these larvae (Lee, 2005).

To determine whether the pins and lgl larval brain phenotypes are due to defects in neuroblast self-renewal, positively marked genetic clones were induced in single neuroblasts to trace their progeny. Clone induction parameters were adjusted to ensure that each clone was derived from a single neuroblast (1.2 clones per lobe). In wild-type brains, neuroblast clones always contained a single Worniu+ Miranda+ nuclear-Prospero- neuroblast and numerous smaller Worniu- Miranda- nuclear-Prospero+ progeny, confirming that wild-type neuroblasts always divide to self-renew and to generate a smaller differentiating GMC. By contrast, lgl mutant brains had an average of 2.3 neuroblasts per clone, with up to six neuroblasts per clone, showing that lgl mutant neuroblasts can divide symmetrically to yield two neuroblasts. The opposite phenotype was seen in pins mutant brains: 72.8% of the clones had no neuroblast and the remainder had a single neuroblast. The neuroblasts did not die in the pins mutants as evidenced by the following: the cell death marker caspase-3 was not upregulated, neuroblast-specific expression of the p35 cell death inhibitor did not rescue the missing neuroblasts, and one clone was observed in which the largest cell coexpressed neuroblast and GMC markers, consistent with an intermediate stage in neuroblast-to-GMC differentiation. It is concluded that wild-type neuroblasts exclusively generate neuroblast/GMC siblings; lgl mutant neuroblasts occasionally undergo ectopic self-renewal to generate neuroblast/neuroblast siblings; and pins mutant neuroblasts occasionally fail to self-renew, resulting in GMC/GMC siblings and termination of the lineage (Lee, 2005).

Next to be examined was whether lgl pins double mutants had fewer neuroblasts (like pins mutants) or extra neuroblasts (like lgl mutants). Unexpectedly, a phenotype was detected in which the larval brain was full of cells expressing the neuroblast markers Worniu, Miranda and Deadpan and lacking expression of the neuronal marker Elav. Additional markers that distinguish neuroblasts and GMCs were examined to determine whether these cells were neuroblasts or a hybrid neuroblast/GMC identity. Both wild-type neuroblasts and lgl pins cells actively transcribed the worniu, deadpan, miranda and prospero genes, maintained proliferation, did not express the Elav neuronal differentiation marker, and did not extend axons. The only potential GMC attribute found in lgl pins neuroblasts was nuclear Prospero protein but, because wild-type neuroblasts and GMCs both contain Prospero protein, which can accumulate in neuroblast nuclei if not properly localized, this protein is not a definitive marker for the GMC cell type. Thus, lgl pins brains contain large numbers of ectopic, proliferating, self-renewing neuroblasts. Combining these lgl, pins and lgl pins mutant data leads to the conclusion that Lgl inhibits self-renewal, whereas Pins has dual functions in promoting and inhibiting self-renewal (Lee, 2005).

To understand how Lgl and Pins regulate neuroblast self-renewal at the cellular level, cortical polarity marker localization was examined in mitotic larval neuroblasts. In wild-type larval neuroblasts, the Par complex (Bazooka-Par6-aPKC) and Pins-Galphai proteins forms an apical crescent at metaphase and are partitioned into the self-renewing neuroblast at telophase, whereas the Miranda and Prospero proteins form a basal crescent at metaphase and are partitioned into the differentiating GMC at telophase. In lgl pins double mutants, in which all neuroblasts divide symmetrically to generate self-renewing neuroblast/neuroblast siblings, most mitotic neuroblasts show uniform cortical aPKC, cytoplasmic Bazooka and Par6, and uniform cortical Miranda at metaphase and telophase. Thus, only aPKC maintained its correct subcellular localization and correlated with neuroblast self-renewal (Lee, 2005).

aPKC localization was examined in lgl and pins single mutants, in which symmetric divisions occurred at lower frequency. In lgl mutants, aPKC showed weak ectopic cortical localization in about half the metaphase neuroblasts, whereas Miranda was delocalized from the cortex; by telophase, however, both proteins appeared to be localized normally. Ectopic cortical aPKC was also observed in dlg mutant larval neuroblasts. A role for Lgl in restricting aPKC to the apical cortex of neuroblasts has not been reported but would be consistent with the observation that basolateral Lgl restricts aPKC to the apical surface of Drosophila and vertebrate epithelia and Xenopus blastomeres. In pins mutants, aPKC and cytoplasmic Miranda showed weak uniform cortical distribution in metaphase neuroblasts, but were properly localized in most telophase neuroblasts Thus, both Lgl and Pins are required to restrict aPKC to the apical cortex in metaphase neuroblasts (Lee, 2005).

Whether aPKC is required for neuroblast self-renewal was examined. aPKC mutant clones in larval mushroom body neuroblasts showed premature lineage termination, consistent with aPKC being required for neuroblast self-renewal. In addition, aPKC null mutants died as second instar larvae with reduced neuroblast numbers. Because this was a relatively mild phenotype and there was no detectable aPKC protein at this stage, it is likely that there are additional pathways for stimulating neuroblast self-renewal. Next, whether aPKC is required for ectopic neuroblast self-renewal in the lgl mutants was tested. lgl aPKC double mutants had normal numbers of neuroblasts, showing that aPKC is required for the ectopic neuroblast self-renewal seen in lgl mutants. aPKC mutants also suppressed ectopic neuroblast self-renewal in several independently isolated lgl mutations, further supporting a role for aPKC in self-renewal. In addition, it was found that aPKC is fully epistatic to lgl in regulating Miranda localization. Thus, aPKC is required for the ectopic neuroblast self-renewal and Miranda delocalization phenotypes seen in lgl mutants (Lee, 2005).

These data are most consistent with a model in which Lgl negatively regulates aPKC, and aPKC directly promotes self-renewal. This model is based on the observations that Lgl restricts aPKC localization to the apical cortex of neuroblasts and that a reduction in aPKC blocks the lgl self-renewal phenotype. To test this model, worniu-Gal4 line was used to drive neuroblast-specific expression of constitutively active aPKC or Lgl proteins, and an increase or decrease in neuroblast numbers was assayed. Neuroblast-specific expression of aPKC targeted to the plasma membrane with a CAAX prenylation motif (UAS-aPKCCAAXWT) resulted in ectopic cortical aPKC localization, loss of cortical Miranda, and a large increase in the number of neuroblasts. These effects were not observed after overexpression of wild-type aPKC or a membrane-targeted kinase-dead aPKC (UAS-aPKCCAAXKD). Expression of a constitutively active aPKC (UAS-aPKCDeltaN) that was predominantly cytoplasmic gave only a slight increase in neuroblast number, showing that cortical localization of aPKC is essential to generate ectopic neuroblasts. By contrast, neuroblast-specific expression of a constitutively active Lgl protein (Lgl3A) resulted in the expected uniform cortical localization of Miranda, but no change in neuroblast numbers. Combined overexpression of both Lgl3A and aPKCCAAXWT, however, resulted in strong suppression of the aPKCCAAXWT ectopic neuroblast phenotype, even though Lgl3A alone had no effect on neuroblast numbers, consistent with Lgl inhibiting aPKC function either directly or through its downstream effectors. Thus, neuroblast-specific overexpression of aPKC can expand the neuroblast population (most probably by promoting symmetric neuroblast/neuroblast cell divisions) without eliminating the ability of these neuroblasts to undergo asymmetric neuroblast/GMC divisions to generate differentiating progeny. It is concluded that aPKC is sufficient to promote neuroblast self-renewal, Lgl can inhibit aPKC function, and membrane-targeting and kinase activity are essential for aPKC function (Lee, 2005).

This study has established Drosophila larval neuroblasts as a model system for studying self-renewal versus differentiation. A simple model is proposed in which Pins anchors aPKC apically and Lgl inhibits aPKC localization basally, thereby restricting aPKC to the apical cortex where it promotes neuroblast self-renewal. In addition, aPKC can phosphorylate and directly inhibit Lgl function, which together with the current data provides evidence for mutual inhibition between Lgl and aPKC in neuroblasts, similar to the mutual inhibition seen between these two proteins in epithelia. Mutual inhibition between aPKC and Lgl would result in stabilization of apical aPKC localization and more reliable partitioning of aPKC into the neuroblast during mitosis. In pins mutants, aPKC is delocalized and nonfunctional owing to Lgl activity, thereby reducing self-renewal; in lgl mutants, aPKC shows weak ectopic cortical localization that increases self-renewal, and in lgl pins double mutants, aPKC is both delocalized and fully active: thus all neuroblasts undergo symmetric self-renewal. Although the targets of aPKC involved in self-renewal are unknown, aPKC may directly phosphorylate and inactivate GMC determinants, and/or phosphorylate and activate neuroblast-specific proteins. Notably, lgl1 mutant mice have neural progenitor hypertrophy and knockdown of a pins mammalian homologue (AGS3) leads to depletion of neural progenitors: phenotypes that are very similar to those described in this study. In the future, it will be important to determine the role of aPKC in mammalian neural progenitor self-renewal and to identify the aPKC-regulated phosphoproteins that regulate neuroblast self-renewal in Drosophila (Lee, 2005).

The PDZ protein Canoe regulates the asymmetric division of Drosophila neuroblasts and muscle progenitors; Cno functions downstream of Insc and Pins-Gαi and upstream of Mud during asymmetric NB division

Asymmetric cell division is a conserved mechanism to generate cellular diversity during animal development and a key process in cancer and stem cell biology. Despite the increasing number of proteins characterized, the complex network of proteins interactions established during asymmetric cell division is still poorly understood. This suggests that additional components must be contributing to orchestrate all the events underlying this tightly modulated process. The PDZ protein Canoe (Cno) and its mammalian counterparts AF-6 and Afadin are critical to regulate intracellular signaling and to organize cell junctions throughout development. Cno functions as a new effector of the apical proteins Inscuteable (Insc)-Partner of Inscuteable (Pins)-Gαi during the asymmetric division of Drosophila neuroblasts (NBs). Cno localizes apically in metaphase NBs and coimmnunoprecipitates with Pins in vivo. Furthermore, Cno functionally interacts with the apical proteins Insc, Gαi, and Mushroom body defect (Mud) to generate correct neuronal lineages. Failures in muscle and heart lineages are also detected in cno mutant embryos. These results strongly support a new function for Cno regulating key processes during asymmetric NB division: the localization of cell-fate determinants, the orientation of the mitotic spindle, and the generation of unequal-sized daughter cells (Speicher, 2008).

NBs delaminate from the neuroectoderm inheriting the apicobasal polarity of the neuroectodermal cells, in which the PDZ proteins Bazooka (Baz)/Par-3 and DmPar-6 and the kinase DaPKC localize apicolaterally. After delamination, NBs maintain the apical localization of Baz/DmPar-6/DaPKC. The cytoplasmic PDZ protein Cno localizes at the adherens junctions of some epithelial cells, and it was asked whether Cno was also present in the neuroectoderm and in the delaminated NBs. Double immunofluorescences with antibodies against Cno and Baz showed that these proteins colocalize both apicolaterally at the adherens junctions of neuroepithelial cells and apically in the delaminated metaphase NBs (mNBs). At later phases of the NB division, Cno was no longer detected (Speicher, 2008).

Apical proteins, such as Baz/Par-3, are critically involved in regulating cell-fate determinants localization and spindle orientation at metaphase. Given that Cno was detected in an apical crescent in mNBs, it was asked whether Cno was also required for modulating those events. In control embryos, the cell-fate determinant Numb was basally located in 95.4% of mNBs. In cno2 zygotic mutants, Numb was uniform or undetectable or was present in nonbasal crescents in 47.9% of the mNBs analyzed. cno2 has been defined as the strongest allele of cno, although the particular lesion associated is unknown. However, cno2 is probably a null allele because cno2 over the Df(3R)6-7 (covering the cno gene) showed a similar percent of Numb localization failures. Additionally, cno3, another strong allele of cno considered as a null displayed defects in Numb localization in comparable cases. The basal distribution of the scaffolding protein Miranda (Mira) was also altered in 16.9% of mNBs of cno2 mutants. Indeed, the localization of two Mira cargo proteins, the cell-fate determinants Prospero (Pros) and Brain Tumor (Brat), was affected in mNBs. The variable penetrance of the cno2 mutant phenotype observed for the different proteins analyzed may reflect, at least in part, the different sensitivity of the antibodies used (Speicher, 2008).

