Plk1 Self-Organization and Priming Phosphorylation of HsCYK-4 at the Spindle Midzone
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ABSTRACT:
PROVIDER: MSV000078497 | MassIVE | Thu Nov 21 11:32:00 GMT 2013
REPOSITORIES: MassIVE
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Project description:Animal cells initiate cytokinesis in parallel with anaphase onset, when an actomyosin ring assembles and constricts through localized activation of the small GTPase RhoA, giving rise to a cleavage furrow. Furrow formation relies on positional cues provided by anaphase spindle microtubules (MTs), but how such cues are generated remains unclear. Using chemical genetics to achieve both temporal and spatial control, we show that the self-organized delivery of Polo-like kinase 1 (Plk1) to the midzone and its local phosphorylation of a MT-bound substrate are critical for generating this furrow-inducing signal. When Plk1 was active but unable to target itself to this equatorial landmark, both cortical RhoA recruitment and furrow induction failed to occur, thus recapitulating the effects of anaphase-specific Plk1 inhibition. Using tandem mass spectrometry and phosphospecific antibodies, we found that Plk1 binds and directly phosphorylates the HsCYK-4 subunit of centralspindlin (also known as MgcRacGAP) at the midzone. At serine 157, this modification creates a major docking site for the tandem BRCT repeats of the Rho GTP exchange factor Ect2. Cells expressing only a nonphosphorylatable form of HsCYK-4 failed to localize Ect2 at the midzone and were severely impaired in cleavage furrow formation, implying that HsCYK-4 is Plk1's rate-limiting target upstream of RhoA. Conversely, tethering an inhibitor-resistant allele of Plk1 to HsCYK-4 allowed furrows to form despite global inhibition of all other Plk1 molecules in the cell. Our findings illuminate two key mechanisms governing the initiation of cytokinesis in human cells and illustrate the power of chemical genetics to probe such regulation both in time and space.
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Project description:The cytoskeleton forms a variety of steady-state, subcellular structures that are maintained by continuous fluxes of molecules and energy. Understanding such self-organizing structures is not only crucial for cell biology but also poses a fundamental challenge for physics, since these systems are active materials that behave drastically differently from matter at or near equilibrium. Active liquid crystal theories have been developed to study the self-organization of cytoskeletal filaments in in vitro systems of purified components. However, it has been unclear how relevant these simplified approaches are for understanding biological structures, which can be composed of hundreds of distinct proteins. Here we show that a suitably constructed active liquid crystal theory produces remarkably accurate predictions of the behaviors of metaphase spindles-the cytoskeletal structure, composed largely of microtubules and associated proteins, that segregates chromosomes during cell division.
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Project description:In anaphase spindles, antiparallel microtubules associate to form tight midzone bundles, as required for functional spindle architecture and correct chromosome segregation. Several proteins selectively bind to these overlaps to control cytokinesis. How midzone bundles assemble is poorly understood. Here, using an in vitro reconstitution approach, we demonstrate that minimal midzone bundles can reliably self-organize in solution from dynamic microtubules, the microtubule crosslinker PRC1, and the motor protein KIF4A. The length of the central antiparallel overlaps in these microtubule bundles is similar to that observed in cells and is controlled by the PRC1/KIF4A ratio. Experiments and computer simulations demonstrate that minimal midzone bundle formation results from promoting antiparallel microtubule crosslinking, stopping microtubule plus-end dynamicity, and motor-driven midzone compaction and alignment. The robustness of this process suggests that a similar self-organization mechanism may contribute to the reorganization of the spindle architecture during the metaphase to anaphase transition in cells.
