Dynein-Dynactin-NuMA clusters generate cortical spindle-pulling forces as a multi-arm ensemble.
ABSTRACT: To position the mitotic spindle within the cell, dynamic plus ends of astral microtubules are pulled by membrane-associated cortical force-generating machinery. However, in contrast to the chromosome-bound kinetochore structure, how the diffusion-prone cortical machinery is organized to generate large spindle-pulling forces remains poorly understood. Here, we develop a light-induced reconstitution system in human cells. We find that induced cortical targeting of NuMA, but not dynein, is sufficient for spindle pulling. This spindle-pulling activity requires dynein-dynactin recruitment by NuMA's N-terminal long arm, dynein-based astral microtubule gliding, and NuMA's direct microtubule-binding activities. Importantly, we demonstrate that cortical NuMA assembles specialized focal structures that cluster multiple force-generating modules to generate cooperative spindle-pulling forces. This clustering activity of NuMA is required for spindle positioning, but not for spindle-pole focusing. We propose that cortical Dynein-Dynactin-NuMA (DDN) clusters act as the core force-generating machinery that organizes a multi-arm ensemble reminiscent of the kinetochore.
Project description:Cortical force generators connect epithelial polarity sites with astral microtubules, allowing dynein movement to orient the mitotic spindle as astral microtubules depolymerize. Complexes of the LGN and NuMA proteins, fundamental components of force generators, are recruited to the cortex by G?i-subunits of heterotrimeric G-proteins. They associate with dynein/dynactin and activate the motor activity pulling on astral microtubules. The architecture of cortical force generators is unknown. Here we report the crystal structure of NuMA:LGN hetero-hexamers, and unveil their role in promoting the assembly of active cortical dynein/dynactin motors that are required in orchestrating oriented divisions in polarized cells. Our work elucidates the basis for the structural organization of essential spindle orientation motors.
Project description:Precise positioning of the mitotic spindle determines the correct cell division axis and is crucial for organism development. Spindle positioning is mediated through a cortical machinery by capturing astral microtubules, thereby generating pushing/pulling forces at the cell cortex. However, the molecular link between these two structures remains elusive. Here we describe a previously uncharacterized protein, MISP (C19orf21), as a substrate of Plk1 that is required for correct mitotic spindle positioning. MISP is an actin-associated protein throughout the cell cycle. MISP depletion led to an impaired metaphase-to-anaphase transition, which depended on phosphorylation by Plk1. Loss of MISP induced mitotic defects including spindle misorientation accompanied by shortened astral microtubules. Furthermore, we find that MISP formed a complex with and regulated the cortical distribution of the +TIP binding protein p150(glued), a subunit of the dynein-dynactin complex. We propose that Plk1 phosphorylates MISP, thus stabilizing cortical and astral microtubule attachments required for proper mitotic spindle positioning.
Project description: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.
Project description:The position of the mitotic spindle determines the plane of cell cleavage, and thereby daughter cell location, size, and content. Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules, which requires an evolutionarily conserved complex of G??GDP, GPR-1/2Pins/LGN, and LIN-5Mud/NuMA proteins. To examine individual functions of the complex components, we developed a genetic strategy for light-controlled localization of endogenous proteins in C. elegans embryos. By replacing G? and GPR-1/2 with a light-inducible membrane anchor, we demonstrate that G??GDP, G??GTP, and GPR-1/2 are not required for pulling-force generation. In the absence of G? and GPR-1/2, cortical recruitment of LIN-5, but not dynein itself, induced high pulling forces. The light-controlled localization of LIN-5 overruled normal cell-cycle and polarity regulation and provided experimental control over the spindle and cell-cleavage plane. Our results define G??GDP-GPR-1/2Pins/LGN as a regulatable membrane anchor, and LIN-5Mud/NuMA as a potent activator of dynein-dependent spindle-positioning forces.
Project description:Oriented cell division is critical for cell fate specification, tissue organization, and tissue homeostasis, and relies on proper orientation of the mitotic spindle. The molecular mechanisms underlying the regulation of spindle orientation remain largely unknown. Herein, we identify a critical role for cylindromatosis (CYLD), a deubiquitinase and regulator of microtubule dynamics, in the control of spindle orientation. CYLD is highly expressed in mitosis and promotes spindle orientation by stabilizing astral microtubules and deubiquitinating the cortical polarity protein dishevelled. The deubiquitination of dishevelled enhances its interaction with nuclear mitotic apparatus, stimulating the cortical localization of nuclear mitotic apparatus and the dynein/dynactin motor complex, a requirement for generating pulling forces on astral microtubules. These findings uncover CYLD as an important player in the orientation of the mitotic spindle and cell division and have important implications in health and disease.
