InteractiveFly: GeneBrief

mushroom body defect : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - mushroom body defect

Synonyms -

Cytological map position- 12E5--6

Function - cytoskeleton

Keywords - asymmetric cell division, spindle orientation, cell cycle, oogenesis, CNS, centrosomal protein

Symbol - mud

FlyBase ID: FBgn0002873

Genetic map position - 1-50

Classification - coiled coil

Cellular location - nuclear and cytoplasmic

NCBI links: Precomputed BLAST | EntrezGene

Recent literature
Christophorou, N., Rubin, T., Bonnet, I., Piolot, T., Arnaud, M. and Huynh, J.R. (2015). Microtubule-driven nuclear rotations promote meiotic chromosome dynamics. Nat Cell Biol [Epub ahead of print]. PubMed ID: 26458247
At the onset of meiosis, each chromosome needs to find its homologue and pair to ensure proper segregation. In Drosophila, pairing occurs during the mitotic cycles preceding meiosis. This study shows that germ cell nuclei undergo marked movements during this developmental window. It was demonstrated that microtubules and Dynein drive nuclear rotations and are required for centromere pairing and clustering. It was further found that Klaroid (SUN) and Klarsicht (KASH) co-localize with centromeres at the nuclear envelope and are required for proper chromosome motions and pairing. Mud (NuMA in vertebrates) was identified as co-localizing with centromeres, Klarsicht and Klaroid. Mud is also required to maintain the integrity of the nuclear envelope and for the correct assembly of the synaptonemal complex. These findings reveal a mechanism for chromosome pairing in Drosophila, and indicate that microtubules, centrosomes and associated proteins play a crucial role in the dynamic organization of chromosomes inside the nucleus.

Dewey, E. B., Sanchez, D. and Johnston, C. A. (2015). Warts phosphorylates Mud to promote Pins-mediated mitotic spindle orientation in Drosophila, independent of Yorkie. Curr Biol 25: 2751-2762. PubMed ID: 26592339
Multicellular animals have evolved conserved signaling pathways that translate cell polarity cues into mitotic spindle positioning to control the orientation of cell division within complex tissue structures. These oriented cell divisions are essential for the development of cell diversity and the maintenance of tissue homeostasis. Despite intense efforts, the molecular mechanisms that control spindle orientation remain incompletely defined. This study describes a role for the Hippo (Hpo) kinase complex in promoting Partner of Inscuteable (Pins)-mediated spindle orientation. Knockdown of Hpo, Salvador (Sav), or Warts (Wts) each result in a partial loss of spindle orientation, a phenotype previously described following loss of the Pins-binding protein Mushroom body defect (Mud). Similar to orthologs spanning yeast to mammals, Wts kinase localizes to mitotic spindle poles, a prominent site of Mud localization. Wts directly phosphorylates Mud in vitro within its C-terminal coiled-coil domain. This Mud coiled-coil domain directly binds the adjacent Pins-binding domain to dampen the Pins/Mud interaction, and Wts-mediated phosphorylation uncouples this intramolecular Mud interaction. Loss of Wts prevents cortical Pins/Mud association without affecting Mud accumulation at spindle poles, suggesting phosphorylation acts as a molecular switch to specifically activate cortical Mud function. Finally, loss of Wts in Drosophila imaginal disc epithelial cells results in diminished cortical Mud and defective planar spindle orientation. These results provide new insights into the molecular basis for dynamic regulation of the cortical Pins/Mud spindle positioning complex and highlight a novel link with an essential, evolutionarily conserved cell proliferation pathway.

Cate, S., Gajendra, S., Alsbury, S., Raabe, T., Tear, G. and Mitchell, K.J. (2016). Mushroom body defect is required in parallel to Netrin for midline axon guidance in Drosophila. Development [Epub ahead of print]. PubMed ID: 26893348
The outgrowth of many neurons within the central nervous system is initially directed towards or away from the cells lying at the midline. Recent genetic evidence suggests that a simple model of differential sensitivity to the conserved Netrin attractants and Slit repellents is not sufficient to explain the guidance of all axons at the midline. In the Drosophila embryonic ventral nerve cord, many axons still cross the midline in the absence of the Netrin genes or their receptor frazzled. This study shows that mutation of mushroom body defect (mud) dramatically enhances the phenotype of Netrin or frazzled mutants, resulting in many more axons failing to cross the midline, though mutations in mud alone have little effect. This suggests that mud, which encodes a microtubule-binding coiled-coil protein homologous to NuMA and Lin-5, is an essential component of a Netrin-independent pathway that acts in parallel to promote midline crossing. This novel role in axon guidance is independent of Mud's previously described role in neural precursor development. These studies identify a parallel pathway controlling midline guidance in Drosophila and highlight a novel role for Mud potentially acting downstream of Frizzled to aid axon guidance.

Bosveld, F., Markova, O., Guirao, B., Martin, C., Wang, Z., Pierre, A., Balakireva, M., Gaugue, I., Ainslie, A., Christophorou, N., Lubensky, D. K., Minc, N. and Bellaiche, Y. (2016). Epithelial tricellular junctions act as interphase cell shape sensors to orient mitosis. Nature 530: 495-498. PubMed ID: 26886796
The orientation of cell division along the long axis of the interphase cell--the century-old Hertwig's rule--has profound roles in tissue proliferation, morphogenesis, architecture and mechanics. In epithelial tissues, the shape of the interphase cell is influenced by cell adhesion, mechanical stress, neighbour topology, and planar polarity pathways. At mitosis, epithelial cells usually adopt a rounded shape to ensure faithful chromosome segregation and to promote morphogenesis. The mechanisms underlying interphase cell shape sensing in tissues are therefore unknown. This study shows that in Drosophila epithelia, tricellular junctions (TCJs) localize force generators, pulling on astral microtubules and orienting cell division via the Dynein-associated protein Mud independently of the classical Pins/Galphai pathway. Moreover, as cells round up during mitosis, TCJs serve as spatial landmarks, encoding information about interphase cell shape anisotropy to orient division in the rounded mitotic cell. Finally, experimental and simulation data show that shape and mechanical strain sensing by the TCJs emerge from a general geometric property of TCJ distributions in epithelial tissues. Thus, in addition to their function as epithelial barrier structures, TCJs serve as polarity cues promoting geometry and mechanical sensing in epithelial tissues.
Tissot, N., Lepesant, J. A., Bernard, F., Legent, K., Bosveld, F., Martin, C., Faklaris, O., Bellaiche, Y., Coppey, M. and Guichet, A. (2017). Distinct molecular cues ensure a robust microtubule-dependent nuclear positioning in the Drosophila oocyte. Nat Commun 8: 15168. PubMed ID: 28447612
Controlling nucleus localization is crucial for a variety of cellular functions. In the Drosophila oocyte, nuclear asymmetric positioning is essential for the reorganization of the microtubule (MT) network that controls the polarized transport of axis determinants. A combination of quantitative three-dimensional live imaging and laser ablation-mediated force analysis reveal that nuclear positioning is ensured with an unexpected level of robustness. The nucleus is pushed to the oocyte antero-dorsal cortex by MTs and that its migration can proceed through distinct tracks. Centrosome-associated MTs favour one migratory route. In addition, the MT-associated protein Mud/NuMA that is asymmetrically localized in an Asp-dependent manner at the nuclear envelope hemisphere where MT nucleation is higher promotes a separate route. These results demonstrate that centrosomes do not provide an obligatory driving force for nuclear movement, but together with Mud, contribute to the mechanisms that ensure the robustness of asymmetric nuclear positioning.

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 Mushroom body defect (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).

Asymmetric cell division makes an important contribution to cell fate diversity in multicellular organisms. In asymmetric division, cells create an internal polarity axis and localize cell fate determinants to one pole. Alignment of the mitotic spindle along the axis of polarity causes the determinants to segregate into one of the two daughter cells, making each daughter cell different from its sibling. How the mitotic spindle coordinates with the polarity axis is unclear (Bowman, 2006).

Drosophila neuroblasts are a well-studied model system for asymmetric cell division. Neuroblasts undergo repeated rounds of asymmetric division, generating a larger apical cell that retains neuroblast characteristics and a smaller, basal ganglion mother cell (GMC) that divides only once more to generate two neurons. During each division, the cell fate determinants Numb, Prospero, and Brat segregate into the GMC with the help of the adaptor proteins Miranda and Pon (Partner of Numb). Asymmetric segregation of all of these proteins requires the PDZ domain proteins Bazooka (Baz, the fly homolog of Par-3) and Par-6. Together with the kinase aPKC, they form the Par complex, which is localized at the apical neuroblast cortex. It is thought that aPKC phosphorylates and inactivates the cytoskeletal protein Lethal (2) Giant Larvae (Lgl) on the apical cell cortex. Since Lgl is required for the cortical localization of cell fate determinants, this could explain why the determinants concentrate at the basal side in a Par complex-dependent manner (Bowman, 2006).

