1,721,028 research outputs found
Chromosome- and spindle-pole-derived signals generate an intrinsic code for spindle position and orientation
No full textMitotic 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.National Institute of General Medical Sciences (U.S.) (Grant GM088313
Distinct Organization and Regulation of the Outer Kinetochore KMN Network Downstream of CENP-C and CENP-T
No full textThe kinetochore provides a vital connection between chromosomes and spindle microtubules [1 and 2]. Defining the molecular architecture of the core kinetochore components is critical for understanding the mechanisms by which the kinetochore directs chromosome segregation. The KNL1/Mis12 complex/Ndc80 complex (KMN) network acts as the primary microtubule-binding interface at kinetochores [3] and provides a platform to recruit regulatory proteins [4]. Recent work found that the inner kinetochore components CENP-C and CENP-T act in parallel to recruit the KMN network to kinetochores [5, 6, 7 and 8]. However, due to the presence of these dual pathways, it has not been possible to distinguish differences in the nature of kinetochore assembly downstream of CENP-C or CENP-T. Here, we separated these pathways by targeting CENP-C and CENP-T independently to an ectopic chromosomal locus in human cells. Our work reveals that the organization of the KMN network components downstream of CENP-C and CENP-T is distinct. CENP-C recruits the Ndc80 complex through its interactions with KNL1 and the Mis12 complex. In contrast, CENP-T directly interacts with Ndc80, which in turn promotes KNL1/Mis12 complex recruitment through a separate region on CENP-T, resulting in functional relationships for KMN network localization that are inverted relative to the CENP-C pathway. We also find that distinct regulatory paradigms control the assembly of these pathways, with Aurora B kinase promoting KMN network recruitment to CENP-C and cyclin-dependent kinase (CDK) regulating KMN network recruitment to CENP-T. This work reveals unexpected complexity for the architecture and regulation of the core components of the kinetochore-microtubule interface.Leukemia & Lymphoma Society of America (Scholar Award)National Institutes of Health (U.S.) (National Institute of General Medical Sciences (U.S.). Grant GM088313)American Cancer Society (Research Scholar Grant 121776
Dynamic regulation of dynein localization revealed by small molecule inhibitors of ubiquitination enzymes
Get PDFCytoplasmic dynein is a minus-end-directed microtubule-based motor that acts at diverse subcellular sites. During mitosis, dynein localizes simultaneously to the mitotic spindle, spindle poles, kinetochores and the cell cortex. However, it is unclear what controls the relative targeting of dynein to these locations. As dynein is heavily post-translationally modified, we sought to test a role for these modifications in regulating dynein localization. We find that dynein rapidly and strongly accumulates at mitotic spindle poles following treatment with NSC697923, a small molecule that inhibits the ubiquitin E2 enzyme, Ubc13, or treatment with PYR-41, a ubiquitin E1 inhibitor. Subsets of dynein regulators such as Lis1, ZW10 and Spindly accumulate at the spindle poles, whereas others do not, suggesting that NSC697923 differentially affects specific dynein populations. We additionally find that dynein relocalization induced by NSC697923 or PYR-41 can be suppressed by simultaneous treatment with the non-selective deubiquitinase inhibitor, PR-619. However, we did not observe altered dynein localization following treatment with the selective E1 inhibitor, TAK-243. Although it is possible that off-target effects of NSC697923 and PYR-41 are responsible for the observed changes in dynein localization, the rapid relocalization upon drug treatment highlights the highly dynamic nature of dynein regulation during mitosis. Keywords:dynein, ubiquitin, NSC697923, PYR-41,
mitosis, microtubule
Polo-like Kinase 1 Licenses CENP-A Deposition at Centromeres
No full textTo ensure the stable transmission of the genome during vertebrate cell division, the mitotic spindle must attach to a single locus on each chromosome, termed the centromere. The fundamental requirement for faithful centromere inheritance is the controlled deposition of the centromere-specifying histone, CENP-A. However, the regulatory mechanisms that ensure the precise control of CENP-A deposition have proven elusive. Here, we identify polo-like kinase 1 (Plk1) as a centromere-localized regulator required to initiate CENP-A deposition in human cells. We demonstrate that faithful CENP-A deposition requires integrated signals from Plk1 and cyclin-dependent kinase (CDK), with Plk1 promoting the localization of the key CENP-A deposition factor, the Mis18 complex, and CDK inhibiting Mis18 complex assembly. By bypassing these regulated steps, we uncoupled CENP-A deposition from cell-cycle progression, resulting in mitotic defects. Thus, CENP-A deposition is controlled by a two-step regulatory paradigm comprised of Plk1 and CDK that is crucial for genomic integrity.Leukemia & Lymphoma Society of America (Scholar Award)National Institute of General Medical Sciences (U.S.) (Grant GM088313)American Cancer Society (Research Scholar Grant 121776
Large-Scale Analysis of CRISPR/Cas9 Cell-Cycle Knockouts Reveals the Diversity of p53-Dependent Responses to Cell-Cycle Defects
No full textDefining the genes that are essential for cellular proliferation is critical for understanding organismal development and identifying high-value targets for disease therapies. However, the requirements for cell-cycle progression in human cells remain incompletely understood. To elucidate the consequences of acute and chronic elimination of cell-cycle proteins, we generated and characterized inducible CRISPR/Cas9 knockout human cell lines targeting 209 genes involved in diverse cell-cycle processes. We performed single-cell microscopic analyses to systematically establish the effects of the knockouts on subcellular architecture. To define variations in cell-cycle requirements between cultured cell lines, we generated knockouts across cell lines of diverse origins. We demonstrate that p53 modulates the phenotype of specific cell-cycle defects through distinct mechanisms, depending on the defect. This work provides a resource to broadly facilitate robust and long-term depletion of cell-cycle proteins and reveals insights into the requirements for cell-cycle progression. Keywords:
