Microtubule-induced Pins/Galphai cortical polarity in Drosophila neuroblasts

Cortical polarity regulates cell division, migration, and differentiation. Microtubules induce cortical polarity in yeast, but few examples are known in metazoans. We show that astral microtubules, kinesin Khc-73, and Discs large (Dlg) induce cortical polarization of Pins/Galphai in Drosophila neuroblasts; this cortical domain is functional for generating spindle asymmetry, daughter-cell-size asymmetry, and distinct sibling fates. Khc-73 localizes to astral microtubule plus ends, and Dlg/Khc-73 and Dlg/Pins coimmunoprecipitate, suggesting that microtubules induce Pins/Galphai cortical polarity through Dlg/Khc-73 interactions. The microtubule/Khc-73/Dlg pathway acts in parallel to the well-characterized Inscuteable/Par pathway, but each provides unique spatial and temporal information: The Inscuteable/Par pathway initiates at prophase to coordinate neuroblast cortical polarity with CNS tissue polarity, whereas the microtubule/Khc-73/Dlg pathway functions at metaphase to coordinate neuroblast cortical polarity with the mitotic spindle axis. These results identify a role for microtubules in polarizing the neuroblast cortex, a fundamental step for generating cell diversity through asymmetric cell division.     

Heterotrimeric G proteins regulate daughter cell size asymmetry in Drosophila neuroblast divisions

Cell division often generates unequally sized daughter cells by off-center cleavages, which are due to either displacement of mitotic spindles or their asymmetry. Drosophila neuroblasts predominantly use the latter mechanism to divide into a large apical neuroblast and a small basal ganglion mother cell (GMC), where the neural fate determinants segregate. Apically localized components regulate both the spindle asymmetry and the localization of the determinants. Here, we show that asymmetric spindle formation depends on signaling mediated by the G beta subunit of heterotrimeric G proteins. G beta 13F distributes throughout the neuroblast cortex. Its lack induces a large symmetric spindle and causes division into nearly equal-sized cells with normal segregation of the determinants. In contrast, elevated G beta 13F activity generates a small spindle, suggesting that this factor suppresses spindle development. Depletion of the apical components also results in the formation of a small symmetric spindle at metaphase. Therefore, the apical components and G beta 13F affect the mitotic spindle shape oppositely. We propose that differential activation of G beta signaling biases spindle development within neuroblasts and thereby causes asymmetric spindles. Furthermore, the multiple equal cleavages of G beta mutant neuroblasts accompany neural defects; this finding suggests indispensable roles of eccentric division in assuring the stem cell properties of neuroblasts.     

The role of centrosomes and astral microtubules during asymmetric division of Drosophila neuroblasts

Drosophila neuroblasts are stem cells that divide asymmetrically to produce another large neuroblast and a smaller ganglion mother cell (GMC). During neuroblast division, several cell fate determinants, such as Miranda, Prospero and Numb, are preferentially segregated into the GMC, ensuring its correct developmental fate. The accurate segregation of these determinants relies on proper orientation of the mitotic spindle within the dividing neuroblast, and on the correct positioning of the cleavage plane. In this study we have analyzed the role of centrosomes and astral microtubules in neuroblast spindle orientation and cytokinesis. We examined neuroblast division in asterless (asl) mutants, which, although devoid of functional centrosomes and astral microtubules, form well-focused anastral spindles that undergo anaphase and telophase. We show that asl neuroblasts assemble a normal cytokinetic ring around the central spindle midzone and undergo unequal cytokinesis. Thus, astral microtubules are not required for either signaling or positioning cytokinesis in Drosophila neuroblasts. Our results indicate that the cleavage plane is dictated by the positioning of the central spindle midzone within the cell, and suggest a model on how the central spindle attains an asymmetric position during neuroblast mitosis. We have also analyzed the localization of Miranda during mitotic division of asl neuroblasts. This protein accumulates in morphologically regular cortical crescents but these crescents are mislocalized with respect to the spindle orientation. This suggests that astral microtubules mediate proper spindle rotation during neuroblast division.     