Intriguingly, the orientation of the mitotic spindle in mNBs of cno2 mutants was randomized in 18.3% of the cases. In control embryos, the spindle is tightly aligned with the center of Numb crescents in mNBs. In cno2 mutants, the spindle was uncoupled with the Numb crescent in 7.7% of the mNBs that showed these crescents (either basal or at other incorrect localizations). The maternal contribution of cno might reduce the penetrance of these phenotypes (Speicher, 2008).

The overexpression of Cno also caused Numb localization failures (45.8%) and aberrant spindle orientations (39%) in mNBs. Hence, the results showed that Cno regulates essential processes during asymmetric NB division: the basal localization of cell-fate determinants and the proper orientation of the mitotic spindle (Speicher, 2008).

Another characteristic feature of asymmetric NB division is the different cell size of the progeny. Hence, whether Cno was also regulating this process was analyzed. Control telophase NBs (tNBs) showed unequal-sized daughter cells in 100% of the cases analyzed. In cno2 mutants, equal-size divisions were observed in 21.3% of tNBs. Two redundant pathways, Baz/DaPKC/Insc and Pins-Gαi, regulate cell size and mitotic-spindle asymmetry at the NB apical pole . Only when both pathways are compromised is the different size of the daughter cells affected. The data suggested that Cno functions downstream of Gαi. Thus, Cno might belong to the Pins-Gαi pathway. Indeed, when both insc and cno were eliminated, 85.2% of tNBs showed equal-sized daughter cells, a much more penetrant phenotype than those displayed by each single mutant. Moreover, ΔGαi, cno2 double mutants showed a much lower percentage of equal-sized divisions (30.4%) than the inscP49; cno2 double mutants. Hence, these results strongly suggest that Cno participates within the Pins-Gαi pathway to regulate NB progeny size (Speicher, 2008).

Given the defects observed in cno2 mutant embryos during NBs division, it was asked whether neuronal lineages were altered in cno2 mutants. The lineage of the ganglion mother cell (GMC) 4-2a has been extensively studied. This GMC expresses the transcription factor Even-Skipped (Eve) and divides asymmetrically to give rise to two different neurons called RP2 and RP2 sibling. Both maintain the expression of Eve initially; however, at later stages of embryogenesis, only the RP2 neuron keeps expressing Eve . In control embryos, 0.9% of the segments analyzed showed defects in the number of RP2s. In cno2 mutants, two or no RP2s were detected per hemisegment in 5.7% of the segments analyzed. Such a result suggested failures in the GMC 4-2a asymmetric division. This phenotype was also observed in cnomis1 hypomorph mutants (4.6%) as well as in mutants for genes that are critical during asymmetric cell division. For example, homozygotes for DaPKCk06403, inscP49, ΔGαi, and mud4 (zygotic null mutant embryos) showed defects in the GMC 4-2a lineage in 6.4%, 13.8%, 2.5%, and 8.3% of the segments analyzed, respectively. Hence, it was next investigated whether Cno was interacting with these proteins to properly generate the GMC 4-2a neuronal lineage. Double heterozygotes DaPKCk06403/+; cno2/+ showed defective RP2 number in 0.8% of segments. This result is consistent with a lack of functional interactions between DaPKC and Cno. However, double heterozygotes inscP49/+; cno2/+ and ΔGαi, +/+, cno2 showed an altered RP2 lineage in 14.4% and 7.6% of the segments analyzed. In addition to the analysis of double heterozygotes, it was found that the cnomis1 phenotype was significantly enhanced in a mud4 zygotic null mutant background. Altogether, these results indicated that Cno functionally interacts with the apical proteins Insc, Gαi, and Mud during the asymmetric cell divisions that generate specific neuronal lineages in the CNS (Speicher, 2008).

Since Cno functionally interacts with Insc, Gαi, and Mud, the epistatic relationships between them were analyzed. To investigate whether Cno was acting upstream of the apical proteins, the localization of Baz, Insc, and Gαi was examined in cno2 mutant embryos. The distribution of all these proteins was normal in cno2 mutants. This result suggested that Cno acts either downstream or in parallel to Baz, Insc, and Gαi. To clarify this point, the distribution of Cno was analyzed in loss- and gain-of-function (lof and gof) mutants for several apical proteins. In inscP49 lof mutants, Cno was untraceable or showed a wrong orientation in 78.8% of the mNBs analyzed. Insc overexpression also caused failures in Cno localization (76%); Cno was either undetectable (13/21) or present in not-apical crescents (3/21). Likewise, in Gβ13F maternal and zygotic null mutant embryos, in which Gαi is lost, Cno was mislocalized or undetectable in 94% of the mNBs. Moreover, the overexpression of Gαi caused a striking mislocalization of Cno in 100% of the mNBs analyzed. The NuMA-related protein Mud binds the apical protein Pins and functions downstream of Pins-Gαi to regulate spindle orientation. In mud mutant NBs, the spindle fails to tightly align with the basal crescent, and this failure is also shown by cno2 mutant NBs. Additionally, Cno and Mud interacted genetically. Hence, it was asked whether Cno functions along with Mud to regulate spindle orientation. In control embryos, Mud localized at the apical cortex of mNBs (97%) and at the two centrosomal regions (100%). In cno2 lof mutants, Mud failed to accumulate apically in 49% of mNBs, and 15% of NBs showed Mud localization in one or none of the two centrosomes. cno gof also caused failures in Mud localization (38%). Altogether, these results strongly support a function of Cno downstream of Insc and Pins-Gαi and upstream of Mud during asymmetric NB division (Speicher, 2008).

Given the functional relationships found between Cno and apical proteins during asymmetric NB division, it was asked whether Cno was physically interacting with some of these proteins. Coimmunoprecipitation experiments from Drosophila embryo extracts showed that Cno is forming a complex with Pins. Cno did not physically interact with DmPar6, Baz, DaPKC, or other apical proteins tested such as Insc, Gαi, and Mud (Speicher, 2008).

Pins also forms a complex in the delaminated metaphase NBs with the tumor-suppressor protein Discs Large (Dlg) and the kinesin Khc-73, an astral microtubule-binding protein. First, at prophase, the DmPar6/Insc pathway is required to polarize Pins/Gαi at the apical pole of the NB. Then, at metaphase, the Pins/Gαi/Dlg/Khc-73 complex forms, and it is key for tightly coupling cortical polarity with spindle orientation. Hence, it was asked whether Cno was also forming part of this complex. Experiments showed that neither Dlg nor Khc-73 coimmunoprecipitate Cno in embryo extracts. This result indicated that Cno is not forming part of the Dlg/Khc-73 complex (Speicher, 2008).

Altogether, a working network of protein interactions is proposed. Analysis of epistatic relationships between apical proteins and Cno showed that Cno is acting downstream of Insc-Pins-Gαi and upstream of Mud. Indeed, genetic analysis suggests that, at least for the control of daughter cells size asymmetry, Cno functions within the Pins-Gαi pathway, in parallel to the DaPKC-Baz-Insc pathway. Accordingly, Cno was found to form a complex with Pins in vivo. Cno did not coimmunoprecipitate with Gαi, though. One possibility is that Cno and Gαi are mutually exclusive in the complex that each of them forms with Pins. Additionally, transient or labile interactions between Cno and Gαi may occur that were not possible to detect. Another Pins interacting partner, the microtubule-binding protein Mud contributes to coordinate spindle orientation with cortical polarity. Given the functional relationships that were found between Cno and Mud, Cno could act in a complex with Pins to modulate Mud localization and, consequently, spindle orientation (Speicher, 2008).

Finally, it was asked whether the function of Cno during asymmetric cell division was conserved in different tissues. Since the NBs of the CNS, the Drosophila somatic muscle and heart progenitors divide asymmetrically to give rise to two different founder cells. Cno is present in the somatic mesoderm and is required for muscle and heart progenitor specification. Hence, it was aked whether Cno was also functioning during the asymmetric division of muscle and heart progenitors. For this analysis, focus was placed on two dorsal progenitors called P2 and P15 that express the transcription factor Eve and whose lineages have been characterized in detail. In this study, it was found that the transcription factor Seven-up (Svp), a characteristic marker of a subset of cardial cells, was expressed in a dorsal founder cell of unknown identity until now, which is here named founder of Svp cardial cells (FSvpCs). With all these markers, specific for individual derivatives, whether dorsal muscle and cardial lineages were altered in cno2 mutants was analyzed. It was found that at late stages (stage 14), 3.1% of hemisegments (n = 96) showed simultaneously either loss of EPCs and gain of DO2 muscle or gain of EPCs and loss of the DO2 muscle (P2 lineage). In control embryos, this phenotype was not observed in any of the hemisegments analyzed. Indeed, Numb localization, which was basal in 100% of the metaphase P2s analyzed in control embryos, was altered in 93% of metaphase P2s in cno2 mutants. Hemisegments showing duplication of DA1 muscle and loss of SvpCs or DA1 muscle loss and gain of SvpCs (P15 lineage) were also detected in cno2 mutants. Hence, Cno was required for the asymmetric division of progenitor cells both in the CNS and in the mesoderm (Speicher, 2008).

In conclusion, the discovery of new modulators of asymmetric cell division, as described in this study, for the PDZ protein Cno, is key to complete understanding of this intricate process. Especially challenging in the future will be unraveling the complete network of connections between all the players required for an accurate asymmetric cell division (Speicher, 2008).

Muscle length and myonuclear position are independently regulated by distinct Dynein pathways

Various muscle diseases present with aberrant muscle cell morphologies characterized by smaller myofibers with mispositioned nuclei. The mechanisms that normally control these processes, whether they are linked, and their contribution to muscle weakness in disease, are not known. This study examined the role of Dynein and Dynein-interacting proteins during Drosophila muscle development and found that several factors, including Dynein heavy chain, Dynein light chain and Partner of inscuteable, contribute to the regulation of both muscle length and myonuclear positioning. However, Lis1 contributes only to Dynein-dependent muscle length determination, whereas CLIP-190 and Glued contribute only to Dynein-dependent myonuclear positioning. Mechanistically, microtubule density at muscle poles is decreased in CLIP-190 mutants, suggesting that microtubule-cortex interactions facilitate myonuclear positioning. In Lis1 mutants, Dynein hyperaccumulates at the muscle poles with a sharper localization pattern, suggesting that retrograde trafficking contributes to muscle length. Both Lis1 and CLIP-190 act downstream of Dynein accumulation at the cortex, suggesting that they specify Dynein function within a single location. Finally, defects in muscle length or myonuclear positioning correlate with impaired muscle function in vivo, suggesting that both processes are essential for muscle function (Folker, 2012).

This study has used the Drosophila musculature to investigate the mechanisms that control muscle size and intracellular organization. Muscle length is regulated independently of the number of fusion events and demonstrate that perturbations that affect embryonic muscle length correlate with decreased larval muscle size and poor muscle function. Additionally, it was found that intracellular organization, specifically myonuclear positioning, is essential for muscle function. Moreover, it was shown that the length and intracellular organization of the myofiber are mechanistically independent. Although a number of factors link both processes, this study identified factors that contribute solely to muscle length or myonuclear position and demonstrates that each fature can be independently manipulate (Folker, 2012).

Dynein regulates both muscle length and myonuclear positioning. Some Dynein-interacting proteins, such as Dlc90F and Pins, are necessary for both processes. That these factors regulate both muscle length and myonuclear positioning suggests that specific aspects of muscle growth and the positioning of myonuclei can indeed be coordinated. However, Lis1 affects muscle length specifically, whereas CLIP-190 and Glued specifically affect myonuclear position. Within the contexts of muscle length and myonuclear position CLIP-190 and Lis1 do not genetically interact, illustrating that, although linked, the two processes are mechanistically distinct (Folker, 2012).

Identification of CLIP-190 and Lis1 as regulators of Dynein is not novel. CLIP-190 and Lis1 are known to interact with Dynein both physically and functionally, and they usually cooperate toward a single goal. This study provides the first example of CLIP-190 and Lis1 serving completely independent, Dynein-dependent functions (Folker, 2012).