In addition to Baz, Par-6, and aPKC, several other proteins are part of the apical complex. The Armadillo repeat protein Inscuteable (Insc) binds to Baz and aPKC. Its main function seems to be in spindle orientation since the ectopic expression of Insc in epithelial cells can trigger a reorientation of the spindle along the apical-basal axis. Insc, in turn, interacts with the N terminus of Pins (Partner of Inscuteable), an adaptor protein that contains three GoLoco motifs in its C terminus. GoLoco motifs are unique in their ability to bind heterotrimeric G protein α subunits in their GDP bound form and catalyze the dissociation of βγ subunits in a receptor-independent manner. Insc, Pins, and its binding partner Gαi, as well as the Par proteins, are all required for apical-basal orientation of the mitotic spindle, suggesting that the apical complex generates an attachment site for astral microtubules to anchor the spindle in an apical-basal orientation. Since the individual members of the complex depend on one another for apical localization, it is not clear which complex member makes the molecular link with spindle microtubules (Bowman, 2006).

The role of heterotrimeric G proteins in spindle positioning is conserved in other organisms. During the first division of the C. elegans zygote, the G proteins GOA-1 and GPA-16 as well as two nearly identical GoLoco motif binding partners called GPR-1 and -2 are essential for posterior displacement of the mitotic spindle. In this case, GPR-1 and -2 bind to LIN-5, a coiled-coil protein that is also required for correct spindle positioning. Since LIN-5 localizes to the mitotic spindle, it is a good candidate for the molecule that connects G proteins to the mitotic spindle in C. elegans. G proteins are also required for spindle orientation in vertebrates. They bind to the vertebrate homolog of Pins, which, in turn, interacts with NuMA (Du, 2004; Du, 2001), a microtubule binding protein that is essential for proper organization of the mitotic spindle (Fant, 2004). NuMA can enhance microtubule polymerization (Du, 2002) and interacts with the minus end-directed motor dynein (Merdes, 1996), and both effects could provide a mechanistic explanation for a potential role in spindle orientation. However, the pleiotropic effects of NuMA in vertebrates and the lack of invertebrate homologs have so far made it impossible to directly address a requirement of NuMA for spindle orientation. It was proposed that Insc might be a functional NuMA homolog in flies, but the recent identification of Insc homologs in C. elegans and mice make this unlikely. This study identifies the Drosophila protein Mushroom Body Defect (Mud) and the C. elegans protein LIN-5 as sequence homologs of NuMA in invertebrates. Mud is shown to bind to Pins and Gαi, and, like NuMA, Mud can enhance microtubule polymerization. mud mutants have defects in spindle orientation leading to missegregation of cell fate determinants and failed asymmetric divisions that produce excess neuroblasts. These results indicate that Mud closes the gap between heterotrimeric G proteins and the mitotic spindle and regulates spindle orientation in asymmetric cell division (Bowman, 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 (Du, 2004). 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 (Srinivasan, 2003), 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 (Lechler, 2005). 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 (Sanada, 2005; Zigman, 2005). Furthermore, NuMA and Pins can create spindle-rocking movements during mitotis (Du, 2004). 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 (Lee, 2006a). 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 (Betschinger, 2006; Lee, 2006a; Lee, 2006b), 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 (Lee, 2006a). 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 (Lee, 2006a). 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).

As in Drosophila, genetic lesions affecting NuMA cause overproliferation and cancer in humans. Acute promyelocytic leukemia (APL) can be caused by a chromosomal translocation that creates a NuMA fusion protein (Wells, 1997). Expression of this fusion protein in mice causes a leukemia indistinguishable from human APL (Sukhai, 2004). In addition, variations in the NuMA gene region on chromosome 11 have been associated with breast cancer susceptibility (Kammerer, 2005). NuMA is part of a conserved heterotrimeric complex that regulates spindle orientation, and, consequently, the NuMA-like protein Mud can influence proliferation in the asymmetrically dividing and self-renewing neuroblasts of Drosophila. In light of the cancer stem cell hypothesis, which proposes that a small fraction of cells in a tumor have the ability to proliferate and self-renew, the evolutionary conservation of protein complexes that regulate spindle orientation and proliferation suggests that Drosophila neuroblasts are useful as a cancer stem cell model (Bowman, 2006).

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).


Protein Interactions

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).

Mud is part of a conserved heterotrimeric complex and interacts with microtubules

To test whether the sequence similarity results in conserved protein interactions, Mud was examined to see if it is part of a ternary complex with Pins and Gαi. Similar to previous studies (Du, 2004; Du, 2001; Du, 2002), C-terminal truncations of Mud (Mud-C) were used, containing the putative Pins and microtubule binding regions. Gαi complexes were immunoprecipitated from S2 cells transfected with myc-tagged Mud-C. In untransfected cells, Gαi can coprecipitate Pins. Upon transfection of Myc-Mud-C, immunoprecipitation of Gαi coprecipitates Pins and Myc-Mud-C. This suggests that Mud-C is in a complex with Pins and Gαi. To determine whether Mud-C binds directly to Pins, these proteins were tested in an in vitro binding assay. In vitro-translated Mud-C can bind to bacterially produced GST-Pins, but not to GST alone, indicating that a Pins-Mud-C complex can form in vitro without additional cofactors. Consistent with this, Mud coimmunoprecipitates with Pins from wild-type embryo extracts. It is concluded that Mud binds to Pins by using a C-terminal region and is part of a ternary complex with Gαi (Bowman, 2006).

Because NuMA binds to the N-terminal TPR repeats of mammalian Pins (Du, 2001), whether Mud behaves similarly in Drosophila was investigated. For this, the N-terminal TPR and C-terminal GoLoco repeats of Drosophila Pins were GFP tagged and expressed in S2 cells with Myc-Mud-C. Immunoprecipitation of GFP shows that Myc-Mud-C binds to Pins-TPR-GFP, but not to GFP-Pins-GoLoco, indicating that Pins binds to Mud by using the TPR repeats. Gαi does not bind to Mud-C in vitro, but earlier work shows that Gαi directly binds to the C-terminal GoLoco repeats of mammalian and Drosophila Pins (Du, 2004; Schaefer, 2001). Because Mud, like NuMA, binds to the N terminus of Pins, this suggests that the geometry of the heterotrimeric NuMA-Pins-Gαi complex is conserved in Drosophila (Bowman, 2006).

The conserved C-terminal fragment of human NuMA can interact with the TPR repeats of Drosophila Pins (Du, 2004), so whether Mud could bind to human Pins was tested. For this, His-HsPins-TPR and GST-Mud-C fusion proteins were produced in bacteria and used in an in vitro binding assay. His-HsPins-TPR binds to GST-Mud-C, but not to GST alone. The binding of human Pins to Drosophila Mud argues for the evolutionary conservation of this interaction. Taken together, these results demonstrate that Mud is part of a heterotrimeric complex that is highly conserved from insects to vertebrates (Bowman, 2006).

NuMA binds to microtubules and can stimulate their polymerization (Du, 2002). To find out if Mud has similar biochemical qualities, whether Mud binds microtubules in a microtubule sedimentation assay was tested. In this experiment, a soluble protein extract was created from S2 cells transfected with Myc-Mud-C. Polymerization of microtubules with GTP and taxol, followed by high-speed centrifugation, separated microtubules and microtubule binding proteins from the supernatant. As expected, α-tubulin and the microtubule binding protein Eb1 remain soluble in the absence of GTP and taxol. When microtubules are stabilized, however, these proteins can be found in the microtubule pellet along with Pins and Myc-Mud-C. It is concluded that Mud and Pins can associate with microtubules. To test whether Mud, like NuMA, can stimulate microtubule polymerization, a solution microtubule formation assay was performed. Tubulin subunits labeled with rhodamine were incubated in an energy-regenerating system with GST, with the GST-Mud-C fusion protein, or in buffer alone. After fixation of this preparation to coverslips, the number of microtubules generated was counted in ten random fields. The average number of microtubules per field formed with GST or buffer alone is less than 20, but when tubulin is incubated with GST-Mud-C, the average number of microtubules formed increases to over 100 per field. This shows that the interaction of Mud-C with tubulin is direct, and, like NuMA-C (Du, 2002), Mud-C can stimulate microtubule formation in vitro. The interaction of Mud with microtubules together with its membership in a ternary complex with Pins and Gαi strongly suggest that Mud is the functional homolog of NuMA in Drosophila (Bowman, 2006).