Project description:Accurate and efficient separation of sister chromatids during anaphase is critical for faithful cell division. It has been proposed that cortical dynein-generated pulling forces on astral microtubules contribute to anaphase spindle elongation and chromosome separation. In mammalian cells, however, definitive evidence for the involvement of cortical dynein in chromosome separation is missing. It is believed that dynein is recruited and anchored at the cell cortex during mitosis by the ? subunit of heterotrimeric G protein (G?)/mammalian homologue of Drosophila Partner of Inscuteable/nuclear mitotic apparatus (NuMA) ternary complex. Here we uncover a G?/LGN-independent lipid- and membrane-binding domain at the C-terminus of NuMA. We show that the membrane binding of NuMA is cell cycle regulated-it is inhibited during prophase and metaphase by cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation and only occurs after anaphase onset when CDK1 activity is down-regulated. Further studies indicate that cell cycle-regulated membrane association of NuMA underlies anaphase-specific enhancement of cortical NuMA and dynein. By replacing endogenous NuMA with membrane-binding-deficient NuMA, we can specifically reduce the cortical accumulation of NuMA and dynein during anaphase and demonstrate that cortical NuMA and dynein contribute to efficient chromosome separation in mammalian cells.
Project description:Accurate positioning of the mitotic spindle is critical to ensure proper distribution of chromosomes during cell division. The small GTPase Ran, which regulates a variety of processes throughout the cell cycle, including interphase nucleocytoplasmic transport and mitotic spindle assembly, was recently shown to also control spindle alignment. Ran is required for the correct cortical localization of LGN and nuclear-mitotic apparatus protein (NuMA), proteins that generate pulling forces on astral microtubules (MTs) through cytoplasmic dynein. Here we use importazole, a small-molecule inhibitor of RanGTP/importin-? function, to study the role of Ran in spindle positioning in human cells. We find that importazole treatment results in defects in astral MT dynamics, as well as in mislocalization of LGN and NuMA, leading to misoriented spindles. Of interest, importazole-induced spindle-centering defects can be rescued by nocodazole treatment, which depolymerizes astral MTs, or by overexpression of CLASP1, which does not restore proper LGN and NuMA localization but stabilizes astral MT interactions with the cortex. Together our data suggest a model for mitotic spindle positioning in which RanGTP and CLASP1 cooperate to align the spindle along the long axis of the dividing cell.
Project description:How dynein motors accurately move cargoes is an important question. In budding yeast, dynein moves the mitotic spindle to the predetermined site of cytokinesis by pulling on astral microtubules. In this study, using high-resolution imaging in living cells, we discover that spindle movement is regulated by changes in microtubule plus-end dynamics that occur when dynein generates force. Mutants that increase plus-end stability increase the frequency and duration of spindle movements, causing positioning errors. We find that dynein plays a primary role in regulating microtubule dynamics by destabilizing microtubules. In contrast, the dynactin complex counteracts dynein and stabilizes microtubules through a mechanism involving the shoulder subcomplex and the cytoskeletal-associated protein glycine-rich domain of Nip100/p150glued Our results support a model in which dynein destabilizes its microtubule substrate by using its motility to deplete dynactin from the plus end. We propose that interplay among dynein, dynactin, and the stability of the microtubule substrate creates a mechanism that regulates accurate spindle positioning.
Project description:The adenomatous polyposis coli (APC) tumor suppressor has dual functions in Wnt/?-catenin signaling and accurate chromosome segregation and is frequently mutated in colorectal cancers. Although APC contributes to proper cell division, the underlying mechanisms remain poorly understood. Here we show that Caenorhabditis elegans APR-1/APC is an attenuator of the pulling forces acting on the mitotic spindle. During asymmetric cell division of the C. elegans zygote, a LIN-5/NuMA protein complex localizes dynein to the cell cortex to generate pulling forces on astral microtubules that position the mitotic spindle. We found that APR-1 localizes to the anterior cell cortex in a Par-aPKC polarity-dependent manner and suppresses anterior centrosome movements. Our combined cell biological and mathematical analyses support the conclusion that cortical APR-1 reduces force generation by stabilizing microtubule plus-ends at the cell cortex. Furthermore, APR-1 functions in coordination with LIN-5 phosphorylation to attenuate spindle-pulling forces. Our results document a physical basis for the attenuation of spindle-pulling force, which may be generally used in asymmetric cell division and, when disrupted, potentially contributes to division defects in cancer.
Project description:Little is known about the mechanisms of mitotic spindle orientation during mammary gland morphogenesis. Here, we report the presence of huntingtin, the protein mutated in Huntington's disease, in mouse mammary basal and luminal cells throughout mammogenesis. Keratin 5-driven depletion of huntingtin results in a decreased pool and specification of basal and luminal progenitors, and altered mammary morphogenesis. Analysis of mitosis in huntingtin-depleted basal progenitors reveals mitotic spindle misorientation. In mammary cell culture, huntingtin regulates spindle orientation in a dynein-dependent manner. Huntingtin is targeted to spindle poles through its interaction with dynein and promotes the accumulation of NUMA and LGN. Huntingtin is also essential for the cortical localization of dynein, dynactin, NUMA, and LGN by regulating their kinesin 1-dependent trafficking along astral microtubules. We thus suggest that huntingtin is a component of the pathway regulating the orientation of mammary stem cell division, with potential implications for their self-renewal and differentiation properties.