InteractiveFly: GeneBrief

wide awake/banderuola: Biological Overview | References

BIOLOGICAL OVERVIEW

Asymmetric cell division (ACD) is a key process that allows different cell types to be generated at precisely defined times and positions. In Drosophila, neural precursor cells rely heavily on ACD to generate the different cell types in the nervous system. A conserved protein machinery that regulates ACD has been identified in Drosophila, but how this machinery acts to allow the establishment of differential cell fates is not entirely understood. To identify additional proteins required for ACD, an in vivo live imaging RNAi screen was carried out for genes affecting the asymmetric segregation of Numb in Drosophila sensory organ precursor cells. Banderuola (Bnd / Wide Awake) was identified an essential regulator of cell polarization, spindle orientation, and asymmetric protein localization in Drosophila neural precursor cells. Genetic and biochemical experiments show that Bnd acts together with the membrane-associated tumor suppressor Discs-large (Dlg) to establish antagonistic cortical domains during ACD. Inhibiting Bnd strongly enhances the dlg phenotype, causing massive brain tumors upon knockdown of both genes. Because the mammalian homologs of Bnd and Dlg are interacting as well, Bnd function might be conserved in vertebrates, and it might also regulate cell polarity in higher organisms. It is concluded that Bnd is a novel regulator of ACD in different types of cells. The data place Bnd at the top of the hierarchy of the factors involved in ACD, suggesting that its main function is to mediate the localization and function of the Dlg tumor suppressor. Bnd has an antioncogenic function that is redundant with Dlg, and the physical interaction between the two proteins is conserved in evolution (Mauri, 2014).

Although most cell divisions are symmetric, some cells can divide asymmetrically into two daughter cells that assume different fates. During development, asymmetric cell division (ACD) allows specific cell types to be generated at precise locations relative to surrounding tissues. To achieve this, the axis of ACD needs to be coordinated with the architecture and polarity of the developing organism. Over the past years, a conserved protein machinery for ACD has been identified, but how this machinery connects to the organism architecture is less clear (Mauri, 2014).

The fruit fly Drosophila melanogaster is one of the best-understood model systems for ACD. In particular, the development of the Drosophila CNS and peripheral nervous system relies heavily on ACD and has contributed much to current understanding of this process. In the peripheral nervous system, external sensory (ES) organs are formed by two outer cells (hair and socket) and two inner cells (neuron and sheath). The four cell types arise from a single sensory organ precursor (SOP) cell, which divides asymmetrically into an anterior pIIb cell and a posterior pIIa cell. In a second round of ACD, pIIa and pIIb generate the outer or inner cells of the ES organ, respectively. The difference between pIIa and pIIb cells arises from different levels of Notch signaling in the two daughter cells. This difference is established by the asymmetric segregation of the Notch inhibitor Numb into the pIIb cell. Numb is known to regulate endocytosis, but how it inhibits Notch signaling is not precisely understood (Mauri, 2014).

In SOP cells, the polarity axis is coordinated with the anterior-posterior planar polarity axis of the overlying epithelium. Planar polarity involves the localization of mutually inhibitory components of a well-characterized machinery to the anterior or posterior plasma membrane. In SOP cells, the planar polarity protein Strabismus (Stbm) localizes to the anterior cortex and initiates the reorganization of plasma membrane domains to establish the axis of ACD. One of the most upstream events of this process is the recruitment of the membrane-associated guanylate kinase (MAGUK) Discs-large (Dlg) to the anterior cortex. This may involve a direct interaction of Dlg with the planar polarity protein Stbm. Dlg was originally identified as a tumor suppressor involved in the regulation of epithelial cell polarity and later shown to play a role in ACD and synaptogenesis. Despite its widespread functions, the biochemical pathways regulated by Dlg in those various cell types are not entirely understood (Mauri, 2014).

In SOP cells, Dlg associates with the adaptor protein Pins to direct the protein Bazooka (Baz) to the basal-posterior side of the dividing SOP cell. Together with Par-6 and aPKC, Baz forms the so-called Par protein complex that plays a pivotal role during ACD in many different cell types. Eventually, the kinase aPKC phosphorylates Numb, mediating its release from the posterior plasma membrane and thereby causing its accumulation to the anterior side (Mauri, 2014).

