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.     

The NuMA-related Mud protein binds Pins and regulates spindle orientation in Drosophila neuroblasts

Asymmetric cell division generates cell diversity during development and regulates stem-cell self-renewal in Drosophila and mammals. In Drosophila, neuroblasts align their spindle with a cortical Partner of Inscuteable (Pins)-G alpha i crescent to divide asymmetrically, but the link between cortical polarity and the mitotic spindle is poorly understood. Here, we show that Pins directly binds, and coimmunoprecipitates with, the NuMA-related Mushroom body defect (Mud) protein. Pins recruits Mud to the neuroblast apical cortex, and Mud is also strongly localized to centrosome/spindle poles, in a similar way to NuMA. In mud mutants, cortical polarity is normal, but the metaphase spindle frequently fails to align with the cortical polarity axis. When spindle orientation is orthogonal to cell polarity, symmetric division occurs. We propose that Mud is a functional orthologue of mammalian NuMA and Caenorhabditis elegans Lin-5, and that Mud coordinates spindle orientation with cortical polarity to promote asymmetric cell division.     

Inscuteable regulates the Pins-Mud spindle orientation pathway

During asymmetric cell division, alignment of the mitotic spindle with the cell polarity axis ensures that the cleavage furrow separates fate determinants into distinct daughter cells. The protein Inscuteable (Insc) is thought to link cell polarity and spindle positioning in diverse systems by binding the polarity protein Bazooka (Baz; aka Par-3) and the spindle orienting protein Partner of Inscuteable (Pins; mPins or LGN in mammals). Here we investigate the mechanism of spindle orientation by the Insc-Pins complex. Previously, we defined two Pins spindle orientation pathways: a complex with Mushroom body defect (Mud; NuMA in mammals) is required for full activity, whereas binding to Discs large (Dlg) is sufficient for partial activity. In the current study, we have examined the role of Inscuteable in mediating downstream Pins-mediated spindle orientation pathways. We find that the Insc-Pins complex requires Gαi for partial activity and that the complex specifically recruits Dlg but not Mud. In vitro competition experiments revealed that Insc and Mud compete for binding to the Pins TPR motifs, while Dlg can form a ternary complex with Insc-Pins. Our results suggest that Insc does not passively couple polarity and spindle orientation but preferentially inhibits the Mud pathway, while allowing the Dlg pathway to remain active. Insc-regulated complex assembly may ensure that the spindle is attached to the cortex (via Dlg) before activation of spindle pulling forces by Dynein/Dynactin (via Mud).     

Robust spindle alignment in Drosophila neuroblasts by ultrasensitive activation of pins

Cellular signaling pathways exhibit complex response profiles with features such as thresholds and steep activation (i.e., ultrasensitivity). In a reconstituted mitotic spindle orientation pathway, activation of Drosophila Pins (LGN in mammals) by Gαi is ultrasensitive (apparent Hill coefficient of 3.1), such that Pins recruitment of the microtubule binding protein Mud (NuMA) occurs over a very narrow Gαi concentration range. Ultrasensitivity is required for Pins function in neuroblasts as a nonultrasensitive Pins mutant fails to robustly couple spindle position to cell polarity. Pins contains three Gαi binding GoLoco domains (GLs); Gαi binding to GL3 activates Pins, whereas GLs 1 and 2 shape the response profile. Although cooperative binding is one mechanism for generating ultrasensitivity, we find GLs 1 and 2 act as "decoys" that compete against activation at GL3. Many signaling proteins contain multiple protein interaction domains, and the decoy mechanism may be a common method for generating ultrasensitivity in regulatory pathways.     

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.     

Mitotic spindle: focus on the function of huntingtin

Mitotic spindle assembly and orientation are tightly regulated to allow the appropriate segregation of genetic material and cell fate determinants during symmetric and asymmetric divisions. Microtubules and many proteins including the dynein/dynactin complex and the large nuclear mitotic apparatus NuMA protein, are fundamental players in these mechanisms. A recent study reported that huntingtin regulates spindle orientation by ensuring the proper localization of the p150(Glued) subunit of dynactin, dynein and NuMA. This function of huntingtin is conserved in Drosophila. Among other events, spindle orientation influences the fate of daughter cells. In agreement with this, huntingtin changes the direction of division of mouse cortical progenitors and promotes neurogenesis in the neocortex. We will also discuss the involvement of mitotic spindle components in neuronal disorders.     

Canoe binds RanGTP to promote Pins(TPR)/Mud-mediated spindle orientation

Regulated spindle orientation maintains epithelial tissue integrity and stem cell asymmetric cell division. In Drosophila melanogaster neural stem cells (neuroblasts), the scaffolding protein Canoe (Afadin/Af-6 in mammals) regulates spindle orientation, but its protein interaction partners and mechanism of action are unknown. In this paper, we use our recently developed induced cell polarity system to dissect the molecular mechanism of Canoe-mediated spindle orientation. We show that a previously uncharacterized portion of Canoe directly binds the Partner of Inscuteable (Pins) tetratricopeptide repeat (TPR) domain. The Canoe-Pins(TPR) interaction recruits Canoe to the cell cortex and is required for activation of the Pins(TPR)-Mud (nuclear mitotic apparatus in mammals) spindle orientation pathway. We show that the Canoe Ras-association (RA) domains directly bind RanGTP and that both the Canoe(RA) domains and RanGTP are required to recruit Mud to the cortex and activate the Pins/Mud/dynein spindle orientation pathway.     