A protein complex containing Inscuteable and the Galpha-binding protein Pins orients asymmetric cell divisions in Drosophila

BACKGROUND: In the fruit fly Drosophila, the Inscuteable protein localises to the apical cell cortex in neuroblasts and directs both the apical-basal orientation of the mitotic spindle and the basal localisation of the protein determinants Numb and Prospero during mitosis. Asymmetric localisation of Inscuteable is initiated during neuroblast delamination by direct binding to Bazooka, an apically localised protein that contains protein-interaction motifs known as PDZ domains. How apically localised Inscuteable directs asymmetric cell divisions is unclear. RESULTS: A novel 70 kDa protein called Partner of Inscuteable (Pins) and a heterotrimeric G-protein alpha subunit were found to bind specifically to the functional domain of Inscuteable in vivo. The predicted sequence of Pins contained tetratrico-peptide repeats (TPRs) and motifs implicated in binding Galpha proteins. Pins colocalised with Inscuteable at the apical cell cortex in interphase and mitotic neuroblasts. Asymmetric localisation of Pins required both Inscuteable and Bazooka. In epithelial cells, which do not express inscuteable, Pins was not apically localised but could be recruited to the apical cortex by ectopic expression of Inscuteable. In pins mutants, these epithelial cells were not affected, but neuroblasts showed defects in the orientation of their mitotic spindle and the basal asymmetric localisation of Numb and Miranda during metaphase. Although localisation of Inscuteable in pins mutants was initiated correctly during neuroblast delamination, Inscuteable became homogeneously distributed in the cytoplasm during mitosis. CONCLUSIONS: Pins and Inscuteable are dependent on each other for asymmetric localisation in delaminated neuroblasts. The binding of Pins to Galpha protein offers the intriguing possibility that Inscuteable and Pins might orient asymmetric cell divisions by localising or locally modulating a heterotrimeric G-protein signalling cascade at the apical cell cortex.     

Differential functions of G protein and Baz-aPKC signaling pathways in Drosophila neuroblast asymmetric division

Drosophila melanogaster neuroblasts (NBs) undergo asymmetric divisions during which cell-fate determinants localize asymmetrically, mitotic spindles orient along the apical-basal axis, and unequal-sized daughter cells appear. We identified here the first Drosophila mutant in the Ggamma1 subunit of heterotrimeric G protein, which produces Ggamma1 lacking its membrane anchor site and exhibits phenotypes identical to those of Gbeta13F, including abnormal spindle asymmetry and spindle orientation in NB divisions. This mutant fails to bind Gbeta13F to the membrane, indicating an essential role of cortical Ggamma1-Gbeta13F signaling in asymmetric divisions. In Ggamma1 and Gbeta13F mutant NBs, Pins-Galphai, which normally localize in the apical cortex, no longer distribute asymmetrically. However, the other apical components, Bazooka-atypical PKC-Par6-Inscuteable, still remain polarized and responsible for asymmetric Miranda localization, suggesting their dominant role in localizing cell-fate determinants. Further analysis of Gbetagamma and other mutants indicates a predominant role of Partner of Inscuteable-Galphai in spindle orientation. We thus suggest that the two apical signaling pathways have overlapping but different roles in asymmetric NB division.     

Apical complex genes control mitotic spindle geometry and relative size of daughter cells in Drosophila neuroblast and pI asymmetric divisions

Drosophila neuroblast asymmetric divisions generate two daughters of unequal size and fate. A complex of apically localized molecules mediates basal localization of cell fate determinants and apicobasal orientation of the mitotic spindle, but how daughter cell size is controlled remains unclear. Here we show that mitotic spindle geometry and unequal daughter cell size are controlled by two parallel pathways (Bazooka/DaPKC and Pins/G alpha i) within the apical complex. While the localized activity of either pathway alone is sufficient to mediate the generation of an asymmetric mitotic spindle and unequal size neuroblast daughters, loss of both pathways results in symmetric divisions. In sensory organ precursors, Bazooka/DaPKC and Pins/G alpha i localize to opposite sides of the cortex and function in opposition to generate a symmetric spindle.     

Drosophila Pins-binding protein Mud regulates spindle-polarity coupling and centrosome organization

The orientation of the mitotic spindle relative to the cell axis determines whether polarized cells undergo symmetric or asymmetric divisions. Drosophila epithelial cells and neuroblasts provide an ideal pair of cells to study the regulatory mechanisms involved. Epithelial cells divide symmetrically, perpendicular to the apical-basal axis. In the asymmetric divisions of neuroblasts, by contrast, the spindle reorients parallel to that axis, leading to the unequal distribution of cell-fate determinants to one daughter cell. Receptor-independent G-protein signalling involving the GoLoco protein Pins is essential for spindle orientation in both cell types. Here, we identify Mushroom body defect (Mud) as a downstream effector in this pathway. Mud directly associates and colocalizes with Pins at the cell cortex overlying the spindle pole(s) in both neuroblasts and epithelial cells. The cortical Mud protein is essential for proper spindle orientation in the two different division modes. Moreover, Mud localizes to centrosomes during mitosis independently of Pins to regulate centrosomal organization. We propose that Drosophila Mud, vertebrate NuMA and Caenorhabditis elegans Lin-5 (refs 5, 6) have conserved roles in the mechanism by which G-proteins regulate the mitotic spindle.     

