The Drosophila NuMA Homolog Mud regulates spindle orientation in asymmetric cell division

During asymmetric cell division, the mitotic spindle must be properly oriented to ensure the asymmetric segregation of cell fate determinants into only one of the two daughter cells. In Drosophila neuroblasts, spindle orientation requires heterotrimeric G proteins and the G alpha binding partner Pins, but how the Pins-G alphai complex interacts with the mitotic spindle is unclear. Here, we show that Pins binds directly to the microtubule binding protein Mud, the Drosophila homolog of NuMA. Like NuMA, Mud can bind to microtubules and enhance microtubule polymerization. In the absence of Mud, mitotic spindles in Drosophila neuroblasts fail to align with the polarity axis. This can lead to symmetric segregation of the cell fate determinants Brat and Prospero, resulting in the mis-specification of daughter cell fates and tumor-like over proliferation in the Drosophila nervous system. Our data suggest a model in which asymmetrically localized Pins-G alphai complexes regulate spindle orientation by directly binding to Mud.     

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.     

Regulation of mitotic spindle orientation: an integrated view

Mitotic spindle orientation is essential for cell fate decisions, epithelial maintenance, and tissue morphogenesis. In most animal cell types, the dynein motor complex is anchored at the cell cortex and exerts pulling forces on astral microtubules to position the spindle. Early studies identified the evolutionarily conserved Gαi/LGN/NuMA complex as a key regulator that polarizes cortical force generators. In recent years, a combination of genetics, biochemistry, modeling, and live imaging has contributed to decipher the mechanisms of spindle orientation. Here, we highlight the dynamic nature of the assembly of this complex and discuss the molecular regulation of its localization. Remarkably, a number of LGN‐independent mechanisms were described recently, whereas NuMA remains central in most pathways involved in recruiting force generators at the cell cortex. We also describe the emerging role of the actin cortex in spindle orientation and discuss how dynamic astral microtubule formation is involved. We further give an overview on instructive external signals that control spindle orientation in tissues. Finally, we discuss the influence of cell geometry and mechanical forces on spindle orientation.     

Cell division orientation and planar cell polarity pathways

The orientation of cell division has a crucial role in early embryo body plan specification, axis determination and cell fate diversity generation, as well as in the morphogenesis of tissues and organs. In many instances, cell division orientation is regulated by the planar cell polarity (PCP) pathways: the Wnt/Frizzled non-canonical pathway or the Fat/Dachsous/Four-jointed pathway. Firstly, using asymmetric cell division in both Drosophila and C. elegans, we describe the central role of the Wnt/Frizzled pathway in the regulation of asymmetric cell division orientation, focusing on its cooperation with either the Src kinase pathway or the heterotrimeric G protein pathway. Secondly, we describe our present understanding of the mechanisms by which the planar cell polarity pathways drive tissue morphogenesis by regulating the orientation of symmetric cell division within a field of cells. Finally, we will discuss the important avenues that need to be explored in the future to better understand how planar cell polarity pathways control embryo body plan determination, cell fate specification or tissue morphogenesis by mitotic spindle orientation.     

Control of spindle polarity and orientation in Saccharomyces cerevisiae

Control of mitotic spindle orientation represents a major strategy for the generation of cell diversity during development of metazoans. Studies in the budding yeast Saccharomyces cerevisiae have contributed towards our present understanding of the general principles underlying the regulation of spindle positioning in an asymmetrically dividing cell. In S. cerevisiae, the mitotic spindle must orient along the cell polarity axis, defined by the site of bud emergence, to ensure correct nuclear division between the mother and daughter cells. Establishment of spindle polarity dictates this process and relies on the concerted control of spindle pole function and a precise program of cues originating from the cell cortex that directs cytoplasmic microtubule attachments during spindle morphogenesis. These cues cross talk with the machinery responsible for bud-site selection, indicating that orientation of the spindle in yeast cells is mechanistically coupled to the definition of a polarity axis and the division plane. Here, we propose a model integrating the inherently asymmetric properties of the spindle pathway with the program of positional information contributing towards orienting the spindle in budding yeast. Because the basic machinery orienting the spindle in higher-eukaryotic cells appears to be conserved, it might be expected that similar principles govern centrosome asymmetry in the course of metazoan development.     

