Transcriptional Regulation

Notch regulates numb: integration of conditional and autonomous cell fate specification

The Notch cell-cell signaling pathway is used extensively in cell fate specification during metazoan development. In many cell lineages, the conditional role of Notch signaling is integrated with the autonomous action of the Numb protein, a Notch pathway antagonist. During Drosophila sensory bristle development, precursor cells segregate Numb asymmetrically to one of their progeny cells, rendering it unresponsive to reciprocal Notch signaling between the two daughters. This ensures that one daughter adopts a Notch-independent, and the other a Notch-dependent, cell fate. In a genome-wide survey for potential Notch pathway targets, the second intron of the numb gene was found to contain a statistically significant cluster of binding sites for Suppressor of Hairless, the transducing transcription factor for the pathway. This region contains a Notch-responsive cis-regulatory module that directs numb transcription in the pIIa and pIIIb cells of the bristle lineage. These are the two precursor cells that do not inherit Numb, yet must make Numb to segregate to one daughter during their own division. These findings reveal a new mechanism by which conditional and autonomous modes of fate specification are integrated within cell lineages (Rebeiz, 2011).

The transcriptional regulation of the numb gene has not previously received much attention because most experimental efforts have been focused on Numb protein localization, asymmetric segregation and function as a Notch pathway inhibitor. The motivation for the present study originated in a computational search of the fly genome for new Notch pathway target genes based on statistically significant clustering of Su(H) binding sites. Although it has been suggested that homotypic site clustering is not a general property of cis-regulatory modules in Drosophila, and therefore that this parameter is of limited utility in computational prediction of enhancers, the data presented in this study and in other reports indicate that this approach can be quite effective in the case of Su(H) and other transcription factors. One beneficial feature of the SCORE method (Rebeiz, 2002) is the use of a largely unbiased window size (100-5000 bp) for the identification of statistically significant binding site clusters. This wide range allows the detection of local maxima that do not necessarily conform to the size expected for a canonical cis-regulatory module. Judging from the present study, the unbiased window-size approach might permit functional enhancer elements to be detected owing to the proximity of multiple enhancers with similar binding inputs. In any case, the SCORE technique successfully identified a functional cis-regulatory module within the ~50 kb of non-coding DNA within and surrounding numb (Rebeiz, 2011).

This study has shown here that a 20 kb genomic DNA fragment is capable of nearly complete phenotypic rescue of two different numb loss-of-function genotypes, and that deletion of the intronic numb CD2 enhancer from this fragment results in widespread 'double socket' and 'double sheath' phenotypes, reflecting a failure to specify the numb-dependent shaft and neuron cell fates. Thus, transcriptional activation of numb in the pIIa and pIIIb precursor cells, in response to the Notch signaling events that specify their respective fates, plays an important role in the proper specification of the Notch-independent progeny cell fate (Rebeiz, 2011).

Given the high proportion of sensory organs in which the shaft and neuron cell fates are correctly specified in the absence of the CD2 enhancer, it seems clear that CD2 is not the only source of Numb for pIIa and pIIIb. This inference was confirmed directly by detecting Numb crescents in dividing pIIa cells in tissue lacking CD2 function, having first demonstrated that the numb796 allele is protein-null (Rebeiz, 2011).

What might be the source of this additional Numb protein? It is, of course, possible that numb is served by a second enhancer module that also contributes to the transcriptional activation of the gene in pIIa and pIIIb in response to Notch signaling; there is substantial precedent for such 'shadow' or 'secondary' enhancers in insects. However, it is very likely that the basal level of Numb protein that is detected in all cells in the epidermis also accumulates in developing sensory organ cells, including pIIa and pIIIb, independently of the CD2 enhancer. This protein would presumably be segregated by the two precursor cells to their shaft and neuron daughter cells, respectively, and might suffice, in most cases, to inhibit Notch signaling in those cells (Rebeiz, 2011).

What, then, would generate the need for the numb CD2 enhancer activity? Integrating all of the current findings, the following evolutionary scenario is favored. Among the cells in the bristle lineage, the pIIa and pIIIb precursors face a unique challenge: because their own fates are specified by Notch signaling, it is crucial that they do not inherit Numb, yet each must make sufficient Numb to distribute asymmetrically to one of their progeny cells. In an ancestral sensory organ lineage, the ubiquitous basal level of Numb accumulation might have been adequate to supply the needs of pIIa and pIIIb. But, perhaps as the execution of the lineage became faster in some rapidly developing insects [the time from birth to division for pIIa and pIIIb is only 3-4 hours in Drosophila, Numb accumulation in these cells failed to meet the required threshold, resulting in unacceptably high failure rates in shaft cell and neuron specification. The emergence of the CD2 enhancer would then have offered the selective advantage of supplementing the basal Numb specifically in these two Notch-dependent precursor cells, without elevating the global activity of the gene. In this scenario, CD2 represents an evolutionary adaptation for ensuring the fidelity of two cell fate decisions during mechanosensory organ development (Rebeiz, 2011).

The Drosophila external sensory organ lineage has stood for many years as an elegant example of the integration of conditional and autonomous mechanisms of cell fate specification. The repeated use of a combination of bi-directional Notch signaling between sister cells and asymmetric segregation of the Notch pathway antagonist Numb is a highly effective strategy for ensuring the proper specification of cell fates in a succession of asymmetric cell divisions. This is particularly so because the orientation of the mitotic spindles and the segregation of Numb are tied to the planar polarity system, such that the appropriate fate is assigned to the appropriate daughter with extremely high fidelity. The results reported in this study bring this Notch-Numb partnership full circle by demonstrating that a reciprocal regulatory linkage also exists: Notch signaling regulates numb (see Model for the Notch-stimulated activation of numb transcription in the pIIa precursor cell) (Rebeiz, 2011).

This study has shown that, although Notch signaling is essential to the activation of the numb bristle enhancer, the transcriptional activation function of Su(H) is not strictly required for enhancer activity. Accordingly, it is suggested that Notch signaling acts here in large part as a trigger, relieving Su(H)-mediated 'default repression' and permitting other activators bound to the enhancer to drive numb transcription. Some or all of these activators are likely to be expressed in both pIIa and pIIb, as implied by the nearly equivalent level of reporter gene activity observed in the two cells when the Su(H) binding sites of the enhancer are mutated. It is further suggested that this regulatory strategy is relevant to the question of timing. Having Notch signaling act as a trigger for the action of a pre-assembled complex of other activators might help to ensure that the transcriptional response is very rapid, allowing sufficient numb mRNA to be accumulated and translated in pIIa and pIIIb before they divide (Rebeiz, 2011).

The bantam microRNA acts through Numb to exert cell growth control and feedback regulation of Notch in tumor-forming stem cells in the Drosophila brain

Notch (N) signaling is central to the self-renewal of neural stem cells (NSCs) and other tissue stem cells. Its deregulation compromises tissue homeostasis and contributes to tumorigenesis and other diseases. How N regulates stem cell behavior in health and disease is not well understood. This study shows that Notch regulates bantam (ban) microRNA to impact cell growth, a process key to NSC maintenance and particularly relied upon by tumor-forming cancer stem cells. Notch signaling directly regulates ban expression at the transcriptional level, and ban in turn feedback regulates N activity through negative regulation of the Notch inhibitor Numb. This feedback regulatory mechanism helps maintain the robustness of N signaling activity and NSC fate. Moreover, this study shows that a Numb-Myc axis mediates the effects of ban on nucleolar and cellular growth independently or downstream of N. These results highlight intricate transcriptional as well as translational control mechanisms and feedback regulation in the N signaling network, with important implications for NSC biology and cancer biology (Wu, 2017).

By revealing the involvement of the miRNA pathway, this study highlights the complexity of the N signaling network in normal NSCs and tumor-forming cancer stem cell (CSC)-like NSCs. Previous studies implicated critical roles for both canonical and non-canonical N signaling pathways in NSCs and CSC-like NSCs, and revealed particular dependence of CSC-like NB growth on non-canonical N signaling, which involves PINK1, mTORC2, and mitochondrial quality control. The current study reveals a particular requirement for ban in CSC-like NBs induced by N hyperactivation. The CSC-like NB overproliferation induced by hyperactivation of N or N pathway component Dpn can all be assumed to be of type II NB origin, since previous studies have clearly established that Notch signaling is essential for the development and/or maintenance of type II NBs, but dispensable for type I NBs, and that hyperactivation of Notch or its downstream effector Dpn induced ectopic CSC-like NB growth by altering the lineage homeostasis of the type II but not type I NBs. It would be interesting to test whether, in addition to ban's role in canonical N signaling, there exists a link between ban and non-canonical N signaling. The data indicate that the ban-Numb signaling motif regulates NSC/CSC behavior through at least two mechanisms. On one hand, it regulates cell growth and particularly nucleolar growth, through Myc, a known regulator of cellular and nucleolar growth. Consistently, negative regulation of Myc protein level by Numb was observed through E3 ubiquitin-protein ligase, Huwe1, and the UPS. c-Myc is an essential regulator of embryonic stem cell (ESC) self-renewal and cellular reprogramming, and Myc level and stability can be controlled in stem cells through targeted degradation by the UPS, suggesting conserved mechanisms. A key function of the nucleolus is the biogenesis of ribosomes, the cellular machinery for mRNA translation, and previous studies in Drosophila have supported the critical role of nucleolar growth in NSC self-renewal and maintenance. On the other hand, the ban-Numb axis feedback regulates the activity of N by a double negative regulation, with the end result being positive feedback regulation. This feedback mechanism may help transform initial not so dramatic differences in N activity between NB and its daughter cell generated by the asymmetric segregation of Numb during NB division [33] into 'all-or-none' decision of cell fates. Feed-forward regulatory loops, both coherent and incoherent, are frequently found in gene regulatory networks, and although ban miRNA is not conserved in mammals, miRNAs have been implicated in an incoherent feed-forward loop in the Numb/Notch signaling network in colon CSCs in mammals (Wu, 2017).

Given the role of ban in a positive feedback regulation of N and the potency of N hyperactivity in inducing tumorigenesis, one may wonder why ban overexpression is not sufficient to cause tumorigenesis. As in any biological systems, feedback regulation is meant to increase the robustness and maintain homeostasis of a pathway. Feedback alone, either negative or positive, should not override the main effect of the signaling pathway. Thus, in the NB system feedback regulation by ban is built on top of the available N signaling activity in a given cell and serving to maintain N activity. Because of ban's 'fine-tuning' rather than 'on/off switching' of Numb expression, its effect on N activity during feedback regulation will also be 'fine-tuning', serving to maintain N activity in NB within a certain range. Overexpression of ban in a wild type background may not be sufficient to cause tumorigenesis because N activity is not be elevated to the level sufficient to induce brain tumor as in N-v5 overexpression condition. Consistent with this, the extent of Numb inhibition by ban is also modest, not reaching the threshold level of Numb inhibition needed to cause tumorigenesis. Consistent with the notion that feedback regulation by ban is built on top of the available N signaling activity in a given cell, and that there is dosage effect of N activity in tumorigenesis, overexpression of ban in N-v5 overexpression background further enhanced N-v5 induced tumorigenesis. It is likely that ban or other miRNAs may participate in additional regulatory mechanisms in the N signaling network in Drosophila. Of particular interest, it would be interesting to test whether miRNAs may impinge on the asymmetric cell division machinery to influence the symmetric vs. asymmetric division pattern, a key mechanism employed by NSCs and transit-amplifying IPs to balance self-renewal with differentiation (Wu, 2017).

The results emphasize the critical role of translational control mechanisms in NSCs and CSC-like NSCs. Compared to the heavily studied transcriptional control, knowledge of the translational control of NSCs and CSCs is rather limited. As fundamental regulators of mRNA translation, miRNAs can interact with both positive and negative regulators of translation to influence gene expression. Thus, miRNA activity can be regulated context-dependently at both the transcriptional and translational levels, which may account for the opposite effect of N on ban activity in the fly brain and wing disc, although the ban genomic locus is bound by Su(H) in both tissues. Whether N regulates the transcription of ban or its activity as a translational repressor in the wing disc remains to be tested. With regard to the translation of numb mRNA, the conserved RNA-binding protein (RNA-BP) Musashi has been shown to critically regulate the level of Numb protein in mammalian hematopoietic SCs and leukemia SCs. Further investigation into the potential interplay between miRNAs and RNA-BPs in the translational control of Numb in NBs and CSC-like NBs promises to reveal new mechanisms and logic in stem cell homeostasis regulation, with important implications for stem cell biology and cancer biology (Wu, 2017).

Targets of Activity

Numb exerts its function at least in part via tramtrack, a target gene. Like numb, ttk acts as a genetic switch. Loss of ttk function causes the sensory organ precursor daughter cell IIa to be transformed into IIb, a phenotype just the opposite of the mutant numb phenotype. However, overexpression of ttk results in the same cell fate transformation as does loss of numb function. Genetic and immunocytochemical studies indicate that ttk is negatively regulated by numb (Guo, 1995).

Interference of human and Drosophila APP and APP-like proteins with PNS development in Drosophila: Numb is a potential target of APP

The view that only the production and deposition of Abeta plays a decisive role in Alzheimer's disease has been challenged by recent evidence from different model systems, which attribute numerous functions to the amyloid precursor protein (APP). To investigate the potential cellular functions of APP and its paralogs, transgenic Drosophila was used as a model. Upon overexpression of the APP-family members, transformations of cell fates during the development of the peripheral nervous system were observed. Genetic analysis showed that APP, APLP1 and APLP2 induce Notch gain-of-function phenotypes, identified Numb as a potential target and provided evidence for a direct involvement of Disabled and Neurotactin in the induction of the phenotypes. The severity of the induced phenotypes not only depended on the dosage and the particular APP-family member but also on particular domains of the molecules. Studies with Drosophila APPL confirmed the results obtained with human proteins and the analysis of flies mutant for the appl gene further supports an involvement of APP-family members in neuronal development and a crosstalk between the APP family and Notch (Merdes, 2004).

These studies show that the ectopic expression of human APP-family members induces Notch gain-of-function phenotypes during the development of the adult PNS. The severity of the induced phenotypes not only depends on the dosage and the particular APP-family member, but also on particular domains of the molecules. This led to the identification of the NPTY motif as the only critical motif within the ICD for the interference with PNS development and for the interaction of APP with Numb/Pon and Dab in vitro and in vivo (Merdes, 2004).

An interaction between APP and Numb has been demonstrated by Roncarati (2002). In mouse brain lysates as well as in cell culture, APP or APP.ICD bind to all four isoforms of Numb and to Numb-like. Surprisingly, in this study, the processing of APP and the release of the ICD of APP resulted in an inhibition of Notch signaling. Numb is a negative regulator of Notch signaling and binds directly via its PTB domain to Notch. Therefore, a direct interaction between APP and the PTB domain of Numb should result in an increase rather than in a decrease of Notch activation. From the known crystal structure of PTB-NPTY interactions, a trimeric complex between Notch, APP and Numb seems unlikely. In this study, the induced Notch gain-of-function phenotypes, the strong genetic interaction, the dependence of the asymmetric localization of APPL on Numb and the direct binding between APP and Numb support a crosstalk between Notch signaling and APP-family members. One explanation for the APP induced Notch gain-of-function phenotypes during mechano-sensory organ (MSO) development would indeed be the sequestration and inactivation of Numb by APP-family members. However, several lines of evidence are provided that (if APP competes with Notch for the binding to Numb) suggest this binding and competition must be highly regulated and requires factors which have not previously been known to be involved in MSO development (Merdes, 2004).

(1) Expression of the human APP-family proteins induces cell fate transformations during MSO development in a dosage- and construct-dependent manner, but the potency in phenotype induction of the different proteins does not correlate with their in vitro and in vivo binding affinity to Numb. Nevertheless, the NPTY motif proves to be essential both for binding to Numb and phenotype induction, suggesting that the binding to Numb might be necessary but not sufficient for phenotype induction. This implies that there is at least one additional factor which plays an important role and which must have different affinities to the APP-family members than Numb, for example, strong binding to APLP2 but weak binding to APP.
(2) Deletion of the ECD of APP results in an inactive molecule, which can no longer induce any phenotypes. This stands in contrast to all in vitro binding studies that have been performed between the NPTY motif of APP and PTB-containing proteins in cell culture. In these assays, the affinity of such a molecule to Fe65, Dab-1/2, X11L, Numb and Numb-like did not change significantly.
(3) APP molecules with a deletion of the NPTY motif could suppress the phenotypes induced by wt APP and induce the loss of macrochaete in wt flies. Such a dominant-negative effect can only be explained if APP-family members have a receptor-like function. In this scenario, APP.DeltaNPTY would compete with wt APP or APPL for ligand binding, but could not relay the 'signal', for example, crosstalk to Notch and/or inactivating Numb. Another possibility would be the necessity of homodimer formation. Such a dimer formation has been postulated, but so far no in vivo data are available. Furthermore, structural data do not provide any evidence for a dimerization of APP molecules prior to the binding of PTB-containing proteins.
(4) Overexpression of Drosophila APPL induces only very weak phenotypes, whereas the overexpression of induces very strong phenotypes. The difference in phenotype induction could not be correlated with significant differences in expression levels, metabolism or processing. This was surprising, since had been generated to impair secretion and therefore processing. As a consequence, it is postulated that the 33 aa deletion in changes the conformation of the ECD, confirming again the important role the ECD plays in determining the potency of the APP-family members for interference with PNS development.
(5) Overexpression of APLP2 results in bold patches, suggesting that presumptive SOPs are transformed into epidermal cells by the induction of a Notch gain-of-function phenotype very early during MSO development. This step during PNS development is known to be independent of Numb and functions via the lateral inhibition mechanism, indicating that APP-family members can also interact with Numb-independent Notch signaling processes. During these processes, so far unknown factors might take over the role of Numb as a negative regulator of Notch to add an additional level of control to the system. From the literature, it seems to be clear that endocytosis is important for inhibition and for the promotion of Notch signaling, but almost nothing is known about the factors directly involved in these events.
(6) Ectopically expressed APPL and as well as APP and APP/APLP2 are asymmetrically localized during MSO development and co-localization and co-immunoprecipitation with Pon has been be demonstrated in vivo. This is an interesting result since APPL and APP induce only weak phenotypes, but and APP/APLP2 induce very strong phenotypes. Nevertheless, both types of proteins are recognized with the same efficiency by the Numb-dependent machinery responsible for the asymmetric distribution of factors during MSO development, thus completely uncoupling this event from phenotype induction. This implies that the phenotype induction occurs after completion of the separation of the SOP siblings and that APP, even if it binds to Numb, does not compete with other binding partners of Numb for asymmetric segregation (Merdes, 2004).

During MSO development, the asymmetric distribution of Numb ensures that the siblings arising from one mother cell show a difference in response to the activation of the Notch receptor. Numb is responsible for the asymmetric segregation of α-adaptin and binds both the ICD of Notch and α-adaptin, suggesting that Numb may regulate Notch by controlled endocytosis. The difference in response to Notch signaling is further amplified by the asymmetric localization of the E3 ubiquitin ligase Neuralized, which upregulates the endocytosis of the Notch ligand Delta. However, one has to take into account that it has also been reported that Numb can (1) bind the ICD of Notch after release, (2) inhibit the ability of this ICD to cause nuclear translocation of Su(H) and (3) can inhibit Notch signaling during wing development by ectopic misexpression. Therefore, even if it is very tempting to suggest that Numb solely regulates Notch by endocytotic mechanisms, there might still be other Numb functions (Merdes, 2004).