Uncovering the link between malfunctions in Drosophila neuroblast asymmetric cell division and tumorigenesis

Asymmetric cell division is a developmental process utilized by several organisms. On the most basic level, an asymmetric division produces two daughter cells, each possessing a different identity or fate. Drosophila melanogaster progenitor cells, referred to as neuroblasts, undergo asymmetric division to produce a daughter neuroblast and another cell known as a ganglion mother cell (GMC). There are several features of asymmetric division in Drosophila that make it a very complex process, and these aspects will be discussed at length. The cell fate determinants that play a role in specifying daughter cell fate, as well as the mechanisms behind setting up cortical polarity within neuroblasts, have proved to be essential to ensuring that neurogenesis occurs properly. The role that mitotic spindle orientation plays in coordinating asymmetric division, as well as how cell cycle regulators influence asymmetric division machinery, will also be addressed. Most significantly, malfunctions during asymmetric cell division have shown to be causally linked with neoplastic growth and tumor formation. Therefore, it is imperative that the developmental repercussions as a result of asymmetric cell division gone awry be understood.     

Rotation and asymmetry of the mitotic spindle direct asymmetric cell division in the developing central nervous system

The asymmetric segregation of cell-fate determinants and the generation of daughter cells of different sizes rely on the correct orientation and position of the mitotic spindle. In the Drosophila embryo, the determinant Prospero is localized basally and is segregated equally to daughters of similar cell size during epidermal cell division. In contrast, during neuroblast division Prospero is segregated asymmetrically to the smaller daughter cell. This simple switch between symmetric and asymmetric segregation is achieved by changing the orientation of cell division: neural cells divide in a plane perpendicular to that of epidermoblast division. Here, by labelling mitotic spindles in living Drosophila embryos, we show that neuroblast spindles are initially formed in the same axis as epidermal cells, but rotate before cell division. We find that daughter cells of different sizes arise because the spindle itself becomes asymmetric at anaphase: apical microtubules elongate, basal microtubules shorten, and the midbody moves basally until it is positioned asymmetrically between the two spindle poles. This observation contradicts the widely held hypothesis that the cleavage furrow is always placed midway between the two centrosomes.     

Lethal giant larvae controls the localization of notch-signaling regulators numb, neuralized, and Sanpodo in Drosophila sensory-organ precursor cells

Asymmetric distribution of fate determinants is a fundamental mechanism underlying the acquisition of distinct cell fates during asymmetric division. In Drosophila neuroblasts, the apical DmPar6/DaPKC complex inhibits Lethal giant larvae (Lgl) to promote the basal localization of fate determinants. In contrast, in the sensory precursor (pI) cells that divide asymmetrically with a planar polarity, Lgl inhibits Notch signaling in the anterior pI daughter cell, pIIb, by a yet-unknown mechanism. We show here that Lgl promotes the cortical recruitment of Partner of Numb (Pon) and regulates the asymmetric distribution of the fate determinants Numb and Neuralized during the pI cell division. Analysis of Pon-GFP and Histone2B-mRFP distribution in two-color movies confirmed that Lgl regulates Pon localization. Moreover, posterior DaPKC restricts Lgl function to the anterior cortex at mitosis. Thus, Lgl functions similarly in neuroblasts and in pI cells. We also show that Lgl promotes the acquisition of the pIIb cell fate by inhibiting the plasma membrane localization of Sanpodo and thereby preventing the activation of Notch signaling in the anterior pI daughter cell. Thus, Lgl regulates cell fate by controlling Pon cortical localization, asymmetric localization of Numb and Neuralized, and plasma-membrane localization of Sandopo.     

DmPAR-6 directs epithelial polarity and asymmetric cell division of neuroblasts in Drosophila

The Drosophila protein Bazooka is required for both apical-basal polarity in epithelial cells and directing asymmetric cell division in neuroblasts. Here we show that the PDZ-domain protein DmPAR-6 cooperates with Bazooka for both of these functions. DmPAR-6 colocalizes with Bazooka at the apical cell cortex of epithelial cells and neuroblasts, and binds to Bazooka in vitro. DmPAR-6 localization requires Bazooka, and mislocalization of Bazooka through overexpression redirects DmPAR-6 to ectopic sites of the cell cortex. In the absence of DmPAR-6, Bazooka fails to localize apically in neuroblasts and epithelial cells, and is distributed in the cytoplasm instead. Epithelial cells lose their apical-basal polarity in DmPAR-6 mutants, asymmetric cell divisions in neuroblasts are misorientated, and the proteins Numb and Miranda do not segregate correctly into the basal daughter cell. Bazooka and DmPAR-6 are Drosophila homologues of proteins that direct asymmetric cell division in early Caenorhabditis elegans embryos, and our results indicate that homologous protein machineries may direct this process in worms and flies.     