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).

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; S├ęgalen, 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, 2000). 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).


To analyze Mud localization in asymmetric cell division, Mud in embryonic neuroblasts were stained by using an anti-Mud antibody (Yu, 2006). At neuroblast delamination, Mud colocalizes with Pins on the apical cell cortex. This cortical localization is maintained through interphase, when alternative methods of fixation also reveal a pool of Mud on the nuclear rim (see also Yu, 2006). At metaphase, when the spindle aligns with the apical crescents of Mud and Pins, Mud can also be observed on spindle poles. At telophase, Mud preferentially segregates into the neuroblast. This localization is consistent with recent work showing that Mud decorates mitotic and meiotic spindle poles and is required for positioning spindles in meiosis II (Yu, 2006). Although Mud is expressed in larval brains, fixation conditions could not be found for analyzing Mud localization in larval tissue. It is concluded that Mud colocalizes with Pins on the cortex of asymmetrically dividing neuroblasts (Bowman, 2006).

To test if the localization of Mud depends on its binding partner Pins, pins Δ50 maternal and zygotic mutant embryos were analyzed. The apical enrichment of Mud is lost in pins mutant neuroblasts in metaphase, and the cortical association is weaker, but Mud remains associated with spindle poles. To test whether Pins is sufficient for directing apical localization of Mud, transgenic inscuteable under the control of the hsp70 promoter was used to express Inscuteable in epithelial cells. Epithelial cells normally divide parallel to the plane of the epithelium. Introduction of ectopic Inscuteable recruits Pins and Gαi from the basolateral to the apical cortex, inducing a spindle reorientation. Mud is also recruited apically and colocalizes with Pins. This suggests that Pins recruits Mud to the apical cortex of epithelial cells in the presence of Inscuteable, and, by extension, that Pins recruits Mud apically in neuroblasts. It is concluded from these experiments that Pins is required and sufficient for the apical recruitment of Mud, but that the spindle pole localization of Mud is independent of Pins. Both the apical localization of Mud and its association with microtubules are consistent with a role in spindle orientation (Bowman, 2006).


In the developing central nervous system of Drosophila, proliferation follows a reproducible and well-described spatial and temporal pattern. This pattern involves a defined number and distribution of neural stem cells (neuroblasts), as well as a precisely regulated time course of division of these neuroblasts. Mutations in the mushroom body defect (mud) gene interfere with the regulation of this pattern in a rather specific manner. In the abdominal neuromeres a subset of neuroblasts prolongs the period of proliferation. Additional daughter cells persist into the imago. Similar defects are expressed in the anterior ventral nerve cord and in the lateral central brain region. In the mushroom body cortex, however, mutations in mud affect the proliferation pattern by increasing the number of neuroblasts. These additional neuroblasts behave like normal mushroom body neuroblasts according to their time course of proliferation and the specification of their progeny (Prokop, 1994).

Mud regulates spindle orientation in asymmetric cell division

A Pins-Gαi interacting protein that also binds microtubules is a good candidate for a regulator of spindle orientation in asymmetric cell division. To find out if Mud controls spindle orientation, larval neuroblast divisions weRE analyzed in animals homozygous for mud4, a presumptive null allele affecting all Mud isoforms (Guan, 2000). For this, third instar larval brains were immunostained for Miranda and Centrosomin. Neuroblasts were defined as Miranda-expressing cells greater than 10 μm in diameter. In wild-type neuroblasts, Miranda forms a crescent in metaphase, and it segregates into a single daughter cell at telophase. In mud zygotic mutants, the Miranda crescent can be bisected by the cleavage plane and inherited by both daughter cells. Missegregation of Miranda could be due to defective spindle orientation, or it could be a secondary consequence of a general loss of polarity. Alternatively, Mud could regulate mitotic spindle morphology or formation. All mud mutant neuroblasts form crescents of aPKC and opposing crescents of Insc and Miranda. Furthermore, spindles in mud mutant neuroblasts appear bipolar with no gross morphological differences from wild-type (see also Yu, 2006). From these data, it is concluded that 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. Therefore, the spindle orientation defect is a direct consequence of Mud loss of function. To quantify this defect, the angle between a line connecting the two centrosomes and a line bisecting the crescent of Miranda was measured in metaphase neuroblasts. A small angle indicates tight coupling of the mitotic spindle with the polarity axis. In wild-type, the measured angle is almost always less than 10°. In mud mutants, the majority of spindles show more oblique orientations, and only a minority of spindles have measured angles of 10° or less. It is concluded that Mud is required for coordinating the mitotic spindle with the axis of polarity. Together, these observations demonstrate that mud mutant neuroblasts polarize correctly, but, in the absence of Mud, the polarized cortical domains cannot direct the orientation of the mitotic spindle. As a result, cell fate determinants can fail to segregate asymmetrically (Bowman, 2006).

mud mutant brains overproliferate due to an increased neuroblast pool

Mud gets its name from defective formation of the mushroom body, an adult brain structure required for olfactory learning and memory. The neurons forming the mushroom body, called Kenyon cells, are generated by four mushroom body neuroblasts that divide repeatedly throughout embryonic, larval, and pupal development. Mushroom body neuroblasts, like all neuroblasts in Drosophila, divide asymmetrically to yield a GMC that produces two neurons and a self-renewing neuroblast. Notably, wild-type mushroom body neuroblasts form crescents of Miranda, which segregate into a single small cell. This shows that neuroblasts of this lineage segregate cell fate determinants asymmetrically. In mud mutants, Miranda also forms a crescent, but spindle misorientation leads to missegregation of Miranda in ~4% of mushroom body neuroblasts. It is proposed that the remaining neuroblasts divide asymmetrically by repositioning either the spindle or the cell polarity axis during telophase. A similar rescue of defects during asymmetric cell division at late stages of mitosis has been described for other mutants, where it is called telophase rescue. Although the vast majority of mud mutant neuroblasts still divide asymmetrically, in those cells that inherit equal amounts of Miranda, and presumably equal amounts of the apical complex members known to regulate cell size asymmetry, the daughter cell size is equal. It is concluded that mushroom body neuroblasts segregate Miranda and therefore its binding partners Brat and Prospero asymmetrically, but faulty spindle orientation leads to occasional missegregation in mud mutants (Bowman, 2006).

Missegregation of cell fate determinants can result in the transformation of GMCs to neuroblasts. Therefore whether symmetric segregation of Miranda resulted in increased numbers of mushroom body neuroblasts in mud mutants was examined. For this, the mushroom body-specific GAL4 line OK107 and UAS CD8-GFP were used to label mushroom body neuroblasts and their progeny, and Miranda, which is present in neuroblasts but rapidly degraded in GMCs, was immunostained. While the number of mushroom body neuroblasts in wild-type late third instar brains is always 4, the average number of mushroom body neuroblasts in mud mutants is nearly 14. Neuroblast number increases over time; an average of around eight mushroom body neuroblasts were observed in the early third instar brains of mud mutants. From these experiments, it is concluded that mud mutants generate excess mushroom body neuroblasts, a conclusion consistent with an earlier study (Prokop, 1994). Increased numbers of neuroblasts were observed in the posterior half of the larval brain hemisphere, ventral nerve cord, and the anterior brain regions of mud mutants. Ectopic neuroblasts in mud mutants express the neuroblast marker Deadpan and incorporate BrdU, showing that they are correctly specified and mitotically active (Bowman, 2006).

In brat mutants, transformation of GMCs to neuroblasts leads to a decrease in the number of neurons. To see if this is true for mud mutants, Kenyon cells were analyzed in late third instar larval brains by using OK107-GAL4-driven CD8-GFP to mark the progeny of the mushroom body neuroblasts. Wild-type larval brains contain around 500 Kenyon cells, but, in mud mutants, the average number of Kenyon cells increases. This suggests that unlike in brat mutants, the ectopic neuroblasts in mud mutants produce ectopic progeny cells. It is proposed that the penetrant symmetric division phenotype in brat mutants makes neuroblasts nearly incapable of producing GMCs, while the frequent asymmetric divisions in mud mutants can still give rise to differentiated progeny (Bowman, 2006).