Nevertheless, more and more evidence is emerging that regulated endocytosis is an important general feature for the modulation of developmental signals. In this respect, it is especially intriguing that Drosophila Dab has been identified as an essential factor for the interaction of APP with Notch signaling. Whereas the overexpression of Dab enhances the phenotype induced by APP, a reduction of the endogenous protein level by RNAi suppresses the phenotype. Notch gain-of-function phenotypes during MSO development can be induced by expression of high levels of Dab alone. This is remarkable since it has been proposed that the mammalian Dab-2 homologs belong to a family of cargo-specific adaptor proteins, which, like Numb and β-Arrestin, regulate cargo selection and pit formation. Accordingly, APP molecules could induce the observed phenotypes during PNS development, influencing endocytosis and processing of Notch with the help of Dab. A function for APP as endocytotic receptor is supported by the finding that full-length APP is internalized via clathrin-coated vesicles. Furthermore, a direct interaction between Drosophila Dab and Notch has been demonstated previously (Giniger, 1998). These binding studies have been reproduced, but the binding of Dab to Notch in vitro was shown to be very weak in comparison to the binding affinity of Su(H) or Numb. However, additional studies suggest not only the presence of a second Notch-binding motif within the C-terminal domain of Dab, but also reveal the presence in vivo of a complex which contains Notch and Dab in Drosophila embryos (LeGall and Giniger, personal communication to Merdes, 2004). The second binding motif could allow a direct interaction between the Notch receptor and APP mediated by Dab, and it will be of great interest to elucidate the role of Dab with respect to Notch and APP signaling in the future. A crosstalk between the APP family and Notch receptors has also been shown to take place in the mammalian system (Merdes, 2004).

Originally, mutations in the dab gene were isolated by genetic interactions with the Drosophila Abl homolog. It has recently been reported that these mutations have been erroneously attributed and that all mutations isolated as dab alleles in fact affect the nrt locus (Liebl, 2003). Nrt is a single-pass type-II transmembrane protein and belongs to the family of neuronal cell adhesion molecules (N-CAMs). Nrt mutants are viable and fertile, but its function in growth cone guidance can be revealed in combination with other N-CAM mutants. Since the originally described dab alleles were used for the first genetic studies, mutations were identified in nrt as dominant suppressors of the APP-induced phenotype and also the overexpression of Nrt itself induces very strong and very specific Notch gain-of-function phenotypes. However, genetic studies ruled out an involvement of Abl in the APP-induced phenotype. Preliminary genetic data suggest a genetic interaction between appl and nrt mutations resulting in lethality of the otherwise viable alleles. Additional experiments will be necessary both in Drosophila and vertebrates to further explore this interaction. Especially, the isolation of new mutants for Drosophila dab and appl generated in a clearly defined genetic background, and their use for genetic interactions with Notch, numb and nrt, should provide insights into the mechanisms underlying the potential functions of APP-family members in endocytosis, Notch signaling and PNS development. However, the identification of appl as a quantitative trait locus already provides evidence for a function of appl during PNS developmen (Merdes, 2004).

Although it has not been established that the binding interactions between APP, Numb and Dab are functionally important in AD, signaling pathways emanating from aberrant APP function, as it occurs in AD, may influence Dab/Numb and thus Notch activity. Also, the use of drugs to lower APP processing and Aβ production could result in altered APP functions and an interference with Notch signaling in the adult brain. As already mentioned, an interaction between APP and Numb and Numb-like in the mouse brain has been demonstrated and there is accumulating evidence for a role of the Notch signaling pathway not only in early events during cell fate specifications but also in stem cells, in already differentiated neuronal cells and in neurodegeneration in the adult vertebrate nervous system. Furthermore, the view that only the production and deposition of Aβ plays a decisive role in AD has been challenged by recent evidence from different model systems that attribute numerous functions to APP and derivatives thereof. These findings together with the current data make it likely that alterations in the processing of APP either during the onset and progression of AD or by the use of therapeutics may result in loss- as well as in gain-of-function phenotypes contributing to the disease or side effects (Merdes, 2004).

Numb is a major downstream target of AurA and aPKC

The choice of self-renewal versus differentiation is a fundamental issue in stem cell and cancer biology. Neural progenitors of the Drosophila post-embryonic brain, larval neuroblasts (NBs), divide asymmetrically in a stem cell-like fashion to generate a self-renewing NB and a ganglion mother cell (GMC), which divides terminally to produce two differentiating neuronal/glial daughters. Aurora-A (AurA) acts as a tumor suppressor by suppressing NB self-renewal and promoting neuronal differentiation. In aurA loss-of-function mutants, supernumerary NBs are produced at the expense of neurons. AurA suppresses tumor formation by asymmetrically localizing atypical protein kinase C (aPKC), an NB proliferation factor. Numb, which also acts as a tumor suppressor in larval brains, is a major downstream target of AurA and aPKC. Notch activity is up-regulated in aurA and numb larval brains, and Notch signaling is necessary and sufficient to promote NB self-renewal and suppress differentiation in larval brains. These data suggest that AurA, aPKC, Numb, and Notch function in a pathway that involved a series of negative genetic interactions. This study has identified a novel mechanism for controlling the balance between self-renewal and neuronal differentiation during the asymmetric division of Drosophila larval NBs (Wang, 2006).

When aurA function is compromised, mutant NBs acquire some features of cancer stem cells. They divide to generate a large number of daughter cells capable of self-renewal. This excessive self-renewal occurs at the expense of neuronal differentiation, suggesting that the normally asymmetric NB divisions have been altered such that the mutant NBs can divide symmetrically to generate two NB-like daughters. Cell cycle regulator CycE and cell growth factor dMyc are expressed in most of these tumor-like cells. Up-regulation of CycE is required for aurA overgrowth phenotype. AurA also regulates proper orientation of the mitotic spindle probably by controlling asymmetric localization of Mud. Both proteins are localized to centrosomes and are required for centrosome function. Centrosome abnormality and chromosome segregation defects in aurA could lead to aneuploidy, and many cancer cells exhibit centrosome defects and chromosome instability. Mammalian AurA when overexpressed can be oncogenic. However, future studies on its possible role as a tumor suppressor will be particularly interesting (Wang, 2006).

The data suggest that aurA negatively regulates aPKC function to regulate NB self-renewal. aPKC appears to act as a NB proliferation factor since overexpression of a modified membrane-targeted version, aPKC-CAAX, which exhibits ectopic cortical localization throughout the NB cortex, leads to overproliferation and tumor formation, similar to loss of aurA. AurA is required for the asymmetric localization of aPKC and restrict aPKC to the cortical region associated with the future NB daughter and loss of aurA results in delocalization of aPKC to the entire cortex. Consistent with and supporting this notion, loss of aPKC can suppress, albeit partially, the aurA mutant overgrowth phenotype (Wang, 2006).

In contrast to the well-studied role of Numb as a cell fate determinant during asymmetric divisions of embryonic GMCs, SOPs, or muscle progenitors, a role for Numb during NB asymmetric divisions has not been described. This study shows that Numb also acts as a tumor suppressor in Drosophila larval brains, and that Numb is a key downstream target of AurA and aPKC in the regulation of NB self-renewal. In both aurA mutant NBs or NBs overexpressing aPKC-CAAX, the asymmetric localization of Numb is compromised and the resultant overgrowth phenotype is consistent with that of numb loss-of-function. numb and aurA mutant NBs also share several common features including excessive self-renewal at the expense of neuronal differentiation as well as the membrane enrichment of Spdo, a positive regulator of Notch signaling. These data suggest that AurA positively regulates Numb function. Genetic analysis is consistent with the notion that this is achieved through the negative regulation of aPKC that in turn negatively regulates Numb (Wang, 2006).

Numb is known to be a negative regulator of Notch signaling. The current findings indicate that Notch is necessary and sufficient for promoting larval NB proliferation and suppressing neuronal differentiation. Genetic epistasis studies suggest that an AurA-aPKC-Numb-Notch genetic hierarchy acts to regulate self-renewal of Drosophila neural progenitor cells. During a wild-type larval NB asymmetric division, aurA acts to negatively regulate aPKC and restrict its localization to the cortical region associated with the future NB daughter; aPKC negatively regulates Numb and ensures that its localization/activity is restricted to the future GMC where Numb acts to antagonize Notch. The net effect is that Notch is asymmetrically activated in the NB daughter where it acts to promote self-renewal and suppress differentiation. Although these data suggest that aurA acts through the aPKC/Numb/Notch pathway, given the partial suppression seen in the double mutants aPKC;aurA and Notchts-1;aurA, the possibility that additional mechanisms may be involved cannot be excluded (Wang, 2006).

Drosophila Aurora-A kinase inhibits neuroblast self-renewal by regulating aPKC/Numb cortical polarity and spindle orientation

Regulation of stem cell self-renewal versus differentiation is critical for embryonic development and adult tissue homeostasis. Drosophila larval neuroblasts divide asymmetrically to self-renew, and are a model system for studying stem cell self-renewal. This study identified three mutations showing increased brain neuroblast numbers that map to the aurora-A gene, which encodes a conserved kinase implicated in human cancer. Clonal analysis and time-lapse imaging in aurora-A mutants show single neuroblasts generate multiple neuroblasts (ectopic self-renewal). This phenotype is due to two independent neuroblast defects: abnormal atypical protein kinase C (aPKC)/Numb cortical polarity and failure to align the mitotic spindle with the cortical polarity axis. numb mutant clones have ectopic neuroblasts, and Numb overexpression partially suppresses aurora-A neuroblast overgrowth (but not spindle misalignment). Conversely, mutations that disrupt spindle alignment but not cortical polarity have increased neuroblasts. It is concluded that Aurora-A and Numb are novel inhibitors of neuroblast self-renewal and that spindle orientation regulates neuroblast self-renewal (Lee, 2006).

Mutations in aurA lead to a massive increase in larval brain neuroblasts. The major cause of this phenotype appears to be misregulation of neuroblast cortical polarity. One cortical polarity defect is increased basal localization of aPKC, which is sufficient to induce ectopic neuroblasts. Consistent with this hypothesis, aPKC aurA double mutants show strong suppression of the aurA supernumerary neuroblast phenotype, consistent with aPKC functioning downstream from AurA. While it is possible that loss of aPKC suppresses the phenotype in a nonspecific way (e.g., by arresting neuroblast cell proliferation or inducing neuroblast apoptosis), ni similarly strong suppression of the brat supernumerary neuroblast phenotype was observed in aPKC brat double mutants. This shows that aPKC functions more specifically in the AurA pathway than in the Brat pathway (Lee, 2006).

The only other detectable cortical polarity defect seen in aurA mutant neuroblasts is a delocalization of Numb from the basal cortex. A similar Numb defect is seen during asymmetric cell division of pupal SOPs in aurA mutants, perhaps reflecting a specific and direct regulation of Numb by AurA, although Numb is not phosphorylated by AurA in vitro. The importance of the Numb delocalization phenotype is revealed by the ability of Numb overexpression in neuroblasts to rescue most of the aurA mutant phenotype (all except the component due to spindle orientation defects). Thus, Numb acts downstream from AurA to inhibit neuroblast self-renewal. Numb joins Mira/Pros/Brat as proteins that are partitioned into the GMC during neuroblast asymmetric cell division, where they function to inhibit neuroblast self-renewal (Lee, 2006).

Where does AurA function to inhibit neuroblast self-renewal? AurA appears to be required in the neuroblast lineage, and not in surrounding glial cells or nonneuronal tissues of the larva, because neuroblast-specific expression of either AurA or the downstream component Numb can rescue most of the aurA supernumerary neuroblast phenotype. This shows that AurA is not required outside the neuroblast lineage to inhibit neuroblast self-renewal. Within the neuroblast, AurA appears to function in the cytoplasm and not at the centrosome, because cnn mutants lack all detectable AurA centrosomal localization yet do not match the aurA supernumerary neuroblast phenotype. It is concluded that AurA acts in the neuroblast cytoplasm to promote aPKC/Numb cortical polarity and spindle-to-cortex alignment (Lee, 2006).

How does Numb inhibit neuroblast self-renewal in the GMC? Numb is a well-characterized inhibitor of Notch signaling that is segregated into the GMC, and Notch signaling is active in larval neuroblasts but not in GMCs. Thus the most obvious model is that Numb blocks Notch receptor signaling in the GMC. However, Notch mutant clones generated in larval neuroblasts do not affect neuroblast survival or clone size. Similarly, no change has been seen in neuroblast number in two different Notch-ts mutants (although the expected small wing imaginal disc phenotype was observed. In addition, no supernumerary neuroblasts were observed in larval neuroblast clones overexpressing the constitutively active Notch intracellular domain, although the same Notch intracellular domain generates the expected sibling neuron phenotype when expressed in the embryonic CNS. Thus, Notch is an excellent candidate for promoting neuroblast self-renewal, but additional experiments will be needed to test this model more rigorously. In this context, it is interesting to note that Notch promotes stem cell self-renewal in mammals (Lee, 2006).

aurA mutant neuroblasts have essentially random orientation of the mitotic spindle relative to the apical/basal cortical polarity axis, resulting in a some neuroblasts dividing symmetrically (in size and cortical polarity markers). This phenotype may arise due to lack of astral microtubule interactions with the neuroblast cortex; aurA mutant neuroblasts have reduced astral microtubule length. Alternatively, AurA may affect spindle orientation by phosphorylating proteins required for spindle orientation, such as Cnn, Pins, or Mud. For example, Mud has a consensus AurA/Ipl1 phosphorylation site within its microtubule-binding domain, and it will be interesting to determine if this site needs to be phosphorylated for Mud to bind microtubules. Spindle orientation defects only generate part of the supernumerary neuroblast phenotype in aurA mutant brains, however, because overexpression of Numb can rescue most of the phenotype without rescuing spindle alignment, and cnn or mud mutants have nearly random spindle alignment but only a modest increase in neuroblast number. Thus, it is proposed that spindle orientation defects and cortical polarity defects combine to generate the dramatic supernumerary neuroblast phenotype seen in aurA mutants (Lee, 2006).

Mammalian aurA has been termed an oncogene due to its overexpression in several cancers, its ability to promote proliferation in certain cell lines, and the fact that reduced levels lead to multiple centrosomes, mitotic delay, and apoptosis. However, an in vivo aurA mutant phenotype has not yet been reported. In contrast, aurA loss-of-function mutations result in a neuroblast 'brain tumor' phenotype, including prolonged neuroblast proliferation during pupal stages when wild-type neuroblasts have stopped proliferating. aurA mutants do not, however, have the imaginal disc epithelial overgrowth seen in other Drosophila tumor suppressor mutants, and aurA mutant neuroblasts have a delay in cell cycle progression. It is proposed that the aurA supernumerary neuroblast phenotype is not due to loss of growth control or a faster cell cycle time, but rather due to a cell fate transformation from a differentiating cell type (GMC) to a proliferating cell type (neuroblast) (Lee, 2006).

It is concluded that AurA restrains neuroblast numbers using two pathways: first by promoting Numb localization into the GMC, and second by promoting alignment of the mitotic spindle with the cortical polarity axis. Absence of the first pathway leads to increased neuroblasts at the expense of GMCs, whereas absence of the second pathway leads to increased neuroblasts due to symmetric cell division. It will be interesting to determine whether mammalian AurA uses one or both pathways to regulate stem cell asymmetric division and self-renewal (Lee, 2006).

Protein phosphatase 2A regulates self-renewal of Drosophila neural stem cells

Drosophila larval brain neuroblasts divide asymmetrically to generate a self-renewing neuroblast and a ganglion mother cell (GMC) that divides terminally to produce two differentiated neurons or glia. Failure of asymmetric cell division can result in hyperproliferation of neuroblasts, a phenotype resembling brain tumors. This study has identified Drosophila Protein phosphatase 2A (PP2A) as a brain tumor-suppressor that can inhibit self-renewal of neuroblasts. Supernumerary larval brain neuroblasts are generated at the expense of differentiated neurons in PP2A mutants. Neuroblast overgrowth was observed in both dorsomedial (DM)/posterior Asense-negative (PAN) neuroblast lineages and non-DM neuroblast lineages. The PP2A heterotrimeric complex, composed of the catalytic subunit (Mts), scaffold subunit (PP2A-29B) and a B-regulatory subunit (Tws), is required for the asymmetric cell division of neuroblasts. The PP2A complex regulates asymmetric localization of Numb, Pon and Atypical protein kinase C, as well as proper mitotic spindle orientation. Interestingly, PP2A and Polo kinase enhance Numb and Pon phosphorylation. PP2A, like Polo, acts to prevent excess neuroblast self-renewal primarily by regulating asymmetric localization and activation of Numb. Reduction of PP2A function in larval brains or S2 cells causes a marked decrease in Polo transcript and protein abundance. Overexpression of Polo or Numb significantly suppresses neuroblast overgrowth in PP2A mutants, suggesting that PP2A inhibits excess neuroblast self-renewal in the Polo/Numb pathway (Wang, 2009).

Mammalian PP2A is a tumor suppressor that participates in malignant transformation by regulating multiple pathways. However, it is unknown whether PP2A controls neural stem cell self-renewal. These data explicitly show that the Drosophila PP2A trimeric complex confers brain tumor-suppressor activity and controls the balance of self-renewal and differentiation of neural stem cells. This study shows that PP2A mutation leads to neural stem cell overproliferation in Drosophila larval brains, which is associated with dramatically reduced neuronal differentiation. Cell cycle genes including CycE, and phospho-Histone H3 and growth factor Myc are upregulated in PP2A mutants, consistent with the neuroblast overgrowth phenotype. Neuroblasts overproliferate in PP2A mutant MARCM clones. When these mutant clones that were generated at larval stages are kept until adulthood, neural stem cells continue to proliferate in adult brains, which is never observed for wild-type clones. Therefore, PP2A can inhibit excess self-renewal and promote neuronal differentiation of neural stem cells (Wang, 2009).

This overgrowth of neural stem cells in PP2A mutants is a consequence of defects in the asymmetric division of neural stem cells. PP2A regulates asymmetric protein localization as well as mitotic spindle orientation. It has been shown that Polo kinase is a brain tumor-suppressor that regulates Numb/Pon and aPKC asymmetric localization, as well as mitotic spindle orientation. Although polo mutants displayed pleiotropic phenotypes during asymmetric divisions, Polo primarily regulates asymmetric division of neural stem cells by regulating Numb asymmetry. Polo directly phosphorylates Pon on Ser611, which leads to the asymmetric localization of Pon and subsequently Numb (Wang, 2007). Strikingly similar to Polo, PP2A also regulates the asymmetric localization of aPKC, Pon and Numb, and is required for Pon phosphorylation on Ser611. Interestingly, both PP2A and Polo are required for Numb phosphorylation, which may be important for Numb asymmetric localization or activity on the cortex. Thus, Numb is a major downstream factor for both PP2A and Polo in regulating neural stem cell self-renewal. Consistent with this, overexpression of Numb, but not PonS611D, a phospho-mimetic form of Pon, in polo mutants significantly rescues the neuroblast overgrowth phenotype (Wang, 2009).

It was further discovered that PP2A functions upstream of Polo/Numb in the same pathway to control self-renewal of neuroblasts. Polo transcript and protein abundance is dependent on PP2A function. The expression of several other genes, including numb, baz and lgl, are not affected by PP2A knockdown, suggesting that the downregulation of polo in PP2A mutants appears to be specific. Moreover, overexpression of GFP-Polo or Numb can largely suppress neuroblast overgrowth in PP2A mutants, suggesting that PP2A primarily acts in the Polo/Numb pathway to inhibit neuroblast overgrowth. This discovery suggests that PP2A and Polo, both of which are crucial brain tumor-suppressors and cell cycle regulators, can function in the same pathway to regulate stem cell self-renewal and tumorigenesis. Currently, it is not clear how PP2A, which is a protein phosphatase, promotes polo expression. It is conceivable that PP2A dephosphorylates a transcription factor and consequently activates it to allow polo transcription. Alternatively, PP2A may dephosphorylate a protein that is required for polo mRNA stabilization (Wang, 2009).

PP2A is involved in a broad range of cellular processes including signal transduction, transcriptional regulation and cell cycle control. PP2A regulates the Wnt/Wingless signaling pathway and affects the degradation of β-catenin, a transcription factor and the central molecule of this pathway. Two of the components of Wnt/Wingless signaling pathway, Adenomatous polyposis coli (APC) and Shaggy (also known as GSK3), do not regulate neuroblast polarity. So it remains to be determined whether Wnt/Wingless signaling plays a role in neuroblast polarity. Mammalian PP2A directly dephosphorylates oncogene cMyc and tumor suppressor p53, both of which are transcription factors. Future studies should identify potential substrate(s) of PP2A that can promote polo expression and control neural stem cell self-renewal (Wang, 2009).