Mechanistically, the data suggest that Dynein function, with respect to the regulation of muscle length and myonuclear positioning, is specified by Lis1 and CLIP-190 downstream of its localization. That Dynein localization to the myofiber pole is important is illustrated by pins (raps193) mutants in which Dynein does not accumulate at the myofiber pole, and defects were observed in both muscle length and myonuclear positioning. This suggests that Pins recruits and stabilizes Dynein at the cortex as it does during mitosis. Lis1 also affects Dynein localization, but in Lis1 mutants Dynein is localized to the muscle pole and is tightly restricted to the pole compared with controls. This is interpreted to mean that Lis1 is necessary for retrograde trafficking of Dynein away from the myofiber pole. It is hypothesized that, in the absence of Dynein retrograde trafficking, cellular components accumulate at the extending muscle end, thus inhibiting the trafficking of factors necessary for further directed growth. Thus, when Dynein is incapable of moving cargo away from the myofiber pole, muscles are shorter than in control embryos. Indeed, a similar correlation between retrograde trafficking and directed growth was recently reported during mechanosensory bristle growth and axonal transport. Interestingly, it was recently reported that decreased retrograde transport of Dynein results in longer processes in both fibroblasts and neurons in culture (Folker, 2012).

This contradictory finding raises several interesting questions. Is there an opposing pathway in muscle or is another aspect of muscle size increased? For example, does the length of the muscle impact its volume? Complications due to muscle contraction in the live embryo make such analysis difficult, however. Likewise, working in vivo on a dynamically changing tissue located 30-150 microm below the surface of the developing embryo presents challenges for imaging the rapid cellular processes that underlie motor activity, microtubule organization, organelle positioning and cell size. Nevertheless, the link between different aspects of cell size and their relationships to each other and to motor activity are being explored (Folker, 2012).

It is not clear why LT muscle growth/length is more sensitive to Dynein activity than the VL muscles in the embryo. A simple explanation is that the maternally loaded Dynein persists longer in the VL muscles than in the LT muscles. Additionally there might be a physical explanation. Although a cluster of potential tendon cells for the VL muscles exist, they are clustered at the segment border. Therefore, the size of the hemisegments, and thus the distance from segment border to segment border, determines muscle length. Conversely, the cluster of potential tendon cells for the LT muscles might be more broadly dispersed and the location of the muscle pole at the time of tendon specification determines the length of the muscle. Under this hypothesis, inefficient extension of the LT muscles would result in the muscles being shorter during tendon specification/maturation and therefore shorter throughout embryonic development. Alternatively, differences in guidance/signaling systems could explain why the LT muscles, but not the VL muscles, are shorter when Dynein activity is compromised. Different signaling mechanisms are employed by these different muscle types. For example, Derailed (Drl) plays a crucial role in the ability of LT muscles to recognize their target. Perhaps, altered trafficking of Drl or another factor in the signaling pathway causes slight, but significant, changes in LT muscle length (Folker, 2012).

It is interesting that the LT muscles are smaller in Dhc64C, Dlc90F, pins and Lis1 mutant embryos, but that other muscles are unaffected, whereas in larvae all muscles appear to be smaller. During the larval stages, muscles remain stably attached to tendon cells and grow through insulin signaling/Foxo- and dMyc-dependent pathways. The observations are intrepreted to mean that Dynein and its regulatory proteins Dlc90F, Pins and Lis1 contribute to insulin receptor-mediated muscle growth. Indeed, it would not be surprising to find that Dynein-dependent cellular trafficking is essential to that signaling pathway (Folker, 2012).

CLIP-190 does not dramatically affect Dynein localization, but it does affect microtubule organization, which, in turn, affects nuclear positioning. CLIP-190 mutant embryos have fewer microtubules at the myofiber pole, suggesting that, similar to its functions in other systems, the role of CLIP-190 is to stabilize microtubule-cortex interactions, which Dynein then uses to move nuclei towards the myofiber pole. The specification of Dynein function, downstream of its localization, is novel. Although phospho-regulation has been shown to alter Dynein function during mitosis and the possibility for competitive regulation of Dynein has been suggested, this is the first example in which Dynein at a single location has its activity modified through interactions with unique binding partners. The ability to specify Dynein function without dramatically altering its localization is likely to be an important factor during development when temporal constraints are high (Folker, 2012).

With regards to physiology, the small, but significant, changes in myonuclear positioning and muscle size seen in the embryo continue throughout larval development and are associated with impaired muscle function. Additionally, that the same defects and impairments are found in larvae that were depleted of Dynein, Lis1 or CLIP-190 specifically in the muscle shows that the effects are, at least in part, muscle specific (Folker, 2012).

Many muscle myopathies are characterized by smaller myofibers and mispositioned nuclei. However, it is unclear whether these pathologies are linked and which of these defects are paramount in causing the muscle weakness associated with these myopathies. This study has shown that in Drosophila these two processes are linked via the requirement for Dynein activity at the muscle pole. It was further shown that these two processes are mechanistically distinct, yet that both are necessary for muscle function. Together, these data suggest that therapeutics aimed at improving the functional capacity of diseased muscles must counteract effects on both muscle size and myonuclear positioning. This highlights Drosophila as an ideal model system with which to identify the genes and mechanisms required for distinct aspects of muscle morphogenesis and to shed light on key features of muscle disease (Folker, 2012).

Synergism between altered cortical polarity and the PI3K/TOR pathway in the suppression of tumour growth

Loss of function of pins (partner of inscuteable) partially disrupts neuroblast (NB) polarity and asymmetric division, results in fewer and smaller NBs and inhibits Drosophila larval brain growth. Food deprivation also inhibits growth. However, this study found that the combination of loss of function of pins and dietary restriction results in loss of NB asymmetry, overproliferation of Miranda-expressing cells, brain overgrowth and increased frequency of tumour growth on allograft transplantation. The same effects are observed in well-fed pins larvae that are mutant for pi3k (phosphatidylinositol 3-kinase) or exposed to the TOR inhibitor rapamycin. Thus, pathways that are sensitive to food deprivation and dependent on PI3K and TOR are essential to suppress tumour growth in Drosophila larval brains with compromised pins function. These results highlight an unexpected crosstalk whereby the normally growth-promoting, nutrient-sensing PI3K/TOR pathway suppresses tumour formation in neural stem cells with compromised cell polarity (Rossi, 2012).

PI3K and TOR activity are necessary for sustained growth and proliferation, and to resume cycling activity after developmentally programmed quiescence. Consistently, there is abundant experimental evidence showing that in most tumour types growth is coupled to activated PI3K, and not to its loss. Evidence showing that low-calorie intake is a protective condition against malignant transformation is also abundant. However, the current results indicate that in Drosophila PI3K and TOR activity and a normal food supply have a function in preventing overgrowth in neural stem cells with compromised cortical polarity. Whether this conclusion applies to vertebrates remains to be ascertained. Interestingly, some PI3Kγ−/− mouse strains have a high incidence of invasive colorectal tumours, which is not due to PI3Kγ loss alone, but is suspected to result from the combined effect of PI3Kγ loss and other unknown factors. Taking into account the fact that the pathways involved in the control of cell polarity and in the response to changing nutrient conditions are largely conserved across species, it is proposed that similar synergistic interactions might take place in vertebrates (Rossi, 2012).

Centrobin controls mother-daughter centriole asymmetry in Drosophila neuroblasts

During interphase in Drosophila neuroblasts, the Centrobin (Cnb)-positive daughter centriole retains pericentriolar material (PCM) and organizes an aster that is a key determinant of the orientation of cell division. This study shows that daughter centrioles depleted of CNB cannot fulfill this function whereas mother centrioles that carry ectopic CNB can. CNB co-precipitates with a set of centrosomal proteins that include gamma-Tub, Ana2, Cnn, Sas-4, Asl, DGRIP71, Polo and Sas-6. Following chemical inhibition of Polo or removal of three Polo phosphorylation sites present in Cnb, the interphase microtubule aster is lost. These results demonstrate that centriolar Cnb localization is both necessary and sufficient to enable centrioles to retain PCM and organize the interphase aster in Drosophila neuroblasts. They also reveal an interphase function for Polo in this process that seems to have co-opted part of the protein network involved in mitotic centrosome maturation (Januschke, 2013).

Since first described in germline stem cells, unequal mother-daughter centriole behaviour has become a major issue in stem cell self-renewing asymmetric division that has now been reported to occur in mice and in other cell types in Drosophila melanogaster including neuroblasts. Drosophila neuroblasts are neural precursors that generate the flys central nervous system through repeated rounds of self-renewing asymmetric divisions. Neuroblast asymmetric division is driven by the polarized localization of protein complexes at the apical and basal cortex and by the orientation of the mitotic spindle along the apico-basal axis. As a result, the apical and basal sides of the cortex are cleaved apart into the renewed neuroblast and the smaller differentiating ganglion mother cell (GMC), respectively. Dysfunction of several of the molecules involved in these processes results in tumour growth (Januschke, 2013).

Neuroblast cortical polarity and mitotic spindle alignment are tightly linked to the centrosome cycle of these cells in which centriole splitting is a very early interphase event and mother and daughter centrosomes exhibit significant differences in structure, function and fate. In Drosophila neuroblasts, the mother centriole, which has no significant microtubule organizing activity and migrates extensively during interphase, is inherited by the GMC after mitosis. In contrast, the daughter centriole, which retains high microtubule organizing activity and remains rather stable near the apical cell cortex throughout interphase, is retained by the neuroblast at mitosis. Thus, unlike most Drosophila cells where centrosomes are rather feeble microtubule organizing centres (MTOCs) during interphase, Drosophila neuroblasts posses a prominent interphase microtubule aster that is organized by the cortex-bound daughter centriole. Drosophila centrioles do not seem to have age-dependent ultrastructural attributes such as the satellites and appendages observed in vertebrates. However, Drosophila CNB, similarly to its homologues in mouse and human cells, binds only to the daughter centriole, revealing a molecular dimorphism that could contribute to daughter-centriole-specific behaviour in Drosophila larval neuroblasts (Januschke, 2013).

This study shows that in Drosophila neuroblasts, daughter centrioles without CNB behave like mother centrioles, and mother centrioles with ectopically localized CNB behave like daughter centrioles. Moreover, by co-immunoprecipitation and tandem mass-spectroscopy (MS/MS) this study shows that CNB co-precipitates with γ-Tub, Ana2, Cnn, Sas-4, Asl, Asl, DGRIP71, Polo and Sas-6. These centriolar and PCM proteins are part of a well-characterized network that drives PCM accumulation during mitotic centrosome maturation (see Gopalakrishnan, 2011). This study also shows that CNB phosphorylation by Polo is essential to maintain the interphase aster, hence revealing a requirement for Polo function during interphase (Januschke, 2013).

In neuroblasts expressing YFP-CNB-PACT (The PACT domain is a conserved centrosomal targeting motif in the coiled-coil proteins AKAP450 and pericentrin), both mother and daughter centrioles carry CNB and behave as daughter centrioles do in wild-type neuroblasts (see Centrobin function and graphical summary). Interestingly, ectopic activation of the interphase PCM retention program on both centrioles forces a canonical centrosome cycle in these cells whereby one centrosome-aster complex splits in two that segregate away from each other. In these cells, spindle assembly frequently occurs at an angle with respect to the apico-basal axis, but eventually rotates to align with it as in delaminating neuroblasts in the Drosophila embryo. In neuroblasts mutant for Cnb or expressing YPF-CNBT4A T9A S82A, the two centrioles behave as mother centrioles do in wild-type neuroblasts; they retain little or no PCM during interphase, but centrosomes mature at mitosis entry. In all three cases the spindle aligns with the apico-basal axis. CNB depletion, similarly to CNB ectopic localization on the two centrioles, randomizes age-dependent centriole fate. Apico-basal orientation can change from one cycle to the next in CNB-mutant cells, but not in neuroblasts that express CNB-PACT, strongly suggesting that the orientation of cortical polarity is critically dependent on daughter centriole behaviour and that whether this is provided by one or two centrioles seems to be irrelevant as long as a cortex-bound microtubule network is maintained during interphase (Januschke, 2013).