To test whether the ectopic neuroblasts and progeny cells develop normally, the morphology of the mushroom body was investigated in mud mutant adults. During development, repeated divisions of mushroom body neuroblasts sequentially generate three types of morphologically distinct Kenyon cells. These neurons project axons that form the characteristic lobed structure of the adult mushroom body. Kenyon cells born from late embryogenesis to the early third instar project their axons into the γ lobe, Kenyon cells born between early third instar and puparium formation project branched axons into the α′ and β′ lobes, and, finally, Kenyon cells born after puparium formation project their branched axons into the α and β lobes. Like larval brains, mud mutant adult brains contain an increased number of Kenyon cells. The number of wild-type Kenyon cells is lower than expected, but OK107 may not detectably label every Kenyon cell. Interestingly, while a small γ lobe is present in mud mutants, the ectopic Kenyon cells are unable to project axons into the α′β′ or αβ lobes. The absence of these lobes could indicate a role for Mud in axon guidance. Alternatively, the presence of the γ lobe suggests that Kenyon cells born early in development, when the maternal supply of Mud is sufficient, are correctly specified. Consistent with this, the mud mutant γ lobe expresses low levels of Fasciclin-II, as in wild-type γ lobes. At later stages, the reduced levels of Mud may result in misspecification of the α′β′ and αβ neurons, resulting in an absence of projections. Taken together, these observations suggest that in mud mutants, the occasional transformation of a GMC into a neuroblast causes overproliferation and cell fate misspecification (Bowman, 2006).

The mushroom body defect gene product is an essential component of the meiosis II spindle apparatus in Drosophila oocytes

In addition to their well-known effects on the development of the mushroom body, mud mutants are also female sterile. This study shows that, although the early steps of ovary development are grossly normal, a defect becomes apparent in meiosis II when the two component spindles fail to cohere and align properly. The products of meiosis are consequently mispositioned within the egg and, with or without fertilization, soon undergo asynchronous and spatially disorganized replication. In wild-type eggs, Mud is found associated with the central spindle pole body that lies between the two spindles of meiosis II. The mutant defect thus implies that Mud should be added to the short list of components that are required for the formation and/or stability of this structure. Mud protein is also normally found in association with other structures during egg development: at the spindle poles of meiosis I, at the spindle poles of early cleavage and syncytial embryos, in the rosettes formed from the unfertilized products of meiosis, with the fusomes and spectrosomes that anchor the spindles of dividing cystoblasts, and at the nuclear rim of the developing oocyte. In contrast to its important role at the central spindle pole body, in none of these cases is it clear that Mud plays an essential role. But the commonalities in its location suggest potential roles for the protein in development of other tissues (Yu, 2006).

This work describes a fully penetrant phenotype that is associated with strong mutations in the mud gene. Specifically, mud mutants have a striking maternal-effect lethal phenotype: although eggs produced by mutant females look normal and accept sperm, they fail to undergo normal cleavage divisions. Instead of forming a well-ordered syncytial blastoderm, these eggs accumulate disordered arrays of replicated material and then necrose. A number of mutants in other genes have been reported to have abnormal early embryonic mitoses. One class of these, comprising strong alleles of the genes pan gu (png), plutonium (plu), and giant nuclei (gnu), reflects defects in a complex with protein kinase activity. It seems unlikely that mud is a member of this class. While png, plu, and gnu mutants typically form one to five giant nuclei, strong alleles of mud characteristically generate dozens of smaller chromatin masses that are scattered throughout the egg. Moreover, in contrast to mutations in genes of the giant nuclei class, mud mutations do not interfere with degradation of maternal mRNA. A closer resemblance to the mud phenotype is shown by mutants that have defects in spindle-associated proteins. In such mutants, as in mud, the meiotic products undergo inappropriate and disorganized mitoses that produce eggs with scattered chromatin masses. The possibility is considered that Mud and the products of one or more of these genes (ncd, alphaTub67C, or gammaTub37C) serve mutually interdependent roles. If so, double heterozygotes might display a maternal-effect phenotype lacking in any of the single heterozygotes. However, when either the mud3 or the mud4 mutation was combined with the ncdD, alphaTub67C1, or gammaTub37C3 mutation, no decrease in fertility was seen over the robust levels seen in the single heterozygotes. Although this outcome is not decisive, it argues against possibilities such as Mud being involved in the localization of these spindle components. In any case, it should be emphasized that the observation that unfertilized mutant eggs undergo the same rounds of disorganized mitoses as do early embryos strongly implies that the mud maternal lethal phenotype is due to a defect in meiosis and not in a subsequent stage of development (Yu, 2006).

What is the basis for the meiotic defect? A central spindle pole body that incorporates several centrosomal proteins together with a diffuse aster of alpha-tubulin is normally found between the two spindles that make up the meiosis II apparatus. As judged by alpha-tubulin staining, this structure is defective or absent in mud mutant eggs. In wild-type eggs, Mud is found associated with the spindle pole body, implying that the protein is needed for the formation or stability of this structure. Because Mud staining appears to surround the alpha-tubulin framework, an attractive possibility is that the former is needed for the latter to be recruited to or be maintained at the central spindle pole body. In any case, just as with weak alleles of polo, which also show defects in the formation of the central spindle pole body, disjoined meiosis II spindles, and disorganized early mitoses, the observations of this study provide a plausible scenario for the female sterility of mud mutants. To wit, without a functional spindle pole body the two meiosis II spindles are not properly held together and the products of meiosis do not get correctly positioned with respect to the egg cortex. It is presumed that positioning is important for condensation of the meiotic products into inactive polar bodies. These structures may normally serve to shield the dead-end meiotic products from the replication machinery, which according to this scenario is competent to operate in unfertilized Drosophila eggs. If so, when proper condensation fails, inappropriate replication ensues. Regardless of the correctness of this model, the similar phenotypes of mud and polo mutations suggest that corresponding proteins might serve interdependent roles in activated eggs. But no decrease in fertility was observed in double heterozygotes of polo1 and either mud3 or mud4. It remains to be seen whether Polo and Mud are similarly distributed in activated oocytes (Yu, 2006).

In addition to its association with the central spindle pole body, Mud protein is readily detected at other spindle poles. However, in those cases where it can be tested, it appears that Mud is not essential for the formation or function of these structures. In this regard, Mud is reminiscent of the fly orthologs of CP190 and pericentrin, which, despite being consistently found at the centrosome, are dispensable for mitosis. It may be that the structures built at the spindle poles are designed to withstand the undersupply of a few ingredients. If so, it might be useful to look for synthetic phenotypes when mud mutations are combined with those of other genes whose products are concentrated at spindle poles. Of course, because eggs that lack a maternal contribution of functional Mud develop so anomalously, it is undecided whether it plays an essential role at the spindle poles of embryonic mitoses or in polar bodies. Insight into these cases will have to await the availability of a conditional mutant that can be shifted to nonpermissive conditions after meiosis II is completed. Despite the doubts about its role at places other than the central spindle pole body, the frequent associations of Mud with microtubular structures in the oocyte and egg suggest that the protein might be regarded as a MAP; direct tests of this hypothesis are underway. Another attractive, albeit speculative, idea is that Mud is functionally related to the vertebrate NuMA protein. Like Mud, NuMA is a large coiled-coil protein that is found at spindle poles. The two proteins are apparently not orthologous in that they cannot be globally aligned outside of their coiled-coil regions; they also differ in subcellular localization during interphase. However, the carboxy terminus of one particular isoform of Mud shares a short region of similarity with NuMA, hinting at a conserved interaction (Yu, 2006).

Two other sites of Mud localization that suggest a connection with microtubules are fusomes and spectrosomes. These are membrane-rich structures that form during the earliest stages of ovary development and are surrounded by microtubules. Mud is present within these structures at a time when they serve to anchor the mitotic spindles of the dividing cystoblasts but not later, when they serve to focus the microtubule network in postmitotic cells. Although this distribution hints at a role for Mud in spindle anchoring, inactivation of the gene causes no obvious defect in fusomes or spectrosomes. These structures not only form normally in mud mutants but the processes they govern during early oogenesis proceed without defect. It is concluded that, if Mud plays a role in building fusomes and spectrosomes or connecting them to spindles, it is a role for which there is adequate redundancy (Yu, 2006).

In cells that are not in metaphase or anaphase, Mud can also be found at the nuclear envelope. It is not clear how Mud gets from the nuclear rim of the oocyte and the early embryo, respectively, to the meiotic and mitotic spindle apparatus. In contrast, Mud might lose contact with the nuclear envelope, where it could have been held by its transmembrane domain, and be directed to the spindle by a distinct targeting signal. But, since the spindles of the Drosophila germ line and pre-syncytial egg are typically enclosed by membranous structures, the movement of Mud might be part of a concerted redistribution of elements of the nuclear envelope. Although the mechanism is thus unclear, it should be pointed out that shuffling between the nuclear envelope and the spindle is not unique to Mud but has been reported for several other proteins. Another open question is whether Mud plays a microtubule-related role at the nuclear rim. However, even though the oocyte nucleus is surrounded by a cage of microtubules and the dynactin complex is concentrated at its rim, the structure and positioning of the oocyte nucleus are not grossly affected by loss of Mud function. Accordingly, the idea is favored that Mud is simply stored at the nuclear rim to ensure a local supply for subsequent delivery to the spindle apparatus but the possibility that a parallel system renders obscure a more active function for Mud at the nuclear envelope cannot be ruled out (Yu, 2006).

