rapsynoid/partner of inscuteable

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Protein Interactions

Interaction of rapsynoid/partner of inscuteable with inscuteable

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The function of Pins during the asymmetric division of the pI cell was analyzed using a viable null allele of pins^Delta1-50 that does not affect epithelial cell polarity. To study the function of Dlg, two hypomorphic alleles, dlg^SW and dlg^1P20 were used that were predicted to encode truncated proteins lacking the C-terminal 14 and 43 amino acids, respectively and which do not perturb apical-basal polarity. The GUK domain of Dlg is partly deleted in the mutant Dlg^1P20 protein, but should be unaffected in the mutant Dlg^SW protein. In the pI cell, the Dlg^SW protein accumulates normally at the anterior cortex, whereas the mutant Dlg1P20 protein is cortical, but fails to accumulate anteriorly (Bellaïche, 2001).

The possible role of Dlg and Pins in regulating the position of the mitotic spindle was investigated. Spindle movements were analyzed in living pupae using Tau-GFP. It was found that the a-p orientation of the pI division does not depend on the activity of pins and is not affected in the dlg^1P20mutant. In wild-type and pins mutant pI cells, the spindle lines up with the planar polarity axis 3-4 min prior to the metaphase-anaphase transition. In contrast, the spindle often rotates throughout metaphase in dlg mutant pI cells. It is concluded that Dlg regulates the localization or the activity of factors responsible for spindle rotation (Bellaïche, 2001).

The roles of Dlg and Pins in the asymmetric localization of Numb and Pon were examined. The interphase localization of Numb at the cortex and of Pon around the nucleus does not depend on the function of the dlg or pins genes. At metaphase, however, the anterior localization of both proteins requires the activity of both dlg and pins. Thus, in pins mutant cells at prometaphase, the crescent of Numb and Pon is either not detected or weak. Nevertheless, both proteins segregate into the anterior cell at anaphase and telophase. In dlg^1P20 mutant pI cells, Numb does not accumulate at the anterior cortex and Pon remains cytoplasmic at metaphase. At telophase, Numb and Pon segregate equally into both daughter cells. These results show that Dlg and Pins are required to localize Numb and Pon at the anterior cortex in the pI cell. Consistently, nonsensory cells are transformed into neurons leading to a bristle loss phenotype in adult flies. Furthermore, the genetic interaction seen between dlg^sw and pins suggests that dlg and pins act in the same process to specify the fate of the pI daughter cells (Bellaïche, 2001).

Pins colocalizes with the anterior accumulation of Dlg and dlg and pins mutations genetically interact. This raises the possibility that the two proteins interact directly. Indeed, in a yeast two-hybrid screen using full-length Dlg as bait, one Pins clone (encoding amino-acid residues 235 to 658) was isolated. To further test for a direct interaction between Dlg and Pins and to identify the Pins interaction domain of Dlg, blot overlay experiments were performed using GST-fusion proteins. A biotinylated Pins protein has been found to interact with the SH3 domain but not with the PDZ1, PDZ2, PDZ3, HOOK, or GUK domains of Dlg. The Dlg-Pins complex is also detected in brains and imaginal discs by coimmunoprecipitation experiments. This interaction is abolished by a single amino-acid substitution (L556P) in the SH3 domain, which does not noticeably affect Dlg stability in dlg^m30 mutant larvae but does result in disc overgrowth and late larval lethality (Bellaïche, 2001).

Consistent with this direct interaction, Pins and Dlg are mutually dependent for their accumulation at the anterior cortex. A very weak crescent of Pins is seen at the anterior cortex in dlg^1P20 mutant pI cells at metaphase, suggesting that the GUK domain might facilitate the interaction between the SH3 domain of Dlg and Pins. Conversely, Dlg does not become enriched at the anterior cortex of pins mutant pI cells at metaphase. It is concluded that Dlg directly interacts with Pins via its SH3 domain, and that this interaction is important for the anterior accumulation of both Dlg and Pins (Bellaïche, 2001).

The role of Pins and Dlg in localizing Baz asymmetrically was examined. In pins mutant pI cells, Baz accumulates at the posterior cortex at metaphase, but the asymmetry is less pronounced than in wild-type cells. This raises the possibility that Pins participates in the asymmetric localization of Baz. In dlg^1P20 mutant pupae, Baz is correctly localized to the apical posterior cortex prior to chromosome condensation, but does not form a cortical crescent below the AJ during late prophase and prometaphase. Instead, Baz accumulates in the cytoplasm and remains cortical only at the level of the AJ. Thus, the initial posterior localization of Baz at the level of the AJ does not depend on the activity of the GUK domain of Dlg, but its cortical localization below the AJ does require dlg activity. It is concluded that planar polarization of the pI cell cannot be maintained without Dlg activity (Bellaïche, 2001).