Interestingly, it was also observed that cMyc protein levels are increased in PP2A mutants, suggesting that PP2A may have a conserved role in modulating cMyc protein and suppressing its function. However, ectopic expression of cMyc alone does not induce brain tumor formation in Drosophila, suggesting that PP2A can regulate multiple pathways to affect neural stem cell self-renewal. However, the PP2A/Numb pathway appears to be one of the major pathways by which PP2A controls the balance of self-renewal and differentiation in Drosophila, as overexpression of Polo or Numb can largely suppress neural stem cell overgrowth in PP2A mutants. Furthermore, PP2A may regulate Numb function and activity by both promoting polo expression and antagonizing aPKC phosphorylation of Numb. Whether mammalian PP2A also regulates stem cell polarity will be of great interest for future study (Wang, 2009).

Protein Interactions

How does Numb oppose Notch signaling? To assess the possibility of a direct physical interaction between Notch and Numb, a yeast two hybrid interaction assay has been carried out. In this experiment genes coding for fragments of each protein are placed in yeast cells, one attached to a coding sequence specifying a DNA binding protein, and the other attached to a coding sequence specifying a transcriptional activator domain. The binding site for the DNA binding domain is placed next to a ß-galactosidase promoter. The fragment attached to the DNA binding domain is thought of as bait; if the bait interacts with the other protein fragment attached to the transcriptional activator, then ß-galactosidase is transcribed. The Notch intracellular domain consists of an N-terminal RAM23 domain, an ankyrin repeat region (serving as a protein interaction domain interacting with Deltex), a C-terminal PEST sequence (serving to promote protein instablity), and a central domain. The N-terminal RAM23 domain does indeed interact with Numb in the yeast two hybrid experiment. The N-terminal phosphotyrosine binding domain of Numb interacts with the N-terminal area of the intracellular region of Notch. The physical interaction of Notch and Numb has been confirmed using an in vitro physical interaction assay (Guo, 1996).

Since Numb can interact with the intracellular domain of Notch, it is presumbed that Numb can interfer with Notch signaling. The next question: what is the target of Notch? A good candidate is tramtrack, which has been shown to be downstream of Numb (Guo, 1995). Is there an alteration of ttk expression due to reduction or overexpression of Notch? Experimental evidence suggests there is. ttk is normally expressed in the sheath cell, one of the products of the sensory organ precursor lineage, but not in the neural cell, the sister of the sheath cell. In Notch mutants, extra neurons have been detected, resulting from a transformation of sheath cells into neurons. ttk is expressed in most cells in the epidermis of the mutant embryo, but not in neurons, including the supernumerary neurons derived from transformation of sheath cells. Thus Notch function is required not only to specify the sheath cell but also to express ttk in this daughter cell of an asymmetric division. In a reciprocal experiment, overexpression of Notch turns on ttk expression in cells that normally do not express ttk. It is concluded that Notch targets ttk presumably downstream of Numb (Guo, 1996).

In Drosophila, action of Inscuteable, a cytoskeleton associated protein, suggests an involvement of cytoskeleton in providing cues for Prospero and Numb localization. Inscuteable localizes to the apical pole of epithelial cells, opposite the pole to which Prospero and Numb localize. In inscuteable mutants both Prospero and Numb form cresents, but the position of the spindle and the position of Prospero and Numb is disrupted, that is, Prospero and Numb do not localize preferentially to basal poles. Cytochalasin D, which disrupts Actin filaments, eliminates Inscuteable cresents and results in incorrect Prospero crescent positioning, while treatment with colcemid, which destroys microtubules (and consequently the spindle) does not effect Inscuteable localization (Kraut, 1996).

Cellular diversity in the Drosophila central nervous system is generated through a series of asymmetric cell divisions in which one progenitor produces two daughter cells with distinct fates. Asymmetric basal cortical localization and segregation of the determinant Prospero during neuroblast cell divisions play a crucial role in effecting distinct cell fates for the progeny sibling neuroblast and ganglion mother cell. Similarly asymmetric localization and segregation of the determinant Numb during ganglion mother cell divisions ensures that the progeny sibling neurons attain distinct fates. The most upstream component identified so far which acts to organize both neuroblast and ganglion mother cell asymmetric divisions, is encoded by inscuteable. The Inscuteable protein is itself asymmetrically localized to the apical cell cortex and is required both for the basal localization of the cell fate determinants during mitosis and for the orientation of the mitotic spindle along the apical/basal axis. The functional domains of Inscuteable have been defined. Amino acids 252-578 appear sufficient to effect all aspects of its function, however, the precise requirements for its various functions differ. The region aa288-497 is necessary and sufficient for apical cortical localization and for mitotic spindle (re)orientation along the apical/basal axis. A larger region (aa288-540) is necessary and sufficient for asymmetric Numb localization and segregation; however, correct localization of Miranda and Prospero requires additional sequences from aa540-578. The requirement for the resolution of distinct sibling neuronal fates appears to coincide with the region necessary and sufficient for Numb localization (aa288-540). These data suggest that apical localization of the Inscuteable protein is a necessary prerequisite for all other aspects of its function. Although Inscuteable RNA is normally apically localized, RNA localization is not required for protein localization or any aspects of inscuteable function (Tio, 1999).

Miranda protein can interact with both Prospero and Numb. The regions of Miranda that interact with these two proteins are distinct from each other and are mapped to the central and C-terminal portions of the Miranda protein, respectively (Shen, 1997).

In embryos deficient for miranda, Prospero is not associated with the membrane, but stays in the cytoplasm in prophase. Prospero remains in the cytoplasm in metaphase and anaphase and then is segregated into both daughter cells. Shortly after cell division, Prospero is translocated into the nuclei of both daughter cells. The orientation of the mitotic spindle in neuroblasts and cells of the procephalic neurogenic region is normal in miranda deficient embryos. A transduced miranda can rescue the Prospero localization defects in neuroblasts of miranda indicating that miranda is required for the correct positioning of Prospero in neuroblasts during mitosis (Shen, 1997).

The Numb protein is asymmetrically localized to the basal cell membrane in neuroblasts and cells of the procephalic neurogenic region during mitosis, in contrast to the cytoplasmic distributions of Prospero. After cell division, Numb is segregated into only the basal daughter cell, whereas Prospero is translocated into the nuclei of both cells (Shen, 1997).

The asymmetric localization of Miranda in neuroblasts of prospero and numb mutants is indistinguishable from that of wild-type embryos. Therefore, the asymmetric localization of Miranda does not require prospero or numb. In embryos homozygous for a null allele of inscuteable, both Miranda and Prospero are unable to form crescents or they form crescents that are randomly localized along the cell membrane. Therefore Miranda crescent formation and localization requires inscuteable (Shen, 1997).

Relevant to a discussion of cell asymmetry is a structure found oogenesis called the fusome. Fusomes consist of cytoskeletal proteins, alpha-Spectrin, ß-Spectrin, Hu-li tai shao (an adducin-like protein) and Ankyrin. Of particular interest is the association of fusomes with the pole of the mitotic spindle (Lin, 1995). During the first cystoblast (cystoblasts are derived from germ line stem cells) division, fusome material is associated with only one pole of the mitotic spindle, revealing that this division is asymmetric. During the subsequent three divisions, the growing fusome always associates with the pole of each mitotic spindle that remains in the mother cell, and only extends through the newly formed ring canals after each division is completed. The association of fusome proteins with the mitotic spindle indicates a direct interaction between cytoskeletal components and only one pole of the mitotic spindle. Surely this must have something to do with the underlying mechanism of asymmetric cell division.

Numb protein, known to interact with the cytoplasmic domain of Notch, interfers with the ability of Notch to cause the nuclear translocation of Suppressor of hairless. Both the C-terminal half of the highly conserved phosphotyrosine binding domain of Numb and the C-terminus of Numb are required to inhibit Notch. Numb prevents the colocalization to the nucleus of cells of the Notch intracellular domain with Su(H) resulting in a cytoplasmic localization. Overexpression of Numb during wing development, which is sensitive to Notch dosage, reveals that Numb is also able to inhibit the Notch receptor in vivo. In the external sense organ lineage, the phosphotyrosine binding domain of Numb is found to be essential for the function but not for asymmetric localization of Numb. These results suggest that Numb determines daughter cell fates in the external sense organ lineage by inhibiting Notch signaling (Frise, 1996).

The phosphotyrosine-binding (PTB) domain is a recently identified protein module that has been characterized as binding to phosphopeptides containing an NPXpY motif (X = any amino acid and pY is phosphotyrosine). A novel peptide sequence is described that is recognized by the PTB domain from Drosophila Numb, a protein involved in cell fate determination and asymmetric cell division during the development of the Drosophila nervous system. Using a Tyr-oriented peptide library to screen for ligands, the Numb PTB domain is found to bind selectively to peptides containing a YIGPYphi motif (phi represents a hydrophobic residue). A synthetic peptide containing this sequence binds specifically to the isolated Numb PTB domain in solution with a dissociation constant (Kd) of 5.78 +/- 0.74 microM. Interestingly, the affinity of this peptide for the Numb PTB domain is increased (Kd = 1.41 +/- 0.10 microM) when the second tyrosine in the sequence is phosphorylated. Amino acid substitution studies of the phosphopeptide demonstrate that a core motif of sequence GP(p)Y is required for high-affinity binding to the Numb PTB domain. The same set of amino acids in the Numb PTB domain is involved in binding both phosphorylated and nonphosphorylated forms of the peptide. The in vitro selectivity of the Numb PTB domain is therefore markedly different from those of the Shc and IRS-1 PTB domains, in that it interacts preferentially with a GP(p)Y motif, rather than NPXpY, and does not absolutely require ligand phosphorylation for binding. These results suggest that the PTB domain is a versatile protein module, capable of exhibiting varied binding specificities (Li, 1997).

Asymmetric cell division is a widespread mechanism in developing tissues that leads to the generation of cell diversity. For the most part the basis of assymetric cell division has been analyzed in neuroblasts in the process by which neuroblast division yields another neuroblast and a secondary precursor cell: the ganglion mother cell (GMC). In the embryonic central nervous system of Drosophila melanogaster, GMCs divide and produce postmitotic neurons that take on different cell fates. The current study analyses the process of binary fate decision of two pairs of sibling neurons that occurs during cell division in GMCs. This process is accomplished through the intrinsic fate determinant, Numb. GMCs have apical-basal polarity; Numb localization and the orientation of division are coordinated to segregate Numb to only one sibling cell. The correct positioning of Numb and the proper orientation of division require Inscuteable (Insc). Loss of insc results in the generation of equivalent sibling cells. These results provide evidence that sibling neuron fate decision is nonstochastic and normally depends on the presence of Numb in one of the two siblings. Moreover, the data suggest that the fate of some sibling neurons may be regulated by signals that do not require lateral interaction between the sibling cells (Buescher, 1998).

The focus for the analysis of the roles of insc, numb, and components of the N-signaling pathway in fate specification, was on the only two pairs of GMC-derived neurons for which sibling relationships have been established: the RP2/RP2sib and the aCC/pCC neurons. These neurons are derived from two GMCs that can be identified unambigously by their specific expression of the nuclear protein Even-skipped (Eve). GMC1-1a divides into the aCC/pCC neurons that have approximately equal size and continue to express Eve. However, at later stages of development, aCC is distinguished from pCC by the expression of Zfh-1 and 22C10 (a membrane associated antigen: see Futsch). aCC is a motoneuron and forms an ipsilateral projection that pioneers the intersegmental nerve. GMC4-2a divides to form the sibling neurons RP2/RP2sib that are morphologically distinguishable. In 88% of the hemisegments, the newborn siblings show a significant difference in the size of their nuclei and cell bodies. This asymmetry appears to be initiated during cell division. In GA1019 mutant embryos, in which GMC4-2a fails to complete cytokinesis, cells are formed that contain one large and one small nucleus. This strongly suggests that the difference in size is generated early, prior to the completion of cytokinesis. The larger cell always adopts the RP2 fate, which is characterized by the expression of Eve, Zfh-1, and 22C10. RP2 forms an antero-ipsilateral projection. The smaller sibling always adopts the RP2sib fate, which is characterized by a further decrease in cell and nuclear size and the loss of Eve immunreactivity. Zfh-1 and 22C10 expression have not been shown in RP2sib. These observations suggest that the cell and nuclear size difference may serve as an early physical marker that will allow one to differentiate between the two progeny of GMC4-2a, irrespective of the molecular markers they express later (Buescher, 1998).

Mutations in mastermind (mam),sanpodo, and Notch equalize aspects of sibling cell fate but retain the difference in cell and nuclear size of sibling neurons. In mam mutant embryos, both progeny of GMC4-2a can adopt the RP2 fate with respect to Eve, Zfh-1, and 22C10 expression. However, despite this apparent change from the RP2sib to the RP2 cell fate, the unequal sizes of the GMC4-2a daughter cells remain; that is, their sizes are unaffected. mam is required for the correct fate specification of RP2sib and pCC but not for that of RP2 and aCC. The requirement for mam suggests that N signaling may be involved in the resolution of distinct sibling neuron cell fate. Mutations in mam and N result in similar defects and support the notion that N signaling is required for the resolution of sibling neuron fate. In inscuteable mutant embryos, GMC1-1a and GMC4-2a are correctly formed and express normal levels of Eve (and in the case of GMC4-2a, also Pdm-1). However, GMC1-1a divides to form two sibling neurons that both adopt the aCC fate (94%) with respect to marker gene expression. Similarly, GMC4-2a division results in two sibling cells, both of which adopt the RP2 fate (96%) with respect to expression of Eve, Zfh-1, and 22C10, as well as axon morphology. This strongly suggests that in wild-type embryos, the divisions of GMC1-1a and GMC4-2a are asymmetric in an insc-dependent manner and produce sibling cells that are intrinsically different; loss of insc function leads to the generation of sibling neurons with equivalent cellular identities. Moreover, in contrast to mam, sanpodo, and Notch mutant embryos, the duplicated RP2s seen in insc mutants are equal with respect to their cell and nuclear size. These observations are consistent with the idea that the size difference seen in wild-type embryos is generated by an insc-dependent process during the GMC cell division and occurs prior to the events mediated by mam, spdo, and N that presumably act at the level of the postmitotic sibling cells. No size asymmetry between the sibling neurons should be generated in an insc background regardless of whether the other functions (e.g., spdo) are present or not (Buescher, 1998).

Staining of wild-type embryos with anti-Insc antibody reveals that Insc is expressed in many, and possibly all, GMCs. During interphase, Insc protein is found cortically. In dividing GMCs, anti-Insc immunoreactivity is always seen as an apical/near apical crescent with respect to the surface of the embryo, that is, Insc is localized in the part of the cell located toward the ventral (outside) portion of the embryo. These observations suggest that GMCs possess apical-basal polarity and that their division may be asymmetric in an insc-dependent manner. If the asymmetry of GMC division leads to the generation of pairs of sibling cells that are nonequivalent, then which are the cell fate determinants that are segregated differentially? One such candidate is Numb: it is widely expressed in the developing CNS and has been shown to act as an intrinsic fate determinant in the SOP and MP2 lineages. Moreover, asymmetric Numb localization has been shown to be Insc-dependent in NBs (Buescher, 1998).

To see if Numb is asymmetrically localized in GMC1-1a and GMC4-2a in an Insc-dependent manner, wild-type and insc mutant embryos were stained with anti-Numb, anti-Eve, and DNA stain. In wild-type embryos, Numb always forms a crescent at or near the basal cortex of dividing GMCs, whereas in insc mutant embryos, crescents are either not formed or appear in basal and occasionally in lateral or apical positions. Although it is difficult to directly visualize the orientation of the mitotic spindle in GMCs, the positioning of the metaphase plate in wild-type strongly suggests a perpendicular orientation with respect to the apical surface of the embryo. Consistent with a horizontally placed cleavage plane, the newborn RP2/RP2sib cells are always oriented perpendicular or nearly perpendicular to the apical surface, with the larger cell located in the more dorsal (basal) position. It is concluded that during GMC4-2a division, Numb will be segregated predominantly or exclusively to the future RP2 neuron. In addition, the orientation of metaphase plate in dividing GMCs other than GMC4-2a is also horizontal with respect to the apical surface, indicating that the division of many, possibly all, GMCs are stereotyped in the same manner as NB divisions. Consistent with its function in NBs, loss of insc alters the orientation of GMC division: In 74% of the samples the metaphase plate is oriented perpendicular or close to perpendicular to the apical surface. Accordingly, the newborn RP2/RP2sib are frequently oriented horizontally with respect to the apical surface. Taken together, these results indicate that the apical-basal polarity, which is found in NBs is maintained in their daughter cells, the GMCs, and that Numb localization as well as spindle orientation are coordinated to ensure asymmetric segregation of Numb into one sibling only. So far, insc appears to be the only gene that is required for the apical-basal polarity of GMCs: the analysis of mam, spdo, and N mutant embryos reveals that none of these mutations alter the plane of GMC division. This observation is in agreement with the notion that these genes act later than insc in sibling neuron fate determination (Buescher, 1998).

It has been shown that the the correct positioning of Numb requires Inscuteable; Numb acts downstream of Insc. Taken together, the data show that asymmetric fate determination in the GMC4-2a lineage involves the same components as fate specification in the MP2 and SOP lineages and may be accomplished through productive (effective) N signaling in one sibling (RP2sib) and inhibited (ineffective N signaling) in the Numb-containing sibling (RP2). However, in the case of asymmetric fate determination in GMCs, Notch function is determined through an intracellular, Numb based mechanism, and not through a lateral inhibition mechanism. Consistent with this notion, the ectopic expression of N-intra (a constitutively active form of N) causes the same RP2 to RP2sib fate transformation as numb loss of function, implying that N-intra ectopic expression can override the effects of Numb (Buescher, 1998).

The loss of size asymmetry in insc mutant embryos is not restricted to the RP2/RP2sib lineage but is also observed in muscle progenitor cell divisions. For example, when the muscle progenitor P15 divides, the daughter cells are not equal in size; it is the larger of the two daughter cells that preferentially inherits the Numb that is asymmetrically localized in the dividing muscle progenitor. Similar to the RP2/RP2sib situation, removing insc function appears to equalize the size of the daughter cells derived from the P15 cell division. In contrast, this equalizing effect does not occur in NBs. NB division is highly asymmetric: each division generates a new NB and a GMC that is several times smaller than the NB. In insc mutants, NBs often bud off GMCs in lateral (rather than basal) positions, but the size asymmetry is retained. At present, it is not understood how size asymmetry is generated during progenitor cell division and if, and how, it might be linked to spindle orientation (Buescher, 1998).

Neural precursors (or neuroblasts) divide in a stem cell lineage to generate a series of ganglion mother cells, each of which divides once to produce a pair of postmitotic neurons or glial cells. An exception to this rule is the MP2 neuroblast, which divides only once to generate two neurons. A screen was carried out for genes expressed in the MP2 neuroblast and its progeny as a means of identifying the factors that specify cell fate in the MP2 lineage. A P-element insertion line was identified that expresses the reporter gene, tau-beta-galactosidase, in the MP2 precursor and its progeny, the vMP2 and dMP2 neurons. The transposon disrupts the neurogenic gene, mastermind, but does not lead to neural hyperplasia. However, the vMP2 neuron is transformed into its sibling cell, dMP2. By contrast, expression of a dominant activated form of the Notch receptor in the MP2 lineage transforms dMP2 to vMP2. Notch signaling requires Mastermind, suggesting that Mastermind acts downstream of Notch to determine the vMP2 cell fate. Mastermind plays a similar role in the neurons derived from ganglion mother cells 1-1a and 4-2a, where it specifies the pCC and RP2sib fates, respectively. This suggests that Notch signaling through Mastermind plays a wider role in specifying neuronal identity in the Drosophila central nervous system. Notch is expressed in both MP2 progeny. Notch signaling is blocked by Numb, which segregates exclusively to dMP2 when the MP2 precursor divides. Numb interacts directly with the intracellular domain of Notch. By antagonizing Notch, Numb promotes the dMP2 cell fate. Thus it is likely that Numb antagonism of Notch signaling in dMP2 confines Mastermind function, acting downstream of Notch, to the vMP2 neuron (Schuldt, 1998).