To ensure the asymmetric segregation of Numb to the anterior pIIb cell, the mitotic spindle has to be oriented along the polarity axis. This function is mediated by Pins through the binding of the microtubule binding protein Mushroom body defect (Mud), which forms a cortical attachment site for astral microtubules, aligning the spindle into the correct orientation. The binding to Pins requires the heterotrimeric G protein Gαi, which associates with Pins to mediate its recruitment to the anterior plasma membrane and switches it to an open conformation in which Pins can bind Mud (Mauri, 2014).

The same protein machinery directs ACD in neuroblasts, the stem cell-like progenitors of the Drosophila CNS. Neuroblasts divide asymmetrically into self-renewing daughter neuroblasts and smaller ganglion mother cells (GMCs) that generate two differentiating neurons through a terminal symmetric division. The asymmetric segregation of the cell fate determinants Numb, Prospero (Pros), and Brat into the GMC is required for proper differentiation. The asymmetric partitioning of Pros and Brat is mediated by the adaptor protein Miranda, and the asymmetric localization of both Miranda and Numb depends on phosphorylation by aPKC. Mutations in any of the three segregating determinants lead to the generation of excessive numbers of neuroblasts and ultimately cause the formation of lethal, transplantable brain tumors. As in SOP cells, Pins, Dlg, and Baz are required for ACD in neuroblasts, but they act in a characteristically different manner. First, neuroblast divisions are oriented along the apical-basal axis and not the planar polarity axis. Second, Pins, Dlg, and Baz colocalize apically in neuroblasts while they occupy opposite domains in SOP cells. In part, those differences can be explained by the recruitment of the adaptor protein Inscuteable (Insc) in the apical complex. Insc is not expressed in SOP cells, but in neuroblasts, it coordinates cortical polarity and spindle alignment by connecting Pins to Baz, ensuring the correct segregation of cell fate determinants in the differentiating daughter cell. In addition, Dlg has a neuroblast-specific role in mediating spindle orientation, acting downstream of Pins to align the spindle pole through the interaction with the kinesin motor Khc-73. Pins, Dlg, and Khc-73 also regulate a pathway called 'telophase rescue' that corrects ACD defects during late mitotic stages. This pathway realigns cortical polarity along the spindle axis independently of the Par complex through a Dlg cortical clustering mechanism to ensure that determinants eventually segregate asymmetrically and daughter cell fates are correctly specified. How Dlg performs those seemingly divergent roles in SOPs and neuroblasts is currently unclear (Mauri, 2014).

Because knowledge about ACD is evidently incomplete, several RNAi screens were performed to identify additional players required for the correct establishment of daughter cell fates. This study used the results from one of those screens to identify Banderuola (Bnd; CG45058, FlyBase name: Wide awake, Wake) Banderuola is a weathervane in the form of a rooster. Bnd is a new key regulator of ACD that acts both in neuroblasts and in SOP cells. Baz, Pins, and Dlg are all mislocalized in bnd mutant SOP cells, placing Bnd at the top of the hierarchy for ACD. In bnd mutant neuroblasts, the asymmetric segregation of cell fate determinants is disrupted because aPKC and Dlg fail to accumulate apically. Importantly, Bnd interacts physically and genetically with Dlg, suggesting that it supports Dlg in performing its divergent functions in various cell types. Because Bnd is conserved in evolution, our data identify a new member of the universal machinery for ACD that might direct cell polarity in vertebrates as well (Mauri, 2014).

These results establish Bnd as a new component of the machinery for asymmetric cell division. bnd RNAi or loss-of-function mutations cause defects in the establishment of polarity and the positioning of the mitotic spindle in mitotic SOP cells. bnd was shown to be required for ACD and continued self-renewal activity in Drosophila larval neuroblasts. Because Bnd interacts both biochemically and genetically with the tumor suppressor protein Dlg, it is proposed that it exerts its function during ACD by regulating the function of Dlg. Moreover, the spindle rotation phenotype that were observed in mitotic SOP cells in bnd mutants is very similar to that of dlg^sw mutants, further strengthening the possibility that the two proteins are functionally connected. Because the mammalian homologs of these two proteins also interact, this function might be conserved in higher organisms as well (Mauri, 2014).