Membrane targeting and asymmetric localization of Drosophila partner of inscuteable are discrete steps controlled by distinct regions of the protein

Asymmetric division of neural progenitors is a key mechanism by which neuronal diversity in the Drosophila central nervous system is generated. The distinct fates of the daughter cells derived from these divisions are achieved through preferential segregation of the cell fate determinants Prospero and Numb to one of the two daughters. This is achieved by coordinating apical and basal mitotic spindle orientation with the basal cortical localization of the cell fate determinants during mitosis. A complex of apically localized proteins, including Inscuteable (Insc), Partner of Inscuteable (Pins), Bazooka (Baz), DmPar-6, DaPKC, and G alpha i, is required to mediate and coordinate basal protein localization with mitotic spindle orientation. Pins, a molecule which directly interacts with Insc, is a key component required for the integrity of this complex; in the absence of Pins, other components become mislocalized or destabilized, and basal protein localization and mitotic spindle orientation are defective. Here we define the functional domains of Pins. We show that the C-terminal region containing the G alpha i binding GoLoco motifs is necessary and sufficient for targeting to the neuroblast cortex, which appears to be a prerequisite for apical localization of Pins. The N-terminal tetratricopeptide repeat-containing region of Pins is required for two processes; TPR repeats 1 to 3 plus the C-terminal region are required for apical localization but are insufficient to recruit Insc to the apical cortex, whereas TPR repeats 1 to 7 plus C-terminal Pins can perform both functions. Hence, the abilities of Pins to cortically localize, to apically localize, and to restore Insc apical localization are all separable, and all three capabilities are necessary to mediate asymmetric division. Moreover, the need for N-terminal Pins can be obviated by fusing a minimal Insc functional domain with the C-terminal region of Pins; this chimeric molecule is apically localized and can fulfill the functions of both Insc and Pins.     

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.     

An ana2/ctp/mud complex regulates spindle orientation in Drosophila neuroblasts

Drosophila neural stem cells, larval brain neuroblasts (NBs), align their mitotic spindles along the apical/basal axis during asymmetric cell division (ACD) to maintain the balance of self-renewal and differentiation. Here, we identified a protein complex composed of the tumor suppressor anastral spindle 2 (Ana2), a dynein light-chain protein Cut up (Ctp), and Mushroom body defect (Mud), which regulates mitotic spindle orientation. We isolated two ana2 alleles that displayed spindle misorientation and NB overgrowth phenotypes in larval brains. The centriolar protein Ana2 anchors Ctp to centrioles during ACD. The centriolar localization of Ctp is important for spindle orientation. Ana2 and Ctp localize Mud to the centrosomes and cell cortex and facilitate/maintain the association of Mud with Pins at the apical cortex. Our findings reveal that the centrosomal proteins Ana2 and Ctp regulate Mud function to?orient the mitotic spindle during NB asymmetric 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.     

Galpha/LGN-mediated asymmetric spindle positioning does not lead to unequal cleavage of the mother cell in 3-D cultured MDCK cells

The position of the mitotic spindle plays a key role in spatial control of cell division. It is generally believed that when a spindle is positioned asymmetrically in a dividing cell, the resulting daughter cells are usually unequal in size due to eccentric cleavage of the mother cell. Molecular mechanisms underlying the generation of unequal sized daughter cells have been extensively studied in Drosophila neuroblast and Caenorhabditis elegans zygote where the Gα subunit of the heterotrimeric G proteins and its binding partner - Pins in Drosophila and GPR-1/2 in C. elegans - are shown to be critical in governing spindle positioning and asymmetric cleavage of the mother cell. In mammalian system, although Gα and LGN (mammalian Pins homolog) are also required for spindle orientation, whether they can mediate asymmetric spindle positioning or asymmetric cleavage of the mother cell is not known. Here, by artificially targeting Gαi to the apical cortex in 3-D cultured MDCK cells, we established a system where asymmetric spindle positioning can be consistently induced. Interestingly, this asymmetrically positioned spindle does not lead to asymmetric cleavage; instead it results in equal sized daughter cells. Live cell time-lapse analysis revealed that anaphase spindle elongation compensated the original asymmetric spindle positioning. Our findings demonstrate that asymmetric spindle positioning does not necessarily lead to unequal sized daughter cells in mammalian system. We discuss potential mechanisms in generating unequal sized daughter cells.     