Numb-associated kinase interacts with the phosphotyrosine binding domain of Numb and antagonizes the function of Numb in vivo

During asymmetric cell division, the membrane-associated Numb protein localizes to a crescent in the mitotic progenitor and is segregated predominantly to one of the two daughter cells. A putative serine/threonine kinase, Numb-associated kinase (Nak), has been identified that interacts physically with the phosphotyrosine binding (PTB) domain of Numb. The PTB domains of Shc and insulin receptor substrate bind to an NPXY motif that is not present in the region of Nak that interacts with Numb PTB domain. The Numb PTB domain but not the Shc PTB domain interacts with Nak through a peptide of 11 amino acids, implicating a novel and specific protein-protein interaction. Overexpression of Nak in the sensory organs causes both daughters of a normally asymmetric cell division to adopt the same cell fate, a transformation similar to the loss of numb function phenotype and opposite the cell fate transformation caused by overexpression of Numb. The frequency of cell fate transformation is sensitive to the numb gene dosage, as expected from the physical interaction between Nak and Numb. These findings indicate that Nak may play a role in cell fate determination during asymmetric cell divisions. The observations made are consistent with the possibility that Nak mediates or modulates the action of asymmetrically distributed Numb. For example, Nak may phosphorylate Numb and negatively regulate Numb function. Alternatively, the interaction of Nak and Numb may prevent the binding of Notch to Numb, thus relieving the inhibition of Notch from Numb. It is also conceivable that Nak may be recruited to the vicinity of Notch due to physical interactions of Numb with Notch and Nak, so that it could phosphorylate Notch or its downstream effectors, thereby inhibiting Notch signaling (Chien, 1998).

Whereas the specific physical interaction between Nak and Numb suggests that Nak may be part of the Numb pathway in specifying daughter cell fate during asymmetric division, it will be necessary to test this possibility by examining both the loss-of-function phenotype and the gain-of-function phenotype of the nak gene. No loss-of-function mutations of the nak gene are currently available. It is worth noting, however, that a number of genes known to be involved in the Numb pathway for asymmetric division exhibit overexpression phenotypes which correspond to cell fate transformations opposite those caused by loss of gene function. Hence, overexpression of the protein products of Delta, Notch,tramtrack, Suppresser of Hairless, orenhancer of split causes transformation of the B cell to the A cell, the hair cell to the socket cell, and the neuron to the sheath cell, opposite their respective loss-of-function phenotypes. Phenotypes due to overexpression of these genes are similar to the numb null mutant phenotype, whereas overexpression of Numb causes the opposite cell fate transformation. The overexpression phenotypes of nak are very similar to those of Notch, tramtrack, and other downstream genes of numb and are therefore highly suggestive of the involvement of nak in asymmetric divisions (Chien, 1998).

The in vivo interaction of Myc-Nak with Numb protein is also indicative of the function of Nak in the asymmetric cell division pathway. In addition to the immunocoprecipitation of Numb and Myc-Nak, it was also observed that the ectopically expressed Myc-Nak localize to the cortical membrane where Numb and Notch are distributed, suggesting that Nak can localize to the site for participation in the asymmetric cell divisions. Due to the overexpression of Myc-Nak, it is difficult to analyze the segregation of Myc-Nak during cell division. Whether Nak is asymmetrically localized during asymmetric cell divisions awaits the availability of an antibody that is suitable for immunocytochemistry (Chien, 1998).

The potential involvement of Nak in asymmetric divisions in Drosophila is reminiscent of the involvement of thepar-1 gene in asymmetric divisions during early embryonic development of C. elegans. Par-1 also contains a serine/threonine kinase domain and a C-terminal region that binds other proteins; whereas the C terminus of Nak binds Numb, the C terminus of Par-1 binds a nonmuscle myosin. A priori, a Numb-binding protein could be involved in asymmetric localization of Numb during asymmetric division or in executing the actions of asymmetrically segregated Numb in specifying daughter cell fate. It appears unlikely that Nak is involved in asymmetric localization of Numb, for the following reasons. First, the Nak overexpression phenotypes could be suppressed by Numb overexpression. This restoration of the proper asymmetric divisions could not have been achieved if overexpression of Nak had abolished asymmetric Numb localization. Second, Nak binds to the PTB domain but not to the rest of the Numb protein. The PTB domain is not necessary for asymmetric localization of Numb in dividing neural precursor cells but is necessary for the ability of Numb to inhibit Notch signaling. Third, both mNumb and mouse Numblike (mNbl; homolog of rNbl) contain PTB domains which are 70 to 75% identical to the PTB domain of dNumb at the amino acid level, and when overexpressed in Drosophila, mNumb and mNbl can transform cell fate in the sensory organ lineages. But only mNumb, not mNbl, is asymmetrically localized in transgenic flies. It thus appears unlikely that Nak plays a role in asymmetric Numb localization (Chien, 1998).

The observation of Nak activity thus far is consistent with the possibility that Nak mediates or modulates the action of asymmetrically distributed Numb. For example, Nak may phosphorylate Numb and negatively regulate Numb function. Alternatively, the interaction of Nak and Numb may prevent the binding of Notch to Numb, thus relieving the inhibition of Notch from Numb. It is also conceivable that Nak may be recruited to the vicinity of Notch due to physical interactions of Numb with Notch and Nak, so that it could phosphorylate Notch or its downstream effectors, thereby inhibiting Notch signaling. These and other possible scenarios may be tested by future genetic and biochemical studies (Chien, 1998).

Multiple modes of peptide recognition by the PTB domain of the cell fate determinant Numb

The phosphotyrosine-binding (PTB) domain of the cell fate determinant Numb is involved in the formation of multiple protein complexes in vivo and can bind a diverse array of peptide sequences in vitro. To investigate the structural basis for the promiscuous nature of this protein module, its solution structure was determined by NMR in a complex with a peptide containing an NMSF sequence derived from the Numb-associated kinase (Nak). The Nak peptide was found to adopt a significantly different structure from that of a GPpY sequence-containing peptide previously determined. In contrast to the helical turn adopted by the GPpY peptide, the Nak peptide forms a beta-turn at the NMSF site followed by another turn near the C-terminus. The Numb PTB domain appears to recognize peptides that differ in both primary and secondary structures by engaging various amounts of the binding surface of the protein. These results suggest a mechanism through which a single PTB domain might interact with multiple distinct target proteins to control a complex biological process such as asymmetric cell division (Zwahlen, 2000; full text of article).

Partner of numb is required for the proper localization of Numb

The proper localization of Numb depends on its interaction with the adapter protein Partner of numb (Pon). In pon mutant embryos, the formation of Numb crescent is delayed in neuroblasts and is disrupted in muscle progenitor cells (Lu, 1998). Pon was isolated on the basis of its physical interaction with Numb. Pon is asymmetrically localized during mitosis and colocalizes with Numb. Ectopically expressed Pon responds to the apical-basal polarity of epithelial cells and is sufficient to localize Numb basally. It is proposed that PON is one component of a multimolecular machinery that localizes Numb by responding to polarity cues conserved in neural precursors and epithelial cells (Lu, 1998 and Lu, 1999).

In principle, the asymmetric localization of Numb/Pon can be accomplished by one or a combination of the following mechanisms: localization and local translation of their mRNAs; active transport of the proteins by motor molecules along the cytoplasmic or cortical cytoskeleton; passive diffusion (3D in the cytosol or 2D along the cortex) and trapping of the proteins by basally localized anchor molecules, or protein targeting to the membrane followed by selective degradation at one side of the cortex. The available method to detect protein localization by immunostaining of fixed embryos only provides static images of the proteins in different cells and is inadequate to distinguish among the above possibilities (Lu, 1999).

The mechanism of asymmetric Pon localization has been shown to operate at the protein level. The asymmetric localization domain of Pon has been mapped to its C-terminal region. Using a fusion between this localization domain and GFP, the entire process of Pon localization was monitored in neuroblasts of living embryos. This in vivo analysis reveals that the asymmetric localization of Pon is a dynamic, multistep process. The protein is first recruited from the cytosol to the cell cortex, a step that requires cell cycle progression into mitosis. Cortically recruited Pon then moves on the cortex and is later restricted to the basal side to form a crescent. The crescent disintegrates upon exit from mitosis. Photobleaching experiments reveal both apical and basal movements of Pon on the cortex. These movements can still occur when myosin motor activity is inhibited by drug treatment. Genetic and pharmacological analyses further reveal that the formation and anchoring of the Pon crescent at the basal cortex require actomyosin and Inscuteable (Lu, 1999).

Inscuteable coordinates mitotic spindle orientation and asymmetric protein localization in neuroblasts. To gain more insight into the mechanism by which Inscuteable directs the asymmetric localization of Pon, Pon-GFP was introduced into an insc mutant background. In insc mutant neuroblasts, cortical recruitment of Pon-GFP is normal, but the Pon-GFP crescent fails to form in 70% of the cases, and, in the remaining 30% of the neuroblasts, the crescent forms but it is randomly positioned. These observations are consistent with previous findings about protein localization in insc mutants. However, the ability to continuously monitor Pon-GFP in living embryos revealed a previously unrecognized function of Inscuteable. In wild-type embryos, the Pon-GFP crescent is stably anchored to the basal cortex once the crescent is formed. In contrast, in insc mutant embryos, the crescent is not stably anchored on the cortex, and it shifts position gradually. Depending on the orientation of the mitotic spindle and the position of the crescent at the time of cytokinesis, the protein can be segregated equally or unequally between the two daughters. Therefore, Inscuteable is required to provide positional cues that direct the formation and the anchoring and maintenance of Pon-GFP crescent at the basal cortex (Lu, 1999).

The gradual recruitment of Pon from the cytosol to the cell cortex at early stages of the cell cycle appears to be coupled to cell cycle progression. To test whether entry into mitosis is a prerequisite for this cortical recruitment, Pon-GFP was introduced into cell cycle mutant backgrounds. In string mutants, postblastoderm cells arrest at the G2 phase of the cell cycle. In neuroblasts of string mutants, the GFP signal is diffuse in the cytoplasm, with some uniform cortical staining. Even after 1 hr of recording, the cytoplasmic signal is not cleared, although the uniform cortical signal seems to increase slightly over time. In contrast, in wild-type neuroblasts, the cytoplasmic signal is cleared within 5-6 min after its appearance. Thus, cortical recruitment of Pon-GFP depends on entry into mitosis (Lu, 1999).

In pebble mutants, cytokinesis of postblastoderm cell divisions is blocked, but other cell cycle events including the asymmetric localization of Numb and Prospero still occur. The initial cortical recruitment and formation of a basal Pon-GFP crescent are normal in pebble mutant neuroblasts. However, by continuously monitoring the Pon-GFP crescent, it was observed that within 10-15 min of its formation, the crescent starts to disintegrate and the protein is dispersed uniformly on the cortex. Gradually, the cortical signal is decreased to background levels, presumably due to degradation or release from the cortex. By comparing the time interval between crescent formation and disintegration in pebble mutants (10-15 minutes) with the interval between crescent formation and the later stages of the neuroblast cell cycle in wild-type embryos, it was determined that the timing of Pon-GFP crescent disintegration in pebble mutants coincides with the end of a normal neuroblast division. This result suggests that at the exit from mitosis certain cell cycle events disassemble or inactivate the localization machinery and that this can occur in the absence of cytokinesis (Lu, 1999).

The defect in the cortical recruitment of Pon-GFP in string mutants suggests that the assembly or proper functioning of the machinery that recruits Pon to the cortex depends on cell cycle progression into mitosis. It is also possible that the cortical recruitment of Pon may depend on its posttranslational modification such as phosphorylation by the p34cdc2 kinase, which is inactive in string mutants. Further biochemical characterization of Pon protein, such as analyzing its posttranslational modification during the cell cycle, should provide more insight into the mechanistic aspects of this regulation (Lu, 1999).

The disintegration of Pon-GFP crescent at the exit from mitosis in pebble mutants implicates a role for the cell cycle machinery in disabling the protein localization machinery. The absence of cytokinesis in pebble mutants allows this step to be observed in more detail. In wild-type neuroblasts, the cleavage furrow coincides with the border of the Pon-GFP crescent at cytokinesis, therefore the GFP signal is distributed all around the GMC cell membrane as soon as the GMC is formed. This makes the crescent disintegration step not observable in wild-type embryos. However, in both wild-type GMC cells and pebble mutant neuroblasts, the uniform cortical GFP signal is gradually decreased to background levels as the cell cycle progresses, suggesting that Pon-GFP is eventually released from the cortex and becomes degraded or delocalized in both wild-type and pebble mutant embryos. In this regard, it will be interesting to test whether the anaphase-promoting complex/cyclosome ubiquitin ligase or components of the mitosis exit signaling pathway are involved in the disintegration of Pon-GFP crescent and the subsequent release of the protein from the cortex (Lu, 1999).

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).

Asymmetric divisions with two different division orientations follow different polarity cues for the asymmetric segregation of determinants in the sensory organ precursor (SOP) lineage. The first asymmetric division depends on frizzled function and has the mitotic spindle of the pI cell in the epithelium oriented along the anterior-posterior axis, giving rise to pIIa and pIIb, which divide in different orientations. Only the pIIb division resembles neuroblast division in daughter-size asymmetry, spindle orientation along the apical-basal axis, basal Numb localization, and requirement for inscuteable function. Because the PDZ domain protein Bazooka is required for spindle orientation and basal localization of Numb in neuroblasts, it was of interest to enquire whether Bazooka plays a similar role in the pIIb in the SOP lineage. Surprisingly, in pI and all subsequent divisions, Bazooka controls asymmetric localization of the Numb-anchoring protein Pon, but not spindle orientation. Bazooka also regulates cell proliferation in the SOP lineage; loss of bazooka function results in supernumerary cell divisions and apoptotic cell death (Roegiers, 2001b).

During embryonic neuroblast divisions, Bazooka is required not only to localize Inscuteable to the apical cortex and Numb, Miranda, Prospero, and Pon to the basal cortex, but also to orient the mitotic spindle along the apical-basal axis. To determine the requirement of bazooka in the asymmetric divisions of the adult SOP lineage, the MARCM system was used to generate baz mutant clones expressing both Pon-GFP (as a reporter for Numb localization) and Tau-GFP (as a reporter for spindle orientation) under the control of scabrous-GAL4, which is strongly expressed in the SOP cell and in the SOP lineage. The movements of Pon-GFP and Tau-GFP were monitored in live tissue throughout all asymmetric divisions of the SOP lineage. In bazxi106 or bazEH171 null mutant clones, pI cells underwent mitosis at ~15 h APF as in wild type. However, in all mutant pI cells observed, Pon-GFP remained uniformly distributed and never formed an anterior crescent as seen in dividing wild-type pI cells. Nor did Pon-GFP crescents form in the subsequent divisions in the lineage. Thus, although only the pIIb resembles the embryonic neuroblast in its orientation of division and requirement for Inscuteable, Bazooka is required for the asymmetric Pon/Numb localization in the pI division, as well as all subsequent divisions (Roegiers, 2001b).

Because Numb functions as an asymmetrically localized cell-fate determinant in the SOP lineage, the absence of Numb crescents in baz mutant clones could lead to cell-fate transformations in the daughters of the pI cell. Thus the anterior daughter cell of the pI in bazooka mutant clones (the pIIb cell in the wild type) is referred to as pIIbb, and the posterior daughter cell as pIIab. It is worth noting, however, that either loss-of-function or misexpression of numb causes cell-fate transformation only in a subset of sensory organs, presumably because the Notch-mediated mutual inhibition may still allow the two daughter cells to adopt different cell fates, albeit without a bias set by the Numb crescent. Transformation of pIIa to pIIb cell fate is known to alter the timing of mitosis of the transformed pIIa cell. Timing of the pIIbb, pIIab, and pIIIbb divisions is indistinguishable from wild-type pIIb, pIIa, and pIIIb cells. In addition, the pI and pIIab spindles align along the A-P axis in all mutant clones. And in eight of the nine clones examined the pIIbb spindles were oriented along the apical-basal axis as in wild type (the remaining pIIbb cell divided before the pIIab, but had its spindle oriented along the anterior-posterior axis). Because an apically localized Inscuteable is required for mitotic spindle positioning in the pIIb cell, Inscuteable localization was also examined in the pIIbb cell in bazooka mutant clones. Inscuteable is localized to an apical stalk in pIIbb, similar to the wild-type pIIb (n = 12). Thus the great majority of pIIbb and pIIab cells resemble wild-type pIIb and pIIa cells in their timing and orientation of division, as well as the expression of Inscuteable in the pIIbb. It therefore appears that these bazooka mutations do not cause detectable cell-fate transformation in most of the pIIbb and pIIab cells, although it remains possible that there are partial transformations and cell-fate changes in a subset of these cells. In light of these observations, the complete loss of Pon-GFP crescents in every mitotic pIIbb and pIIab cell examined strongly supports a model wherein Bazooka controls Pon/Numb asymmetric localization in not only pI but also pIIb and pIIa cells (Roegiers, 2001b).

Partner of Inscuteable/Discs-large complex is required for the location of Numb and Partner of numb during establishment planar polarity during asymmetric cell division

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 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).

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

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).

Domina/jumeau is required for the location of Numb and Partner of numb

The gene Domina (Dom) has been identifed as a Drosophila member of the Forkhead/winged-helix (FKH/WH) gene family; it is a suppressor of position effect variegation, and affects and regulates eye and bristle development. Domina [termed jumeau (Jumu) by Cheah, 2000] is required for generating asymmetric sibling neuronal cell fates. The Dom/Jumu protein is expressed in the developing embryonic CNS, including the neuroblast GMC4-2a. Dom/Jumu appears to play a role in fate determination during CNS lineage development. Dom/Jumu is dispensable for Inscuteable apical localization but necessary for the basal localization of Partner of numb and Numb These results suggest that in addition to the correct formation of an apical complex, transcription mediated by molecules like Dom/Jumu is also required to facilitate the correct asymmetric localization and segregation of cell fate determinants like Numb (Cheah, 2000).

jumeau was identified in a mutant screen of lethal P-element insertions that affect the number of RP2 motorneurons. jumu L40, a strong hypomorph which deletes sequences from the transcribed region of jumu, increases the expressivity of an RP2 duplication phenotype 4 fold; jumu L70, that shows a seven-fold increase in expressivity appears to be a total loss-of-function allele. What might be the underlying mechanism responsible for the RP2 duplication phenotype associated with the jumu loss-of-function mutants? Several observations suggest that Jumu may be acting at the level of the GMC4-2a cell division. Jumu is not expressed in NB4-2 prior to its first divisions, making it unlikley that it would be acting at the level of the NB4-2 cell divisions. Moreover, it is only transiently detected in the postmitotic RP2 and RP2sib and it is not asymmetrically segregated to one of these cells. Therefore it seems unlikely that jumu would be acting at the level of the postmitotic neurons. Since nuclear Jumu can be detected in GMC4-2a, the possibility was examined that jumu may be required for the asymmetric localization of the cell fate determinant Numb during the GMC4-2a cell division. Since Numb always colocalizes with Pon, which acts to facilitate its localization, an anti-Pon antibody was used to illustrate the localization of Numb. Examination of late prophase to metaphase GMC4-2a cells triple labelled with anti-Eve, DNA stain and anti-Pon indicates that Pon localization is defective in dividing jumu GMC4-2a cells. In essentially all of the dividing wild-type GMC4-2a cells, Pon and Numb always form basal cortical crescents; it has been suggested that the more basal progeny, which preferentially inherits Numb, becomes the RP2 neuron. However, in jumu mutant embryos, many dividing GMC4-2a cells fail to localize Pon as a basal crescent: about 36% show either cortical Pon distribution or misplaced crescents. The frequency of the Pon mislocalization roughly coincides with the frequency of hemisegments showing the RP2 duplication phenotype (29%). Similar conclusions can be drawn using anti-Numb. These data are therefore consistent with the notion that the duplication of RP2 neurons in jumu embryos arises as a result of the symmetric segregation of Numb to both the postmitotic RP2 and RP2sib leading to a RP2sib to RP2 cell fate transformation. In contrast to Pon/Numb, Insc localization does not appear to be affected in jumu embryos. Essentially all of the dividing GMC4-2a cells in both wild-type and jumu embryos localize Insc as an apical cortical crescent. Hence, the loss of jumu function does not exert a general effect on the protein localization machinery per se but appears to specifically affect the localization of Pon/Numb. Loss of jumu also does not alter the localization of any of the asymmetrically localized proteins, i.e. Miranda, Pros, Insc, during NB divisions (Cheah, 2000).

lethal (2) giant larvae and discs large are involved in localization of Numb

Drosophila neuroblasts are a model system for studying asymmetric cell division: they divide unequally to produce an apical neuroblast and a basal ganglion mother cell that differ in size, mitotic activity and developmental potential. During neuroblast mitosis, an apical protein complex orients the mitotic spindle and targets determinants of cell fate to the basal cortex, but the mechanisms of these two processes are unknown. The tumor-suppressor genes lethal (2) giant larvae (lgl) and discs large (dlg) regulate basal protein targeting, but not apical complex formation or spindle orientation, in both embryonic and larval neuroblasts. Dlg protein is apically enriched and is required for maintaining cortical localization of Lgl protein. Basal protein targeting requires microfilament and myosin function, yet the lgl phenotype is strongly suppressed by reducing levels of myosin II. It is concluded that Dlg and Lgl promote, and myosin II inhibits, actomyosin-dependent basal protein targeting in neuroblasts (Peng, 2000).