kinetochore; centromere; mitosis; DNA replication; spindle; microtubule; multipolarity; p53; CRISPR/Cas
Aurora B phosphorylates spatially distinct targets to differentially regulate the kinetochore-microtubule interface
Get PDFAccurate chromosome segregation requires carefully regulated interactions between kinetochores and microtubules, but how plasticity is achieved to correct diverse attachment defects remains unclear. Here we demonstrate that Aurora B kinase phosphorylates three spatially distinct targets within the conserved outer kinetochore KNL1/Mis12 complex/Ndc80 complex (KMN) network, the key player in kinetochore-microtubule attachments. The combinatorial phosphorylation of the KMN network generates graded levels of microtubule-binding activity, with full phosphorylation severely compromising microtubule binding. Altering the phosphorylation state of each protein causes corresponding chromosome segregation defects. Importantly, the spatial distribution of these targets along the kinetochore axis leads to their differential phosphorylation in response to changes in tension and attachment state. In total, rather than generating exclusively binary changes in microtubule binding, our results suggest a mechanism for the tension-dependent fine-tuning of kinetochore-microtubule interactions.Smith Family FoundationMassachusetts Life Sciences CenterKinship Foundation. Searle Scholars ProgramNational Institute of General Medical Sciences (U.S.) (Grant number GM088313
Resonance assignments of the microtubule-binding domain of the C. elegans spindle and kinetochore-associated protein 1
Get PDFDuring mitosis, kinetochores coordinate the attachment of centromeric DNA to the dynamic plus ends of microtubules, which is hypothesized to pull sister chromatids toward opposing poles of the mitotic spindle. The outer kinetochore Ndc80 complex acts synergistically with the Ska (spindle and kinetochore-associated) complex to harness the energy of depolymerizing microtubules and power chromosome movement. The Ska complex is a hexamer consisting of two copies of the proteins Ska1, Ska2 and Ska3, respectively. The C-terminal domain of the spindle and kinetochore-associated protein 1 (Ska1) is the microtubule-binding domain of the Ska complex. We solved the solution structure of the C. elegans microtubule-binding domain (MTBD) of the protein Ska1 using NMR spectroscopy. Here, we report the resonance assignments of the MTBD of C. elegans Ska1.Austrian Science Fund (project P22170, and the doctoral school ‘‘DK Molecular Enzymology’’ (W901-B05)
Affinity Purification of Protein Complexes in C. elegans
No full textC. elegans is a powerful metazoan model system to address fundamental questions in cell and developmental biology. Research in C. elegans has traditionally focused on genetic, physiological, and cell biological approaches. However, C. elegans is also a facile system for biochemistry: worms are easy to grow in large quantities, the functionality of tagged fusion proteins can be assessed using mutants or RNAi, and the relevance of putative interaction partners can be rapidly tested in vivo. Combining biochemistry with function-based genetic and RNA interference screens can rapidly accelerate the delineation of protein networks and pathways in diverse contexts. In this chapter, we focus on two strategies to identify protein-protein interactions: single-step immunoprecipitation and tandem affinity purification. We describe methods for growth of worms in large-scale liquid culture, preparation of worm and embryo extracts, immunoprecipitation, and tandem affinity purification. In addition, we describe methods to test specificity of antibodies, strategies for optimizing starting material, and approaches to distinguish specific from non-specific interactions. Keywords: Affinity; Culture; Epitope; Immunoblotting; Immunoprecipitation; Polyclona
A Regulatory Switch Alters Chromosome Motions at the Metaphase-to-Anaphase Transition
Get PDFSummaryTo achieve chromosome segregation during mitosis, sister chromatids must undergo a dramatic change in their behavior to switch from balanced oscillations at the metaphase plate to directed poleward motion during anaphase. However, the factors that alter chromosome behavior at the metaphase-to-anaphase transition remain incompletely understood. Here, we perform time-lapse imaging to analyze anaphase chromosome dynamics in human cells. Using multiple directed biochemical, genetic, and physical perturbations, our results demonstrate that differences in the global phosphorylation states between metaphase and anaphase are the major determinant of chromosome motion dynamics. Indeed, causing a mitotic phosphorylation state to persist into anaphase produces dramatic metaphase-like oscillations. These induced oscillations depend on both kinetochore-derived and polar ejection forces that oppose poleward motion. Thus, our analysis of anaphase chromosome motion reveals that dephosphorylation of multiple mitotic substrates is required to suppress metaphase chromosome oscillatory motions and achieve directed poleward motion for successful chromosome segregation
The CENP-L-N Complex Forms a Critical Node in an Integrated Meshwork of Interactions at the Centromere-Kinetochore Interface
No full textDuring mitosis, the macromolecular kinetochore complex assembles on the centromere to orchestrate chromosome segregation. The properties and architecture of the 16-subunit Constitutive Centromere-Associated Network (CCAN) that allow it to build a robust platform for kinetochore assembly are poorly understood. Here, we use inducible CRISPR knockouts and biochemical reconstitutions to define the interactions between the human CCAN proteins. We find that the CCAN does not assemble as a linear hierarchy, and instead, each sub-complex requires multiple non-redundant interactions for its localization to centromeres and the structural integrity of the overall assembly. We demonstrate that the CENP-L-N complex plays a crucial role at the core of this assembly through interactions with CENP-C and CENP-H-I-K-M. Finally, we show that the CCAN is remodeled over the cell cycle such that sub-complexes depend on their interactions differentially. Thus, an interdependent meshwork within the CCAN underlies the centromere specificity and stability of the kinetochore.United States. National Institutes of Health (GM088313)United States. National Institutes of Health (GM108718)American Cancer Society (121776
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