To what extent do the observations on the female sterility shed light on the other phenotypes of mud mutants, particularly the role of Mud in the development of the adult nervous system? It is easy to imagine that a protein that can associate with the nuclear envelope might be part of the mechanism that prepares the way for exit from the cell cycle. And a protein that is commonly found around spindle poles might be involved in the regulation of spindle orientation that governs the transition from symmetric to asymmetric division of neuroblasts and thus the switch from neuroblast proliferation to stem cell behavior. Similarly, a protein that associates with microtubules might play a role in the precision of growth cone movement that is needed for proper axon pathfinding. Thus, although speculative, concrete suggestions for places to look for Mud action in the nervous system can be gleaned from the insights gained from a description of this protein in the oocyte and early embryo (Yu, 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).

Apical/basal spindle orientation is required for neuroblast homeostasis and neuronal differentiation in Drosophila

Precise regulation of stem cell self-renewal/differentiation is essential for embryogenesis and tumor suppression. Drosophila neural progenitors (neuroblasts) align their spindle along an apical/basal polarity axis to generate a self-renewed apical neuroblast and a differentiating basal cell. This study genetically disrupted spindle orientation without altering cell polarity to test the role of spindle orientation in self-renewal/differentiation. Correlative live imaging of polarity markers and spindle orientation over multiple divisions were performed within intact brains, followed by molecular marker analysis of cell fate. It was found that spindle alignment orthogonal to apical/basal polarity always segregates apical determinants into both siblings, which invariably assume a neuroblast identity. Basal determinants can all be localized into one sibling without inducing neuronal differentiation, but overexpression of the basal determinant Prospero can deplete neuroblasts. It is concluded that the ratio of apical/basal determinants specifies neuroblast/GMC identity, and that apical/basal spindle orientation is required for neuroblast homeostasis and neuronal differentiation (Cabernard, 2009).

It is critical for these studies to use mutants that affect spindle orientation but not cortical polarity, so it was first confirmed that the spindle orientation mutants mud and cnn had no detectable effect on apical/basal cortical polarity. It was found that 12-13% of the metaphase neuroblasts in these mutants showed aberrant spindle orientation orthogonal to the apical/basal cortical polarity axis, and that both had an increased number of brain neuroblasts. Thus, these two mutants are appropriate tools for studying the role of spindle orientation in regulating neuroblast self-renewal versus differentiation (Cabernard, 2009).

To determine if mud and cnn mutants act autonomously within neuroblast lineages to increase neuroblast number, as predicted if neuroblast spindle orientation defects lead to increased brain neuroblast numbers, GFP-marked mutant clones were generated within single neuroblasts using the MARCM technique. Clones were induced in first instar larvae and analyzed in third instar larvae. Mutant clones were identified by GFP expression and scored for the neuroblast markers Deadpan (Dpn) or Mira and the GMC/neuron marker nuclear Pros (nPros). Wild-type single neuroblast clones always contained a single large Dpn+ nPros neuroblast and several smaller nPros+ GMC/neurons. In contrast, mud and cnn mutant single neuroblast clones often contained two or more Dpn+ nPros neuroblasts; the multiple neuroblasts in a clone were of similar size and were always tightly adjacent. Importantly, mutant clones containing zero neuroblasts were never observed, which would be expected if defects in spindle orientation resulted in some divisions producing GMC/GMC siblings, and live imaging confirms that GMC/GMC siblings are never generated. These data are consistent with a model in which mud and cnn mutant neuroblasts generate neuroblast/neuroblast sibling cells, but not GMC/GMC sibling cells (Cabernard, 2009).

To determine whether ectopic neuroblasts in mud and cnn mutants arise occasionally or invariably from neuroblast orthogonal divisions, live imaging of neuroblast cell lineages was performed within intact larval brains. This method allowed tracking of individual neuroblasts from mitotic spindle orientation through to subsequent sibling cell fates. Spindle orientation was monitored with a microtubule-associated Cherry::Jupiter fusion protein, cortical polarity was monitored using the basal marker GFP::Mira, and neuroblast/GMC cell fates were determined by multiple cell biological criteria (subsequent cell division profile, cell lineage, cell cycle length, and cell size. Wild-type neuroblasts always showed apical/basal spindle orientation, production of unequally sized daughter cells, and partitioning of the basal cortical marker GFP::Mira into the smaller daughter cell. As expected, cnn and mud mutant neuroblasts also frequently showed normal apical/basal spindle orientation, divided asymmetrically, and generated neuroblast/GMC siblings (Cabernard, 2009).

Importantly, a subset of cnn and mud mutant neuroblast divisions showed spindle orientation orthogonal to the apical/basal polarity axis, allowing determination of the role of spindle orientation in neuroblast self-renewal versus differentiation. Live imaging showed that neuroblasts undergoing orthogonal divisions always generated equally sized siblings that both invariably assumed a neuroblast identity based on their ability to maintain a neuroblast-like short cell cycle and ability to subsequently undergo asymmetric cell division. To provide an independent molecular assay of sibling cell identity, correlative microscopy was performed in which live imaging was used to identify orthogonal neuroblast cell divisions and then subsequently the identical neuroblast lineage were fixed and stained for molecular marker expression. It was found that neuroblast orthogonal divisions always generated two siblings that expressed the neuroblast marker Deadpan (Dpn) and lacked the differentiation marker nPros. It is concluded that neuroblast orthogonal divisions always generate two equally sized cells that assume a neuroblast identity: they have a short cell cycle, can divide asymmetrically, express the neuroblast marker Dpn, and lack the GMC/neuronal marker nPros. Thus, altering neuroblast spindle orientation from apical/basal to orthogonal results in the invariant production of two sibling neuroblasts, based on both cell biological and molecular criteria (Cabernard, 2009).

Neuroblasts dividing orthogonally to the apical/basal polarity axis invariably generate two sibling neuroblasts. To determine how apical/basal cortical determinants correlate with cell fate specification -- if they correlate at all, the partitioning of apical or basal cortical domains was quantitated in cnn or mud mutant orthogonal neuroblast divisions. As expected, wild-type or mutant neuroblasts with apical/basal spindle orientation always segregated the majority of the apical marker Baz::GFP into the neuroblast, and the majority of the basal marker Cherry::Mira into the GMC. In contrast, mud mutant neuroblasts with orthogonal spindle orientation always segregated the apical marker Baz::GFP equally into both sibling cells. The apical protein aPKC is also symmetrically partitioned during orthogonal divisions. The basal marker Cherry::Mira could also be segregated equally to both siblings, but surprisingly was more frequently partitioned unequally to only one sibling. Similar results were obtained with cnn mutant neuroblasts. Clearly the segregation of all basal determinants into just one sibling was insufficient to induce neuronal differentiation, as all orthogonal divisions generated two sibling neuroblasts. It is concluded that the apical cortical domain is perfectly correlated with acquisition of neuroblast identity, whereas the basal cortical domain is insufficient to specify GMC identity (Cabernard, 2009).

Orthogonal neuroblast divisions always partition apical proteins into both siblings and always generate two neuroblasts; basal proteins can all be localized into one sibling without inducing differentiation. It is thus tempting to conclude that only apical proteins are used to specify cell fate. However, an alternative model is that cell fate is determined by the ratio of apical:basal proteins and that a sibling containing half the apical proteins and all of the basal proteins still has an apical:basal ratio high enough to promote neuroblast identity (Cabernard, 2009).

It is possible to distinguish between these two models by increasing the amount of the basal cell fate determinant Prospero: the 'apical dominant' model predicts no effect on neuroblast identity, whereas the 'apical:basal ratio' model predicts at least some loss of neuroblast identity. It was found that overexpressing Prospero in neuroblasts results in coexpression of nuclear Prospero and the neuroblast marker Deadpan and a striking depletion of larval neuroblasts. Importantly, no change was observed in the localization or function of apical cortical proteins: Baz::GFP formed an apical crescent, and aPKC was able to exclude Miranda from the apical cortex. Thus, increasing the amount of the cell fate determinant Prospero, without altering apical cortical proteins, is sufficient to block neuroblast specification or maintenance, resulting in a decrease in neuroblast numbers. It is concluded that the ratio of apical:basal cortical polarity markers is important for determining neuroblast/GMC identity and that apical/basal spindle orientation maintains neuroblast homeostasis and promotes neuronal differentiation by allowing the production of a basal cell with a high basal:apical ratio of cell fate determinants (Cabernard, 2009).

