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 (K_d = 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 K_d = ~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).

GENE STRUCTURE

Bases in 5' UTR - 367

Bases in 3' UTR - 908

PROTEIN STRUCTURE

Amino Acids - 658

Structural Domains

Analyses of the predicted protein reveals in the N-terminal half of Raps the presence of seven TPR repeats and a general protein-protein interaction motif. A putative human homolog of unknown function, LGN (Mochizuki, 1996) bears 46% identity and 63% similarity over the entire length of the coding region (Yu, 2000).

The carboxy-terminal half contains three GoLoco domains, which have been implicated in binding and regulating Gi and Go proteins (see Drosophila Loco). Raps is the apparent ortholog of LGN, a human protein identified on the basis of an interaction with Gi (Mochizuki, 1996) and Go (Luo, 1999) in a yeast two-hybrid screen. In addition, a predicted C. elegans open reading frame (wormpep accession number F32A6.4) shows significant homology to Raps (Schaefer, 2000).

To identify proteins that interact with Drosophila Galphai, a yeast two-hybrid interaction screen was performed using this protein as a bait. The interacting clones that were identified included eight overlapping clones that encoded a new Drosophila protein called Raps. rapsynoid cDNA encodes a 659-amino acid protein that contains seven tetratricopeptide repeats (TPRs), which serve as protein interaction motifs and regulatory domains in various proteins. These repeats are similar (23% identity and 43% similarity) to those existing in rapsyn, a mouse protein, hence the name rapsynoid. However, Raps is not a Drosophila homolog of rapsyn. It is more similar to the human protein LGN (48% identity) and the rat protein activator of G-protein signaling 3 (AGS3) (45% identity) and lacks homology with the C-terminal region of rapsyn. As with Raps, LGN was identified because of its ability to interact with Galphai₂ in a two-hybrid assay (Mochizuki, 1996). AGS3 has been shown to activate Galphai proteins independently of G-protein-coupled receptors (Takesono, 1999). LGN and AGS3 possess, at their C terminus, four motifs, called GoLoco sequences (Siderovski, 1999) found in numerous proteins that interact with Galphai/o. Raps contains three such GoLoco consensus sequences near its C terminus. The smallest Raps sequence that is able to interact with Galphai (from aa 560 to 659) in a two-hybrid assay contains only the most C-terminal complete GoLoco motif, suggesting that this copy of the motif is sufficient to allow interaction with Galphai (Parmentier, 2000).

The functional domains of Pins have been studied. The C-terminal region containing the G alpha i binding GoLoco motifs is necessary and sufficient for targeting to the neuroblast cortex, which appears to be a prerequisite for apical localization of Pins. The N-terminal tetratricopeptide repeat-containing region of Pins is required for two processes: TPR repeats 1 to 3 plus the C-terminal region are required for apical localization but are insufficient to recruit Insc to the apical cortex, whereas TPR repeats 1 to 7 plus C-terminal Pins can perform both functions. Hence, the abilities of Pins to cortically localize, to apically localize, and to restore Insc apical localization are all separable, and all three capabilities are necessary to mediate asymmetric division. Moreover, the need for N-terminal Pins can be obviated by fusing a minimal Insc functional domain with the C-terminal region of Pins; this chimeric molecule is apically localized and can fulfill the functions of both Insc and Pins (Yu, 2002).

date revised: 20 January 2005

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