Mammalian Pins is a conformational switch that links NuMA to heterotrimeric G proteins

During asymmetric cell divisions, mitotic spindles align along the axis of polarization. In invertebrates, spindle positioning requires Pins or related proteins and a G protein alpha subunit. A mammalian Pins, called LGN, binds Galphai and also interacts through an N-terminal domain with the microtubule binding protein NuMA. During mitosis, LGN recruits NuMA to the cell cortex, while cortical association of LGN itself requires the C-terminal Galpha binding domain. Using a FRET biosensor, we find that LGN behaves as a conformational switch: in its closed state, the N and C termini interact, but NuMA or Galphai can disrupt this association, allowing LGN to interact simultaneously with both proteins, resulting in their cortical localization. Overexpression of Galphai or YFP-LGN causes a pronounced oscillation of metaphase spindles, and NuMA binding to LGN is required for these spindle movements. We propose that a related switch mechanism might operate in asymmetric cell divisions in the fly and nematode.     

The PDZ protein Canoe regulates the asymmetric division of Drosophila neuroblasts and muscle progenitors

Asymmetric cell division is a conserved mechanism to generate cellular diversity during animal development and a key process in cancer and stem cell biology. Despite the increasing number of proteins characterized, the complex network of proteins interactions established during asymmetric cell division is still poorly understood. This suggests that additional components must be contributing to orchestrate all the events underlying this tightly modulated process. The PDZ protein Canoe (Cno) and its mammalian counterparts AF-6 and Afadin are critical to regulate intracellular signaling and to organize cell junctions throughout development. Here, we show that Cno functions as a new effector of the apical proteins Inscuteable (Insc)-Partner of Inscuteable (Pins)-Galphai during the asymmetric division of Drosophila neuroblasts (NBs). Cno localizes apically in metaphase NBs and coimmunoprecipitates with Pins in vivo. Furthermore, Cno functionally interacts with the apical proteins Insc, Galphai, and Mushroom body defect (Mud) to generate correct neuronal lineages. Failures in muscle and heart lineages are also detected in cno mutant embryos. Our results strongly support a new function for Cno regulating key processes during asymmetric NB division: the localization of cell-fate determinants, the orientation of the mitotic spindle, and the generation of unequal-sized daughter cells.     

Epithelial polarity and spindle orientation: intersecting pathways

During asymmetric stem cell divisions, the mitotic spindle must be correctly oriented and positioned with respect to the axis of cell polarity to ensure that cell fate determinants are appropriately segregated into only one daughter cell. By contrast, epithelial cells divide symmetrically and orient their mitotic spindles perpendicular to the main apical–basal polarity axis, so that both daughter cells remain within the epithelium. Work in the past 20 years has defined a core ternary complex consisting of Pins, Mud and Gαi that participates in spindle orientation in both asymmetric and symmetric divisions. As additional factors that interact with this complex continue to be identified, a theme has emerged: there is substantial overlap between the mechanisms that orient the spindle and those that establish and maintain apical–basal polarity in epithelial cells. In this review, we examine several factors implicated in both processes, namely Canoe, Bazooka, aPKC and Discs large, and consider the implications of this work on how the spindle is oriented during epithelial cell divisions.     

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.     

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.     

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.     

Cortical aPKC kinase activity distinguishes neural stem cells from progenitor cells by ensuring asymmetric segregation of Numb

During asymmetric stem cell division, polarization of the cell cortex targets fate determinants unequally into the sibling daughters, leading to regeneration of a stem cell and production of a progenitor cell with restricted developmental potential. In mitotic neural stem cells (neuroblasts) in fly larval brains, the antagonistic interaction between the polarity proteins Lethal (2) giant larvae (Lgl) and atypical Protein Kinase C (aPKC) ensures self-renewal of a daughter neuroblast and generation of a progenitor cell by regulating asymmetric segregation of fate determinants. In the absence of lgl function, elevated cortical aPKC kinase activity perturbs unequal partitioning of the fate determinants including Numb and induces supernumerary neuroblasts in larval brains. However, whether increased aPKC function triggers formation of excess neuroblasts by inactivating Numb remains controversial. To investigate how increased cortical aPKC function induces formation of excess neuroblasts, we analyzed the fate of cells in neuroblast lineage clones in lgl mutant brains. Surprisingly, our analyses revealed that neuroblasts in lgl mutant brains undergo asymmetric division to produce progenitor cells, which then revert back into neuroblasts. In lgl mutant brains, Numb remained localized in the cortex of mitotic neuroblasts and failed to segregate exclusively into the progenitor cell following completion of asymmetric division. These results led us to propose that elevated aPKC function in the cortex of mitotic neuroblasts reduces the function of Numb in the future progenitor cells. We identified that the acyl-CoA binding domain containing 3 protein (ACBD3) binding region is essential for asymmetric segregation of Numb in mitotic neuroblasts and suppression of the supernumerary neuroblast phenotype induced by increased aPKC function. The ACBD3 binding region of Numb harbors two aPKC phosphorylation sites, serines 48 and 52. Surprisingly, while the phosphorylation status at these two sites directly impinged on asymmetric segregation of Numb in mitotic neuroblasts, both the phosphomimetic and non-phosphorylatable forms of Numb suppressed formation of excess neuroblasts triggered by increased cortical aPKC function. Thus, we propose that precise regulation of cortical aPKC kinase activity distinguishes the sibling cell identity in part by ensuring asymmetric partitioning of Numb into the future progenitor cell where Numb maintains restricted potential independently of regulation by aPKC.