This study used a combination of genetic mutants that specifically disrupt spindle orientation without affecting cell polarity, live imaging of apical/basal spindle orientation for multiple neuroblast divisions within intact larval brains, and correlative microscopy to determine the molecular profile of terminal progeny of the imaged lineages. The results show that apical/basal spindle orientation is essential for maintaining neuroblast pool size and promoting neuronal differentiation: direct observation shows that all mutant neuroblasts with orthogonal spindle orientation generate two neuroblast siblings, whereas all mutant and wild-type neuroblasts with apical/basal spindle orientation generate neuroblast/GMC siblings. This provides strong evidence that spindle orientation defects in these mutants lead to the observed increase in neuroblast numbers, rather than other possible defects including brain patterning, nonautonomous effects in glia or GMCs, or altered cell polarity (Cabernard, 2009).

Analysis of orthogonal divisions reveals that only apical proteins are correlated with cell fate (being 100% correlated with neuroblast identity), whereas inheritance of all the basal proteins by one sibling is insufficient to induce neuronal differentiation. This is strikingly similar to mammalian embryonic neural stem cells, where only the apical cortical domain is correlated with self-renewal, while the basolateral and adherens junctional domains distribute independently of cell fate. Nevertheless, this study shows that the apical cortical domain is not the sole determinant of cell fate, but rather it is the ratio of apical:basal proteins that specifies neuroblast/GMC identity. This model is supported by the observation that increasing levels of the apical determinant aPKC can switch GMCs into neuroblasts, and that decreasing the levels of basal determinants can turn GMCs into neuroblasts. A high apical:basal ratio may promote neuroblast identity by inactivating basal proteins, increasing cell size, promoting cell proliferation, or altering centrosome composition/function. Conversely, a high basal:apical ratio may promote differentiation via Prospero repression of genes promoting cell proliferation or neuroblast identity, by Brain tumor suppression of Myc-dependent cell growth, and/or by Numb inhibition of Notch-dependent neuroblast self-renewal (Cabernard, 2009).

In wild-type Drosophila neuroblasts, the mitotic spindle is always aligned with the apical/basal axis, which maintains neuroblast pool size and allows neuronal differentiation. In other insects and mammals, regulated spindle orientation may allow switching between neural progenitor expansion and homeostasis. In the honeybee Apis, mushroom body neuroblasts expand via symmetric divisions prior to switching to an asymmetric division mode to generate neurons. Neuroblast expansion may be due to an increased apical:basal determinant ratio or a phase of orthogonal spindle orientation. Similarly, mammalian neural progenitors switch between phases of progenitor expansion, homeostasis, and depletion. Clues that spindle orientation plays an important role come from the analysis of mammalian mutants CDK5RAP2 and lis1, which cause microcephaly in mammals; the orthologous Drosophila mutants cnn and lis1 both disrupt spindle orientation, but not cortical polarity, and lead to an increase in neuroblast numbers. However, the respective contribution of apical/basal determinant ratio and spindle orientation remains to be determined in mammals, primarily due to the lack of candidate cell fate determinants and the difficulty of performing correlative microscopy within intact brain tissue. These results suggest that concurrent live imaging of cell polarity, spindle orientation, and sibling cell fate will be necessary to determine the role of spindle orientation in regulating mammalian neural stem cell self-renewal versus differentiation (Cabernard, 2009).

Microtubule-driven nuclear rotations promote meiotic chromosome dynamics

At the onset of meiosis, each chromosome needs to find its homologue and pair to ensure proper segregation. In Drosophila, pairing occurs during the mitotic cycles preceding meiosis. This study shows that germ cell nuclei undergo marked movements during this developmental window. It was demonstrated that microtubules and Dynein drive nuclear rotations and are required for centromere pairing and clustering. It was further found that Klaroid (SUN) and Klarsicht (KASH) co-localize with centromeres at the nuclear envelope and are required for proper chromosome motions and pairing. Mud (NuMA in vertebrates) was identified as co-localizing with centromeres, Klarsicht and Klaroid. Mud is also required to maintain the integrity of the nuclear envelope and for the correct assembly of the synaptonemal complex. These findings reveal a mechanism for chromosome pairing in Drosophila, and indicate that microtubules, centrosomes and associated proteins play a crucial role in the dynamic organization of chromosomes inside the nucleus (Christophorou, 2015).

Rotations of nuclei have been described previously in somatic cells; their function remains however unclear. In germ cells, meiotic chromosome movements are thought to be required for homologue pairing, removing chromosome entanglements, promoting maturation of recombination intermediates, or for assessing chromosome homology before synapsis, in different model organisms. In Drosophila, a high temporal correlation was found between nuclear rotations and chromosome pairing occurring mainly in 8-cell cysts. This work uncovered a second interesting correlation between the speed of nuclear rotation and the degree of centromere pairing and clustering. Indeed, mutations in klaroid affected the least nuclear rotations and disrupted the least centromere associations and synapsis. Rotations were slowed down more significantly in klarsicht, sas-4 and asl mutant germ cells. Accordingly, strong defects were observed in the initial pairing of centromeres and in synaptonemal complex formation. Finally, nuclear rotations were completely abolished in the absence of Dynein or dynamic microtubules. In dynein mutant germ cells, an average of six centromeres were distinguished during pre-meiotic pairing, which is higher than any mutants tested previously, including null alleles of c(3)G. Similarly, five centromeres on average were coundted during clustering in region 2a, a mutant phenotype that is comparable to the strongest orientation disruptor (ord) or c(3)G mutations (lateral and central elements of synaptonemal complex respectively). Nuclear rotations thus play an important role in homologue chromosome pairing and synaptonemal complex formation (Christophorou, 2015).

It was found that microtubules could be nucleated from the fusome, the nuclear envelope and the centrosome in region 1 germ cells. On the basis of these observations and centrosome mutant analysis, it is speculated that the whip-like movements of microtubules could be the main forces creating cytoplasmic flows, as observed in many biological systems and demonstrated theoretically. In addition, microtubules nucleated by the centrosomes could also push on the nucleus and the cell membrane, which could bias nuclear movement towards one direction of rotation as proposed for the migration of this same oocyte. These two forces depend on microtubules and dynein, and would act redundantly for efficient and unidirectional nuclear rotations. However, even in the absence of dynamic microtubules, centromeres ended up paired, albeit much later in region 2b. Synapsis, on the other hand, was completely disrupted. It is thus believed that, as in yeast and worms, these movements are there to facilitate pairing, synapsis or recombination, but that at least chromosome pairing could occur slowly without motions by redundant mechanisms. In flies, Spag4 is a second SUN-domain protein, but it is only expressed in male testis and is thus not likely to play a role during oogenesis. There is also a second KASH-domain protein called MSP-300/Nesprin, which interacts with the actin cytoskeleton. In the absence of microtubules, nuclei were not ‘rolling anymore; however, they still showed some back and forth ‘rocking movements. It will be interesting to investigate whether MSP300/Nesprin and the actin cytoskeleton are involved in these rocking movements (Christophorou, 2015).

This study found that although mud mutant ovaries showed only mild defects in centromere dynamics, significant genetic interactions were uncovered with klaroid and klarsicht in this same process. Striking features of Mud in this study were its co-localization with centromeres in interphasic germline cysts and the formation of polycomplexes in mud mutant cysts. The formation of polycomplexes was associated with a lack of nuclear membrane and diffused DNA in the cytoplasm, suggesting that Mud is required to maintain nuclear envelope integrity. It is proposed that the disappearance of the NE in mud cysts is the primary defect leading first to the de-localization of DNA into the cytoplasm and then the formation of polycomplexes. Polycomplexes could thus be the result of self-assembly of synaptonemal complex components polymerizing in the absence of chromatin. Polycomplexes were also observed in klaroid and klarsicht mutants although at a lower penetrance than in mud mutants. Interestingly, large distortions of the NE were also observed in muscle cell nuclei mutant for unc-84, which encodes a C. elegans SUN protein. These deformations were particularly strong in these cells, because muscle cell nuclei are subjected to mechanical stress. It is likely that rolling nuclei of 8-cell cysts are also exposed to some mechanical forces. Klarsicht, Klaroid and Mud may all participate in maintaining the integrity of the nuclear envelope in these conditions. In their absence, the NE is weakened and cannot resist mechanical forces, which also leads to synaptonemal complex assembly defects. In the most extreme cases the NE completely disappears causing the formation of polycomplexes. Interestingly, Mud initially localizes at the NE of all germline cells in region 1, but then becomes localized only to the cells remaining in meiosis in region 2a, and finally only specifically at the NE of the oocyte. This may hint that the meiotic nucleus is subjected to specific mechanical forces during oogenesis (Christophorou, 2015).