Embryonic Drosophila neuroblasts develop from an apical/basal polarized epithelium. Individual cells delaminate into the embryo, enlarge to form neuroblasts, and begin a series of asymmetric cell divisions; these divisions result in the production of a large mitotically active apical cell (neuroblast), and a smaller basal cell (ganglion mother cell, GMC) that differentiates into two neurons or glia. A growing number of proteins are known to be asymmetrically localized in mitotic neuroblasts: apically localized proteins include Bazooka (Baz), Inscuteable (Insc) and Partner of Inscuteable (Pins); basally targeted proteins include Miranda, Prospero, Partner of Numb (Pon) and Numb, which are important for GMC development. Miranda and Prospero are apically localized at late interphase before their mitosis-dependent transport to the basal cortex. The Baz/Insc/Pins apical complex is required for both apical/basal spindle orientation and basal protein targeting, but little is known about how this complex regulates either process (Peng, 2000).

To identify genes required for apical/basal protein targeting in neuroblasts, deficiency stocks were screened looking for defects in Prospero basal localization in neuroblasts. This screen identified the lgl gene, which encodes a WD-40 repeat protein with homologues in many species, including the closely related 'Lgl family' genes Lgl1/Lgl2 (human), Lgl1 (mouse), U51993 (Caenorhabditis elegans); the slightly more divergent 'Tomosyn family' genes Tomosyn (rat), KIAA1006 (human), C617762 (Drosophila ), and M01A10 (C. elegans); and recently duplicated genes similar to both families: sro7/sro77 (budding yeast). In Drosophila, lgl mutations affect protein targeting to epithelial apical junctions, epidermal cell-shape changes, and produce tumors of the brain and the imaginal disc. This spectrum of phenotypes has been noted for another tumor-suppressor gene, discs large. This study explores the role of Lgl and Dlg in regulating neuroblast cell polarity (Peng, 2000).

Apical and basal protein targeting are compared in neuroblasts from wild-type embryos and embryos that lack all maternal and zygotic Lgl or Dlg function (called lglGLC or dlgGLC embryos). Wild-type metaphase neuroblasts show apical Insc/Pins localization, and basal Miranda/Prospero/Pon crescents. In addition, Miranda and Prospero proteins can be observed around the apical centrosome and weakly on the mitotic spindle in wild-type neuroblasts. In contrast, all lglGLC and dlgGLC metaphase neuroblasts show cytoplasmic Pon and uniformly cortical and strongly spindle-associated Miranda/Prospero; the apical proteins Insc/Pins are normal or slightly expanded. Although lglGLC and dlgGLC embryos show striking defects in neuroblast basal protein localization, they also show an early loss of embryonic epithelial apical/basal polarity, which could indirectly cause the observed neuroblast defects (Peng, 2000).

To determine the neuroblast-specific function of Lgl and Dlg, Lgl- or Dlg-depleted neuroblasts were studied in embryos or larvae where epithelial development occurs normally. Initially, homozygous null lgl4 embryos were studied, in which maternal Lgl protein allows normal embryonic epithelial development (including Armadillo, Crumbs and Dlg localization). In stage 16-17 lgl4 embryos, mitotic neuroblasts show normal Baz/Insc/Pins apical crescents, and normal spindle orientation, but Miranda/Prospero are delocalized onto the spindle and around the cortex and Pon is cytoplasmic. This phenotype is less severe in early embryos but fully penetrant in older embryos, presumably due to progressive loss of maternal Lgl protein. Next, neuroblasts were assayed in lgl3344 or dlgv55 homozygous larvae -- these larval neuroblasts are persistent embryonic neuroblasts that develop from a normal embryonic epithelium due to maternal Lgl and Dlg protein function. Wild-type larval metaphase neuroblasts have Insc/Pins crescents the opposite of Miranda/Prospero/Pon/Numb crescents, whereas homozygous lgl3344 or dlgv55 larval metaphase neuroblasts show normal Insc/Pins crescents but Miranda/Prospero/Pon proteins are cytoplasmic, uniformly cortical, and weakly spindle-associated. It is concluded that Lgl and Dlg are required specifically in neuroblasts for basal protein targeting, without affecting apical protein localization or spindle orientation (Peng, 2000).

Delocalization of the Prospero and Numb proteins produces defects in the nervous system and other tissues, so lgl mutant embryos were scored for cell fate defects. lglGLC embryos have severe morphological defects that preclude analysis, and lgl4 embryos can only be scored for late embryonic phenotypes, due to persistence of maternal Lgl protein. lgl4 embryos show a decrease in Even-skipped lateral (EL) neuron number at stage 17. A similar but stronger phenotype is seen in numb mutants, suggesting that the lgl phenotype may be due to delocalization of Numb during the GMC divisions that produce the EL neurons. The relatively mild lgl phenotype could be due to 'telophase rescue' of Numb protein in these GMCs, or to maternal Lgl protein (Peng, 2000).

How does Lgl regulate basal protein targeting? Lgl binds non-muscle myosin II in all organisms tested, and sro7/77 and myo1 (encoding Lgl-related proteins and myosin II, respectively) show strong negative genetic interactions in yeast. Tests were performed for genetic interactions between lgl4 and two different null mutations in zipper (encoding myosin II), scoring Miranda basal localization in stage 17 neuroblasts, when maternal Lgl and Myosin II protein levels are lowest. Wild-type and zip embryos have normal basal protein localization, whereas lgl4 embryos show complete delocalization of basal proteins. However, lgl4 embryos lacking one copy of myosin II show a significant increase in basal protein targeting; and lgl4;zip1 mutant embryos show virtually normal basal protein targeting. Thus, reducing myosin II levels strongly suppresses the lgl phenotype, indicating that myosin II can inhibit basal targeting when Lgl levels are low (Peng, 2000).

In addition, the general myosin inhibitor 2,3-butanedione monoxime (BDM) can suppress the lgl phenotype: stage 10 lgl4 embryos treated with BDM show a significant increase in basal protein localization compared with sham-treated stage 10 lgl4 embryos. Wild-type or lgl4 embryos treated with 50 mM BDM show delocalization of Miranda, Prospero and Pon. These data indicate that a myosin that is sensitive to 25 mM BDM inhibits basal protein localization in lgl embryos (probably myosin II), and at least one myosin that is sensitive to 50 mM BDM promotes basal protein targeting in mitotic neuroblasts (Peng, 2000).

Thus, in neuroblasts Lgl and Dlg regulate targeting of all known basal proteins without affecting apical protein localization or spindle orientation. In epithelia, Lgl and Dlg are necessary to restrict proteins to the apical membrane domain. Lgl could promote protein targeting to specific membrane domains in both neuroblasts (basal) and epithelia (apical), similar to the role of Lgl-related proteins in facilitating secretory vesicle fusion at specific membrane domains in yeast and mammals. If so, Lgl must act in neuroblasts via a secretory pathway that is independent of brefeldin A, because it has been shown that treatment with brefeldin A disrupts Golgi, inhibits Wingless secretion, but does not block basal protein targeting. Alternatively, Lgl may actively promote actomyosin-dependent localization of basal proteins and/or function to keep myosin II levels low so that they do not interfere with myosin-dependent basal localization. A general function of the Lgl protein family may be to increase the fidelity of protein targeting to specific domains of the plasma membrane (Peng, 2000).

aurora is required for the asymmetic distribution of Numb

During asymmetric cell division in the Drosophila nervous system, Numb segregates into one of two daughter cells where it is required for the establishment of the correct cell fate. Numb is uniformly cortical in interphase, but in late prophase, the protein concentrates in the cortical area overlying one of two centrosomes in an actin/myosin-dependent manner. What triggers the asymmetric localization of Numb at the onset of mitosis is unclear. The mitotic kinase Aurora-A is required for the asymmetric localization of Numb. In Drosophila sensory organ precursor (SOP) cells mutant for the aurora-A allele aurA37, Numb is uniformly localized around the cell cortex during mitosis and segregates into both daughter cells, leading to cell fate transformations in the SOP lineage. aurA37 mutant cells also fail to recruit Centrosomin (Cnn) and gamma-Tubulin to centrosomes during mitosis, leading to spindle morphology defects. However, Numb still localizes asymmetrically in cnn mutants or after disruption of microtubules, indicating that there are two independent functions for Aurora-A in centrosome maturation and asymmetric protein localization during mitosis. Using photobleaching of a GFP-Aurora fusion protein, it has been shown that two rapidly exchanging pools of Aurora-A are present in the cytoplasm and at the centrosome and might carry out these two functions. These results suggest that activation of the Aurora-A kinase at the onset of mitosis is required for the actin-dependent asymmetric localization of Numb. Aurora-A is also involved in centrosome maturation and spindle assembly, indicating that it regulates both actin- and microtubule-dependent processes in mitotic cells (Berdnik, 2002a).

In a screen for mutations that affect asymmetric localization of Numb, aurA37, a mutant in aurora-A in which bipolar mitotic spindles are formed and chromosomes segregate, but Numb fails to localize asymmetrically, was identified. aurA37 mutants also have defects in centrosome maturation and fail to recruit the proteins Cnn and gamma-Tubulin to centrosomes during mitosis. However, Numb still localizes asymmetrically in cnn mutants that were reported to lack functional mitotic centrosomes, suggesting that the centrosome maturation defects are not responsible for the failure to localize Numb asymmetrically. Aurora-A is concentrated at centrosomes, but photobleaching experiments reveal rapid exchange with a cytoplasmic pool of the protein. These results suggest that two rapidly interchanging pools of Aurora-A kinase are involved in spindle assembly and asymmetric protein localization during mitosis (Berdnik, 2002a).

To identify mutations that cause defects in Numb localization, it was predicted that such mutations would lead to cell fate transformations in the SOP lineage and cause morphological abnormalities in fly bristles. In a genetic screen, the eyeless-Flp/FRT system was used to generate heterozygous flies that become homozygous by mitotic recombination in all tissues that are derived from the eye imaginal disk. These include most of the head cuticle, and therefore mutant phenotypes can be analyzed in bristles on the head. One of the mutations identified (termed aurA37) causes the frequent formation of morphologically abnormal bristles that contain two hairs and two sockets, indicating that inner cells are transformed into additional outer cells. The mutation was mapped to the cytological interval 87A7-9 based on lethality over Df(3R)P-58. Df(3R)P-58 includes the Drosophila aurora-A gene, and, indeed, aurA37 is lethal over known alleles of aurora-A. Flies homozygous for aurA37, transheterozygous for aurA37 and the strong allele aur87Ac-3 (Glover, 1995), or transheterozygous for aurA37 and the deficiency Df(3R)M-Kx1 (which includes aurora-A) die during pupal stages with bristle abnormalities similar to the ones observed in aurA37 mutant clones. aurA37 has no dominant phenotype, indicating that the protein has not acquired a novel activity. aurA37 mutants carry a single G-to-A nucleotide exchange that is predicted to exchange a conserved arginine (amino acid 201) into histidine. The affected residue is located in the catalytic domain and is conserved between all kinases of the Aurora-family but not in all other protein kinases. It is concluded that aurA37 is a new allele of Drosophila aurora-A (Berdnik, 2002a).

To characterize the cellular defects that cause the bristle phenotypes in aurA37 mutants, the SOP lineage in these mutants was analyzed. The four cell types in Drosophila ES organs can be distinguished by their characteristic size and marker gene expression: of the two larger outer cells, only the socket cell expresses the transcription factor Suppressor of Hairless (Su[H]). The two smaller inner cells can be distinguished based on expression of Prospero only in the sheath cell. One of each of these four cell types is found in control ES organs. Most aurA37 mutant ES organs, in contrast, consist of four large cells, two of which express Su(H), which indicates that the two inner cells are transformed into additional outer cells. Thus, instead of dividing asymmetrically, aurA37 mutant SOP cells divide symmetrically into two pIIa cells that then each generate one hair and one socket. The Numb protein is crucial for asymmetric SOP cell division, and, therefore, whether this lineage defect is due to missegregation of Numb was tested by staining homozygous aurA37 mutant pupae for Numb and DNA. While the Numb protein accumulates at the anterior SOP cell cortex during late prophase in wild-type and segregates into the anterior pIIb cell, no asymmetric localization of Numb is observed in aurA37 mutants. The protein is uniformly distributed around the cell cortex throughout mitosis and segregates into both daughter cells. Asymmetric localization of Gαi to the anterior cell cortex and localization of Bazooka to the posterior cell cortex is unaffected in aurA37 mutants, indicating that cell polarity is set up correctly. Thus, aurora-A is required for the asymmetric localization of Numb during mitosis (Berdnik, 2002a).

A mitotic function for Drosophila Aurora-A has been described before. In strong alleles of aurora-A, centrosomes fail to separate, leading to the generation of abnormal monopolar mitotic spindles, defects in chromosome segregation, and the formation of polyploid cells. aurA37 mutant cells, in contrast, complete mitosis and divide into two daughter cells and can therefore be used to characterize other aspects of Aurora-A function. To analyze mitotic spindles in aurA37 mutants, control and mutant pupae were stained for DNA and alpha-Tubulin. Bipolar mitotic spindles are formed in aurA37 mutants, but while microtubule minus ends converge on the centrosome in wild-type, they are less focused in aurA37 mutants. This spindle morphology phenotype could reflect a defect in centrosome function, and therefore aurA37 mutants were stained for the centrosomal marker gamma-Tubulin. In control SOP cells, gamma-Tubulin staining is weak during interphase, but two strong dots appear during mitosis, indicating that gamma-Tubulin is recruited to centrosomes. In 67% of the aurA37 mutant mitotic SOP cells, no strong dots of gamma-Tubulin staining were visible, indicating a failure to recruit the protein to centrosomes. This defect is not completely penetrant, since one dot was observed in 17% of the cells, and, in another 17%, two closely spaced dots were seen. Defects in mitotic recruitment of gamma-Tubulin have been described before in flies mutant for cnn, a centrosomal core component that is dispersed in interphase but localized to centrosomes during mitosis. aurA37 mutant pupae were therefore double stained for Cnn and gamma-Tubulin. In contrast to wild-type, where Cnn is detected in two strong dots at either spindle pole, the protein is undetectable on centrosomes of most aurA37 mutant SOP cells. Thus, Aurora-A is required for recruiting both gamma-Tubulin and Cnn to centrosomes during mitosis, and these defects in centrosome maturation might be the cause of the abnormal spindle morphology. Despite the spindle defects in aurA37 mutants, however, the two daughter cells of SOPs are still preferentially arranged along the anterior-posterior axis, indicating that spindle orientation is unaffected (Berdnik, 2002a).

Cnn has been shown to be required for localization of gamma-Tubulin to centrosomes during mitosis. The defects in gamma-Tubulin localization in aurA37 mutants could therefore be an indirect consequence of the failure to recruit Cnn. To test whether the defects in Numb localization are also caused by the failure to recruit Cnn, cnn null mutant Drosophila pupae were stained for Numb, gamma-Tubulin, and DNA. Even though no Cnn protein could be detected, these flies are viable. As in wild-type, Numb localizes into a cortical crescent during late prophase in all cnn mutant SOP cells even though, in 9% of the mitotic SOP cells (n = 22), the Numb crescent is mispositioned and does not correlate with the orientation of the metaphase plate. Since gamma-Tubulin is not recruited to centrosomes in these mutants, it is concluded that neither Cnn nor gamma-Tubulin recruitment to mitotic centrosomes is required for the asymmetric localization of Numb (Berdnik, 2002a).

The failure to localize Numb asymmetrically in aurA37 mutants could still be caused by the spindle defects. In neuroblasts, Numb localization still occurs after complete disruption of the mitotic spindle. To test the requirement of a mitotic spindle for Numb localization in SOP cells, wild-type pupae were incubated in 20 µg/ml colcemid for 1 or 2 hr and stained for Numb and gamma-Tubulin. After 1 hr of treatment, on average, nine SOP cells per pupal notum showed the mitotic arrest phenotype typical of microtubule inhibitors: chromosomes were no longer aligned in the metaphase plate, and centrosomes were distributed at random positions. Numb was still asymmetrically localized in 78% of these colcemid-arrested SOP cells. After 2 hr of treatment, the average number of metaphase-arrested SOP cells per notum increased to 27, and Numb was asymmetrically localized in 81% of them, indicating that new Numb crescents can be formed in the absence of a functional mitotic spindle. No effect on centrosome position was observed in a control experiment in which colcemid was omitted and Numb crescents were observed in 71% of the mitotic SOP cells. Thus, neither a functional mitotic spindle nor recruitment of Centrosomin and gamma-Tubulin to centrosomes are required for asymmetric localization of Numb. It is concluded that the defects in Numb localization observed in aurA37 mutants are not indirect consequences of the spindle or centrosome defects. Rather, they indicate an independent role for Aurora-A in asymmetric protein localization during mitosis (Berdnik, 2002a).

In C. elegans and vertebrates, Aurora-A proteins localize to centrosomes and the mitotic spindle (Nigg, 2001). The subcellular localization of Aurora-A in Drosophila has not been described, but if Aurora-A is exclusively localized to centrosomes, a function in asymmetric protein localization at the cell cortex is hard to imagine. Antibody was therefore generated against Aurora-A and it was used to stain wild-type and aurA37 mutant Drosophila pupae. As in other organisms, Aurora-A is concentrated at centrosomes and the mitotic spindle in prophase and metaphase of wild-type Drosophila cells. In contrast, no centrosomal staining is detected in aurA37 mutant cells. In addition to the centrosomal staining, significant staining was detected in the cytoplasm of both wild-type and aurA37 mutant cells that can be blocked by preincubation of the Aurora-A antibody with the immunogenic peptide. To better analyze the dynamics of Aurora-A localization in living cells, transgenic flies were generated expressing a GFP-Aurora-A fusion protein (GFP-AurA). Using the UAS/Gal4 system, GFP-AurA was expressed in pupal SOP cells, and its distribution during mitosis was followed using confocal time-lapse microscopy. Like the endogenous protein, GFP-AurA is concentrated at centrosomes and the mitotic spindle during mitosis, but a fraction of the protein was detected in the cytoplasm. To determine the exchange rate between the centrosomal and the cytoplasmic pool of Aurora-A, photobleaching experiments were performed. When one of the two centrosomes was bleached by intense laser light, centrosomal staining was completely recovered within 10 s. No recovery was observed, however, when both the cytoplasm and the centrosomes were bleached using the same bleaching protocol. Thus, centrosomal localization of Aurora-A is transient, and the centrosomal pool rapidly interchanges with Aurora-A in the cytoplasm (Berdnik, 2002a).