The results show that interphase PCM retention in neuroblasts requires Polo to act on CNB-decorated centrioles to mediate Cnn and PCM recruitment. Which centriole and where exactly on the centriole CNB is positioned do not seem to be essential because ectopic localization of CNB by the PACT domain works just as well. Indeed, the interaction of CNB with proteins such as Ana2 and Sas6 that are essential to build the centriolar cartwheel, and with Cnn and Polo that are key regulators of PCM assembly, strongly suggests that CNB might serve its function by providing a critical bridge between centrioles and PCM. It also suggests that the neuroblast and daughter-centriole-specific process of PCM retention by the CNB-containing centriole has co-opted at least some of the proteins that drive PCM enrichment on centrosomes during mitosis in many cell types. Nevertheless, the fact that mitotic centrosome maturation does not require CNB and is hardly affected by 20 nM of the Polo kinase inhibitor BI2536 strongly suggests that important differences apply between interphase PCM retention in Drosophila neuroblasts and the ubiquitous process of mitotic centrosome maturation (Januschke, 2013).

Interphase PCM retention by CNB-containing centrioles also requires Pins, but seems to require neither Mud, nor the Khc-73/Dlg pathway. Centriole migration to the cortex shortly after mitosis in neuroblasts mutant for pins suggests that Pins function is required at a later stage. Such a function is unlikely to require cortical Pins because CNB-decorated centrioles detached from the cortex by microtubule poisons efficiently retain PCM and, indeed, no evidence of Pins cortical localization during interphase has been reported in Drosophila larval neuroblasts. The seemingly normal mitotic centrosome maturation in >pins-mutant neuroblasts further substantiates that despite shared molecular factors, important differences apply between interphase PCM retention in Drosophila neuroblasts and mitotic centrosome maturation (Januschke, 2013).

Neither cell size asymmetry nor Mira cortical polarization seem perturbed by CNB depletion or by CNB ectopic localization in both centrioles. However, given the extraordinary complexity of cell fate determination, which is only partially understood, these observations cannot substantiate any solid conclusion on the effect that CNB dysfunction might have on cell fate determination and brain development. CNB is a ubiquitous protein that is one of the two centrioles in different cell types in Drosophila. This study also found that some CNB allelic combinations are lethal, which strongly suggests that CNB might have functions in other tissues. In vertebrates, Centrobin has been reported to bind to the tumour suppressor BRCA2, and some common genetic variants of human Centrobin have been associated with breast cancer susceptibility (Januschke, 2013 and references therein).

A recent study has revealed that in human neuroblastoma cell lines, the sister cell that retains the NuMA crescent, which is thought to have more self-renewal potential, frequently inherits the daughter centrosome. These results suggest the tantalizing possibility that the age-dependent centrosome segregation demonstrated in Drosophila neuroblasts might also occur in mammals and may have relevance in human disease (Januschke, 2013).


EVOLUTIONARY HOMOLOGS

GPR-1 and GPR-2, Partner of inscuteable homologs in C. elegans

The Caenorhabditis elegans coiled-coil protein LIN-5 mediates several processes in cell division that depend on spindle forces, including alignment and segregation of chromosomes and positioning of the spindle. Two closely related proteins, GPR-1 and GPR-2 (G protein regulator), associate with LIN-5 in vivo and in vitro and depend on LIN-5 for localization to the spindle and cell cortex. GPR-1/GPR-2 contain a GoLoco/GPR motif that mediates interaction with GDP-bound Galphai/o. A number of proteins in other metazoans contain GoLoco motifs in various numbers, and several GoLoco motif proteins, including mammalian AGS3 and Drosophila Pins, have been shown to interact with Galphai/o subunits of heterotrimeric G proteins. Inactivation of lin-5, gpr-1/gpr-2, or the Galphai/o genes goa-1 and gpa-16 all cause highly similar chromosome segregation and spindle positioning defects, indicating a positive role for the LIN-5 and GPR proteins in G protein signaling. The lin-5 and gpr-1/gpr-2 genes appear to act downstream of the par polarity genes in the one- and two-cell stages and downstream of the tyrosine kinase-related genes mes-1 and src-1 at the four-cell stage. Together, these results indicate that GPR-1/GPR-2 in association with LIN-5 activate G protein signaling to affect spindle force. Polarity determinants may regulate LIN-5/GPR/Galpha locally to create the asymmetric forces that drive spindle movement. Results in C. elegans and other species are consistent with a novel model for receptor-independent activation of Galphai/o signaling (Srinivasan, 2003).

In both Drosophila and C. elegans, a conserved PAR protein complex establishes cell polarity and spindle position but is not required for chromosome movements. This PAR-determined polarity directs spindle positioning possibly through activation of G protein signaling mediated by Pins/Inscuteable (Insc) in Drosophila neuroblasts, Pins/Discs large (Dlg) in Drosophila SOP cells, and GPR/LIN-5 in C. elegans embryos. Although Insc, Dlg, and LIN-5 all act to localize GoLoco proteins, their functions and localizations differ. LIN-5, GPR-1/GPR-2, and Galphai/o interactions appear to be required for cell division and chromosome segregation, whereas no such role has been shown for Drosophila Galphai or Pins. Consistent with a role in chromosome movements, GPR-1/GPR-2 proteins localize to the spindle apparatus, whereas Pins does not. This may indicate that an additional spindle-associated GoLoco protein exists, and/or possibly that in Drosophila multiple Galpha subunits act redundantly in mitosis, as in C. elegans. Consistent with the former hypothesis, a mammalian homolog of Pins, LGN, is required for spindle assembly and localizes to spindle asters (Srinivasan, 2003 and references therein).

G-protein signaling plays important roles in asymmetric cell division. In C. elegans embryos, homologs of receptor-independent G protein activators, GPR-1 and GPR-2 (GPR-1/2, homologs of Drosophila PINS), function together with Galpha (GOA-1 and GPA-16) to generate asymmetric spindle pole elongation during divisions in the P lineage. Although Galpha is uniformly localized at the cell cortex, the cortical localization of GPR-1/2 is asymmetric in dividing P cells. The asymmetry of GPR-1/2 localization depends on PAR-3 and its downstream intermediate LET-99 (a novel protein that acts downstream of PAR-3 and PAR-2 to determine spindle positioning, potentially through the asymmetric regulation of forces on the spindle). Furthermore, in addition to its involvement in spindle elongation, Galpha is required for the intrinsically programmed nuclear rotation event that orients the spindle in the one-cell. LET-99 functions antagonistically to the Galpha/GPR-1/2 signaling pathway, providing an explanation for how Galpha-dependent force is regulated asymmetrically by PAR polarity cues during both nuclear rotation and anaphase spindle elongation. In addition, Galpha and LET-99 are required for spindle orientation during the extrinsically polarized division of EMS cells. In this cell, both GPR-1/2 and LET-99 are asymmetrically localized in response to the MES-1/SRC-1 signaling pathway. Their localization patterns at the EMS/P2 cell boundary are complementary, suggesting that the signaling of LET-99 and Galpha/GPR-1/2 functions in opposite ways during this cell division as well. These results provide insight into how polarity cues are transmitted into specific spindle positions in both extrinsic and intrinsic pathways of asymmetric cell division (Tsou, 2003).

Forces must be polarized in response to PAR polarity cues in order to achieve proper spindle positioning. The localization of GPR-1/2 has led to the model that the enrichment of GPR-1/2 at the posterior provides higher pulling forces on the posterior spindle pole, thus mediating anaphase spindle positioning. This model does not address a role for GPR in nuclear rotation, however. Posterior enrichment of GPR-1/2 was seen in only some embryos during nuclear rotation. Such asymmetry at this time is actually predicted to be counter-productive, as it would potentially hold the nucleus at the posterior and prevent centration and rotation (Tsou, 2003).

It has been proposed that the asymmetric enrichment of LET-99 in a cortical band provides the asymmetric cue to polarize forces during both rotation and anaphase. Loss of LET-99 results in an absence of nuclear rotation and an absence of the normal asymmetric spindle pole movements during anaphase. Based on the hyperactive movements of nuclei and metaphase spindles, it is proposed that the ultimate effect of LET-99 activity is a downregulation of cortical forces that act on centrosomes. Because LET-99 is enriched in a cortical band that encircles P lineage cells, downregulation of cortical forces in this region during prophase would result in higher net anterior and posterior forces that would produce a rotational movement of the nuclear-centrosome complex. After rotation, the posterior centrosome/spindle pole lies partially underneath the LET-99 band. Downregulation of cortical forces in the LET-99 band region at this stage would affect lateral astral microtubule interactions, producing higher net forces directed towards the posterior and thus asymmetric anaphase spindle elongation. The results reported here on the genetic interactions between LET-99 and Galpha/GPR signaling are consistent with this model. Loss of LET-99 causes gain of Galpha/GPR-1/2-like phenotypes, hyperactive nuclear and spindle movements. These hyperactive movements are completely suppressed in Galpha(RNAi); let-99 or gpr-1/2(RNAi); let-99 mutant embryos, suggesting that LET-99 opposes Galpha/GPR-1/2 signaling. The antagonistic role of let-99 to Galpha/GPR-1/2 signaling is further supported by the observation that partially reducing let-99 activity suppresses the lethality caused by loss of gpa-16 activity alone. Finally, the weak asymmetry of spindle positioning observed in gpr-1/2(RNAi) embryos is no longer observed in gpr-1/2(RNAi); let-99 double mutant embryos. These results suggest that let-99 not only functions oppositely to Galpha/GPR-1/2 signaling, but also indeed provides an asymmetric cue. Based on these results and the pattern of cortical LET-99 localization, it is proposed that LET-99 antagonizes Galpha/GPR-1/2 signaling, thus downregulating cortical forces asymmetrically during both rotation and anaphase spindle elongation (Tsou, 2003).

Spindle positioning during an asymmetric cell division is of fundamental importance to ensure correct size of daughter cells and segregation of determinants. In the C. elegans embryo, the first spindle is asymmetrically positioned, and this asymmetry is controlled redundantly by two heterotrimeric G? subunits, GOA-1 and GPA-16. The Galpha subunits act downstream of the PAR polarity proteins, which control the relative pulling forces acting on the poles. How these heterotrimeric G proteins are regulated and how they control spindle position is still unknown. The Galpha subunits are regulated by a receptor-independent mechanism. RNAi depletion of gpr-1 and gpr-2, homologs of mammalian AGS3 and Drosophila PINS (receptor-independent G protein regulators), results in a phenotype identical to that of embryos depleted of both GPA-16 and GOA-1; the first cleavage is symmetric, but polarity is not affected. The loss of spindle asymmetry after RNAi of gpr-1 and gpr-2 appears to be the result of weakened pulling forces acting on the poles. The GPR protein(s) localize around the cortex of one-cell embryos and are enriched at the posterior. Thus, asymmetric G protein regulation could explain the posterior displacement of the spindle. Posterior enrichment is abolished in the absence of the PAR polarity proteins PAR-2 or PAR-3. In addition, LIN-5, a coiled-coil protein also required for spindle positioning, binds to and is required for cortical association of the GPR protein(s). The GPR domain of GPR-1 and GPR-2 behaves as a GDP dissociation inhibitor for GOA-1, and its activity is thus similar to that of mammalian AGS3. These results suggest that GPR-1 and/or GPR-2 control an asymmetry in forces exerted on the spindle poles by asymmetrically modulating the activity of the heterotrimeric G protein in response to a signal from the PAR proteins (Gotta, 2003).

Asymmetric divisions are crucial for generating cell diversity; they rely on coupling between polarity cues and spindle positioning, but how this coupling is achieved is poorly understood. In one-cell stage C. elegans embryos, polarity cues set by the PAR proteins mediate asymmetric spindle positioning by governing an imbalance of net pulling forces acting on spindle poles. The GoLoco-containing proteins GPR-1 and GPR-2, as well as the Galpha subunits GOA-1 and GPA-16, are essential for generation of proper pulling forces. GPR-1/2 interact with guanosine diphosphate-bound GOA-1 and are enriched on the posterior cortex in a par-3- and par-2-dependent manner. Thus, the extent of net pulling forces may depend on cortical Galpha activity, which is regulated by anterior-posterior polarity cues through GPR-1/2 (Colombo, 2003).