LIN-4, the Mud homolog of C. elegans

Successful divisions of eukaryotic cells require accurate and coordinated cycles of DNA replication, spindle formation, chromosome segregation, and cytoplasmic cleavage. The Caenorhabditis elegans gene lin-5 is essential for multiple aspects of cell division. Cells in lin-5 null mutants enter mitosis at the normal time and form bipolar spindles, but fail chromosome alignment at the metaphase plate, sister chromatid separation, and cytokinesis. Despite these defects, cells exit from mitosis without delay and progress through subsequent rounds of DNA replication, centrosome duplication, and abortive mitoses. In addition, early embryos that lack lin-5 function show defects in spindle positioning and cleavage plane specification. The lin-5 gene encodes a novel protein with a central coiled-coil domain. This protein localizes to the spindle apparatus in a cell cycle- and microtubule-dependent manner. The LIN-5 protein is located at the centrosomes throughout mitosis, at the kinetochore microtubules in metaphase cells, and at the spindle during meiosis. These results show that LIN-5 is a novel component of the spindle apparatus required for chromosome and spindle movements, cytoplasmic cleavage, and correct alternation of the S and M phases of the cell cycle (Lorson, 2000).

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).

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).

Successful cell division requires proper assembly, placement and functioning of the spindle apparatus that segregates the chromosomes. The Caenorhabditis elegans gene lin-5 encodes a novel coiled-coil component of the spindle required for spindle positioning and chromosome segregation. To gain further insights into lin-5 function, a screen was performed for dominant suppressors of the partial loss-of-function phenotype associated with the mutation lin-5ev571ts, and 68 suppressing mutations were isolated. Eight out of the ten suppressors sequenced contained intragenic missense mutations immediately upstream of the lesion in lin-5ev571ts. These probably help to stabilize protein-protein interactions mediated by the coiled-coil domain. This domain was found to be required for binding to several putative LIN-5 interacting (LFI) proteins identified in yeast two-hybrid screens. Interestingly, interaction with the coiled-coil protein LFI-1 was specifically reduced by the lin-5ev571ts mutation and restored by a representative intragenic suppressor mutation. Immunostaining experiments showed that LIN-5 and LFI-1 may co-localize around the kinetochore microtubules during metaphase, indicating potential interaction in vivo. The coiled-coil domain of LIN-5 was also found to mediate homodimerization, while the C-terminal region of LIN-5 was sufficient for interaction with GPR-1, a recently identified component of a LIN-5 spindle-regulatory complex. A single amino-acid substitution in the N-terminal region of LIN-5, encoded by the e1457 allele, abolished all LIN-5 interactions. Taken together, these results indicate that the spindle functions of LIN-5 depend on interactions with multiple protein partners, and that these interactions are mediated through several different domains of LIN-5 (Fisk Green, 2004).

NuMA interaction with LGN/AGS3, a mammalian homolog of Drosophila Pins

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, 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).

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 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 8A catalyzes release of Galphai-GTP and nuclear mitotic apparatus protein (NuMA) from NuMA/LGN/Galphai-GDP complexes

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).

NuMA is a substrate for covalent modification by tankyrase-1

Poly(ADP-ribose) (PAR) is a large, negatively charged post-translational modification that is produced by polymerization of NAD+ by PAR polymerases (PARPs). There are at least 18 PARPs in the human genome, several of which have functions that are unknown. PAR modifications are dynamic; PAR structure depends on the balance between synthesis and hydrolysis by PAR glycohydrolase. PAR is enriched in vertebrate somatic-cell mitotic spindles and a requirement for PAR in the assembly of Xenopus egg extract spindles has been demonstrated. This study performed a knockdown of all characterized PARPs using RNA interference (RNAi), and tankyrase-1 was identified as the PARP that is required for mitosis. Tankyrase-1 localizes to mitotic spindle poles, to telomeres and to the Golgi apparatus. Tankyrase-1 RNAi was recently shown to result in mitotic arrest, with abnormal chromosome distributions and spindle morphology observed--data that is interpreted as evidence of post-anaphase arrest induced by failure of telomere separation. Tankyrase-1 RNAi results in pre-anaphase arrest, with intact sister-chromatid cohesion. A requirement for tankyrase-1 has been demonstrated in the assembly of bipolar spindles, and the spindle-pole protein NuMA has been identified as a substrate for covalent modification by tankyrase-1 (Chang, 2005).

NuMA interaction with microtubules

In mitosis, NuMA localises to spindle poles where it contributes to the formation and maintenance of focussed microtubule arrays. Previous work has shown that NuMA is transported to the poles by dynein and dynactin. So far, it is unclear how NuMA accumulates at the spindle poles following transport and how it remains associated throughout mitosis. This study shows that NuMA can bind to microtubules independently of dynein/dynactin. A 100-residue domain located within the C-terminal tail of NuMA has been characterized that mediates a direct interaction with tubulin in vitro and that is necessary for NuMA association with tubulin in vivo. Moreover, this domain induces bundling and stabilisation of microtubules when expressed in cultured cells and leads to formation of abnormal mitotic spindles with increased microtubule asters or multiple poles. These results suggest that NuMA organises the poles by stable crosslinking of the microtubule fibers (Haren, 2002).

NuMA interaction with spindle poles

The large coiled-coil protein NuMA plays an essential role in organizing microtubule minus ends at spindle poles in vertebrate cells. This study used both in vivo and in vitro methods to examine NuMA dynamics at mitotic spindle poles. Using fluorescence recovery after photobleaching, an exogenously expressed green-fluorescent-protein/NuMA fusion was shown to undergo continuous exchange between soluble and spindle-associated pools in living cells. These dynamics require cellular energy and display an average half-time for fluorescence recovery of approximately 3 minutes. To explore how NuMA dynamics at spindle poles is regulated, the association was exploited of NuMA with microtubule asters formed in mammalian mitotic extracts. Using a monoclonal antibody specific for human NuMA, the fate of human NuMA associated with microtubule asters was followed upon dilution with a hamster mitotic extract. Consistent with in vivo data, this assay shows that NuMA can be displaced from the core of pre-assembled asters into the soluble pool. The half-time of NuMA displacement from asters under these conditions is approximately 5 minutes. Using this assay, it was shown that protein kinase activity and the NuMA-binding protein LGN regulate the dynamic exchange of NuMA on microtubule asters. Thus, the dynamic properties of NuMA are regulated by multiple mechanisms including protein phosphorylation and binding to the LGN protein, and the rate of exchange between soluble and microtubule-associated pools suggests that NuMA associates with an insoluble matrix at spindle poles (Kisurina-Evgenieva, 2004).

NuMA interaction with dynein/dynactin

NuMA is a nuclear protein during interphase but redistributes to the spindle poles early in mitosis. To investigate its role during spindle formation, spindle assembly was tested in frog egg extracts from which NuMA was immunodepleted. Immunodepletion revealed that NuMA forms a complex with cytoplasmic dynein and dynactin. The depleted extracts failed to assemble normal mitotic spindles, producing, instead, chromatin-associated irregular arrays of microtubules lacking characteristic spindle poles. A subdomain of the NuMA tail was shown to induce microtubule aster formation by mediating microtubule bundling. These findings suggest that NuMA forms bifunctional complexes with cytoplasmic dynein and dynactin that can tether microtubules at the spindle poles and that are essential for mitotic spindle pole assembly and stabilization (Merdes, 1996).

NuMA is a large nuclear protein whose relocation to the spindle poles is required for bipolar mitotic spindle assembly. This process depends on directed NuMA transport toward microtubule minus ends powered by cytoplasmic dynein and its activator dynactin. Upon nuclear envelope breakdown, large cytoplasmic aggregates of green fluorescent protein (GFP)-tagged NuMA stream poleward along spindle fibers in association with the actin-related protein 1 (Arp1) protein of the dynactin complex and cytoplasmic dynein. Immunoprecipitations and gel filtration demonstrate the assembly of a reversible, mitosis-specific complex of NuMA with dynein and dynactin. NuMA transport is required for spindle pole assembly and maintenance, since disruption of the dynactin complex (by increasing the amount of the dynamitin subunit) or dynein function (with an antibody) strongly inhibits NuMA translocation and accumulation and disrupts spindle pole assembly (Merdes, 2000).