Thus the mitotic kinase Aurora-A is required for the asymmetric localization of Numb. Aurora-A is also involved in centrosome maturation and spindle assembly. However, neither functional centrosomes nor the mitotic spindle are required for Numb localization. Numb localization is an actin/myosin-dependent process, suggesting that Aurora-A regulates both actin- and microtubule-dependent processes during mitosis. Two rapidly interchanging pools of Aurora-A are found in the cytoplasm and at centrosomes and might carry out these two functions (Berdnik, 2002a).

The results are consistent with the functions of Aurora-A described in other systems. In C. elegans, inactivation of the Aurora-A homolog air-1 by RNAi leads to defects in spindle morphology and failure to recruit the proteins CeGrip, ZYG-9, and gamma-Tubulin to centrosomes in mitosis (Schumacher, 1998; Hannak, 2001). Interestingly, air-1(RNAi) embryos also have defects in asymmetric distribution of cellular determinants: P-granules and the cytoplasmic protein Pie-1, both markers for the C. elegans germline, fail to segregate into one of the two daughter cells and are frequently found in both cells instead (Schumacker, 1998). Since Aurora-A is localized to centrosomes, this was thought to indicate a role for microtubules in asymmetric protein segregation (Schumacher, 1998). However, the asymmetric localization of both P-granules and the Pie-1 protein are actin dependent and microtubule independent. The results presented in this paper indicate that centrosomal localization of Aurora-A is transient and the protein is rapidly exchanging with a cytoplasmic pool. Assuming that Aurora-A localization is similarly dynamic in C. elegans, the defects in P-granule and Pie-1 localization could actually indicate an evolutionarily conserved function of Aurora-A in actin-dependent asymmetric protein localization during mitosis (Berdnik, 2002a).

Mutations in Drosophila aurora-A were shown before to have defects in centrosome separation and chromosome segregation, leading to the formation of polyploid cells. Defects in centrosome separation are seen in a fraction of aurA37 mutant cells, but monopolar mitotic spindles or polyploid cells are not observed. aurA37 could be a hypomorphic allele that affects some Aurora-A-dependent processes more than others. aurA37 affects an arginine in kinase subdomain III that is found in all Aurora- and MAP-kinases but is not generally conserved between all protein kinases. In MAP kinases, the equivalent residue is predicted to make contact with threonine 183, a residue in the activation loop that needs to be phosphorylated to activate the kinase. Phosphorylation of the residue equivalent to threonine 183 can also activate Aurora-A kinase (Walter, 2000), and aurA37 could prevent the conformational change needed for full activation of the kinase (Berdnik, 2002a).

How could Aurora-A function in asymmetric cell division? Aurora-A is not required for setting up polarity during interphase since both Galphai and Bazooka are asymmetrically localized in aurA37 mutants. Instead, it is needed for interpreting this polarity to initiate the asymmetric localization of Numb at the onset of mitosis. In vertebrates, Aurora-A activity peaks at the G2/M transition (Bischoff, 1998), and phosphorylation of either Numb itself or a component of the Numb localization machinery could be required for Numb localization. So far, Lgl (Lethal [2] giant larvae) is the only other protein required for Numb localization but not polarity establishment. Since phosphorylation of Numb or Lgl by Aurora-A in vitro could not be detected, another, yet to be identified, component of the Numb localization machinery seems to be phosphorylated by Aurora-A at the onset of mitosis (Berdnik, 2002a).

Numb localization also requires activation of the Cdc2 kinase, and, in hypomorphic cdc2 mutants, cells enter mitosis but have defects in asymmetric protein localization, a phenotype that is remarkably similar to aurA37. How the two kinases act together is unclear. In vertebrates, Aurora-A activity peaks before activation of Cdc2, suggesting that Cdc2 is not required for Aurora-A activation. Conversely, Cdc2 activation is Aurora-A independent, since many Cdc2-dependent events do not require Aurora-A. Thus, it is likely that activation of the Numb localization machinery requires both Cdc2 and Aurora-A-dependent phosphorylation events (Berdnik, 2002a).

The human homolog of Aurora-A, Aurora2, is amplified in colorectal cancer, suggesting that it is involved in carcinogenesis. How Aurora2 causes cancer is unclear. Overexpression of Aurora-A could force quiescent cells to reenter the cell cycle or cause defects in chromosome segregation and aneuploidy. Assuming that Aurora-A is also involved in asymmetric cell division in vertebrates, cell lineage defects are an interesting alternative possibility (Berdnik, 2002a).

Quantitative analysis of protein dynamics during asymmetric cell division

In dividing Drosophila sensory organ precursor (SOP) cells, the fate determinant Numb and its associated adaptor protein Partner of numb (Pon) localize asymmetrically and segregate into the anterior daughter cell, where Numb influences cell fate by repressing Notch signaling. Asymmetric localization of both proteins requires the protein kinase aPKC and its substrate Lethal (2) giant larvae (Lgl). Because both Numb and Pon localization require actin and myosin, lateral transport along the cell cortex has been proposed as a possible mechanism for their asymmetric distribution. This study used quantitative live analysis of GFP-Pon and Numb-GFP fluorescence and fluorescence recovery after photobleaching (FRAP) to characterize the dynamics of Numb and Pon localization during SOP division. It was demonstrated that Numb and Pon rapidly exchange between a cytoplasmic pool and the cell cortex and that preferential recruitment from the cytoplasm is responsible for their asymmetric distribution during mitosis. Expression of a constitutively active form of aPKC impairs membrane recruitment of GFP-Pon. This defect can be rescued by coexpression of nonphosphorylatable Lgl, indicating that Lgl is the main target of aPKC. It is proposed that a high-affinity binding site is asymmetrically distributed by aPKC and Lgl and is responsible for asymmetric localization of cell-fate determinants during mitosis (Mayer, 2005).

In order to study the dynamics of asymmetric protein localization, a time series of the division of an SOP cell expressing GFP-Pon and Histone2B-RFP was recorded under the control of a specific promoter. Histone2B-RFP was used to visualize DNA, thus allowing correlation of distinct steps of GFP-Pon localization with other mitotic events. In interphase, some GFP-Pon is cortical, but a large part localizes to the cytoplasm. As the cell enters mitosis, it rounds up and undergoes strong membrane blebbings, indicative of local rearrangements of the cortical cytoskeleton. Interestingly, similar blebbing events have also been observed in the first division of the C. elegans zygote. Unlike in SOP cells, however, they only occur on the anterior side of the C. elegans zygote, where Par-3/6 localize. Shortly after blebbing has started, chromosomes condense and GFP-Pon accumulates on random sites of the cell cortex. The accumulations are transient and do not necessarily predict the position of the final Pon crescent. This suggests that the process leading to Pon accumulation can take place all around the cell but is reinforced specifically in the crescent region. Some GFP-Pon was also observed at the nucleus. This signal might be due to GFP-Pon binding to the nuclear envelope or to the endoplasmic reticulum, and it disappears slowly after nuclear-envelope breakdown. At nuclear-envelope breakdown, cortical blebbing ceases, the cell cortex smoothes, and first signs of asymmetric localization of GFP-Pon into an anterior cortical crescent are observed. As the cell progressed into metaphase, the GFP-Pon signal in the crescent area becomes stronger. Surprisingly, the intensity of the cortical area opposite of the crescent is almost not changed during this process. Thus, GFP-Pon might actually be recruited to the crescent directly from the cytoplasm rather than being transported along the cell cortex. Indeed, quantification of fluorescence intensity showed that GFP-Pon recruitment at the cell cortex is accompanied by a comparable loss of cytoplasmic GFP-Pon. Note that local degradation of GFP-Pon in the cytoplasm is not responsible for this reduction because total GFP-Pon remains unchanged (Mayer, 2005).

Subsequently, the metaphase plate was oriented with respect to the crescent, and during cytokinesis, GFP-Pon segregated largely into the anterior daughter cell. It is proposed that GFP-Pon localization is a two-step process involving the establishment of a cortical area where the crescent will form and the progressive recruitment of protein to the predefined site until metaphase (Mayer, 2005).

Asymmetry of Numb and Pon could be created by lateral movement along the cell cortex or by direct recruitment from the cytoplasm to one side of the cell cortex. To quantify the exchange of Numb and Pon between the cell cortex and the cytoplasm, fluorescence recovery after photobleaching (FRAP) was used of GFP fusions to Numb and Pon. Numb-GFP can partially rescue the numb mutant phenotype, indicating that it is functional. GFP-Pon contains just the asymmetric-localization domain. Its rescue behavior is unknown, but it colocalizes with endogenous Pon throughout mitosis. When cytoplasmic GFP-Pon is photobleached, fluorescence recovers with a half-time of 0.48 s, indicating that diffusion is not limiting. Recovery of cortical GFP-Pon fluorescence occurred with single exponential kinetics and a half-time of 35 s, whereas the half-time for Numb-GFP was 27 s. Therefore, Numb and Pon showed a surprisingly dynamic association with the cell cortex (Mayer, 2005).

Either cortical recruitment of cytoplasmic GFP-Pon or lateral diffusion/transport of cortical GFP-Pon could be responsible for fluorescence recovery. To measure the exchange between cortical and cytoplasmic Pon, an area covering approximately 40% of the cytoplasm was repeatedly photobleached in an SOP cell expressing GFP-Pon. Fluorescence intensity was simultaneously recorded at the cortex. Cortical fluorescence intensity dropped to less than 5% with a half-time of 52 s. Thus, the cortical and cytoplasmic pools of GFP-Pon rapidly interchange with a mobile fraction of more than 95% (Mayer, 2005).

When the dynamic association with the cell cortex is taken into account, Pon asymmetry could be explained either by fast and continuous lateral transport or by directed recruitment to an asymmetric cortical binding site. To determine the contribution of lateral transport, FRAP rates were compared on the edge and in the center of a photobleached region within the GFP-Pon crescent. The bleached region was defined such that a region of nonbleached molecules was left behind at the edges of the crescent after photobleaching. To avoid recovery from above and below the image plane, a protocol was used in which the region of interest was bleached in several planes. The efficiency of this procedure was confirmed by 3D reconstruction after photobleaching in fixed tissue. FRAP curves from ten experiments were averaged. Their superposition shows that the two regions recover nearly identically with half-times of 32 s for a region close to nonbleached GFP-Pon and of 35 s for a region farther away. Taken together, these observations suggest a model where Pon is preferentially recruited from the cytoplasm to the site of crescent formation. It is proposed that a cortical high-affinity binding site for Pon is established during mitosis and mediates specific recruitment of Pon to one side of the cell cortex (Mayer, 2005).

To test the role of Lgl in asymmetric protein localization in SOP cells, cortical recruitment of GFP-Pon was measured in lgl1 mutant clones. In a similar experiment, Lgl has been shown to be dispensable for Pon localization, although Pon recruitment seemed to be delayed. The ratio between total cortical and total cytoplasmic fluorescence was calculated. Because GFP fluorescence intensity is proportional to GFP-Pon concentration, this ratio should give a good estimate of the fraction of GFP-Pon localized at the cell cortex. Although GFP-Pon was still asymmetric, quantitative analysis revealed that the cortical GFP-Pon fraction was slightly but significantly reduced in lgl1 mutant clones. This might be a hypomorphic phenotype caused by small residual amounts of Lgl protein present in the mutant clones. Therefore expression of deregulated aPKC (aPKC-deltaN) was used as another means to inactivate Lgl. Expression of aPKC-deltaN was shown to phenocopy lgl mutants in embryonic tissues, presumably because it phosphorylates and inactivates Lgl all around the cell. In contrast to lgl1 mutant SOP cells, a much stronger decrease of cortical GFP-Pon recruitment was observed upon aPKC-deltaN expression. Still, a slight cortical asymmetry was observed, which is thought is due to the presence of endogenous aPKC. Even at anaphase, the degree of recruitment hardly reached that of control cells in prophase. To test whether Lgl phosphorylation was responsible for this phenotype, aPKC-deltaN was coexpressed with nonphosphorylatable lgl3A. Expression of lgl3A completely rescued the cortical-recruitment defect. The observed differences are not due to increased protein levels because total cellular GFP-Pon fluorescence remains constant (Mayer, 2005).

Thus, active, nonphosphorylated Lgl is needed for cortical recruitment of GFP-Pon although lgl1 mutant clones did not show a very strong phenotype. The easiest explanation for the discrepancy between the lgl1 mutant and ectopic Lgl phosphorylation is the perdurance of residual Lgl protein in mutant tissue. This is supported by previous observations describing Numb-localization defects in temperature-sensitive alleles of lgl. It is possible that Lgl can mediate its effects even at protein concentrations below the detection limit of the antibody. Thus, Lgl may not be needed at stoichiometric levels for asymmetric protein localization in SOP cells, but it instead plays a catalytic or signaling role (Mayer, 2005).

How could Lgl recruit Pon to the cell cortex? Formally, it is possible that Pon simply binds Lgl in a phosphorylation-dependent manner. However, no direct interaction has been described and such a model would not explain why Pon is cortical even when Lgl levels are strongly reduced. Two other models are more likely: Either cortical binding sites for Numb and Pon are present all around the cell, but their affinity depends on Lgl and its phosphorylation status and therefore varies along the cell cortex (Model 1); or a limiting number of cortical binding sites are present only on one side of the cell, and Lgl is responsible for their asymmetric distribution (Model 2). To distinguish between these models, FRAP rates were measured for cortical GFP-Pon in different genetic backgrounds. The FRAP rate is a function of the rate constants for both association and dissociation of GFP-Pon with its postulated cortical binding site. In Model 1, expression of activated lgl (lgl3A) or deregulated aPKC (aPKC-?N) should alter the affinity of the binding site and therefore change the rate constants, resulting in a variation of the FRAP rate. Because the FRAP rate is independent of receptor concentration, however, it would remain constant under the same conditions in Model 2. Cortical GFP-Pon FRAP rates were measured in wild-type SOP cells, in cells expressing lgl3A, and in cells where Lgl was inactivated by expression of aPKC-deltaN. Although expression of aPKC-deltaN dramatically reduced the amount of GFP-Pon present at the cortex, it did not influence the kinetics of GFP-Pon binding to the cortical binding site. Thus, the number of Pon binding sites at the cell cortex, and not their affinity for Pon, seems to be reduced by aPKC-deltaN expression (Mayer, 2005).

To gain independent evidence for the two models, the fraction of GFP-Pon present at the cell cortex was quantitated. If Lgl regulated GFP-Pon binding site affinity, expression of lgl3A would change the entire SOP cell cortex to high affinity, and therefore it would increase the cortical GFP-Pon fraction. If Lgl regulated only the distribution of binding sites, however, the cortical fraction of GFP-Pon should remain the same. Cortical recruitment was quantified by measuring the ratio of cortical to cytoplasmic fluorescence for GFP-Pon and Numb-GFP at different time points in mitosis. Compared to wild-type cells, expression of lgl3A did not cause a significant increase in cortical recruitment. This is not because cytoplasmic GFP-Pon is limiting; increased GFP-Pon expression predominantly increased the cytoplasmic signal. Taken together, these results favor Model 2, in which Lgl acts by asymmetrically distributing a limiting number of cortical GFP-Pon binding sites. The loss of cortical fluorescence upon aPKC-deltaN expression indicates that lgl is also required for binding site formation, in addition to binding site positioning. However, this second role of lgl does not seem to be rate limiting under normal conditions because lgl3A expression does not increase the cortical GFP-Pon fraction. Although these results are most consistent with Model 2, more-complex models cannot be excluded. For example, lgl could distribute a limiting adaptor protein that links Pon to a receptor but is not the receptor itself (Mayer, 2005).

The direct cortical binding partners for Pon or Numb have not yet been identified. Thus, it is only possible to speculate on the molecular mechanisms of their postulated asymmetric distribution. Although the results are inconsistent with lateral transport of GFP-Pon, they do not exclude lateral transport of its cortical anchor. Similar to what has been proposed for asymmetric cell division in C. elegans, a possible mechanism could be local tearing and contraction of the cortical actin cytoskeleton. Lgl was shown to inhibit the cortical localization of myosin II, and it has been proposed that cortical myosin II might exclude asymmetrically segregating proteins. These data could be integrated with the model if myosin II excludes the cortical binding sites rather than influencing determinant localization directly. Alternatively, transmembrane receptors for Pon or Numb could be delivered to the position of crescent formation by vesicle transport. Such a mechanism in which transmembrane receptors are present on vesicles that dock at the membrane in an Lgl-dependent fashion would be consistent with the quantitative observations. It would also explain why Lgl is essential for crescent formation but not needed in metaphase for maintenance of asymmetric protein localization. It is remarkable that lateral diffusion of transmembrane proteins is slow enough to allow a stable asymmetric distribution, if the delivery of the protein is asymmetric, both in yeast and in SOP cells. The yeast Lgl orthologs Sro7p and Sro77p have been implicated in plasma-membrane fusion of secretory vesicles, and Lgl has been proposed to regulate vesicular targeting to specific membrane domains. Furthermore, asymmetric protein localization in Drosophila requires myosin VI, a motor whose main function is vesicle movement, suggesting that vesicle trafficking plays some role (Mayer, 2005).

These data provide insight into the dynamic protein movements of cell-fate determinants and their associated adaptor proteins during asymmetric cell division. It is proposed that these determinants are preferentially recruited from the cytoplasm to a high-affinity binding site during late prophase. Establishment of this binding site is regulated by the phosphorylation status of Lgl. The role of Lgl is more to concentrate binding sites on one side of the cell than to act as a receptor itself or change the affinity of another Numb or Pon binding site (Mayer, 2005).

Dual roles for the trimeric G protein Go in asymmetric cell division in Drosophila; The trimeric G protein Go plays a role in the anterior localization of Numb and daughter cell fate specification

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

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

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

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

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

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

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

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

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

Polo inhibits progenitor self-renewal and regulates Numb asymmetry by phosphorylating Pon

Self-renewal and differentiation are cardinal features of stem cells. Asymmetric cell division provides one fundamental mechanism by which stem cell self-renewal and differentiation are balanced. A failure of this balance could lead to diseases such as cancer. During asymmetric division of stem cells, factors controlling their self-renewal and differentiation are unequally segregated between daughter cells. Numb is one such factor that is segregated to the differentiating daughter cell during the stem-cell-like neuroblast divisions in Drosophila, where it inhibits self-renewal. The localization and function of Numb is cell-cycle-dependent. This study shows that Polo acts as a tumour suppressor in the larval brain. Supernumerary neuroblasts are produced at the expense of neurons in polo mutants. Polo directly phosphorylates Partner of Numb (Pon), an adaptor protein for Numb, and this phosphorylation event is important for Pon to localize Numb. In polo mutants, the asymmetric localization of Pon, Numb and atypical protein kinase C are disrupted, whereas other polarity markers are largely unaffected. Overexpression of Numb suppresses neuroblast overproliferation caused by polo mutations, suggesting that Numb has a major role in mediating this effect of Polo. These results reveal a biochemical link between the cell cycle and the asymmetric protein localization machinery, and indicate that Polo can inhibit progenitor self-renewal by regulating the localization and function of Numb (Wang, 2007).

Asymmetric localization of Numb depends on its adaptor protein Pon. The Pon localization domain (Pon-LD) is located at the carboxy terminus of the protein. The Ser 611 (S611) residue in this domain matches the consensus phosphorylation site for Polo. Because the localization of Pon is cell-cycle-dependent, tests were perfomred to see whether Polo can directly phosphorylate Pon. Pon-LD, but not Pon(S611A)-LD, in which S611 was mutated to Ala, was readily phosphorylated by mammalian Polo-like kinase 1 (Plk1) in vitro, demonstrating that Pon S611 is a Polo phosphorylation site (Wang, 2007).