Distinct roles of Galphai and Gbeta13F subunits of the heterotrimeric G protein complex in the mediation of Drosophila neuroblast asymmetric divisions

The asymmetric division of Drosophila neuroblasts involves the basal localization of cell fate determinants and the generation of an asymmetric, apicobasally oriented mitotic spindle that leads to the formation of two daughter cells of unequal size. These features are thought to be controlled by an apically localized protein complex comprising of two signaling pathways: Bazooka/Drosophila atypical PKC/Inscuteable/DmPar6 and Partner of inscuteable (Pins)/Galphai; in addition, Gbeta13F is also required. However, the role of Galphai and the hierarchical relationship between the G protein subunits and apical components are not well defined. Here we describe the isolation of Galphai mutants and show that Galphai and Gbeta13F play distinct roles. Galphai is required for Pins to localize to the cortex, and the effects of loss of Galphai or pins are highly similar, supporting the idea that Pins/Galphai act together to mediate various aspects of neuroblast asymmetric division. In contrast, Gbeta13F appears to regulate the asymmetric localization/stability of all apical components, and Gbeta13F loss of function exhibits phenotypes resembling those seen when both apical pathways have been compromised, suggesting that it acts upstream of the apical pathways. Importantly, our results have also revealed a novel aspect of apical complex function, that is, the two apical pathways act redundantly to suppress the formation of basal astral microtubules in neuroblasts.     

Deletion analysis of the Drosophila Inscuteable protein reveals domains for cortical localization and asymmetric localization

The Drosophila Inscuteable protein acts as a key regulator of asymmetric cell division during the development of the nervous system [1] [2]. In neuroblasts, Inscuteable localizes into an apical cortical crescent during late interphase and most of mitosis. During mitosis, Inscuteable is required for the correct apical-basal orientation of the mitotic spindle and for the asymmetric segregation of the proteins Numb [3] [4] [5], Prospero [5] [6] [7] and Miranda [8] [9] into the basal daughter cell. When Inscuteable is ectopically expressed in epidermal cells, which normally orient their mitotic spindle parallel to the embryo surface, these cells reorient their mitotic spindle and divide perpendicularly to the surface [1]. Like the Inscuteable protein, the inscuteable RNA is asymmetrically localized [10]. We show here that inscuteable RNA localization is not required for Inscuteable protein localization. We found that a central 364 amino acid domain - the Inscuteable asymmetry domain - was necessary and sufficient for Inscuteable localization and function. Within this domain, a separate 100 amino acid region was required for asymmetric localization along the cortex, whereas a 158 amino acid region directed localization to the cell cortex. The same 158 amino acid fragment could localize asymmetrically when coexpressed with the full-length protein, however, and could bind to Inscuteable in vitro, suggesting that this domain may be involved in the self-association of Inscuteable in vivo.     

Extrinsic cues orient the cell division axis in Drosophila embryonic neuroblasts

Cell polarity must be integrated with tissue polarity for proper development. The Drosophila embryonic central nervous system (CNS) is a highly polarized tissue; neuroblasts occupy the most apical layer of cells within the CNS, and lie just basal to the neural epithelium. Neuroblasts are the CNS progenitor cells and undergo multiple rounds of asymmetric cell division, ;budding off' smaller daughter cells (GMCs) from the side opposite the epithelium, thereby positioning neuronal/glial progeny towards the embryo interior. It is unknown whether this highly stereotypical orientation of neuroblast divisions is controlled by an intrinsic cue (e.g. cortical mark) or an extrinsic cue (e.g. cell-cell signal). Using live imaging and in vitro culture, we find that neuroblasts in contact with epithelial cells always ;bud off' GMCs in the same direction, opposite from the epithelia-neuroblast contact site, identical to what is observed in vivo. By contrast, isolated neuroblasts 'bud off' GMCs at random positions. Imaging of centrosome/spindle dynamics and cortical polarity shows that in neuroblasts contacting epithelial cells, centrosomes remained anchored and cortical polarity proteins localize at the same epithelia-neuroblast contact site over subsequent cell cycles. In isolated neuroblasts, centrosomes drifted between cell cycles and cortical polarity proteins showed a delay in polarization and random positioning. We conclude that embryonic neuroblasts require an extrinsic signal from the overlying epithelium to anchor the centrosome/centrosome pair at the site of epithelial-neuroblast contact and for proper temporal and spatial localization of cortical Par proteins. This ensures the proper coordination between neuroblast cell polarity and CNS tissue polarity.     

