Molecular mechanisms controlling kinetochore-microtubule attachments during mitosis

项目摘要：有丝分裂细胞组装两个关键的细胞结构，以平均分离染色体 在两个子细胞之间--有丝分裂纺锤体和着丝点。动点是由多种蛋白质组成的 在有丝分裂过程中在染色体着丝粒处形成的复合体，用作 有丝分裂纺锤体的微管[MT(S)]。动粒-微管(KMT)界面产生的力 推动染色体配对和分离。Varma实验室目前的重点是了解 KMT附着的分子机制及其对精确染色体分离的贡献。 在有丝分裂早中期，动点最初横向附着在MT晶格上。这些横向的 当姐妹动点变得稳定时，附件随后被转换为端接附件 中期着生于纺锤体MTS的正端。研究表明，初始捕获和横向捕获 运动中枢在MTS上的滑动是由动力蛋白驱动的，动力蛋白是一种负端定向马达。最后--在动粒上-- 微管(KMT)附着的形成及其稳定性是由MT结合的动粒复合体介导的， 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 MT依恋坐标生成适当所需的动态国民党依恋的方式 染色体比对。我们最近的研究为两国之间的对立关系提供了证据 人类有丝分裂过程中的动力蛋白和NDC80复合体。我们未发表的结果表明，动力蛋白和 Ndc80复合体具有协同作用，可有效捕获染色体并稳定KMT在 中期，但这种协调的机制尚不清楚。 Ndc80复合体的着丝粒-远端区域位于Hec1亚基的N-末端结构域 被确定为稳定国民党依恋所需的MT结合部位。该区域的磷酸化通过 极光B激酶负性调节KMT依附的强度。我们的工作发现，除了 对于N-末端结构域，更多的内部环结构域在附着中起主要作用。附属品 需要环域介导的动粒招募复制许可蛋白CDT1，我们 Find是一种新的动态平衡MT结合蛋白。我们的工作还证明了CDT1与MTS的结合 是受Aurora B负调控的。我们未发表的研究表明，CDT1与另一个 运动中枢的MT相关蛋白(MAP)，已被证明促进有效结合的Ska复合体 从Ndc80复合体到kmts。我们的第二个主要目标是确定不同的动粒图谱如何调节 Ndc80复合体和MTS之间的强健相互作用对国民党附着体的稳定 中期，以驱动准确的染色体分离。从长远来看，我们实验室的目标是识别小说 控制国民党依恋的机制和调控这一过程的途径，同时也建立了 研究这些过程的新模型系统和方法。

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.