A casein kinase 1 and PAR proteins regulate asymmetry of a PIP(2) synthesis enzyme for asymmetric spindle positioning

Spindle positioning is an essential feature of asymmetric cell division. The conserved PAR proteins together with heterotrimeric G proteins control spindle positioning in animal cells, but how these are linked is not known. In C. elegans, PAR protein activity leads to asymmetric spindle placement through cortical asymmetry of Gα regulators GPR-1/2 (Drosophila homolog: Pins). The casein kinase 1 gamma CSNK-1 and a PIP2 synthesis enzyme (PPK-1) transduce PAR polarity to asymmetric Gα regulation. PPK-1 is posteriorly enriched in the one-celled embryo through PAR and CSNK-1 activities. Loss of CSNK-1 causes uniformly high PPK-1 levels, high symmetric cortical levels of GPR-1/2 and LIN-5, and increased spindle pulling forces. In contrast, knockdown of ppk-1 leads to low GPR-1/2 levels and decreased spindle forces. Furthermore, loss of CSNK-1 leads to increased levels of the lipid PIP2. It is proposed that asymmetric generation of PIP2 by PPK-1 directs the posterior enrichment of GPR-1/2 and LIN-5, leading to posterior spindle displacement (Panbianco, 2008).

In multiple different systems, spindle position and/or orientation during asymmetric cell division is controlled by conserved PAR polarity proteins and their regulation of heterotrimeric G protein activity. This study has uncovered a connection between these pathways involving a casein kinase 1 and PI(4)P5-kinase, a PIP2 synthesis enzyme (Panbianco, 2008).

Gα subunits are key effectors of spindle positioning in animal cells and are regulated by two components which are proposed to form a complex with Gα: a large coiled-coil proposed scaffolding protein (LIN-5 [C. elegans], Mud [Drosophila], or NuMA [mammals]) and GDP dissociation inhibitors that act as receptor-independent G protein regulators (GPR-1/2 [C. elegans], Pins [Drosophila], or LGN [mammals]). How these proteins respond to PAR polarity is unknown in any system. CSNK-1 regulation of PPK-1 links the conserved PAR and G protein pathways in the control of asymmetric spindle positioning in C. elegans by controlling cortical levels of GPR-1/2 and LIN-5 (Panbianco, 2008).

csnk-1(RNAi) embryos have normal PAR polarity but increased levels and loss of asymmetry of GPR-1/2 and LIN-5 at the cortex, causing excessive spindle and pronuclear movements. ppk-1(RNAi) embryos show the opposite phenotype: decreased GPR-1/2 and reduced spindle pulling forces. Together with the finding that CSNK-1 inhibits anterior localization of PPK-1 and downregulates PIP2 levels, the results indicate that CSNK-1 negatively regulates PPK-1. This is likely to be a direct interaction, because CSNK-1 orthologs of S. cerevisiae and S. pombe phosphorylate PPK-1 orthologs (Panbianco, 2008).

The results support a model whereby CSNK-1 links PAR asymmetry to asymmetric forces acting on the spindle by regulating GPR-1/2 and LIN-5 localization at the cortex through PPK-1. The link between PAR polarity and CSNK-1 appears to be via anterior enrichment of CSNK-1. PPK-1 also appears to be regulated by a PAR-dependent but CSNK-1-independent mechanism, since early asymmetry of PPK-1 is disrupted in par-3 mutant, but not csnk-1(RNAi), embryos (Panbianco, 2008).

It is proposed that enrichment of PPK-1 at the posterior would lead to asymmetric generation of the lipid PIP2, which in turn would lead to posterior enrichment, in an unknown manner, of LIN-5 and GPR-1/2. In the absence of CSNK-1 and its inhibitory role, PPK-1 is uniformly high at the cortex, which would lead to high cortical levels of the lipid PIP2, high cortical enrichment of GPR-1/2 and LIN-5, and increased spindle pulling forces. As yet, it is not known what responds to PIP2. It is possible that either GPR-1/2 or LIN-5 could bind this lipid, but neither protein has a known PIP2-binding domain. Another possibility is that one of these proteins could bind to an as yet unidentified PIP2-binding protein. Alternatively, a different phosphoinositide might be more directly relevant for spindle positioning, and interfering with PIP2 disrupts its levels. Additionally, despite PPK-1 being a PI(4)P5-kinase, other models whereby PPK-1 controls spindle positioning by directly binding downstream effectors rather than by producing PIP2 cannot be ruled out. A key goal for the future is to identify the mode of action of PPK-1 (Panbianco, 2008).

Controlled localization of proteins to specific membranes at particular times is critical in the regulation of many intracellular processes. Such localization is often driven by reversible association with particular membrane lipids. This is the first study showing that asymmetric enrichment of a phosphoinositide synthesis enzyme is important for asymmetric cell division. However, the importance of phosphoinositide asymmetries in polarized events have been described in other systems (Panbianco, 2008).

In Dyctostelium, in response to chemoattractant concentration, receptor G protein signaling directs PI3-kinases and the lipid phosphatase PTEN to relocate to discrete regions of the membrane that are exposed to higher and lower chemoattractant concentrations, respectively. This leads to a gradient of PIP3 important for pseudopodia formation. While the mechanisms of enzyme activation/inhibition have not been established, a similar local accumulation of PIP3 controls polarity in other cells, including neutrophils and fibroblasts (Panbianco, 2008).

Other studies have described links between the PAR-3 complex and phospohinosotide-generating enzymes. PI3-kinase and PTEN affect the polarization of hippocampal neurons in culture and the localization of PAR-3 and aPKC to the tip of the neurite that is going to become the axon. Recently, it was shown that PTEN directly binds the Drosophila PAR-3 homolog, Bazooka (Baz), and colocalizes with it at the apical membrane of epithelia and neuroblasts (von Stein, 2005). In Drosophila photoreceptors, PTEN is recruited to cell junctions by PAR-3/Bazooka and is important for apical membrane morphogenesis (Pinal, 2006). In MDCK cells, PTEN localizes to the apical plasma membrane to mediate the enrichment of PIP2, which in turn recruits Annexin2, Cdc42, and aPKC, important for the apicobasal membrane formation (Panbianco, 2008).

Links between phosphoinositide asymmetries and polarity in different organisms and processes suggest widespread roles for phosphoinositides in polarity regulation. In the case of spindle positioning, conservation of involvement of PAR and heterotrimeric G proteins suggests a common transduction mechanism between these pathways. It is proposed that a central part of such a mechanism involves casein kinase 1 regulation of PI(4)P5-kinases (Panbianco, 2008).

Dynamic localization of C. elegans TPR-GoLoco proteins mediates mitotic spindle orientation by extrinsic signaling

Cell divisions are sometimes oriented by extrinsic signals, by mechanisms that are poorly understood. Proteins containing TPR and GoLoco-domains (C. elegans GPR-1/2, Drosophila Pins, vertebrate LGN and AGS3) are candidates for mediating mitotic spindle orientation by extrinsic signals, but the mechanisms by which TPR-GoLoco proteins may localize in response to extrinsic cues are not well defined. The C. elegans TPR-GoLoco protein pair GPR-1/2 is enriched at a site of contact between two cells - the endomesodermal precursor EMS and the germline precursor P(2) - and both cells align their divisions toward this shared cell-cell contact. To determine whether GPR-1/2 is enriched at this site within both cells, mosaic embryos were generated with GPR-1/2 bearing a different fluorescent tag in different cells. It was surprising to find that GPR-1/2 distribution is symmetric in EMS, where GPR-1/2 had been proposed to function as an asymmetric cue for spindle orientation. Instead, GPR-1/2 is asymmetrically distributed only in P(2). A role for normal GPR-1/2 localization was demonstrated in P(2) division orientation. MES-1/Src signaling plays an instructive role in P(2) for asymmetric GPR-1/2 localization and normal spindle orientation. A model in which signaling localizes GPR-1/2 by locally inhibiting LET-99, a GPR-1/2 antagonist, was ruled out. Instead, asymmetric GPR-1/2 distribution is established by destabilization at one cell contact, diffusion, and trapping at another cell contact. Once the mitotic spindle of P(2) is oriented normally, microtubule-dependent removal of GPR-1/2 prevented excess accumulation, in an apparent negative-feedback loop. These results highlight the role of dynamic TPR-GoLoco protein localization as a key mediator of mitotic spindle alignment in response to instructive, external cues (Werts, 2011).

Partner of inscuteable homologs in vertebrates

The Purkinje cell protein-2 (Pcp2, also known as L7) gene is abundantly expressed only in Purkinje cells of the cerebellum and bipolar neurons of the retina. The yspatio-temporal expression pattern of this gene suggests a role for PCP2 in Purkinje cell development or normal cell physiology. A PCP2-deficient mouse was created by gene targeting to test the hypothesis that it is required for Purkinje cell development or function. Although normally present in abundance, the absence of PCP2 in null animals causes no observable cerebellar abnormalities. Behavioral analysis reveals normal abilities for balance and coordination. Null cerebellum has normal Purkinje cell numbers, morphology, and ultrastructure. Retinal bipolar neurons appear similarly unaffected. Aged null animals (22 months) were also examined and no deficits were detected using the same behavioral and histologic analyses. Although the null animal does not reveal the function of PCP2, it does rule out an essential role for PCP2 in Purkinje cell development, in Purkinje cell survival, and in at least some aspects of cerebellar function (Mohn, 1997).

The yeast two-hybrid system has been used to identify proteins that interact with the alpha-subunit of the heterotrimeric GTP-binding protein, Gi2. A human B cell cDNA library was screened with full-length G alpha i2 and four positive colonies were isolated, one of which expresses the 44-kDa COOH terminus of a previously unrecognized 677-amino acid (aa) protein. A full-length clone was isolated from a HeLa cell cDNA library. The deduced protein contains 10 Leu-Gly-Asn repeats, and thus it has been named LGN. Computer analysis indicates that LGN is a mosaic protein with seven repeated sequences of about 40 aa in length at its N-terminal end, and four repeated sequences of about 34 aa at its C-terminal end. Each of the two repeat regions shows substantial similarity to proteins found in other organisms. RT-PCR analysis of human tissues shows that the mRNA of LGN is ubiquitously expressed. The specificity of interaction between G alpha i2 and LGN was confirmed by an in vitro binding assay using recombinant proteins. These data indicate that the yeast two-hybrid system can identify novel proteins, such as LGN, that interact with G alpha proteins. As a mosaic protein, LGN shows similarity with portions of proteins from many species and thus may define a new protein family (Mochizuki, 1997).

The heterotrimeric G protein Galphao is ubiquitously expressed throughout the central nervous system, but many of its functions remain to be defined. To search for novel proteins that interact with Galphao, a mouse brain library was screened using the yeast two-hybrid interaction system. Pcp2 (Purkinje cell protein-2) was identified as a partner for Galphao in this system. Pcp2 is expressed in cerebellar Purkinje cells and retinal bipolar neurons, two locations where Galphao is also expressed. Pcp2 was first identified as a candidate gene to explain Purkinje cell degeneration in pcd mice, but its function remains unknown because Pcp2 knockout mice are normal. Galphao and Pcp2 binding was confirmed in vitro using glutathione S-transferase-Pcp2 fusion proteins and in vitro translated [35S]methionine-labeled Galphao. In addition, when Galphao and Pcp2 are cotransfected into COS cells, Galphao is detected in immunoprecipitates of Pcp2. To determine whether Pcp2 could modulate Galphao function, kinetic constants kcat and koff of bovine brain Galphao were determined in the presence and absence of Pcp2. Pcp2 stimulates GDP release from Galphao more than 5-fold without affecting kcat. These findings define a novel nucleotide exchange function for Pcp2 and suggest that the interaction between Pcp2 and Galphao is important to Purkinje cell function (Luo, 1999).