To test whether Pon S611 is normally phosphorylated in vivo, an antibody was generated against S611-phosphorylated (p-S611) Pon. The specificity of this antibody was shown by its ability to recognize a glutathione S-transferase-Pon-LD fusion protein (GST-Pon-LD) only after the fusion protein was pre-phosphorylated by Plk1. It did not recognize GST-Pon(S611A)-LD in the same assay. Next, larval brain extracts prepared from wild type as well as heterozygotes [polo9(+/-) and polo10(+/-)], and homozygotes [polo9(-/-) and polo10(-/-)] of two different polo loss-of-function alleles were analysed by western blotting using this p-S611-specific antibody. p-S611-positive Pon was clearly detected in wild-type animals and in heterozygotes, but was barely detectable in homozygous mutant animals, demonstrating that Pon is phosphorylated at S611 in vivo in a Polo-dependent fashion (Wang, 2007).

Immunohistochemistry was used to verify S611 phosphorylation in vivo and to visualize phospho-Pon localization. p-S611-positive endogenous Pon was detected in wild-type larval brains as a crescent in metaphase neuroblasts, and was segregated to the ganglion mother cell (GMC, the daughter committed to the differentiation pathway) after division. In the polo9 mutant, however, p-S611-positive Pon was undetectable. The p-S611 antibody also reacted with Pon-LD, but not with Pon(S611A)-LD, in transgenic larval brain (Wang, 2007).

To test for a functional role of S611 phosphorylation, S611 was mutated to a non-phosphorylatable Ala residue (S611A) or to a phospho-mimetic Asp residue (S611D). Wild-type and phospho-mutant Pon-LD were fused to green fluorescent protein (GFP) and expressed in embryonic neuroblasts. Both GFP-Pon-LD and GFP-Pon(S611D)-LD showed the expected basal localization. In contrast, the localization of GFP-Pon(S611A)-LD was defective. At prometaphase and metaphase, it showed either uniformly cortical (80%) or basally enriched but apically detectable cortical (20%) distribution. At anaphase and telophase, however, it formed basal crescents in most neuroblasts. This 'telophase rescue' seemed to be partially mediated by endogenous Pon, because less rescue was observed in pon mutant neuroblasts, with 58 neuroblasts mis-segregating GFP-Pon(S611A)-LD at late anaphase/telophase. It is unlikely that the S611A mutation affects Pon localization by altering the charge or global conformation and folding of the protein, because mutation of an adjacent Ser residue (S616) or triple mutations at three potential atypical protein kinase C (aPKC) phosphorylation sites (S644A/S648A/S652A) had no effect on Pon-LD localization, suggesting that S611 represents a unique regulatory point in Pon localization (Wang, 2007).

To assess whether Polo has a role in neuroblast self-renewal and/or asymmetric division, central brain neuroblast numbers were quantified in two strong hypomorphic alleles, polo9 and polo10, using Deadpan (Dpn) and Miranda (Mira) as neuroblast markers. Wild-type larval central brains averaged 37 neuroblasts 24 h after larval hatching (ALH) and 10 neuroblasts 96 h ALH. polo9 larval central brains averaged 36 neuroblasts 24 h ALH. However, the number increased dramatically to 254 96 h ALH. Consistent with this increase in neuroblast number, the numbers of BrdU-labelled, CycE-positive or phospho-histone-H3-positive proliferating cells were also increased in polo9 mutant brains compared to wild type. Concomitantly, a dramatic reduction of differentiated cells expressing neuronal markers, Embryonic Lethal Abnormal Vision (Elav) or nuclear Prospero (Pros), was observed in polo9 mutant brains. A similar neuroblast overproliferation phenotype was observed in polo10 and in trans-heterozygotes between polo9 and a deficiency that deletes polo. A Polo-GFP genomic construct fully rescued this polo mutant phenotype, verifying that these defects are caused by polo loss-of-function. Excess self-renewal and proliferation at the expense of neuronal differentiation was also observed in MARCM (mosaic analysis with a repressible cell marker) clones derived from single polo9 mutant neuroblasts. These results indicate that Polo behaves like a tumour suppressor to inhibit neuroblast self-renewal and to promote differentiation. polo mutant GMCs may revert to neuroblast-like cells, as has been shown for brat (brain tumor) mutants (Wang, 2007).

The physiological role of Polo in regulating Pon localization and function was analyzed. Most larval neuroblasts were found at metaphase in polo9 mutant brains, and both Pon and Numb were uniformly distributed. In late anaphase/telophase neuroblasts, Pon and Numb were mis-segregated to both daughter cells. Defective Pon and Numb localization in the polo mutant is unlikely to be a secondary consequence of cell cycle arrest, because arrest of wild-type neuroblasts at metaphase with microtubule-depolymerizing drugs actually increased the number of cells possessing a Numb crescent (Wang, 2007).

To test whether Polo is specifically required for Pon/Numb localization, other apical and basal proteins were analyzed. In polo9 mutant neuroblasts, the basal localization of Brat and Pros was relatively normal. Moreover, double-labelling of the same mutant neuroblast showed that the localization of Mira, the adaptor protein for Pros and Brat, was normal, whereas Pon localization was abnormal. Introduction of a Polo-GFP transgene into polo9 effectively rescued the Pon localization and cell-cycle defects. Apical proteins such as Insc, Baz, Pins and Discs Large 1 (Dlg1) were localized normally in polo9 mutant neuroblasts. The only apical protein showing abnormal localization was aPKC, which became distributed uniformly on the cortex and showed cytoplasmic localization. During telophase, aPKC could be mis-segregated into both daughter cells (Wang, 2007).

Polo is localized to centrosomes and is required for centrosome organization and separation. Whether Polo has a role in orienting neuroblast mitotic spindles was tested. The tight coupling of spindle orientation with crescent formation seen in wild-type neuroblasts was disrupted in polo9 metaphase neuroblasts with two centrosomes. Therefore, Polo kinase is also required for coupling mitotic spindle orientation with cortical protein asymmetry. This spindle phenotype was fully rescued by the Polo-GFP transgene (Wang, 2007).

Next the role of Pon phosphorylation in mediating Numb localization was probed. Full-length Pon containing the S611A or S611D mutation was used to assess the effects of S611 phosphorylation. In pon mutant neuroblasts, Numb localization was defective. Introducing wild-type Pon restored Numb asymmetric localization at metaphase and later stages. Most pon mutant neuroblasts expressing Pon(S611D) also showed rescue. In contrast, pon mutant neuroblasts expressing Pon(S611A) showed largely abnormal Numb localization. Polo-mediated phosphorylation is therefore important for Pon to localize Numb. The function of Pon in neuroblast self-renewal was tested by generating pon MARCM clones. Interestingly, compared to wild-type clones, ectopic neuroblast self-renewal was observed in pon mutant clones (Wang, 2007).

This study has shown that polo mutations affect Numb and aPKC localization as well as spindle orientation -- processes known to affect neuroblast self-renewal to various degrees. Strikingly, overexpression of either Numb-GFP or Numb effectively suppressed the ectopic neuroblast self-renewal phenotype seen in the polo9 mutant. This effect was not caused by increased neuroblast apoptosis, and overexpression of Numb-GFP or Numb in a wild-type background did not affect the neuroblast number. These results indicate that Numb is a principal player downstream of Polo in regulating neuroblast self-renewal. Numb overexpression did not rescue the aPKC mislocalization and spindle misorientation phenotypes of polo mutants. These defects could also contribute to the neuroblast overgrowth phenotype of polo mutants, but their effects might have been masked by Numb overexpression. Consistent with this, introduction of Pon(S611D) into a polo mutant neuroblast did not significantly rescue the neuroblast overgrowth phenotype, despite the partial restoration of Numb localization. Because aPKC localization and spindle orientation defects were not rescued by Pon(S611D), these defects may account for the inability of Pon(S611D) to rescue the overgrowth phenotype of polo. aPKC has been shown to phosphorylate Numb. Delocalized aPKC at the basal side may be sufficient to inactivate endogenous Numb, but not overexpressed Numb, owing to titration by the overexpressed protein (Wang, 2007).

Numb was previously shown to inhibit neuroblast self-renewal by antagonizing Notch signalling. Reducing Notch significantly suppressed the neuroblast overgrowth phenotype of the polo9 mutant. Reducing Notch in a wild-type background also led to a partial loss of neuroblasts, consistent with Notch being required for progenitor self-renewal. It is envisioned that in polo or pon mutants, owing to the symmetric distribution of Numb, the GMCs receive insufficient Numb to inhibit Notch, thereby causing them to adopt a neuroblast-like fate. To test further the importance of Numb asymmetric localization in neuroblast self-renewal versus differentiation, the numbS52F mutation, which apparently affects Numb asymmetric localization but not its stability or activity, was tested. In numbS52F neuroblast clones, ectopic neuroblast self-renewal similar to that seen in polo or pon clones was observed. Thus, loss of Numb asymmetric localization is sufficient to cause neuroblast overgrowth (Wang, 2007).

These results indicate that Polo controls the self-renewal versus differentiation decision of neural progenitors by regulating the localization and activity of Numb and the orientation of mitotic spindles. Polo regulates the localization of Numb by means of Pon. Although immunofluorescence shows that Polo is primarily localized to the centrosomes, a cytosolic pool of Polo probably exists because Polo exhibits dynamic in vivo association with the mitotic apparatus and many non-centrosomal Polo substrates have been identified. Cytosolic localization of the centrosomal kinase Aurora-A has been demonstrated. How Polo regulates the localization of aPKC, the activity of Numb and the orientation of mitotic spindles awaits further investigation. In addition to the Numb/Notch pathway, other factors such as Pros and Brat are necessary for preventing GMCs from undergoing self-renewing divisions. Because these factors are segregated normally in polo neuroblasts, it seems that they are not sufficient to prevent progenitor self-renewal or that activation of Notch is able to override their effects. Intriguingly, some Plks behave as tumour suppressors in mammals, and loss of Numb has also been implicated in the hyperactivation of Notch signalling in breast cancer cells. These results and future studies in models like Drosophila will provide mechanistic insights into these observed tumour-suppressor roles of Polo and Numb (Wang, 2007).

aPKC-mediated phosphorylation regulates asymmetric membrane localization of the cell fate determinant Numb

In Drosophila, the partition defective (Par) complex containing Par3, Par6 and atypical protein kinase C (aPKC) directs the polarized distribution and unequal segregation of the cell fate determinant Numb during asymmetric cell divisions. Unequal segregation of mammalian Numb has also been observed, but the factors involved are unknown. This study identified in vivo phosphorylation sites of mammalian Numb, and showed that both mammalian and Drosophila Numb interact with, and are substrates for aPKC in vitro. A form of mammalian Numb lacking two protein kinase C (PKC) phosphorylation sites (Numb2A) accumulates at the cell membrane and is refractory to PKC activation. In epithelial cells, mammalian Numb localizes to the basolateral membrane and is excluded from the apical domain, which accumulates aPKC. In contrast, Numb2A is distributed uniformly around the cell cortex. Mutational analysis of conserved aPKC phosphorylation sites in Drosophila Numb suggests that phosphorylation contributes to asymmetric localization of Numb, opposite to aPKC in dividing sensory organ precursor cells. These results suggest a model in which phosphorylation of Numb by aPKC regulates its polarized distribution in epithelial cells as well as during asymmetric cell divisions (Smith, 2007).

To establish whether aPKC-dependent phosphorylation is a conserved mechanism for regulating the cortical membrane localization of Numb, whether a myc-tagged version of Drosophila Numb forms a complex with PKCzeta in HEK293 cells was examined. Co-immunoprecipitation of Drosophila Numb with PKCzeta indicates that this interaction is conserved. The sequence of Drosophila Numb was examined. A total of five evolutionarily conserved aPKC phosphorylation sites were revealed including Ser52 and Ser304, corresponding to Ser7 and Ser295 in murine Numb (isoform p66). Whether PKC could phosphorylate Drosophila Numb was examined in an in vitro kinase assay. Both PKCα and PKCzeta, the human orthologue of Drosophila aPKC, phosphorylated Numb in an immune-complex assay. PKCzeta also phosphorylated a GST-Numb fusion protein. Mutations of all five of the conserved aPKC sites (Numb5A) reduced the in vitro phosphorylation by PKCzeta, indicating that some of these sites are the targets of PKCzeta. A form of Numb in which Ser52 is left intact, while the other four serines were mutated to alanine (Numb4A), was still efficiently phosphorylated by PKCzeta indicating that Ser52 is one of the acceptor sites in vitro. However, mutation of Ser52 into alanine did not significantly reduce the in vitro phosphorylation of GST-Numb, suggesting that PKCzeta phosphorylates additional sites. NanoLC-MS-MS analyses of the in vitro phosphorylated GST-Numb identified a total of eight aPKC sites. Confirmation of the phosphorylated Ser52 residue was obtained from the MS-MS spectrum of m/z 497.7. Five PKCzeta phosphorylation sites that do not appear conserved were identified in this analysis (Ser31, Ser35, Ser48, Ser161, and Ser297). These sites likely account for the residual phosphorylation of GST-Numb5A. Although these analyses provide direct identification of aPKC phosphorylated residues, other potential phosphorylation sites remained elusive. For example, the early eluting tryptic peptide QMS304LR was observed only in the control sample. Its absence in the aPKC-treated sample strongly suggests that Ser304 is in fact phosphorylated and could not be detected owing to nonretention of this hydrophilic peptide during reverse phase LC. It is concluded that aPKC phosphorylates Numb at several sites in both Drosophila and mouse, including at the conserved Ser7 and Ser295 sites (Ser52 and Ser304 in Drosophila Numb) (Smith, 2007).

The localization was examined of Drosophila Numb in dividing sensory organ precursor pI cells. The pI cells divide asymmetrically within the plane of the notum epithelium and along the body axis. In these cells, Numb localizes at the anterior cortex, opposite to aPKC, which relocalizes from the apical cortex to the lateral posterior cortex upon mitosis. The possible role of phosphorylation in the regulation of Drosophila Numb localization was examined by studying the distribution of Myc-tagged versions of Numb, Numb4A, NumbS52A, and Numb5A that were expressed in pI cells using the neurPGAL4 driver. Importantly, overexpression of Numb4A, NumbS52A, or Numb5A in pI cells led to cell-fate transformation in the bristle lineage indicative of gain of Numb function. This indicates that these Numb mutant proteins are functional. Similar to endogenous Numb, Myc-Numb localized at the anterior cortex, opposite to aPKC, in all cells at prometaphase and metaphase. Consistently, myc-Numb colocalized with Pins. In contrast, the crescent formed by Numb5A appeared to extend posteriorly in 91% of the dividing pI cells at prometaphase. Interestingly, a recent study has shown that a mutant Numb protein, NumbS52F, fails to localize properly in dividing pI cells. Thus, one possible interpretation of the data is that the mislocalization of Numb5A is due to the S52A mutation. Therefore the localization of NumbS52A was studied and it was found to localize asymmetrically in 84% of the pI cells at prometaphase. Thus, the S52A mutation alone cannot be responsible for the defective localization of Numb5A. Additionally, mutations of the four other serine residues in Numb4A did not significantly change the asymmetric distribution of Numb. Therefore, it is concluded that the defects in Numb5A distribution in dividing pI cells depends on the combination of at least two mutations, S52A and a mutation in one of the four conserved aPKC consensus sites, possibly Ser304. Thus, these data support the notion that aPKC-mediated phosphorylation of Drosophila Numb contributes to the asymmetric distribution of Numb in dividing pI cells (Smith, 2007)..

This study provides evidence for a conserved mechanism regulating the asymmetric distribution of the cell-fate determinant Numb. Mammalian and Drosophila Numb proteins are substrates for aPKC and phosphorylation regulates Numb localization at the cortical membrane. The data also indicate that aPKC-dependent phosphorylation regulates the polarized distribution of Numb in mammalian epithelial cells and Drosophila sensory organ precursor cells (Smith, 2007).

The aPKC/Par3/Par6 complex plays a conserved role in establishing polarity in a variety of cellular contexts, including during asymmetric cell divisions in C. elegans and Drosophila, and in apical-basal polarity of epithelial tissues. In mammalian epithelial cells, aPKC is required for the establishment and maintenance of apical-basal polarity. In this context, several targets of aPKC have been identified, including the conserved proteins, Lgl and Par1, whose activities also contribute to cell polarity. In mammalian cells, Lgl plays a role in adherens junction disassembly and phosphorylation of Lgl by aPKC restricts its localization to the lateral cell membrane. Similarily, aPKC-dependent phosphorylation of Par1 restricts its localization to the basolateral membrane of polarized MDCK cells. These data indicate that Numb is also a downstream target of aPKC in polarized cells, and that phosphorylation at Ser7 and 295 mediates exclusion from the apical domain and accumulation at the lateral domain (Smith, 2007).

A role for mammalian Numb in receptor endocytosis and recycling has been established. The current findings suggest that in polarized epithelial cells the trafficking function of Numb may be restricted to the basolateral membrane by aPKC-dependent phosphorylation. Thus, Numb may serve as a link between the Par/aPKC polarity complex and the endocytic machinery, and function to regulate the trafficking of membrane proteins at the basolateral membrane. In agreement with such a model, Numb has previously been implicated in the polarized endocytosis of the neuronal cell adhesion molecule L1. Although the relevant membrane targets of Numb in epithelial cells are currently unknown, components of the Notch pathway are attractive candidates; Numb antagonizes Notch receptor signaling pathway in both Drosophila and in mammalian cells (Smith, 2007).

In Drosophila, the Par complex has been shown to direct the asymmetric localization of Numb, Pon, and Miranda via the aPKC-mediated inhibitory phosphorylation of Lgl. However, Numb asymmetric localization could still be observed in 30% of lgl mutant pI cells, suggesting that additional mechanisms may exist to regulate the asymmetric localization of Numb. Thus, it is proposed that the aPKC-dependent phosphorylation of Numb may account for the observed Lgl-independent asymmetric localization of Numb. This proposal implicitly assumes that this Lgl-independent process is aPKC-dependent. To verify this assumption, clones of apkc mutant cells were generated. Unfortunately, large apkc mutant clones could not easily be recovered in the pupal notum, preventing studying of the distribution of Numb in apkc mutant pI cells. A mutation in one of the Numb sites shown to be phosphorylated by aPKC, Ser52, has been characterized. The mutant protein, NumbS52F, fails to localize asymmetrically in pI at mitosis. The defective localization of NumbS52F contrasts with the asymmetric localization of NumbS52A. One possible interpretation is that the S52F, but not the S52A, mutation alters the conformation of Numb such that it prevents the phosphorylation of other essential aPKC sites or inhibits the actin-dependent cortical localization of Numb that is mediated by the N-terminal region of Numb (Smith, 2007).

In addition to the aPKC-dependent regulation of Numb localization, the results raise the possibility that a hierarchy of phosphorylation sites may be responsible for controlling additional aspects of Numb localization and function. In addition to serines 7 and 295, seven additional in vivo phosphorylation sites were have identified on mammalian Numb. Several of these do not conform to PKC consensus sites yet are conserved in Drosophila. Ser276 has been described as a target of CaMK, and this site was identified in mass spectral analysis. Although the functional consequences of phosphorylation at this site were not addressed, the authors demonstrate that phosphorylation confers binding to 14-3-3 proteins suggesting this site has a regulatory role. In addition, the Drosophila Numb-associated kinase (NAK), which was isolated in a yeast two-hybrid screen as a Numb interactor (Chien, 1998), is highly related to mammalian adaptin-associated kinase (AAK), raising the possibility that members of this family of protein kinases might also phosphorylate Numb in a manner that regulates its association with α-adaptin or other endocytic proteins. Further functional analysis of Numb phosphorylation site mutants and identification of upstream kinases will yield insight into the conserved signaling pathways that regulate the localization and function of Numb and also will reveal areas of divergence (Smith, 2007).

Linking cell cycle to asymmetric division: Aurora-A phosphorylates the Par complex to regulate Numb localization

Drosophila neural precursor cells divide asymmetrically by segregating the Numb protein into one of the two daughter cells. Numb is uniformly cortical in interphase but assumes a polarized localization in mitosis. This study shows that a phosphorylation cascade triggered by the activation of Aurora-A is responsible for the asymmetric localization of Numb in mitosis. Aurora-A phosphorylates Par-6, a regulatory subunit of atypical protein kinase C (aPKC). This activates aPKC, which initially phosphorylates Lethal (2) giant larvae (Lgl), a cytoskeletal protein that binds and inhibits aPKC during interphase. Phosphorylated Lgl is released from aPKC and thereby allows the PDZ domain protein Bazooka to enter the complex. This changes substrate specificity and allows aPKC to phosphorylate Numb and release the protein from one side of the cell cortex. These data reveal a molecular mechanism for the asymmetric localization of Numb and show how cell polarity can be coupled to cell-cycle progression (Wirtz-Peitz, 2008).