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

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

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

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

Pins interacts with Galphai to direct asymmetric cell division

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

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

To test whether Insc is required for asymmetric Galphai localization in neuroblasts, insc^P72 mutant embryos were stained for Galphai and DNA. During neuroblast delamination, Galphai fails to localize apically in insc mutants and in 87% of insc mutant metaphase neuroblasts, the protein is distributed around the whole cell cortex. To test whether ectopic expression of Insc is sufficient for the apical localization of Galphai, insc was ubiquitously expressed from a heat-inducible transgene. While Galphai is localized basolaterally in epidermal cells of heat-shocked control embryos, heat-shock-induced ectopic expression of insc in these cells results in apical concentration of Galphai. Thus, expression of insc is both required and sufficient for apical recruitment of Galphai (Schaefer, 2001).

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

Genetic analysis of Galphai is complicated by the presence of another gene within the first intron and the lack of identified P-element insertions near the gene. However, a P-element inserted into the 5' untranslated region of the Gß13F gene was identified and this was used to generate mutants by imprecise excision. Two lethal imprecise excisions were isolated, one of which (Gß13F^Delta1-96A) removes the entire coding region, can be rescued to viability by a transgene containing the Gß13F genomic region, and was used in all experiments (Schaefer, 2001).

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

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

Heterotrimeric G proteins can interact with their downstream targets either via the Gßgamma subunit or the GTP-bound Galpha subunit. Overexpression of wild-type Galphai and Galphai^Q205L, a GTPase-deficient mutant form, should distinguish between these possibilities. Wild-type Galphai should bind and deplete free Gßgamma and inhibit its downstream interactions. Galphai^Q205L, in contrast, should be in the GTP-bound form that does not bind Gßgamma and should not interfere with Gßgamma signaling. Signaling via the alpha subunit, however, should be enhanced by Galphai^Q205L, but not be affected by the wild-type form (Schaefer, 2001).

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

If the defects observed upon Galphai overexpression are due to depletion of free Gßgamma, overexpression of Galphai^Q205L should be without effect. Asymmetric cell division was therefore analyzed in neuroblasts after overexpression of Galphai^Q205L under the same ubiquitous maternal promoter. Like wild-type Galphai, Galphai^Q205L localizes around the cell cortex when overexpressed in neuroblasts. However, the mutant form fails to recruit Pins to the cell cortex and has no effect on Pins localization. No defects in spindle orientation or basal localization of Numb and Miranda were observed and Insc was still localized into an apical crescent in 91% of the metaphase neuroblasts. This suggests that the phenotypes caused by Galphai overexpression might be a consequence of Gßgamma depletion (Schaefer, 2001).

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

The strong overexpression phenotypes caused by wild-type Galphai but not by Galphai^Q205L suggest that the GDP-bound form of Galphai may have a function in asymmetric cell division. To test whether Pins interacts preferentially with the GTP- or the GDP-bound form, Galphai was immunoprecipitated in the presence or absence of the slowly hydrolyzable GTP-analog GTPgammaS. While Pins can be readily coimmunoprecipitated with Galphai in the presence of GDP, only trace amounts of Pins can be coimmunoprecipitated in the presence of GTPgammaS, suggesting that Pins preferentially interacts with the GDP-bound form of Galphai (Schaefer, 2001).

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

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

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

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

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

In neuroblasts, Galphai function does not seem to involve the GTP-bound form of Galphai. To test whether this is also the case in SOP cells, wild-type Galphai and Galphai^Q205L were overexpressed in SOP cells. Upon overexpression, Galphai is no longer asymmetrically localized and Numb is uniformly distributed around the cell cortex. Thirty-eight percent of the Galphai overexpressing SOP cells (n = 149) but only 9% of the controls divided at an angle that deviated more than 45° from the anterior-posterior axis. However, unlike in neuroblasts, in this case similar defects can be generated by overexpression of the activated Galphai^Q205L mutant form. The different effects of Galphai^Q205L overexpression in neuroblasts and SOP cells suggest that distinct pathways might function downstream of G proteins in the two cell types (Schaefer, 2001).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Pins functions in the asymmetric localization of Neuralized