The G-protein regulatory (GPR) motif in AGS3 is a region for protein binding to heterotrimeric G-protein alpha subunits. To define the properties of this approximately 20-amino acid motif, a GPR consensus peptide was designed and its influence on the activation state of G-protein and receptor coupling to G-protein was determined. The GPR peptide sequence (28 amino acids) encompasses the consensus sequence defined by the four GPR motifs conserved in the family of AGS3 proteins. The GPR consensus peptide effectively prevents the binding of AGS3 to Gialpha1,2 in protein interaction assays, inhibits guanosine 5'-O-(3-thiotriphosphate) binding to Gialpha, and stabilizes the GDP-bound conformation of Gialpha. The GPR peptide has little effect on nucleotide binding to Goalpha and brain G-protein indicating selective regulation of Gialpha. Thus, the GPR peptide functions as a guanine nucleotide dissociation inhibitor for Gialpha. The GPR consensus peptide also blocks receptor coupling to Gialphabetagamma, indicating that although the AGS3-GPR peptide stabilizes the GDP-bound conformation of Gialpha, this conformation of Gialpha(GDP) is not recognized by a G-protein coupled receptor. The AGS3-GPR motif presents an opportunity for selective control of Gialpha- and Gbetagamma-regulated effector systems, and the GPR motif allows for alternative modes of signal input to G-protein signaling systems (Peterson, 2000).

Asymmetric cell division requires the orientation of mitotic spindles along the cell-polarity axis. In Drosophila neuroblasts, this involves the interaction of the proteins Inscuteable (Insc) and Partner of inscuteable (Pins). A human Pins-related protein, called LGN, is instead essential for the assembly and organization of the mitotic spindle. LGN is cytoplasmic in interphase cells, but associates with the spindle poles during mitosis. Ectopic expression of LGN disrupts spindle-pole organization and chromosome segregation. Silencing of LGN expression by RNA interference also disrupts spindle-pole organization and prevents normal chromosome segregation. LGN binds the nuclear mitotic apparatus protein NuMA (Drosophila homolog: Mushroom body defect), which tethers spindles at the poles, and this interaction is required for the LGN phenotype. Anti-LGN antibodies and the LGN-binding domain of NuMA both trigger microtubule aster formation in mitotic Xenopus egg extracts, and the NuMA-binding domain of LGN blocks aster assembly in egg extracts treated with taxol. Thus, a mammalian Pins homolog has been identified as a key regulator of spindle organization during mitosis (Du, 2001).

LGN is closely related to a Drosophila protein, Partner of inscuteable (Pins), that is required for polarity establishment and asymmetric cell divisions during embryonic development. In mammalian cells, LGN binds with high affinity to the C-terminal tail of NuMA, a large nuclear protein that is required for spindle organization, and accumulates at the spindle poles during mitosis. LGN also regulates spindle organization, possibly through inhibition of NuMA function, but the mechanism of this effect has not yet been understood. Using mammalian cells, frog egg extracts, and in vitro assays, it is shown that a small domain within the C terminus of NuMA stabilizes microtubules (MTs), and that LGN blocks stabilization. The nuclear localization signal adjacent to this domain is not involved in stabilization. NuMA can interact directly with MTs, and the MT binding domain on NuMA overlaps by ten amino acid residues with the LGN binding domain. It is therefore proposed that a simple steric exclusion model can explain the inhibitory effect of LGN on NuMA-dependent mitotic spindle organization (Du, 2002).

Mammalian LGN/AGS3 proteins and their Drosophila Pins ortholog are cytoplasmic regulators of G-protein signaling. In Drosophila, Pins localizes to the lateral cortex of polarized epithelial cells and to the apical cortex of neuroblasts where it plays important roles in neuroblast asymmetric division. Using overexpression studies in different cell line systems, it has been demonstrated that like Drosophila Pins, LGN can exhibit enriched localization at the cell cortex, depending on the cell cycle and the culture system used. In WISH, PC12, and NRK but not COS cells, LGN is largely directed to the cell cortex during mitosis. Overexpression of truncated protein domains further identified the Galpha-binding C-terminal portion of LGN as a sufficient domain for cortical localization in cell culture. In mitotic COS cells that normally do not exhibit cortical LGN localization, LGN is redirected to the cell cortex upon overexpression of Galpha subunits of heterotrimeric G-proteins. The results also show that the cortical localization of LGN is dependent on microfilaments and that interfering with LGN function in cultured cell lines causes early disruption to cell cycle progression (Kaushik, 2003).

Asymmetric cell division is a fundamental mechanism used to generate cellular diversity in invertebrates and vertebrates. In Drosophila, asymmetric division of neuroblasts is achieved by the asymmetric segregation of cell fate determinants Prospero and Numb into the basal daughter cell. Asymmetric segregation of cell fate determinants requires an apically localized protein complex that includes Inscuteable, Pins, Bazooka, DmPar-6, DaPKC and Galphai. Pins acts to stabilize the apical complex during neuroblast divisions. Pins interacts and colocalizes with Inscuteable, as well as maintaining its apical localization. A mouse homolog of pins (Pins) has been isolated and its expression profile has been characterized. Mouse PINS shares high similarity in sequence and structure with Pins and other Pins-like proteins from mammals. Pins is expressed in many mouse tissues but its expression is enriched in the ventricular zone of the developing central nervous systems. PINS localizes asymmetrically to the apical cortex of mitotic neuroblasts when ectopically expressed in Drosophila embryos. Like Pins, its N-terminal tetratricopeptide repeats can directly interact with the asymmetric localization domain of Insc, and its C-terminal GoLoco-containing region can direct localization to the neuroblast cortex. Pins can fulfill all aspects of pins function in Drosophila neuroblast asymmetric cell divisions. These results suggest a conservation of function between the fly and mammalian Pins homologues (Yu, 2003a).

Database searches of the mouse genome with the fly Pins amino acid sequence identified EST clones that encode two Pins-like proteins with varying homologies to Pins. The mouse protein showing a higher percentage of homology to Drosophila Pins is referred to as PINS. PINS shows a higher level of homology to human LGN than to rat AGS3. The second mouse protein is more closely related to AGS3 than to LGN and is therefore referred to as mouse AGS3. Hence, there are at least two homologues of Drosophila Pins in mouse, PINS and mouse AGS3. Similarly, the human genome project also identifies two Pins-like sequences, LGN and AGS3. Hence, PINS/LGN and mouse AGS3/AGS3 appear to be paralogues, formed by duplication after divergence of mammals and flies. The two Pins-like proteins identified in the mammalian genomes have different features. In situ hybridization of mouse Pins and Ags3 shows a distinct distribution in the neural tube: Pins is enriched in a layer of cortical precursors, whereas Ags3 is uniformly distributed in the neural tube, suggesting distinct roles for these proteins during neurogenesis. This is reminiscent of the localization profiles of mouse numb and numb-like in the neural tube of the mouse embryo (Yu, 2003a).

During asymmetric cell divisions, mitotic spindles align along the axis of polarization. In invertebrates, spindle positioning requires Pins or related proteins and a G protein alpha subunit. A mammalian Pins, called LGN, binds Galphai (see Drosophila Gαi) and also interacts through an N-terminal domain with the microtubule binding protein NuMA. During mitosis, LGN recruits NuMA to the cell cortex, while cortical association of LGN itself requires the C-terminal Galpha binding domain. Using a FRET biosensor, it was found that LGN behaves as a conformational switch: in its closed state, the N and C termini interact, but NuMA or Galphai can disrupt this association, allowing LGN to interact simultaneously with both proteins, resulting in their cortical localization. Overexpression of Galphai or YFP-LGN causes a pronounced oscillation of metaphase spindles, and NuMA binding to LGN is required for these spindle movements. It is proposed that a related switch mechanism might operate in asymmetric cell divisions in the fly and nematode (Du, 2004).

Resistance to inhibitors of cholinesterase (Ric) 8A is a guanine nucleotide exchange factor that activates certain G protein alpha-subunits. Genetic studies in C. elegans and Drosophila have placed RIC-8 (Ric8a in Drosophila) in a previously uncharacterized G protein signaling pathway that regulates centrosome movements during cell division. Components of this pathway include G protein subunits of the G alphai class, GPR or GoLoco domain-containing proteins, RGS (regulator of G protein signaling) proteins, and accessory factors. These proteins interact to regulate microtubule pulling forces during mitotic movement of chromosomes. It is unclear how the GTP-binding and hydrolysis cycle of G alphai functions in the context of this pathway. In mammals, the GoLoco domain-containing protein LGN (GPSM2), the LGN- and microtubule-binding nuclear mitotic apparatus protein (NuMA), and G alphai regulate a similar process. Mammalian Ric-8A dissociates G alphai-GDP/LGN/NuMA complexes catalytically, releasing activated G alphai-GTP in vitro. Ric-8A-stimulated activation of G alphai causes concomitant liberation of NuMA from LGN. It is concluded that Ric-8A efficiently utilizes GoLoco/G alphai-GDP complexes as substrates in vitro and suggest that Ric-8A-stimulated release of Galphai-GTP and/or NuMA regulates the microtubule pulling forces on centrosomes during cell division (Tall, 2005).

Models are envisioned in which one cellular function of Ric-8A is to dissociate Galphai-GDP/GoLoco complexes by stimulation of nucleotide exchange. G protein control of asymmetric cell division involves cycling of Galphai between its GDP- and GTP-bound forms, as evidenced by the fact that (in C. elegans) both RIC-8 and RGS7 influence the pathway in opposed fashion. It remains speculative whether Galphai-GDP/GoLoco or the production of Galphai-GTP from a GoLoco scaffold activates signaling. It stands to reason that Galphai-GTP must dissociate from GoLoco at some point during signaling. If multiple rounds of cycling between Galphai-GDP/GoLoco and liberated Galphai-GTP are required to complete cell division, then Ric-8A-stimulated dissociation of a Galphai/GoLoco complex could be responsible for either terminating or activating the signal. In either context, RGS-facilitated hydrolysis of GTP by Galpha ensues. The resultant Galphai-GDP could rebind to GoLoco (and not betagamma) to complete one round of the cycle. Rapid cycling of this process may be necessary to regulate the pulling forces on microtubules appropriately during a round of chromosome segregation (supporting information on the PNAS web site, for these proposed models). Regulation of other Galpha or Galpha/GoLoco-mediated signaling pathways by Ric-8A is also worth considering, given the number of distinct Galpha binding partners of mammalian Ric-8A and Ric-8B and the many processes that appear to be regulated by RIC-8 in C. elegans (Tall, 2005).

In cell polarization of Drosophila neuroblasts, Inscuteable (Insc) functions via tethering Partner of Insc (Pins) to Bazooka, homologous to human cell polarity protein Par3. However, little has been known about mammalian homologues of Insc. Two distinct cDNAs have been cloned from human Insc gene, which is differentially expressed from alternative first exons: one encodes 579 amino acids, whereas the other lacks the N-terminal 47 amino acids. In contrast to human homologues for Pins and Par3, human Insc exhibits a weak homology with the Drosophila counterpart. Nevertheless, human Insc proteins bind to the human Pins homologues LGN and AGS3, and also to human Par3 and its related protein Par3beta. Although LGN by itself is incapable of interacting with Par3, coexpression of human Insc leads to the interaction between LGN and Par3, indicating that human Insc plays an evolutionarily conserved role as an adaptor protein that links Pins to Par3 (Izaki, 2006).

