Molecular mechanisms controlling kinetochore-microtubule attachments during mitosis

有丝分裂过程中控制动粒-微管附着的分子机制

• 批准号: 10294230
• 负责人: Dileep Varma
• 金额: $ 33.6万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-10 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10294230
• 关键词: Aneuploidy; Binding; Binding Proteins; Binding Sites; Biogenesis; Biological Models; Cells; Cellular Structures; Centromere; Chromosome Segregation; Chromosomes; Complex; Data; Defect; Distal; Dynein ATPase; Goals; Human; Individual; Kinetochores; Lateral; Lead; Mediating; Metaphase; Microtubule Stabilization; Microtubule-Associated Proteins; Microtubules; Mitosis; Mitotic; Mitotic spindle; Molecular; Motor; Multiprotein Complexes; N-terminal; Pathway interactions; Phosphorylation; Phosphotransferases; Process; Prometaphase; Proteins; Publishing; Regulation; Replication Licensing; Role; Sister; Site; Slide; Testing; Work; aurora B kinase; chromosome conformation capture; chromosome missegregation; daughter cell; novel; prevent; protein function; recruit; segregation; synergism; tumor progression

Project Summary: Mitotic cells assemble two key cellular structures to segregate the chromosomes equally between the two daughter cells - the mitotic spindle and the kinetochores. Kinetochores are multi-protein complexes that form at the centromeres of the chromosomes during mitosis and serve as attachment sites for microtubules [MT(s)] of the mitotic spindle. The kinetochore-microtubule (kMT) interface generates force that drives chromosome alignment and segregation. The current focus of the Varma lab is on understanding the molecular mechanisms involved in kMT attachments and their contribution to accurate chromosome segregation. During early mitotic prometaphase, kinetochores initially attach to the MT lattice laterally. These lateral attachments are subsequently converted into end-on attachments when sister kinetochores become stably attached to the plus-ends of spindle MTs in metaphase. Studies have shown that the initial capture and lateral sliding of kinetochores on MTs is driven by dynein, a minus-end-directed motor. The end-on kinetochore- microtubule (kMT) attachment formation and its stabilization is mediated by the MT-binding kinetochore complex, Ndc80. Our 1st major goal is to determine how the kinetochore complexes required for these two alternate modes of MT attachment coordinate to produce dynamic kMT attachments required for proper chromosome alignment. Our recent work has provided evidence for an antagonistic relationship between dynein and the Ndc80 complexes in humans during mitosis. Our unpublished results suggest that dynein and the Ndc80 complex synergize for efficient chromosome capture and for stabilizing kMT attachments during metaphase, but the mechanism for this coordination is unclear. The centromere-distal region of the Ndc80 complex at the N-terminal domain of the Hec1 subunit has been identified as the MT-binding site required to stabilize kMT attachments. Phosphorylation of this region by Aurora B kinase negatively regulates the strength of kMT attachments. Our work has discovered that in addition to the N-terminal domain, the more internal loop domain has a major role in attachment. The attachment requires the loop domain-mediated kinetochore recruitment of the replication licensing protein Cdt1, which we find is a novel MT-binding protein at kinetochores. Our work also demonstrates that the binding of Cdt1 to MTs is negatively regulated by Aurora B. Our unpublished studies demonstrate that Cdt1 synergizes with another MT-associated protein (MAP) at kinetochores, the Ska complex that has been shown to promote efficient binding of the Ndc80 complex to kMTs. Our 2nd major goal is to determine how different kinetochore MAPs mediate robust interaction between the Ndc80 complex and MTs for the stabilization of kMT attachments during metaphase to drive accurate chromosome segregation. In the long term, our lab aims to identify novel mechanisms controlling kMT attachments and the pathways that regulate this process, while also establishing novel model systems and approaches to study these processes.

项目概述：有丝分裂细胞组装两个关键的细胞结构，以平等地分离染色体 有丝分裂纺锤体和动粒之间的联系。动粒是多蛋白质的 在有丝分裂过程中在染色体的着丝粒处形成的复合物，作为 有丝分裂纺锤体的微管[MT（s）]。运动舞蹈-微管（kMT）界面产生力， 驱动染色体排列和分离。Varma实验室目前的重点是了解 kMT附着的分子机制及其对染色体精确分离的贡献。 在早期有丝分裂前中期，动粒最初附着到MT晶格横向。这些横向 当姐妹动粒稳定时， 在中期附着在纺锤体MT的正端。研究表明，初始捕获和横向 动粒在MT上的滑动由动力蛋白（负端定向马达）驱动。端部着丝粒- 微管（kMT）附着的形成及其稳定化由MT结合动粒复合物介导， Ndc80。我们的第一个主要目标是确定这两个交替所需的动粒复合体如何 MT连接模式协调，以产生适当的所需的动态kMT连接 染色体排列我们最近的工作提供了证据， 动力蛋白和Ndc 80复合物在人类有丝分裂过程中。我们未发表的结果表明，动力蛋白和 Ndc 80复合物协同有效地捕获染色体和稳定kMT附着 中期，但这种协调的机制尚不清楚。 Ndc 80复合物的着丝粒远端区域位于Hec 1亚基的N-末端结构域， 已被确定为稳定kMT附件所需的MT结合位点。这一区域的磷酸化， 极光B激酶负调节kMT附着的强度。我们的研究还发现， 对于N-末端结构域，更内环结构域在连接中具有主要作用。附接 需要复制许可蛋白Cdt 1的环结构域介导的动粒募集，我们 find是一种新的位于着丝粒的MT结合蛋白。我们的工作还表明Cdt 1与MT的结合 由极光B负调控。我们未发表的研究表明，Cdt 1与另一个 着丝粒处的MT相关蛋白（MAP），已显示促进有效结合的Ska复合物 的Ndc 80络合物的KMT。我们的第二个主要目标是确定不同的动粒MAP如何介导 Ndc 80复合物和MT之间的强大相互作用，用于稳定kMT附件， 中期分裂以驱动精确的染色体分离。从长远来看，我们的实验室旨在确定新的 控制kMT附着的机制和调节这一过程的途径，同时也建立 新的模型系统和方法来研究这些过程。