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Project description:Plk1 is a key mitotic kinase that localizes to distinct subcellular structures to promote accurate mitotic progression. Plk1 recruitment depends on direct interaction between polo-box domain (PBD) on Plk1 and PBD binding motif (PBD BM) on the interactors. However, recent study showed that PBD BM alone is not enough for stable binding between CENP-U and Plk1 highlighting the complexity of the interaction which warrants further investigation. An important interactor for Plk1 during mitosis is the checkpoint protein BubR1. Plk1 bound to BubR1 via PBD interaction with pT620 phosphorylates BubR1 S676/T680 to promote BubR1-PP2A/B56 interaction. The BubR1-PP2A/B56 complex counteracts the destablizing effect on kinetochore-microtubule attachments by mitotic kinases to promote mitotic progression.
Here we show that Plk1 phosphorylates T600/T608 on BubR1 and the double phosphorylation is critical for BubR1-Plk1 interaction. A similar mechanism for Plk1-Bub1 interaction also exists indicating a general principle for Plk1 kinetochore recruitment through self-priming. Mechanistically preventing BubR1 T600/T608 phosphorylation impairs chromosome congression and checkpoint silencing by reducing Plk1 and PP2A/B56 binding to BubR1. Increasing the binding affinity towards Plk1 and PP2A/B56 in BubR1 through protein engineering bypasses the requirement of T600/T608 phosphorylation for mitotic progression. These results reveal a new layer of regulation for accurate mitotic progression.
2024-10-31 | MSV000096264 | MassIVE
Project description:The spindle is a dynamic intracellular structure self-organized from microtubules and microtubule-associated proteins. The spindle's bipolar morphology is essential for the faithful segregation of chromosomes during cell division, and it is robustly maintained by multifaceted mechanisms. However, abnormally shaped spindles, such as multipolar spindles, can stochastically arise in a cell population and cause chromosome segregation errors. The physical basis of how microtubules fail in bipolarization and occasionally favor nonbipolar assembly is poorly understood. Here, using live fluorescence imaging and quantitative shape analysis in Xenopus egg extracts, we find that spindles of varied shape morphologies emerge through nonrandom, bistable self-organization paths, one leading to a bipolar and the other leading to a multipolar phenotype. The bistability defines the spindle's unique morphological growth dynamics linked to each shape phenotype and can be promoted by a locally distorted microtubule flow that arises within premature structures. We also find that bipolar and multipolar spindles are stable at the steady-state in bulk but can infrequently switch between the two phenotypes. Our microneedle-based physical manipulation further demonstrates that a transient force perturbation applied near the assembled pole can trigger the phenotypic switching, revealing the mechanical plasticity of the spindle. Together with molecular perturbation of kinesin-5 and augmin, our data propose the physical and molecular bases underlying the emergence of spindle-shape variation, which influences chromosome segregation fidelity during cell division.
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Project description:Polo-like kinases regulate many aspects of mitotic and meiotic progression from yeast to man. In early mitosis, mammalian Polo-like kinase 1 (Plk1) controls centrosome maturation, spindle assembly, and microtubule attachment to kinetochores. However, despite the essential and diverse functions of Plk1, the full range of Plk1 substrates remains to be explored. To investigate the Plk1-dependent phosphoproteome of the human mitotic spindle, we combined stable isotope labeling by amino acids in cell culture with Plk1 inactivation or depletion followed by spindle isolation and mass spectrometry. Our study identified 358 unique Plk1-dependent phosphorylation sites on spindle proteins, including novel substrates, illustrating the complexity of the Plk1-dependent signaling network. Over 100 sites were validated by in vitro phosphorylation of peptide arrays, resulting in a broadening of the Plk1 consensus motif. Collectively, our data provide a rich source of information on Plk1-dependent phosphorylation, Plk1 docking to substrates, the influence of phosphorylation on protein localization, and the functional interaction between Plk1 and Aurora A on the early mitotic spindle.