Par3 controls epithelial spindle orientation by aPKC-mediated phosphorylation of apical Pins

Formation of epithelial sheets requires that cell division occurs in the plane of the sheet. During mitosis, spindle poles align so the astral microtubules contact the lateral cortex. Confinement of the mammalian Pins protein to the lateral cortex is essential for this process. Defects in signaling through Cdc42 and atypical protein kinase C (aPKC) also cause spindle misorientation. When epithelial cysts are grown in 3D cultures, misorientation creates multiple lumens. This study shows that silencing of the polarity protein Par3 causes spindle misorientation in Madin-Darby canine kidney cell cysts. Silencing of Par3 also disrupts aPKC association with the apical cortex, but expression of an apically tethered aPKC rescues normal lumen formation. During mitosis, Pins is mislocalized to the apical surface in the absence of Par3 or by inhibition of aPKC. Active aPKC increases Pins phosphorylation on Ser401, which recruits 14-3-3 protein. 14-3-3 binding inhibits association of Pins with Gαi, through which Pins attaches to the cortex. A Pins S401A mutant mislocalizes over the cell cortex and causes spindle orientation and lumen defects. It is concluded that the Par3 and aPKC polarity proteins ensure correct spindle pole orientation during epithelial cell division by excluding Pins from the apical cortex. Apical aPKC phosphorylates Pins, which results in the recruitment of 14-3-3 and inhibition of binding to Gαi, so the Pins falls off the cortex. In the absence of a functional exclusion mechanism, astral microtubules can associate with Pins over the entire epithelial cortex, resulting in randomized spindle pole orientation (Hao, 2010).

mPins modulates PSD-95 and SAP102 trafficking and influences NMDA receptor surface expression

Appropriate trafficking and targeting of glutamate receptors (GluRs) to the postsynaptic density is crucial for synaptic function. mPins (mammalian homologue of Drosophila Partner of inscuteable) interacts with SAP102 and PSD-95 (two PDZ proteins present in neurons), and functions in the formation of the NMDAR - MAGUK (N-methyl-D-aspartate receptor - membrane-associated guanylate kinase) complex. mPins enhances trafficking of SAP102 and NMDARs to the plasma membrane in neurons. Expression of dominant-negative constructs and short-interfering RNA (siRNA)-mediated knockdown of mPins decreases SAP102 in dendrites and modifies surface expression of NMDARs. mPins changes the number and morphology of dendritic spines and these effects depend on its Galphai interaction domain, thus implicating G-protein signalling in the regulation of postsynaptic structure and trafficking of GluRs (Sans, 2005).

mPins is a ubiquitously expressed protein that is critical for the regulation of mitotic spindle organization in dividing cells. mPins interacts with several functionally distinct proteins, including NuMA, Ras, LKB1 and Galphai. The finding that mPins interacts with the PSD-95 family adds another group of important proteins to those whose trafficking depends on mPins. Drosophila Pins is required for asymmetric division of sensory organ precursor cells (pI) and dividing neuroblasts. Whereas the roles of Pins in cell division are relatively well-characterized, the function of mPins in the mature mammalian central nervous system remains enigmatic. The related protein, AGS3, may affect cocaine-induced plasticity by regulating G-protein signalling in the prefrontal cortex. The data show that mPins and AGS3 are both expressed in the developing hippocampus but have different subcellular localizations, perhaps because mPins, but not AGS3, interacts with SAP102. Moreover, AGS3 is down-regulated in adult hippocampus and seems to be absent from the PSD, whereas mPins is expressed throughout development and is enriched in synaptic membranes. mPins and AGS3 are found in different domains throughout the cell body and dendrites in primary cultures of hippocampal neurons. mPins, but not AGS3, redistributes into punctate structures after ionomycin or NMDA treatment, suggesting that calcium signalling functions in trafficking of mPins complexes. These findings strongly suggest that these two orthologues of Drosophila Pins have different functions in neurons (Sans, 2005).

The MAGUKs do not compete with the other known interacting proteins of mPins suggesting that the association of these other interacting proteins may indirectly influence the trafficking of the MAGUK and its associated proteins, such as NMDARs. Both Ras and Galphai are particularly interesting in this context. Ras has been implicated in the trafficking of GluRs. Characterized as molecular switches that alternate between GTP-bound ('on') and GDP-bound ('off') forms, these proteins are involved in the reorganization of synaptic structure. G-proteins, such as Galphai, influence NMDAR trafficking through metabotropic GluRs. In this study, it is shown that Galphai proteins function in NMDAR trafficking through a direct interaction with the mPins-SAP102 complex. mPins mediates G-protein signalling through binding to Galphai1-3GDP, thereby inhibiting binding of Galphai to Gßγ (and consequently enhancing Gßγ signalling in the absence of a G-protein-coupled receptor). mPins shifts between a closed state, when the N- and C-terminal halves of the protein bind to one another, and an open state when NuMA binds to mPins to switch it open, allowing the binding of Galphai. SAP102, similarly to SAP97, may exist in the cytoplasm as a folded molecule in which the GK domain is folded onto the SH3 domain. The data suggest that SAP102 binds to mPins in its closed state, as the two proteins localized in ring-like structures in COS cells. Therefore, mPins could be required upstream of, or in parallel to, the NR2B-SAP102 interaction. It is also shown that SAP102-mPins complexes have a different fate from that of NR2B-SAP102-mPins complexes, since the three proteins form clusters in COS cells and synaptic clusters in spines. These data suggest that NMDARs can open the SAP102-mPins complexes. Interestingly, cotransfection of the linker region of mPins with NR2B and SAP102 results in the formation of ternary complexes that are rapidly degraded, suggesting that interaction of Galphai with GoLoco domains (or an unidentified protein with TPR domains) is important for stabilization of the complex. mPins can bind four Galphai molecules, and it is unclear at present whether all of the sites need to be occupied for proper folding and targeting of mPins. As a modulator of G-protein signalling, the possibility cannot be excluded that Galphai binds to the NMDAR-MAGUK-mPins complex at synapses after activation of a G-protein-coupled receptor. Studies have suggested that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) can exhibit some of their effects through interactions with heterotrimeric G-proteins in addition to their ionic channel function. For instance, it has been shown that AMPA can induce dissociation of Galphai1 from the Galphai1/ß heterotrimeric complex and its association with GluR1 through an adaptor protein. In light of the current data, the possibility exists that Galphai signalling proteins may also be recruited to certain MAGUK-mPins complexes through simultaneous dissociation from AMPARs (Sans, 2005).

The results suggest that the NMDAR associates indirectly through SAP102 with two molecular complexes -- the exocyst and mPins-Galphai complexes -- and that these associations are necessary for proper trafficking of receptors in neurons. The results also suggest that this complex is formed in the ER in heterologous cells and early in the secretory pathway in neurons. Although this has not been demonstrated directly for native proteins, an association of MAGUK with AMPARs in the ER (or cis-Golgi) has been shown for native AMPARs in brain by using the endo-H sensitivity of immature AMPARs, so such an association is not unprecedented. These results suggest that NMDARs are trafficked as part of a large complex from their site of synthesis in the cell body to the postsynaptic membrane, presumably in a transport vesicle. The identification of other components of the SAP102 cargo complex (containing NMDARs, the exocyst and mPins-Galphai complexes) will undoubtedly help to clarify the steps involved in trafficking of NMDARs from assembly and ER exit to transport in dendrites and spines in normal and disease states (Sans, 2005).

Modulation of basal and receptor-induced GIRK potassium channel activity and neuronal excitability by the mammalian PINS homolog LGN

G protein-activated inwardly rectifying potassium (GIRK) channels mediate slow synaptic inhibition and control neuronal excitability. It is unknown whether GIRK channels are subject to regulation by guanine dissociation inhibitor (GDI) proteins like LGN, a mammalian homolog of Drosophila Partner of Inscuteable (mPINS). This study reports that LGN increases basal GIRK current but reduces GIRK activation by metabotropic transmitter receptors coupled to Gi or Go, but not Gs. Moreover, expression of its N-terminal, TPR-containing protein interaction domains mimics the effects of LGN in mammalian cells, probably by releasing sequestered endogenous LGN. In hippocampal neurons, expression of LGN, or LGN fragments that mimic or enhance LGN activity, hyperpolarizes the resting potential due to increased basal GIRK activity and reduces excitability. Using Lenti virus for LGN RNAi to reduce endogenous LGN levels in hippocampal neurons, an essential role was demonstrated of LGN for maintaining basal GIRK channel activity and for harnessing neuronal excitability (Wiser, 2006).

In Drosophila, the GDI proteins Partner of Inscuteable (PINS) and Locomotion Defects (LOCO) act synergistically in neuroblasts to release Gβγ subunits, thereby ensuring asymmetric division. The mammalian homologs of these proteins are LGN, also known as mPINS, and Activator of G protein Signaling 3 (AGS3). These proteins contain three or four GPR motifs in the C-terminal GPR domain, seven tetratricopeptide (TPR) motifs in the N-terminal TPR domain, and a linker peptide in between; the linker as well as the TPR domain interact with other proteins. Both LGN and AGS3 bind Gαi more strongly than Gαo, and exhibit much stronger GDI activity for Gαi than Gαo. Both LGN and AGS3 are expressed in neurons, though likely in different subcellular compartments; these GDI proteins may also be subjected to different regulation (Wiser, 2006).

The importance of GDI proteins in neuronal signaling is exemplified by the identification of AGS3 as a gatekeeper for the behavioral manifestation of drug dependence. Whereas AGS3 expression has no detectable direct effect on GPCR-mediated activation or inhibition of adenylyl cyclase, AGS3 is required for the paradoxical activation of protein kinase A (PKA) by opiates acting on Gi-coupled receptors. Indeed, AGS3 at the nucleus accumbens core plays an instrumental role in the relapse of heroin-seeking behavior (Wiser, 2006 and references therein).

The functional role of LGN in neurons is an interesting open question. In proliferating cells, LGN regulates mitotic spindle organization, analogous to the function of its Drosophila and C. elegans counterparts. This function involves interaction of its TPR domain with its GPR domain as well as the Nuclear Mitotic Apparatus (NuMA) protein, and the ability of its GPR domain to bind Gα-GDP, apparently without the involvement of any GPCR. Whether LGN contributes to GPCR signaling has not been explored, notwithstanding the broad LGN expression in postmitotic neurons, the interesting redistribution of LGN upon activation of NMDA receptors in hippocampal neurons, and its likely involvement in NMDA receptor trafficking due to its ability to bind the MAGUK protein SAP102. How might GDIs like LGN regulate GPCR signaling? Do they reduce GPCR coupling to G proteins and hence diminish GPCR signaling? Or do they enhance activation of Gβγ effectors by stabilizing and sequestering Gα-GDP so as to prolong the action of Gβγ? Could LGN regulate Go-coupled receptors (Wiser, 2006 and references therein)?

This study characterizes LGN modulation of the Gβγ effector, the GIRK (Kir3) channels that mediate slow synaptic inhibition in mammalian brain. LGN was found to increase basal GIRK current but reduce GPCR-induced GIRK current in expression systems such as Xenopus oocytes and HEK293 cells; these functions have been verified by RNAi-mediated knockdown of LGN endogenous to HEK293 cells. This study demonstrates LGN modulated signaling of Go-coupled receptors as well as Gi-coupled receptors, but not Gs-coupled receptors. Moreover, reducing LGN endogenous to hippocampal neurons abolished basal GIRK current and increased excitability. Whereas elevating or reducing LGN protein level had opposite effects on basal GIRK channel activity, the bidirectional effects on neuronal excitability likely involve other proteins under LGN modulation as well. Finally, expression of the TPR-containing protein interaction domains of LGN in mammalian HEK293 cells or hippocampal neurons mimics the effects of LGN and GPR, indicating that LGN may be modulated not only by self interaction but also by sequestration in the postmitotic neurons (Wiser, 2006).

Activator of G protein signaling 3: a gatekeeper of cocaine sensitization and drug seeking

Chronic cocaine administration reduces G protein signaling efficacy. The expression of AGS3, which binds to GialphaGDP and inhibits GDP dissociation, is upregulated in the prefrontal cortex (PFC) during late withdrawal from repeated cocaine administration. Increased AGS3 is mimicked in the PFC of drug-naive rats by microinjecting a peptide containing the Giα binding domain (GPR) of AGS3 fused to the cell permeability domain of HIV-Tat. Infusion of Tat-GPR mimicked the phenotype of chronic cocaine-treated rats by manifesting sensitized locomotor behavior and drug seeking and by increasing glutamate transmission in nucleus accumbens. By preventing cocaine withdrawal-induced AGS3 expression with antisense oligonucleotides, signaling through Giα was normalized, and both cocaine-induced relapse to drug seeking and locomotor sensitization were prevented. When antisense oligonucleotide infusion was discontinued, drug seeking and sensitization were restored. It is proposed that AGS3 gates the expression of cocaine-induced plasticity by regulating G protein signaling in the PFC (Bowers, 2004).