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

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

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

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

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

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

How does Stbm direct the localization of Pins to the anterior cortex? One hypothesis is that Stbm directs the anterior localization of Pins via the regulated assembly of a Stbm-Dlg-Pins complex. The anterior accumulation of Pins depends on its interaction with Dlg. Evidence is provided that Stbm may bind Dlg. (1) In vitro binding studies indicate that Stbm interacts with Dlg. It is noted, however, that PDZ-containing proteins other than Dlg may also bind Stbm in this assay. (2) The localization of Stbm overlaps with the distribution of Pins and Dlg in dividing pI cells. (3) The PBM motif of Stbm appears to regulate the re-localization of Stbm in pI cells. The data are therefore consistent with a model in which, upon mitosis, the binding of Stbm to Dlg in turn promotes the binding of Pins to Dlg and, hence, localization of Pins at the anterior cortex where Stbm and Dlg accumulations overlap. This model predicts that the PBM of Stbm should be required for the anterior localization of Pins. It was found, however, that StbmDeltaPBM is fully functional and that Pins is properly recruited at the anterior cortex in stbm^6c mutant pI cells expressing StbmDeltaPBM. One interpretation of this result is that Stbm regulates the localization of Pins not only via the PBM-dependent assembly of the Dlg-Pins complex but also via a second PBM-independent mechanism. Since Dsh acts redundantly with Baz to localize Pins asymmetrically, it is suggested that this second mechanism may involve Dsh. Accordingly, in stbm^6c mutant pI cells, uniformly distributed Dsh activity would prevent Pins cortical localization. By contrast, since the PCP function of stbm does not depend on its PBM, the activity of Dsh should be restricted to the posterior cortex in stbm^6c mutant pI cells expressing StbmDeltaPBM. Dsh should therefore restrict Pins localization to the anterior cortex in this mutant background. Another interpretation of the correct localization of Pins in stbm^6c mutant pI cells expressing StbmDeltaPBM is that Stbm recruits Pins via a mechanism that does not involve an interaction with Dlg (or any other PDZ-containing proteins). Future studies will address how the Stbm-Pk complex regulates the localization of Pins in the pI cell (Bellaïch, 2004).

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

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

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

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

Because the Gß13F-Ggamma1 complex, which distributes uniformly in the cortex, functions in asymmetric organization of the spindle, differential activation or inactivation of Gßgamma signaling must occur in the apical-basal direction. Two apical signaling pathways are implicated in the apical-basal difference in spindle development in a redundant fashion. What is the relationship between the apical signals and the Gßgamma signal? Spindle size is reduced by an increase in the amount of Gßgamma, but a lack of Gßgamma results in formation of a large, symmetric spindle. These findings raise the possibility that spindle development is suppressed by the Gßgamma signal, which is repressed by the presence of an apical complex on the apical side in the wild-type cells, resulting in a large apical and small basal spindle. This model suggests that the apical complex acts upstream of the Gßgamma signal. In contrast, elimination of Gß13F affects the localization of the apical components: Pins becomes uniformly distributed and Galphai becomes undetectable. In addition, Ggamma1^N159 and G圩F mutations appear to destabilize the localization of the components in the Baz-DaPKC pathway, as judged by the reduced staining by their antibodies (although this may be an indirect consequence of the mislocalization of Pins-Galphai). The Gßgamma signal is thus required for normal distribution of the components of both apical pathways, consistent with the idea that the apical pathways acts downstream of the Gßgamma signal in regulating spindle asymmetry. Tests for epistasis between the apical pathways and the Gßgamma signal are needed to clarify their relationship in the regulation of spindle organization (Izumi, 2004).

The effects of Ggamma1^N159 and G圩F mutations on cell-size asymmetry are remarkable but different from those in double mutants in which both apical pathways are disrupted simultaneously, where daughter cell sizes are completely equal. The cell-size ratio of GMCs to their sibling NBs shows a broad distribution: from 0.6 to 1 in the Ggamma1 (and G圩F) mutants. This residual asymmetry in daughter cell size is due to Baz-DaPKC activity. The components of this pathway indeed distribute asymmetrically in Ggamma1 (and G圩F) mutant NBs in which Pins-Galphai activity is no longer asymmetric (Pins is uniformly distributed and Galphai is absent) (Izumi, 2004).

Why does this polarized Baz-DaPKC activity cause less asymmetry in daughter cell size in spite of the redundant function of the Baz-DaPKC pathway and Pins-Galphai? Antibody staining for Baz, DaPKC, and DmPar-6 suggests that their levels and their polarized distribution are weakened in Ggamma1 (and G圩F) mutants. A possible explanation is that low levels of polarized Baz-DaPKC activity confer only low levels of asymmetry to the daughter cell size in the absence of polarized Pins-Galphai. Thus, the degree of cell-size asymmetry resulting from NB divisions may depend on the dosage of the components of one apical pathway when the other is absent or uniformly distributed. In contrast, Miranda localization does not appear to be severely impaired in Ggamma1^N159 and G圩F mutants until late embryonic stages, indicating that the polarized Baz-DaPKC activity in these mutants is sufficient to localize Miranda. Therefore, full asymmetry in daughter cell size may require relatively higher levels of Baz-DaPKC activity than does polarized distribution of cell-fate determinants does (Izumi, 2004).

In Ggamma1^N159 and G圩F mutants, Insc has a distribution different from the other components of the Baz-DaPKC pathway. In most of these mutant NBs, Insc distributes broadly to both the cytoplasm and the cortex in a slightly asymmetric way, but Baz, DaPKC, and DmPar-6 localize asymmetrically in the cortex. The cytoplasmic distribution of Insc is also slightly asymmetric in pins mutant NBs. It is not known whether cytoplasmic Insc is functional. Interestingly, Insc distribution often appears to correlate better with the asymmetry in daught