The process of ACD involves the establishment of a polarity axis, the orientation of the mitotic spindle, the polarized distribution of cell fate determinants, and, ultimately, the establishment of different daughter cell fates. In SOP cells, the axis of polarity is established when Dlg and Pins interact with components of the planar polarity pathway to concentrate anteriorly. Because Bnd binds to Dlg and is required for Pins and Dlg localization, but not for planar polarity, the data indicate that it acts at the very top of this hierarchy. Because Dlg is also mislocalized in bnd mutant neuroblasts, the role of Bnd in this tissue appears to be similar. Nevertheless, because the defect in asymmetry establishment is not completely penetrant, it is plausible that bnd function is partially redundant. Alternatively, it might also be that the residual protein derived from maternal contribution is sufficient to maintain, at least partially, the asymmetric partitioning of determinants. Further experiments will be needed to address these issues and clarify the instructive role of Bnd in establishing cell asymmetry (Mauri, 2014).

How could Bnd perform its function on a molecular level? Bnd::GFP localizes at the centrosomes, on the spindle, and, transiently, at the cell cortex. Because Bnd contains both Ankyrin repeats and an FN3 domain, it could mediate protein-protein interactions leading to the anterior localization of Dlg downstream of the PCP pathway. The localization of Dlg and Pins to the anterior side of dividing SOP cells is regulated by Strabismus (Stbm) and Dishevelled (Dsh). It is thought that Dsh excludes Dlg/Pins from the posterior side, whereas Stbm binds Dlg at the anterior cortex, promoting the association with Pins. This hypothesis is reinforced by the fact that Dlg interacts directly with the PDZ binding motif (PBM) of Stbm in Drosophila embryos. However, Pins is localized to the anterior cortex in stbm mutant SOP cells expressing a Stbm protein lacking the PBM domain. Hence, the localization of Dlg/Pins can be regulated independently of a direct binding to Stbm. It is tempting to speculate that Bnd could be a mediator between the PCP pathway and the establishment of the asymmetry axis in mitotic SOP cells (Mauri, 2014).

Alternatively, however, Bnd could also affect the function of Dlg and other cortical proteins through its RA domain. RA domains mediate binding to small GTPases and regulate their activity. Small GTPases are involved in the modification of the actomyosin network, and the establishment of polarity is influenced by myosin activity and by the contractility of the actomyosin mesh. In particular, Cdc42, a small GTPase of the Rho family, plays a central role in the establishment of polarity in a wide variety of biological contexts, including the localization of Par6/aPKC to the apical cortex of neuroblasts. More recent data have also implicated small Ras-like GTPases in regulating cortical polarity and spindle orientation. The Rap1/Rgl/Ral signaling network was shown to mediate those events through the regulation of the PDZ domain protein Canoe, which is a known binding partner of Pins. It is intriguing to hypothesize that Bnd could be part of a similar signaling network impinging on Dlg. Because the RA domain of Banderuola is not conserved in higher organisms, however, the first hypothesis is favored that rests on the conserved domains of the protein (ANK domains, FN3 domain, and Bnd motif). Hence, Bnd could act as an adaptor that mediates protein-protein interactions and regulates the function of binding partners such as Dlg (Mauri, 2014).

Why Bnd is also found at centrosomes and at the spindle is harder to explain. In fact, Bnd is the only known protein apart from Mud that localizes to both the centrosome and the cell cortex during ACD. It could help in promoting the alignment of the spindle through the interaction with the Pins/Gαi/Mud complex, but this cannot explain the entire phenotype because microtubules are not strictly required for polarity establishment during ACD. Although no biochemical interaction were detected between Bnd and Pins, Gαi, or Mud, this interaction could be transient, or it could depend on polymerized microtubules. It will be compelling to verify the localization of the endogenous protein because this would consolidate the data derived from the protein overexpression experiments. Furthermore, this could allow unraveling in detail the dynamics of Bnd cortical localization and its alignment with the SOP polarity axis, which could be concealed in overexpression conditions (Mauri, 2014).