LGN and and vertebrate neurogenesis

Neurons in the developing mammalian brain are generated from progenitor cells in the proliferative ventricular zone, and control of progenitor division is essential to produce the correct number of neurons during neurogenesis. This study establishes that GΔgamma subunits of heterotrimeric G proteins are required for proper mitotic-spindle orientation of neural progenitors in the developing neocortex. Interfering with Gbetagamma function in progenitors causes a shift in spindle orientation from apical-basal divisions to planar divisions. This results in hyperdifferentiation of progenitors into neurons as a consequence of both daughter cells adopting a neural fate instead of the normal asymmetric cell fates. Silencing AGS3, a nonreceptor activator of Gbetagamma, results in defects similar to the impairment of Gbetagamma, providing evidence that AGS3-Gbetagamma signaling in progenitors regulates apical-basal division and asymmetric cell-fate decisions. Furthermore, the observations indicate that the cell-fate decision of daughter cells is coupled to mitotic-spindle orientation in progenitors (Sanada, 2005).

The spatio-temporal regulation of symmetrical as opposed to asymmetric cell divisions directs the fate and location of cells in the developing CNS. In invertebrates, G-protein regulators control spindle orientation in asymmetric divisions, which generate progeny with different identities. This study investigated the role of the G-protein regulator LGN (also called Gpsm2) in spindle orientation and cell-fate determination in the spinal cord neuroepithelium of the developing chick embryo. LGN was shown to be located at the cell cortex and spindle poles of neural progenitors, and it regulates spindle movements and orientation. LGN promotes planar divisions in the early spinal cord. Interfering with LGN function randomizes the plane of division. Notably, this does not affect cell fate, but frequently leads one daughter of proliferative symmetric divisions to exit the neuroepithelium prematurely and to proliferate aberrantly in the mantle zone. Hence, tight control of planar spindle orientation maintains neural progenitors in the neuroepithelium, and regulates the proper development of the nervous system (Morin, 2007).

During mammalian development, neuroepithelial cells function as mitotic progenitors, which self-renew and generate neurons. Although spindle orientation is important for such polarized cells to undergo symmetric or asymmetric divisions, its role in mammalian neurogenesis remains unclear. This study shows that control of spindle orientation is essential in maintaining the population of neuroepithelial cells, but dispensable for the decision to either proliferate or differentiate. Knocking out LGN, (the G protein regulator), randomizes the orientation of normally planar neuroepithelial divisions. The resultant loss of the apical membrane from daughter cells frequently converts them into abnormally localized progenitors without affecting neuronal production rate. Furthermore, overexpression of Inscuteable to induce vertical neuroepithelial divisions shifts the fate of daughter cells. These results suggest that planar mitosis ensures the self-renewal of neuroepithelial progenitors by one daughter inheriting both apical and basal compartments during neurogenesis (Konno, 2008).

The LGN protein promotes planar proliferative divisions in the neocortex but apicobasal asymmetric terminal divisions in the retina

Cell division orientation is critical to control segregation of polarized fate determinants in the daughter cells to produce symmetric or asymmetric fate outcomes. While most studies in vertebrates have focused on the role of mitotic spindle orientation in proliferative asymmetric divisions, it remains unclear whether altering spindle orientation is required for the production of asymmetric fates in differentiative terminal divisions. This study shows that the GoLoco motif protein LGN, which interacts with Gαi to control apicobasal division orientation in Drosophila neuroblasts, is excluded from the apical domain of retinal progenitors undergoing planar divisions, but not in those undergoing apicobasal divisions. Inactivation of LGN reduces the number of apicobasal divisions in mouse retinal progenitors, whereas it conversely increases these divisions in cortical progenitors. While LGN inactivation increases the number of progenitors outside the ventricular zone in the developing neocortex, it has no effect on the position or number of progenitors in the retina. Retinal progenitor cell lineage analysis in LGN mutant mice, however, shows an increase in symmetric terminal divisions producing two photoreceptors, at the expense of asymmetric terminal divisions producing a photoreceptor and a bipolar or amacrine cell. Similarly, inactivating Gαi decreases asymmetric terminal divisions, suggesting that LGN function with Gαi to control division orientation in retinal progenitors. Together, these results show a context-dependent function for LGN and indicate that apicobasal divisions are not involved in proliferative asymmetric divisions in the mouse retina, but are instead essential to generate binary fates at terminal divisions (Lacomme, 2016).

LGN-dependent orientation of cell divisions in the dermomyotome controls lineage segregation into muscle and dermis

The plane of cell divisions is pivotal for differential fate acquisition. Dermomyotome development provides an excellent system with which to investigate the link between these processes. In the central sheet of the early dermomyotome, single epithelial cells divide with a planar orientation. This study reports that in the avian embryo, in addition to self-renewing, a subset of progenitors translocates into the myotome where they generate differentiated myocytes. By contrast, in the late epithelium, individual progenitors divide perpendicularly to produce both mitotic myoblasts and dermis. To examine whether spindle orientations influence fate segregation, early planar divisions were randomized and/or shifted to a perpendicular orientation by interfering with LGN function or by overexpressing inscuteable. Clones derived from single transfected cells exhibited an enhanced proportion of mixed dermomyotome/myotome progeny at the expense of 'like' daughter cells in either domain. Loss of LGN or Gαi1 function in the late epithelium randomized otherwise perpendicular mitoses and favored muscle development at the expense of dermis. Hence, LGN-dependent early planar divisions are required for the proper allocation of progenitors into either dermomyotome or myotome, whereas late perpendicular divisions are necessary for the normal balance between muscle and dermis production (Ben-Yair, 2011).

Multisite phosphorylation of NuMA-related LIN-5 controls mitotic spindle positioning in C. elegans

During cell division, the mitotic spindle segregates replicated chromosomes to opposite poles of the cell, while the position of the spindle determines the plane of cleavage. Spindle positioning and chromosome segregation depend on pulling forces on microtubules extending from the centrosomes to the cell cortex. Critical in pulling force generation is the cortical anchoring of cytoplasmic dynein (see Drosophila Dynein) by a conserved ternary complex of Gα (see Drosophila G-iα65A), GPR-1/2 (see Drosophila Pins), and LIN-5 (see Drosophila Mushroom body defect) proteins in C. elegans (Galpha-LGN-NuMA in mammals). Previous studies showed that the polarity kinase PKC-3 (see Drosophila aPKC) phosphorylates LIN-5 to control spindle positioning in early C. elegans embryos. This study investigated whether additional LIN-5 phosphorylations regulate cortical pulling forces, making use of targeted alteration of in vivo phosphorylated residues by CRISPR/Cas9-mediated genetic engineering. Four distinct in vivo phosphorylated LIN-5 residues were found to have critical functions in spindle positioning. Two of these residues form part of a 30 amino acid binding site for GPR-1, which was identified by reverse two-hybrid screening. Evidence is provided for a dual-kinase mechanism, involving GSK3 phosphorylation of S659 followed by phosphorylation of S662 by casein kinase 1. These LIN-5 phosphorylations promote LIN-5-GPR-1/2 interaction and contribute to cortical pulling forces. The other two critical residues, T168 and T181, form part of a cyclin-dependent kinase consensus site and are phosphorylated by CDK1-cyclin B (see Drosophila CyclinB) in vitro. This study applied a novel strategy to characterize early embryonic defects in lethal T168,T181 knockin substitution mutants, and evidence is provided for sequential LIN-5 N-terminal phosphorylation and dephosphorylation in dynein recruitment. These data support that phosphorylation of multiple LIN-5 domains by different kinases contributes to a mechanism for spatiotemporal control of spindle positioning and chromosome segregation (Portegijs, 2016).

A link between planar polarity and staircase-like bundle architecture in hair cells

Sensory perception in the inner ear relies on the hair bundle, the highly polarized brush of movement detectors that crowns hair cells. Previous studies have shown that, in the mouse cochlea, the edge of the forming bundle is defined by the 'bare zone', a microvilli-free sub-region of apical membrane specified by the Insc-LGN-Gαi protein complex. This study reports that LGN and Gαi (see Drosophila G protein αi subunit 65A) also occupy the very tip of stereocilia that directly abut the bare zone. LGN and Gαi are both essential for promoting the elongation and differential identity of stereocilia across rows. Interestingly, it was also revealed that total LGN-Gαi protein amounts are actively balanced between the bare zone and stereocilia tips, suggesting that early planar asymmetry of protein enrichment at the bare zone confers adjacent stereocilia their tallest identity. It is proposed that LGN and Gαi participate in a long-inferred signal that originates outside the bundle to model its staircase-like architecture, a property that is essential for direction sensitivity to mechanical deflection and hearing (Tarchini, 2016).

Loss of the canonical spindle orientation function in the Pins/LGN homolog AGS3

In many cell types, mitotic spindle orientation relies on the canonical "LGN complex" composed of Pins/LGN, Mud/NuMA, and Gαi subunits. Membrane localization of this complex recruits motor force generators that pull on astral microtubules to orient the spindle. Drosophila Pins shares highly conserved functional domains with its two vertebrate homologs LGN and AGS3. Whereas the role of Pins and LGN in oriented divisions is extensively documented, involvement of AGS3 remains controversial. This study shows that AGS3 is not required for planar divisions of neural progenitors in the mouse neocortex. AGS3 is not recruited to the cell cortex and does not rescue LGN loss of function. Despite conserved interactions with NuMA and Gαi in vitro, comparison of LGN and AGS3 functional domains in vivo reveals unexpected differences in the ability of these interactions to mediate spindle orientation functions. Finally, Drosophila Pins was found to be is unable to substitute for LGN loss of function in vertebrates, highlighting that species-specific modulations of the interactions between components of the Pins/LGN complex are crucial in vivo for spindle orientation (Saadaoui, 2017).


REFERENCES

Search PubMed for articles about Drosophila partner of inscuteable

Afshar, K. et al. (2004). RIC-8 is required for GPR-1/2-dependent G function during asymmetric division of C. elegans embryos. Cell 119: 219-230. 15479639

Andersen, R. O., Turnbull, D. W., Johnson, E. A. and Doe, C. Q. (2012). Sgt1 acts via an LKB1/AMPK pathway to establish cortical polarity in larval neuroblasts. Dev Biol 363: 258-265. Pubmed: 22248825

Asaba, N., Hanada, T., Takeuchi, A. and Chishti, A. H. (2003). Direct interaction with a kinesin-related motor mediates transport of mammalian discs large tumor suppressor homologue in epithelial cells. J. Biol. Chem. 278(10): 8395-400. 12496241

Bansal, P. K., Mishra, A., High, A. A., Abdulle, R. and Kitagawa, K. (2009). Sgt1 dimerization is negatively regulated by protein kinase CK2-mediated phosphorylation at Ser361. J Biol Chem 284: 18692-18698. Pubmed: 19398558

Bell, G. P., Fletcher, G. C., Brain, R. and Thompson, B. J. (2014). Aurora kinases phosphorylate Lgl to induce mitotic spindle orientation in Drosophila epithelia. Curr Biol 25(1):61-8. PubMed ID: 25484300

Bellaïche, Y., et al. (2001). The Partner of Inscuteable/Discs-large complex is required to establish planar polarity during asymmetric cell division in Drosophila. Cell 106: 355-366. 11509184

Bellaïche, Y., et al. (2004). The planar cell polarity protein Strabismus promotes Pins anterior localization during asymmetric division of sensory organ precursor cells in Drosophila. Development 131: 469-478. 14701683

Ben-Yair, R., Kahane, N. and Kalcheim, C. (2011). LGN-dependent orientation of cell divisions in the dermomyotome controls lineage segregation into muscle and dermis. Development 138(19): 4155-66. PubMed Citation: 21852400

Bergstralh, D. T., Lovegrove, H. E., St Johnston, D. (2013) Discs Large Links Spindle Orientation to Apical-Basal Polarity in Drosophila Epithelia. Curr Biol. PubMed ID: 23891112

Bowers, M. S., et al. (2004). Activator of G protein signaling 3: a gatekeeper of cocaine sensitization and drug seeking. Neuron 42(2): 269-81. Medline abstract: 15091342

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date revised: 22 May 2023

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