The protein NuMA localizes to mitotic spindle poles where it contributes to the organization of microtubules. NuMA loses its stable association with the spindle poles after anaphase onset. Using extracts from Xenopus laevis eggs, this study shows that NuMA is dephosphorylated in anaphase and released from dynein and dynactin. In the presence of a nondegradable form of cyclin B (Delta90), NuMA remains phosphorylated and associated with dynein and dynactin, and remains localized to stable spindle poles that fail to disassemble at the end of mitosis. Inhibition of NuMA or dynein allows completion of mitosis, despite inducing spindle pole abnormalities. It is proposed that NuMA functions early in mitosis during the formation of spindle poles, but is released from the spindle after anaphase, to allow spindle disassembly and remodelling of the microtubule network (Gehmlich, 2004).

NuMA function in meiosis

During the maturation of Xenopus oocytes, a transient microtubule array (TMA) is nucleated from a novel MTOC near the base of the germinal vesicle. The MTOC-TMA transports the meiotic chromosomes to the animal cortex, where it serves as the precursor to the first meiotic spindle. To understand more fully the assembly of the MTOC-TMA, confocal immunofluorescence microscopy was used to examine the localization and function of XMAP215, XKCM1, NuMA, and cytoplasmic dynein during oocyte maturation. XMAP215, XKCM1, and NuMA were all localized to the base of the MTOC-TMA and the meiotic spindle. Microinjection of anti-XMAP215 inhibits microtubule (MT) assembly during oocyte maturation, disrupting assembly of the MTOC-TMA and subsequent assembly of the first meiotic spindle. In contrast, microinjection of anti-XKCM1 promotes MT assembly throughout the cytoplasm, disrupting organization of the MTOC-TMA and meiotic spindle. Finally, microinjection of anti-dynein or anti-NuMA disrupts the organization of the MTOC-TMA and subsequent assembly of the meiotic spindles. These results suggest that XMAP215 and XKCM1 act antagonistically to regulate MT assembly and organization during maturation of Xenopus oocytes, and that dynein and NuMA are required for organization of the MTOC-TMA (Becker, 2003).

NuMA and epidermis

The epidermis is a stratified squamous epithelium forming the barrier that excludes harmful microbes and retains body fluids. To perform these functions, proliferative basal cells in the innermost layer periodically detach from an underlying basement membrane of extracellular matrix, move outward and eventually die. Once suprabasal, cells stop dividing and enter a differentiation program to form the barrier. The mechanism of stratification is poorly understood. Although studies in vitro have led to the view that stratification occurs through the delamination and subsequent movement of epidermal cells, most culture conditions favour keratinocytes that lack the polarity and cuboidal morphology of basal keratinocytes in tissue. These features could be important in considering an alternative mechanism, that stratification occurs through asymmetric cell divisions in which the mitotic spindle orients perpendicularly to the basement membrane. This study shows that basal epidermal cells use their polarity to divide asymmetrically, generating a committed suprabasal cell and a proliferative basal cell. It is further demonstrated that integrins and cadherins are essential for the apical localization of atypical protein kinase C, the Par3-LGN-Inscuteable complex and NuMA-dynactin to align the spindle (Leuchler, 2005).

NuMA and tumors

Acute promyelocytic leukaemia (APL) is uniquely associated with chromosomal translocations that disrupt the gene encoding the retinoic acid receptor, RARA. In more than 99% of cases, this disruption results in the formation of a PML-RARA gene fusion. Two rare variants of APL have been described, in which RARA is fused to one of two other genes, PLZF and NPM. Although RARA dysregulation is evidently important in APL, the role of the various fusion partners remains unclear. A fourth APL gene fusion has been characterized that links exons encoding the retinoic acid and DNA-binding domains of RARA to 5' exons of NuMA, a gene that encodes the nuclear mitotic apparatus protein. The NuMA-RARA fusion protein exists in sheet-like nuclear aggregates with which normal NuMA partly co-localizes. In contrast to t(15;17) APL, the intracellular distribution of PML is normal in these cells. These results suggest that interference with retinoid signalling, and not disruption of PML organization, is essential to the APL phenotype and implicates for the first time an element of the mitotic apparatus in the molecular pathogenesis of human malignancy (Wells, 1997).

Most tumor cells are characterized by increased genomic instability and chromosome segregational defects, often associated with hyperamplification of the centrosome and the formation of multipolar spindles. However, extra centrosomes do not always lead to multipolarity. This study describe a process of centrosomal clustering that prevents the formation of multipolar spindles in noncancer cells. Noncancer cells needed to overcome this clustering mechanism to allow multipolar spindles to form at a high frequency. The microtubule motor cytoplasmic dynein is a critical part of this coalescing machinery, and in some tumor cells overexpression of the spindle protein NuMA interfers with dynein localization, promoting multipolarity (Quintyne, 2005).

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).


Search PubMed for articles about Drosophila mushroom body defect

Becker, B. E., Romney, S. J. and Gard, D. L. (2003). XMAP215, XKCM1, NuMA, and cytoplasmic dynein are required for the assembly and organization of the transient microtubule array during the maturation of Xenopus oocytes. Dev. Biol. 261(2): 488-505. 14499655

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

Betschinger, J., Mechtler, K. and Knoblich, J. A. (2006). Asymmetric segregation of the tumor suppressor brat regulates self-renewal in Drosophila neural stem cells. Cell 124(6): 1241-53. 16564014

Bowman, S. K., Neumuller, R. A., Novatchkova, M., Du, Q. and Knoblich, J. A. (2006). The Drosophila NuMA Homolog Mud regulates spindle orientation in asymmetric cell division. Dev. Cell 10(6): 731-42. 16740476

Cabernard, C. and Doe, C. Q. (2009). Apical/basal spindle orientation is required for neuroblast homeostasis and neuronal differentiation in Drosophila. Dev. Cell 17(1): 134-41. PubMed Citation: 19619498

Chang, P., Coughlin, M. and Mitchison, T. J. (2005). Tankyrase-1 polymerization of poly(ADP-ribose) is required for spindle structure and function. Nat. Cell Biol. 7(11): 1133-9. 16244666

Christophorou, N., Rubin, T., Bonnet, I., Piolot, T., Arnaud, M. and Huynh, J.R. (2015). Microtubule-driven nuclear rotations promote meiotic chromosome dynamics. Nat Cell Biol [Epub ahead of print]. PubMed ID: 26458247

Culurgioni, S., Alfieri, A., Pendolino, V., Laddomada, F. and Mapelli, M. (2011). Inscuteable and NuMA proteins bind competitively to Leu-Gly-Asn repeat-enriched protein (LGN) during asymmetric cell divisions. Proc. Natl. Acad. Sci. 108(52): 20998-1003. PubMed Citation: 22171003

Du, Q., Stukenberg, P. T. and Macara, I. G. (2001). A mammalian Partner of inscuteable binds NuMA and regulates mitotic spindle organization. Nat. Cell Biol. 3(12): 1069-75. 11781568

Du, Q., Taylor, L., Compton, D. A. and Macara, I. G. (2002). LGN blocks the ability of NuMA to bind and stabilize microtubules. A mechanism for mitotic spindle assembly regulation. Curr. Biol. 12(22): 1928-33. 12445386

Du, Q. and Macara, I. G. (2004). Mammalian Pins is a conformational switch that links NuMA to heterotrimeric G proteins. Cell 119(4): 503-16. 15537540

Fant, X., Merdes, A. and Haren, L. (2004). Cell and molecular biology of spindle poles and NuMA, Int. Rev. Cytol. 238: 1-57. 15364196

Fisk Green, R., et al. (2004) Identification of critical domains and putative partners for the Caenorhabditis elegans spindle component LIN-5. Mol. Genet. Genomics 271(5): 532-44. 15138888

Gehmlich, K., Haren, L., Merdes, A. (2004). Cyclin B degradation leads to NuMA release from dynein/dynactin and from spindle poles. EMBO Rep. 5(1): 97-103. 14710193

Gotta, M., Dong, Y., Peterson, Y. K., Lanier, S. M., and Ahringer, J. (2003). Asymmetrically distributed C. elegans homologs of AGS3/PINS control spindle position in the early embryo. Curr. Biol. 13: 1029-1037. 12814548

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Portegijs, V., et al. (2016). Multisite phosphorylation of NuMA-related LIN-5 controls mitotic spindle positioning in C. elegans. PLoS Genet 12: e1006291. PubMed ID: 27711157

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Biological Overview

date revised: 11 November 2016

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