Spindle orientation and epidermal morphogenesis

Asymmetric cell divisions (ACDs) result in two unequal daughter cells and are a hallmark of stem cells. ACDs can be achieved either by asymmetric partitioning of proteins and organelles or by asymmetric cell fate acquisition due to the microenvironment in which the daughters are placed. Increasing evidence suggests that in the mammalian epidermis, both of these processes occur. During embryonic epidermal development, changes occur in the orientation of the mitotic spindle in relation to the underlying basement membrane. These changes are guided by conserved molecular machinery that is operative in lower eukaryotes and dictates asymmetric partitioning of proteins during cell divisions. That said, the shift in spindle alignment also determines whether a division will be parallel or perpendicular to the basement membrane, and this in turn provides a differential microenvironment for the resulting daughter cells. Here, we review how oriented divisions of progenitors contribute to the development and stratification of the epidermis.     

NuMA localization,stability, and function in spindle orientation involve 4.1 and Cdk1 interactions

The epidermis is a multilayered epithelium that requires asymmetric divisions for stratification. A conserved cortical protein complex, including LGN, nuclear mitotic apparatus (NuMA), and dynein/dynactin, plays a key role in establishing proper spindle orientation during asymmetric divisions. The requirements for the cortical recruitment of these proteins, however, remain unclear. In this work, we show that NuMA is required to recruit dynactin to the cell cortex of keratinocytes. NuMA''s cortical recruitment requires LGN; however, LGN interactions are not sufficient for this localization. Using fluorescence recovery after photobleaching, we find that the 4.1-binding domain of NuMA is important for stabilizing its interaction with the cell cortex. This is functionally important, as loss of 4.1/NuMA interaction results in spindle orientation defects, using two distinct assays. Furthermore, we observe an increase in cortical NuMA localization as cells enter anaphase. Inhibition of Cdk1 or mutation of a single residue in NuMA mimics this effect. NuMA''s anaphase localization is independent of LGN and 4.1 interactions, revealing two distinct mechanisms responsible for NuMA cortical recruitment at different stages of mitosis. This work highlights the complexity of NuMA localization and reveals the importance of NuMA cortical stability for productive force generation during spindle orientation.     

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.     

Two protein 4.1 domains essential for mitotic spindle and aster microtubule dynamics and organization in vitro

Multifunctional structural proteins belonging to the 4.1 family are components of nuclei, spindles, and centrosomes in vertebrate cells. Here we report that 4.1 is critical for spindle assembly and the formation of centrosome-nucleated and motor-dependent self-organized microtubule asters in metaphase-arrested Xenopus egg extracts. Immunodepletion of 4.1 disrupted microtubule arrays and mislocalized the spindle pole protein NuMA. Remarkably, assembly was completely rescued by supplementation with a recombinant 4.1R isoform. We identified two 4.1 domains critical for its function in microtubule polymerization and organization utilizing dominant negative peptides. The 4.1 spectrin-actin binding domain or NuMA binding C-terminal domain peptides caused morphologically disorganized structures. Control peptides with low homology or variant spectrin-actin binding domain peptides that were incapable of binding actin had no deleterious effects. Unexpectedly, the addition of C-terminal domain peptides with reduced NuMA binding caused severe microtubule destabilization in extracts, dramatically inhibiting aster and spindle assembly and also depolymerizing preformed structures. However, the mutant C-terminal peptides did not directly inhibit or destabilize microtubule polymerization from pure tubulin in a microtubule pelleting assay. Our data showing that 4.1 is a crucial factor for assembly and maintenance of mitotic spindles and self-organized and centrosome-nucleated microtubule asters indicates that 4.1 is involved in regulating both microtubule dynamics and organization. These investigations underscore an important functional context for protein 4.1 in microtubule morphogenesis and highlight a previously unappreciated role for 4.1 in cell division.     

Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate

Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Loss of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium.     

细胞极性蛋白复合物PAR/aPKC的研究进展

不对称细胞分裂是动物发育过程中用以调控细胞分化的一种进化上保守的基本模式。极性的祖细胞通过不对称分裂产生两个不同命运的子细胞,这一过程涉及细胞命运决定因子的不对称分布、纺锤体的旋转定位等,而这些过程都必须依赖特定细胞极性的存在才能得以正常进行。简要综述了高度保守的蛋白复合物PAR／aPKC在细胞极性建立和维持中的重要作用,以及它如何调控纺锤体定位和命运决定因子不对称分配,并讨论了在该领域的一些新发现和研究进展。     

Regulation of asymmetric cell division in the epidermis

For proper tissue morphogenesis, cell divisions and cell fate decisions must be tightly and coordinately regulated. One elegant way to accomplish this is to couple them with asymmetric cell divisions. Progenitor cells in the developing epidermis undergo both symmetric and asymmetric cell divisions to balance surface area growth with the generation of differentiated cell layers. Here we review the molecular machinery implicated in controlling asymmetric cell division. In addition, we discuss the ability of epidermal progenitors to choose between symmetric and asymmetric divisions and the key regulatory points that control this decision.     