In neuroblasts, Bnd is required for self-renewal and asymmetric protein segregation and has an antioncogenic function that is redundant with Dlg. In bnd mutants, defects were observed leading to neuroblast loss. The remaining neuroblasts are misshapen, displaying abnormalities in the asymmetric protein segregation and reduced mitotic activity. Although the FRT site remaining in the bnd mutants prevents addressing this question through a clonal analysis, the hypothesis is favored that the phenotype is cell autonomous and is due to premature differentiation of neuroblasts. Indeed, this is consistent with the phenotype in the SOP lineage because genetic manipulations resulting in a pIIa to pIIb transformation (like Numb overexpression or Notch loss of function) often cause neuroblasts to divide symmetrically into two differentiating daughter cells (Mauri, 2014).

The localization of both the basal determinants and Dlg itself are affected in bnd mutants. Dlg is known to mediate the basal localization of cell fate determinants in Drosophila neuroblasts. The abnormal localization of aPKC in bnd mutant neuroblasts could also be explained as an effect of dlg LOF because aPKC localization is affected in dlg mutants. Thus, the various protein mislocalization phenotypes in bnd mutant neuroblasts could be explained by a model in which Bnd exerts its function solely by localizing Dlg (Mauri, 2014).

The tumor phenotypes, on the other hand, suggest that the two genes act in parallel. Overproliferation phenotypes are observed only upon LOF of both genes, and bnd LOF enhances the dlg RNAi phenotype. In fact, this type of genetic interaction has been described for pins and lgl before: whereas pins mutant neuroblasts underproliferate due to self-renewal failure, pins lgl double mutants have a massive overproliferation of neuroblasts due to an aberrant self-renewal program triggered by aPKC. A similar mechanism could underlie the overproliferation was observed upon double RNAi of bnd and dlg. An alternative explanation for the double knockdown phenotype is provided by the additional role that Dlg has in the telophase rescue pathway, which might be independent from bnd. This pathway is known to mediate the establishment of Pins/Gαi cortical polarity, even in the absence of the Par complex, through a Dlg-dependent mechanism. The pathway is active in wild-type neuroblasts but becomes essential only when components of the apical Par complex are missing. It is possible that the telophase rescue pathway ensures the asymmetric segregation of cell fate determinants upon bnd RNAi. When dlg is inhibited as well, however, this pathway could be compromised, resulting in overproliferation and tumor formation (Mauri, 2014).

Dlg has four mammalian homologs. Like the Drosophila protein, they localize at the basolateral cortex in epithelia and have been shown to regulate cell polarity in various cell types. During rat astrocyte migration, for example, Dlg1 is required in association with APC for the polarization of the microtubule cytoskeleton at the leading edge of the migrating cell. Dlg-mediated polarity can be also considered a gatekeeper against tumor progression: Dlg1 is a target of oncoviral proteins and is often mislocalized or downregulated in late-stage tumors, implicating a causal connection between Dlg1 and cancer. As the interaction between Bnd and Dlg is conserved, Banderuola could be an evolutionarily conserved regulator of Dlg activity, and these studies may therefore be relevant for a variety of biological processes in higher organisms as well (Mauri, 2014).

Wide awake mediates the circadian timing of sleep onset

How the circadian clock regulates the timing of sleep is poorly understood. This study identifies a Drosophila mutant, wide awake (wake), that exhibits a marked delay in sleep onset at dusk. Loss of Wake in a set of arousal-promoting clock neurons, the large ventrolateral neurons (l-LNvs), impairs sleep onset. Wake levels cycle, peaking near dusk, and the expression of Wake in l-LNvs is Clock dependent. Strikingly, Clock and cycle mutants also exhibit a profound delay in sleep onset, which can be rescued by restoring Wake expression in LNvs. Wake interacts with the GABAA receptor Resistant to Dieldrin (Rdl), upregulating its levels and promoting its localization to the plasma membrane. In wake mutant l-LNvs, GABA sensitivity is decreased and excitability is increased at dusk. It is proposed that Wake acts as a clock output molecule specifically for sleep, inhibiting LNvs at dusk to promote the transition from wake to sleep (Liu, 2014).