Since the discovery of Numb asymmetry, several proteins required for Numb localization have been identified, but how they cooperate remained unclear. This paper describes a cascade of interactions among these proteins that culminates in the asymmetric localization of Numb in mitosis. In interphase, Lgl localizes to the cell cortex, where it forms a complex with Par-6 and aPKC. At the onset of mitosis, AurA phosphorylates Par-6 in this complex, thereby releasing aPKC from inhibition by Par-6. Activated aPKC phosphorylates Lgl, causing its release from the cell cortex. Since Baz competes with Lgl for entry into the Par complex, the disassembly of the Lgl/Par-6/aPKC complex allows for the assembly of the Baz/Par-6/aPKC complex. Baz is a specificity factor that allows aPKC to phosphorylate Numb on one side of the cell cortex. Since p-Numb is released from the cortex (Nishimura, 2007; Smith, 2007), these events restrict Numb into a cortical crescent on the opposite side (Wirtz-Peitz, 2008).

The data show that Lgl acts as an inhibitory subunit of the Par complex. Given that Par-6 inhibits aPKC activity until the onset of mitosis, why would an additional layer of regulation be required? Like all phosphoproteins Numb is in a dynamic equilibrium between the phosphorylated and unphosphorylated states. Too high a rate of phosphorylation shifts this equilibrium toward the phosphorylated state, mislocalizing Numb into the cytoplasm. Too low a rate shifts it toward the unphosphorylated state, mislocalizing Numb around the cell cortex. Importantly, these data show that only the Baz complex can phosphorylate Numb. Assuming an abundance of Lgl over cortical Par-6, an increase in aPKC activity would translate into a comparatively small increase in the levels of Baz complex. This is because assembly of the Baz complex requires free subunits of Par-6 and aPKC, which become available only once the pool of cortical Lgl has been completely phosphorylated. Therefore, it is proposed that Lgl acts as a molecular buffer for the activity of the Par complex toward Numb. This maintains Numb phosphorylation within a range that is sufficiently high to exclude Numb from one side of the cell cortex but sufficiently low to permit the cortical localization of Numb to the other side (Wirtz-Peitz, 2008).

What is the evidence for this model? Lgl3A, a nonphosphorylatable mutant of Lgl in which the three aPKC phosphorylation sites are mutated to Ala, infinite buffering capacity, induces the mislocalization of Numb around the cell cortex. Conversely, in lgl mutants, having no buffering capacity, Numb is mislocalized into the cytoplasm. Moreover, the model predicts the loss of buffering capacity in the lgl mutant to be offset by an increase in the amount of substrate, since this would render the excess activity of the Par complex limiting. Indeed, overexpression of Numb in lgl mutants restores the cortical localization of Numb as well as its cortical asymmetry (Wirtz-Peitz, 2008).

The results indicate that Lgl gain- and loss-of-function phenotypes are entirely accounted for by the role of Lgl in inhibiting the assembly of the Baz complex. Previously, however, it was thought that the asymmetric phosphorylation of Lgl by aPKC restricts an activity of Lgl to the opposite side of the cell cortex. Based on this model, it was subsequently proposed that Lgl mediates the asymmetric localization of cell fate determinants by inhibiting the cortical localization of myosin-II. In addition, the role of the yeast orthologs of Lgl in exocytosis led to speculation that Lgl establishes an asymmetric binding site for cell fate determinants by promoting targeted vesicle fusion. However, the data show that Lgl asymmetry is extremely transient, and that the protein is completely cytoplasmic from NEBD onward. Lgl cannot therefore interact with any cortical proteins in prometaphase or metaphase, when myosin-II was reported to localize asymmetrically, or establish a stable landmark for vesicle fusion. Interestingly, a recent study demonstrated that yeast Lgl inhibits the assembly of SNARE complexes by sequestering a plasma membrane SNARE (Hattendorf, 2007). This mechanism is reminiscent of fly Lgl sequestering Par-6 and aPKC from interaction with Baz, suggesting that the defining property of Lgl-family members is not a specific role in exocytosis, but a more generic role in regulating the assembly of protein complexes (Wirtz-Peitz, 2008).

The data identify Numb as a key target of aPKC in tumor formation and suggest that Lgl acts as a tumor suppressor in the larval brain by inhibiting the aPKC-dependent phosphorylation of Numb. Although it is tempting to conclude that tumor formation in lgl mutants results from the missegregation of Numb, missegregation of Numb in numbS52F or upon expression of Lgl3A does not cause neuroblast tumors. How might this be explained? During mitosis, unphosphorylated cortical Numb is inherited by the differentiating daughter. At the same time, Baz and aPKC are excluded from this daughter, which limits Numb phosphorylation after exit from mitosis. In the subsequent interphase, some differentiating daughters reexpress members of the Baz complex (Bowman, 2008), but Numb continues to be protected from phosphorylation since cortical Lgl prevents the reassembly of the Baz complex. Thus, Lgl acts both in mitosis and interphase to maximize the amount of unphosphorylated Numb in the differentiating daughter cell (Wirtz-Peitz, 2008).

In lgl mutants, Numb phosphorylation is increased in mitosis, and less unphosphorylated Numb is segregated into the basal daughter cell. Moreover, the assembly of the Baz complex is unrestrained in the subsequent interphase, which is exacerbated by the missegregation of aPKC into both daughter cells. Together, these defects minimize the amount of unphosphorylated Numb in the differentiating daughter cell (Wirtz-Peitz, 2008).

Why is the amount of unphosphorylated Numb critical for differentiation? Recently, it was shown that aPKC-dependent phosphorylation of Numb inhibits not only its cortical localization, but also its activity, owing to the reduced affinity of p-Numb for its endocytic targets (Nishimura, 2007). Therefore, ectopic phosphorylation of Numb leads to its inactivation, transforming the basal daughter cell into a neuroblast in a manner similar to mutation of numb. Consistent with this model, studies in SOP cells have documented ectopic Notch signaling in lgl mutants. Although the numbS52F mutant and Lgl3A overexpression also lead to missegregation of Numb, the levels of active unphosphorylated Numb are increased rather than decreased in these cases and are sufficient to support differentiation (Wirtz-Peitz, 2008).

The data also provide additional insight into the mechanism of tumor formation in aurA mutants. In aurA mutants, the differentiating daughter cell inherits less Numb because Numb is mislocalized around the cell cortex. At the same time, aPKC is missegregated into the differentiating daughter cell, where it promotes Numb phosphorylation in the subsequent interphase. Together, these events result in subthreshold amounts of unphosphorylated Numb in some basal daughter cells, transforming these into neuroblasts. This model explains why aurA mutants are characterized by reduced aPKC activity in mitosis, but are nonetheless suppressed by aPKC mutations, since a lack of aPKC in the differentiating daughter cell restores threshold amounts of unphosphorylated Numb (Wirtz-Peitz, 2008).

The data reveal that Lgl inhibits Numb phosphorylation to maintain Numb activity, whereas AurA promotes Numb phosphorylation in mitosis to ensure its asymmetric segregation. It is concluded that Lgl and AurA act on opposite ends of a regulatory network that maintains appropriate levels of Numb phosphorylation at the appropriate time in the cell cycle (Wirtz-Peitz, 2008).

Dronc caspase exerts a non-apoptotic function to restrain phospho-Numb-induced ectopic neuroblast formation in Drosophila

Drosophila neuroblasts have served as a model to understand how the balance of stem cell self-renewal versus differentiation is achieved. Drosophila Numb protein regulates this process through its preferential segregation into the differentiating daughter cell. How Numb restricts the proliferation and self-renewal potentials of the recipient cell remains enigmatic. This study shows that phosphorylation at conserved sites regulates the tumor suppressor activity of Numb. Enforced expression of a phospho-mimetic form of Numb (Numb-TS4D) or genetic manipulation that boosts phospho-Numb levels, attenuates endogenous Numb activity and causes ectopic neuroblast formation (ENF). This effect on neuroblast homeostasis occurs only in the type II neuroblast lineage, which generates intermediate neural progenitors (INPs). INPs undergo a maturation process and multiple rounds of asymmetric division to produce GMCs and differentiated progenies. This study identified Dronc caspase as a novel binding partner of Numb, and demonstrates that overexpression of Dronc suppresses the effects of Numb-TS4D in a non-apoptotic and possibly non-catalytic manner. Reduction of Dronc activity facilitates ENF induced by phospho-Numb. These findings uncover a molecular mechanism that regulates Numb activity and suggest a novel role for Dronc caspase in regulating neural stem cell homeostasis (Ouyang, 2011).

Proper balance of the self-renewal versus differentiation of stem cells is crucial for tissue homeostasis. Disruption of this process could contribute to tumorigenesis. Numb has been identified as a key player that limits the proliferation potential of neuroblasts and INPs. This study elucidates the mechanisms of Numb action in this process and uncover a novel mechanism by which Numb activity is regulated at the post-translational level. The results suggest a model in which phosphorylation of Numb at conserved sites within its functionally important PTB domain impairs its association with the caspase Dronc and attenuates its tumor suppressor activity in type II neuroblasts (Ouyang, 2011).

As a defining feature of Numb protein is its asymmetric localization in stem cells and progenitors, previous studies of Numb have been focused on the control of its asymmetric localization. A number of factors have been identified to regulate Numb localization, including its binding partner Pon and kinases such as aPKC, Aurora A and Polo. This presents evidence that phosphorylation of Numb at the putative Polo sites primarily affect Numb activity in negatively regulating Notch signaling through promoting the endocytosis of Spdo. Although not all the identified Polo phosphorylation sites in Numb perfectly match the optimal consensus sequence initially defined for Polo, the Polo consensus sequence being defined is evolving, and specific characterized phosphorylation sites in other Polo substrates actually do not conform to the above consensus sequences. A common feature appears to be negatively charged residues surrounding the S/T residues; all five sites identified in Numb have this feature. Moreover, evidence is provided that the sites that were identified are responsive to phosphorylation controlled by Polo and PP2A. More importantly, phosphorylation of Numb at these sites has a significant effect on NSC homeostasis (Ouyang, 2011).

Polo kinase was shown to also control Numb asymmetric localization by phosphorylating Pon, an adaptor protein for Numb. Loss of Numb asymmetry in polo mutants contributes to ENF. The increased neuroblasts in polo mutants largely occur in the type I lineage. This study demonstrates that overexpression of Polo impairs Numb activity and leads to ENF in type II lineage. In this situation, Pon is presumably also phosphorylated by Polo. However, its positive effect on Numb asymmetric localization is likely to be overridden by impairment of Numb activity by Polo. This underlines the importance of Numb activity regulation in vivo and further indicates that Polo kinase acts on diverse targets to control neuroblast homeostasis. This study shows that phosphorylation of Numb by Polo is probably antagonized by PP2A action in type II lineage, which presumably serves to fine-tune Numb activity through dephosphorylation. Interestingly, the relationships between Polo and PP2A in type I lineage is different from that in type II lineage. In type I neuroblasts, overexpression of Polo can rescue PP2A loss-of-function phenotype, consistent with Polo being positively regulated by PP2A at the transcription level. Elucidation of the mechanisms mediating these differential effects will help lead to an understanding of the distinct behaviors of neuroblasts in these two lineages (Ouyang, 2011).

Deregulation of Numb phosphorylation contributes to loss of Numb activity and eventually leads to unrestrained ENF. Given the conservation of the phospho-sites identified in this study and the potential role of Numb in tumor suppression in mammals, mutations in Numb itself or in some kinases/phosphatases that affect Numb phosphorylation under pathophysiological conditions could contribute to cancer in humans. In lung and breast cancer tissues, Polo expression is upregulated. It is possible that under such pathophysiological conditions, Numb becomes hyperphosphorylated and consequently loses its antagonistic effect on Notch signaling, which could have detrimental consequences on tissue homeostasis. The phosphorylation sites of Numb identified in this study are conserved in mammals. It would be interesting to test in the future whether this phospho-epitope could be detected in human tumor samples (Ouyang, 2011).

This study demonstrated that ENF induced by phospho-Numb occurs specifically in type II lineages, consistent with Numb primarily acting in type II lineage to restrict the proliferation of INPs. It is conceivable that Numb is also phosphorylated by Polo kinase in type I lineage. However, certain unidentified factors might block the effect of phospho-Numb on type I neuroblasts. It is also possible that type I and type II lineages might employ different molecular mechanisms to control their stem cell self-renewal and differentiation, considering their different origin and modes of neurogenesis. Consistent with this notion, the Numb/Notch pathway has been suggested to be dispensable in the type I lineage (Ouyang, 2011).

The prominent brain tumor phenotype induced by Numb-TS4D provides an excellent system with which to identify novel molecules involved in controlling NSC homeostasis. This study shows that Dronc, a newly identified binding partner of Numb, is involved in regulating neuroblast homeostasis. Overexpression of Dronc is sufficient to attenuate Numb-TS4D-induced ENF without promoting neuroblast apoptosis. At the mechanistic level, this study shows that Dronc appears to act upstream of Notch to regulate Numb function, apparently in a process that does not strictly depend on its catalytic activity. Importantly, reduction of dronc function results in neuroblasts being more susceptible to the effect of phospho-Numb on neuroblast homeostasis. In addition, Dronc RNAi is able to further increase ectopic neuroblasts in numbS52F mutant, indicating that Dronc-Numb interaction is normally involved in regulating neuroblast homeostasis. Accumulating evidence suggests that caspases, in addition to their pro-apoptotic functions, also participate in other developmental process without inducing cell death. For example, Dronc has been implicated in a non-autonomous role in compensatory proliferation. It would be interesting to examine in the future whether Dronc transduces a signal from the neighboring niche cells via cell-cell interaction to establish neuroblast homeostatic control. It is also worth noting that mice deficient for caspase 2 (Casp2), which is closely related to Dronc in Drosophila, develop normally as their wild-type siblings; however, the fibroblasts from Casp2 null animals are easily transformed when challenged with oncogenic insults (Ho, 2009). The downstream effectors mediating this effect are not known. It would therefore be interesting to test whether the Numb/Dronc pathway identified here is generally involved in stem cell and cancer biology (Ouyang, 2011).

Endocytosis by Numb breaks Notch symmetry at cytokinesis

Cell-fate diversity can be generated by the unequal segregation of the Notch regulator Numb at mitosis in both vertebrates and invertebrates. Whereas the mechanisms underlying unequal inheritance of Numb are understood, how Numb antagonizes Notch has remained unsolved. Live imaging of Notch in sensory organ precursor cells revealed that nuclear Notch is detected at cytokinesis in the daughter cell that does not inherit Numb. Numb and Sanpodo act together to regulate Notch trafficking and establish directional Notch signalling at cytokinesis. It is proposed that unequal segregation of Numb results in increased endocytosis in one daughter cell, hence asymmetry of Notch at the cytokinetic furrow, directional signalling and binary fate choice (Couturier, 2012).

This analysis supports the following model for the control of Notch by Numb and Spdo in the context of asymmetric cell division. Before mitosis, endocytosis of Notch by Spdo decreases cortical Notch levels. At mitosis, cortical Notch moves along the apical cortex towards the apical pIIa/pIIb interface whereas internalized Notch is delivered to the newly formed plasma membrane along the pIIa/pIIb interface. At cytokinesis, Numb acts in pIIb to regulate the endocytosis of Notch, thereby removing Notch from the pIIb membrane. As a result, Notch is activated only in pIIa. In the absence of Spdo, Notch accumulates at the apical cortex before mitosis, resulting in increased cortical Notch at the apical pIIa/pIIb interface as well as decreased levels of Notch in endosomes, hence reduced levels of Notch delivered to the cytokinetic furrow. In the absence of Numb, similar amounts of Notch localize in pIIb and pIIa at the pIIa/pIIb interface, hence resulting in symmetric activation. In this model, the unequal segregation of Numb at mitosis results in an early asymmetry of Notch localization at the pIIa/pIIb interface, hence leading to asymmetric signalling and binary fate choice (Couturier, 2012).

As Numb interacts with Spdo through its phosphotyrosine-binding domain and with the ear domain of the α-adaptin through its NPF motif, Numb probably regulates the endocytosis, that is internalization and/or endosomal sorting, of Spdo-Notch complexes. Numb has also been proposed to interact directly with Notch, suggesting that Numb may also regulate the endocytosis of Notch receptors that are not in a complex with Spdo. Thus, these molecular interactions suggest that Numb may link both Notch and Spdo-Notch complexes to the AP-2 endocytic machinery to promote their internalization. Alternatively, but non-exclusively, Numb could block the recycling of both Notch and Spdo-Notch complexes back to the pIIa/pIIb interface. In both molecular models, the presence of Numb in pIIb would lead to the depletion of Notch from the pIIb membrane (Couturier, 2012).

How Spdo positively regulates Notch is not entirely clear. It is proposed that Spdo may act positively by increasing the pool of endosomal Notch in SOPs before mitosis to ensure that an appropriate number of receptors are targeted towards the cytokinetic furrow to localize along the basal pIIa/pIIb interface at cytokinesis. Alternatively, the data showing that Spdo interacts and co-traffics with Notch in pIIa indicate that Spdo may regulate endocytosis and activation of Notch in pIIa, that is in the absence of Numb. Spdo could, for instance, regulate the trafficking of active membrane-tethered S2-cleaved Notch towards a compartment where it is further processed by the γ-secretase. As extracellular epitopes are separated from activated membrane-tethered Notch on S2 cleavage, this potential role of Spdo was not examined in the antibody uptake assay (Couturier, 2012).

A recent study has suggested that Notch is directionally trafficked into pIIa at cytokinesis. This conclusion was based on the live-imaging analysis of anti-NECD/anti-Mouse Fab complexes that had been internalized in SOPs just before mitosis. However, this study did not provide evidence that endogenous Notch localized into the pool of endosomes that are directionally trafficked at cytokinesis. This study did not observe directional trafficking of NiGFP at cytokinesis. Thus, these data do not support the notion that Notch is directionally trafficked towards pIIa (Couturier, 2012).

In conclusion, this live-imaging study of Notch in Drosophila has revealed that directional Notch signalling is established at the end of mitosis through the regulated internalization and/or endosomal sorting of Notch-Spdo complexes by Numb in one of the two daughter cells. This model provides a simple and possibly general mechanism for the role of Numb in asymmetric division in animal cells (Couturier, 2012).

Couturier, L., Trylinski, M., Mazouni, K., Darnet, L. and Schweisguth, F. (2014). A fluorescent tagging approach in Drosophila reveals late endosomal trafficking of Notch and Sanpodo. J Cell Biol 207(3): 351-63. PubMed ID: 25365996

A fluorescent tagging approach in Drosophila reveals late endosomal trafficking of Notch and Sanpodo

Signaling and endocytosis are highly integrated processes that regulate cell fate. In the Drosophila melanogaster sensory bristle lineages, Numb inhibits the recycling of Notch and its trafficking partner Sanpodo (Spdo) to regulate cell fate after asymmetric cell division. This paper used a dual GFP/Cherry tagging approach to study the distribution and endosomal sorting of Notch and Spdo in living pupae. The specific properties of GFP, i.e., quenching at low pH, and Cherry, i.e., slow maturation time, revealed distinct pools of Notch and Spdo: cargoes exhibiting high GFP/low Cherry fluorescence intensities localized mostly at the plasma membrane and early/sorting endosomes, whereas low GFP/high Cherry cargoes accumulated in late acidic endosomes. These properties were used to show that Spdo is sorted toward late endosomes in a Numb-dependent manner. This dual-tagging approach should be generally applicable to study the trafficking dynamics of membrane proteins in living cells and tissues (Couturier, 2014).

numb: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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