Eya1 controls cell polarity, spindle orientation, cell fate and Notch signaling in distal embryonic lung epithelium

Cell polarity, mitotic spindle orientation and asymmetric division play a crucial role in the self-renewal/differentiation of epithelial cells, yet little is known about these processes and the molecular programs that control them in embryonic lung distal epithelium. Herein, we provide the first evidence that embryonic lung distal epithelium is polarized with characteristic perpendicular cell divisions. Consistent with these findings, spindle orientation-regulatory proteins Insc, LGN (Gpsm2) and NuMA, and the cell fate determinant Numb are asymmetrically localized in embryonic lung distal epithelium. Interfering with the function of these proteins in vitro randomizes spindle orientation and changes cell fate. We further show that Eya1 protein regulates cell polarity, spindle orientation and the localization of Numb, which inhibits Notch signaling. Hence, Eya1 promotes both perpendicular division as well as Numb asymmetric segregation to one daughter in mitotic distal lung epithelium, probably by controlling aPKCζ phosphorylation. Thus, epithelial cell polarity and mitotic spindle orientation are defective after interfering with Eya1 function in vivo or in vitro. In addition, in Eya1(-/-) lungs, perpendicular division is not maintained and Numb is segregated to both daughter cells in mitotic epithelial cells, leading to inactivation of Notch signaling. As Notch signaling promotes progenitor cell identity at the expense of differentiated cell phenotypes, we test whether genetic activation of Notch could rescue the Eya1(-/-) lung phenotype, which is characterized by loss of epithelial progenitors, increased epithelial differentiation but reduced branching. Indeed, genetic activation of Notch partially rescues Eya1(-/-) lung epithelial defects. These findings uncover novel functions for Eya1 as a crucial regulator of the complex behavior of distal embryonic lung epithelium.     

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).     

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.     

Chromosome- and spindle-pole-derived signals generate an intrinsic code for spindle position and orientation

Mitotic spindle positioning by cortical pulling forces defines the cell division axis and location, which is critical for proper cell division and development. Although recent work has identified developmental and extrinsic cues that regulate spindle orientation, the contribution of intrinsic signals to spindle positioning and orientation remains unclear. Here, we demonstrate that cortical force generation in human cells is controlled by distinct spindle-pole- and chromosome-derived signals that regulate cytoplasmic dynein localization. First, dynein exhibits a dynamic asymmetric cortical localization that is negatively regulated by spindle-pole proximity, resulting in spindle oscillations to centre the spindle within the cell. We find that this signal comprises the spindle-pole-localized polo-like kinase (Plk1), which regulates dynein localization by controlling the interaction between dynein-dynactin and its upstream cortical targeting factors NuMA and LGN. Second, a chromosome-derived RanGTP gradient restricts the localization of NuMA-LGN to the lateral cell cortex to define and maintain the spindle orientation axis. RanGTP acts in part through the nuclear localization sequence of NuMA to locally alter the ability of NuMA-LGN to associate with the cell cortex in the vicinity of chromosomes. We propose that these chromosome- and spindle-pole-derived gradients generate an intrinsic code to control spindle position and orientation.     

Asymmetric centrosome behavior and the mechanisms of stem cell division

The ability of dividing cells to produce daughters with different fates is an important developmental mechanism conserved from bacteria to fungi, plants, and metazoan animals. Asymmetric outcomes of a cell division can be specified by two general mechanisms: asymmetric segregation of intrinsic fate determinants or asymmetric placement of daughter cells into microenvironments that provide extrinsic signals that direct cells to different states. For both, spindle orientation must be coordinated with the localization of intrinsic determinants or source of extrinsic signals to achieve the proper asymmetric outcome. Recent work on spindle orientation in Drosophila melanogaster male germline stem cells and neuroblasts has brought into sharp focus the key role of differential centrosome behavior in developmentally programmed asymmetric division (for reviews see Cabernard, C., and C.Q. Doe. 2007. Curr. Biol. 17:R465-R467; Gonzalez, C. 2007. Nat. Rev. Genet. 8:462-472). These findings provide new insights and suggest intriguing new models for how cells coordinate spindle orientation with their cellular microenvironment to regulate and direct cell fate decisions within tissues.