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Project description:AMBRA1 is a crucial factor for nervous system development, and its function has been mainly associated with autophagy. It has been also linked to cell proliferation control, through its ability to regulate c-Myc and D-type cyclins protein levels, thus regulating G1-S transition. However, it remains still unknown whether AMBRA1 is differentially regulated during the cell cycle, and if this pro-autophagy protein exerts a direct role in controlling mitosis too. Here we show that AMBRA1 is phosphorylated during mitosis on multiple sites by CDK1 and PLK1, two mitotic kinases. Moreover, we demonstrate that AMBRA1 phosphorylation at mitosis is required for a proper spindle function and orientation, driven by NUMA1 protein. Indeed, we show that the localization and/or dynamics of NUMA1 are strictly dependent on AMBRA1 presence, phosphorylation and binding ability. Since spindle orientation is critical for tissue morphogenesis and differentiation, our findings could account for an additional role of AMBRA1 in development and cancer ontogenesis.
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Project description:Proper orientation of the mitotic spindle defines the correct division plane and is essential for accurate cell division and development. In metazoans, an evolutionarily conserved complex comprising of NuMA/LGN/Gαi regulates proper orientation of the mitotic spindle by orchestrating cortical dynein levels during metaphase. However, the molecular mechanisms that modulate the spatiotemporal dynamics of this complex during mitosis remain elusive. Here, we report that acute inactivation of Polo-like kinase 1 (Plk1) during metaphase enriches cortical levels of dynein/NuMA/LGN and thus influences spindle orientation. We establish that this impact of Plk1 on cortical levels of dynein/NuMA/LGN is through NuMA, but not via dynein/LGN. Moreover, we reveal that Plk1 inhibition alters the dynamic behavior of NuMA at the cell cortex. We further show that Plk1 directly interacts and phosphorylates NuMA. Notably, NuMA-phosphorylation by Plk1 impacts its cortical localization, and this is needed for precise spindle orientation during metaphase. Overall, our finding connects spindle-pole pool of Plk1 with cortical NuMA and answers a long-standing puzzle about how spindle-pole Plk1 gradient dictates proper spindle orientation for error-free mitosis.
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Project description:Preneoplastic mammary tissues from human female BRCA1 mutation carriers, or Brca1-mutant mice, display unexplained abnormalities in luminal differentiation. We now study the division characteristics of human mammary cells purified from female BRCA1 mutation carriers or non-carrier donors. We show primary BRCA1 mutant/+ cells exhibit defective BRCA1 localization, high radiosensitivity and an accelerated entry into cell division, but fail to orient their cell division axis. We also analyse 15 genetically-edited BRCA1 mutant/+ human mammary cell-lines and find that cells carrying pathogenic BRCA1 mutations acquire an analogous defect in their division axis accompanied by deficient expression of features of mature luminal cells. Importantly, these alterations are independent of accumulated DNA damage, and specifically dependent on elevated PLK1 activity induced by reduced BRCA1 function. This essential PLK1-mediated role of BRCA1 in controlling the cell division axis provides insight into the phenotypes expressed during BRCA1 tumorigenesis.
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Project description:During cell division, chromosome segregation is orchestrated by the interaction of spindle microtubules with the centromere. Accurate attachment of spindle microtubules to kinetochore requires the chromosomal passenger of Aurora B kinase complex with borealin, INCENP and survivin (SUR). The current working model argues that SUR is responsible for docking Aurora B to the centromere whereas its precise role in Aurora B activation has been unclear. Here, we show that Aurora B kinase activation requires SUR priming phosphorylation at Ser20 which is catalyzed by polo-like kinase 1 (PLK1). Inhibition of PLK1 kinase activity or expression of non-phosphorylatable SUR mutant prevents Aurora B activation and correct spindle microtubule attachment. The PLK1-mediated regulation of Aurora B kinase activity was examined in real-time mitosis using fluorescence resonance energy transfer-based reporter and quantitative analysis of native Aurora B substrate phosphorylation. We reason that the PLK1-mediated priming phosphorylation is critical for orchestrating Aurora B activity in centromere which is essential for accurate chromosome segregation and faithful completion of cytokinesis.
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