The molecular pathways by which the circadian clock modulates the timing of sleep are unknown. This study identified a molecule, Wide Awake, that promotes sleep and is required for circadian timing of sleep onset. The data argue for a direct role for the circadian oscillator in regulating sleep and support a model whereby Wake acts as a molecular intermediary between the circadian clock and sleep. In this model, Wake transmits timing information from the circadian clock to inhibit arousal circuits at dusk, thus facilitating the transition from wake to sleep. wake is transcriptionally upregulated by Clk activity, specifically in LNv clock neurons. Wake levels in l-LNvs rise during the day and peak at the early night, near the wake/sleep transition. This increase in Wake levels upregulates Rdl in l-LNvs, enhancing their sensitivity to GABA signaling and serving to inhibit the l-LNv arousal circuit. In this manner, cycling of Wake promotes cycling of the excitability of l-LNv cells. In wake mutants, l-LNvs lose this circadian electrical cycling; the higher firing rate of these cells at dusk leads to increased release of Pdf, which would act on Pdfr on downstream neurons to inhibit sleep onset. The identity of the GABAergic neurons signaling to the l-LNvs is currently unknown, but if they serve to convey information about sleep pressure from homeostatic circuits, the l-LNvs could serve as a site of integration for homeostatic and circadian sleep regulatory signals (Liu, 2014).

Although Wake is expressed in clock neurons and its levels vary throughout the day, Wake itself is not a core clock molecule, since period length and activity rhythm strength are intact in wake mutants in constant darkness. The effects of Wake on sleep latency are not attributable to alterations in core clock function. In addition, because locomotor rhythm strength is intact in wake mutants, Wake is not a clock output molecule for locomotor rhythms. Rather, Wake is the first clock output molecule shown to specifically regulate sleep timing (Liu, 2014).

Previous studies have demonstrated that Rdl in LNvs regulates sleep in Drosophila. This work further implicates Rdl as a key factor in the circadian modulation of sleep. In mammals, the localization and function of GABAA receptors are regulated by a variety of cytosolic accessory proteins, some of which are associated with the plasma membrane and cytoskeletal elements. The data suggest that Wake acts as an accessory protein for Rdl, upregulating its levels and promoting its targeting to the plasma membrane. Rdl is broadly expressed throughout the adult Drosophila brain, whereas Wake appears more spatially restricted. It is likely that Rdl is regulated by Wake in specific cells (e.g., Wake+ cells), while in other cells that express Rdl but not Wake, other factors are involved. Together, these data suggest a model in which increased GABA sensitivity is required in specific arousal circuits to facilitate rapid and complete switching between sleep/wake states at the appropriate circadian time (Liu, 2014).

Intriguingly, the data, as well as data from the Allen Brain Atlas, suggest that the putative mouse homolog of Wake (ANKFN1) is enriched in the mouse SCN, the master circadian pacemaker in mammals. Specifically, ANKFN1 is expressed in the 'core' region of the SCN, which is analogous to the large LNvs in flies, in that it receives light input and its molecular oscillator does not cycle or cycles weakly in DD. These observations support a potential conservation of Wake function in regulating clock-dependent timing of sleep onset, which will be evaluated by ongoing genetic analysis in mice. The pronounced difficulty of wake flies to fall asleep at lights off is reminiscent of sleep-onset insomnia in humans. Moreover, the most widely used medications for the treatment of insomnia are GABA agonists. Thus, the identification of a molecule that mediates circadian timing of sleep onset by promoting GABA signaling may lead to a deeper understanding of mechanisms underlying insomnia and its potential therapies (Liu, 2014).

Search PubMed for articles about Drosophila Wide awake/Banderuola

Liu, S., Lamaze, A., Liu, Q., Tabuchi, M., Yang, Y., Fowler, M., Bharadwaj, R., Zhang, J., Bedont, J., Blackshaw, S., Lloyd, T. E., Montell, C., Sehgal, A., Koh, K. and Wu, M. N. (2014). WIDE AWAKE mediates the circadian timing of sleep onset. Neuron 82(1):151-66. PubMed ID: 24631345

Mauri, F., Reichardt, I., Mummery-Widmer, J. L., Yamazaki, M., Knoblich, J. A. (2014) The conserved Discs-large binding partner Banderuola regulates asymmetric cell division in Drosophila. Curr Biol 24(16): 1811-25. PubMed ID: 25088559

date revised: 11 September 2022

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