Twins/PP2A regulates aPKC to control neuroblast cell polarity and self-renewal

Asymmetric cell division is a mechanism for generating cell diversity as well as maintaining stem cell homeostasis in both Drosophila and mammals. In Drosophila, larval neuroblasts are stem cell-like progenitors that divide asymmetrically to generate neurons of the adult brain. Mitotic neuroblasts localize atypical protein kinase C (aPKC) to their apical cortex. Cortical aPKC excludes cortical localization of Miranda and its cargo proteins Prospero and Brain tumor, resulting in their partitioning into the differentiating, smaller ganglion mother cell (GMC) where they are required for neuronal differentiation. In addition to aPKC, the kinases Aurora-A and Polo also regulate neuroblast self-renewal, but the phosphatases involved in neuroblast self-renewal have not been identified. Here we report that aPKC is in a protein complex in vivo with Twins, a Drosophila B-type protein phosphatase 2A (PP2A) subunit, and that Twins and the catalytic subunit of PP2A, called Microtubule star (Mts), are detected in larval neuroblasts. Both Twins and Mts are required to exclude aPKC from the basal neuroblast cortex: twins mutant brains, twins mutant single neuroblast mutant clones, or mts dominant negative single neuroblast clones all show ectopic basal cortical localization of aPKC. Consistent with ectopic basal aPKC is the appearance of supernumerary neuroblasts in twins mutant brains or twins mutant clones. We conclude that Twins/PP2A is required to maintain aPKC at the apical cortex of mitotic neuroblasts, keeping it out of the differentiating GMC, and thereby maintaining neuroblast homeostasis.     

Drosophila neural progenitor polarity and asymmetric division

In the Drosophila embryonic central nervous system, the neural precursor cells called neuroblasts undergo a number of asymmetric divisions along the apical-basal axis to give rise to different daughter cells of distinct fates. This review summarizes recent progress in understanding the mechanisms of these asymmetric cell divisions. We discuss proteins that are localized at distinct domains of cortex in the neuroblasts and their role in generating asymmetry. We also review uniformly cortical localized factors and actin cytoskeleton-associated motor proteins with regard to their potential role to serve as a link between distinct cortical domains in the neuroblasts. In this review, asymmetric divisions of sensory organ precursor and larval neuroblasts are also briefly discussed.     

Asymmetric cortical extension shifts cleavage furrow position in Drosophila neuroblasts

The cytokinetic cleavage furrow is typically positioned symmetrically relative to the cortical cell boundaries, but it can also be asymmetric. The mechanisms that control furrow site specification have been intensively studied, but how polar cortex movements influence ultimate furrow position remains poorly understood. We measured the position of the apical and the basal cortex in asymmetrically dividing Drosophila neuroblasts and observed preferential displacement of the apical cortex that becomes the larger daughter cell during anaphase, effectively shifting the cleavage furrow toward the smaller daughter cell. Asymmetric cortical extension is correlated with the presence of cortical myosin II, which is polarized in neuroblasts. Loss of myosin II asymmetry by perturbing heterotrimeric G-protein signaling results in symmetric extension and equal-sized daughter cells. We propose a model in which contraction-driven asymmetric polar extension of the neuroblast cortex during anaphase contributes to asymmetric furrow position and daughter cell size.     

Dlg,Scribble and Lgl in cell polarity,cell proliferation and cancer

Dlg (Discs large), Scrib (Scribble) and Lgl (Lethal giant larvae) are evolutionarily conserved components of a common genetic pathway that link the seemingly disparate functions of cell polarity and cell proliferation in epithelial cells. dlg, scrib and lgl have been identified as tumour suppressor genes in Drosophila, mutations of which cause similar phenotypes, involving disruption of cell polarity and neoplastic overgrowth of tissues. The molecular mechanisms by which Dlg, Scrib and Lgl proteins regulate cell proliferation are not clear, but there is some evidence that epithelial polarisation is required for this regulation. Dlg, Scrib and Lgl are highly conserved between human and Drosophila, and we discuss evidence that these proteins also play a role in cancer progression in humans.