Kinetochore Function in Vertebrate Cells

描述（由申请人提供）：本研究的长期目标是使用细胞生物学和生物化学技术来了解脊椎动物细胞中运动编排-微管（MT）界面的分子性质。为了使染色体在有丝分裂过程中正确分离，它们必须牢固地附着在动态生长和缩短的MT正末端上。它们通过动粒这样做，动粒是一种在有丝分裂染色体的初级收缩处构建的大型蛋白质组合。这种连接可能是复杂的，因为它必须是坚固的以抵抗染色体双取向的力，而又是灵活的以允许结合的MT+末端的流体生长和缩短。Kinetochores还必须调节这种连接的强度，因为必须释放不正确连接的MT，并且必须稳定那些正确连接的MT。在真核细胞中产生稳定的着丝粒-MT附着需要着丝粒相关的NDC 80复合物，但是该复合物如何构建和调节纺锤体MT的正末端的结合位点仍然是有丝分裂领域中最重要的未回答的问题之一。这个建议的目的是回答以下问题：NDC 80复合物的哪些结构域构成了与MT加端的直接接触点？复合物是否在一个“套管”中拴在一起，以便MT加端插入？如果是这样的话，这些复合体是如何结合在一起的？弱结合的NDC 80复合物是否沿着MT晶格扩散以促进染色体的聚集？或者，动粒和MT之间的接触点是否由高亲和力结合相互作用组成，需要持续释放和重新结合以驱动染色体运动？极光B激酶对NDC 80复合物的磷酸化是否调节了激动素-MT结合强度？什么磷酸酶抵消激酶活性，以确保kinetochore-MT稳定？这些问题将使用以下方法来回答：首先，将在PtK 1细胞中开发NDC 80复合物组分的基因沉默/拯救策略，以明确评估表达突变NDC 80复合物的细胞中的着丝粒-MT附着表型。其次，将进行使用NDC 80突变体的生物化学和生物物理实验，以机械地理解NDC 80复合物如何结合到MT并沿着MT易位，以及复合物的哪些特征负责物理耦合加端MT动力学以迫使产生染色体运动。第三，蛋白质-蛋白质相互作用将映射在kinetochore-MT接口的第一次在体内通过kinetochore-specific荧光相互作用测定的发展。这些研究将为有丝分裂领域中一系列尚未解决的关键问题提供答案，所开发的技术将适用于有丝分裂蛋白和过程的进一步研究。相关性：通过有丝分裂与不正确的着丝粒-MT附着的进展是非整倍体的主要原因，其与人类肿瘤的起始和进展以及出生缺陷的形成有关。因此，了解细胞如何产生和调节kinetochore-MT附件对人类健康至关重要。 公共卫生相关性：在有丝分裂过程中，染色体必须正确分离，以防止形成非整倍体细胞，其中含有不正确的染色体数目。这对人类健康至关重要，因为众所周知，非整倍体会导致出生缺陷，并被认为是肿瘤发生和发展的致病因素。了解细胞将其染色体正确地平均分成两个子细胞的机制对于了解导致非整倍体细胞出现的途径至关重要。

DESCRIPTION (provided by applicant): The long-term objective of this research is to use cell biological and biochemical techniques to understand the molecular nature of the kinetochore-microtubule (MT) interface in vertebrate cells. For chromosomes to properly segregate during mitosis, they must firmly attach to dynamically growing and shortening MT plus- ends. They do so via the kinetochore, a large protein assemblage built at the primary constriction of mitotic chromosomes. This linkage is likely complicated, as it must be robust to resist the forces of chromosome bi- orientation, yet flexible to allow for fluid growth and shortening of bound MT plus-ends. Kinetochores must also regulate the strength of this attachment, since incorrectly attached MTs must be released, and those that are correctly attached must be stabilized. The kinetochore-associated NDC80 complex is required for generating stable kinetochore-MT attachments in eukaryotic cells, but how this complex builds and regulates binding sites for the plus-ends of spindle MTs remains one of the most important unanswered questions in the mitosis field. This proposal is designed to answer the following questions: What domains of the NDC80 complex make up the direct points of contact with MT plus-ends? Are the complexes tethered together in a "sleeve" for the MT plus-ends to insert into? If so, how are the complexes tethered together? Do weakly-associated NDC80 complexes diffuse along the MT lattice to facilitate chromosome congression? Or, alternatively, are the points of contact between kinetochores and MTs made up of high affinity binding interactions that require continual release and re-binding to drive chromosome movement? Is kinetochore-MT binding strength regulated through phosphorylation of the NDC80 complex by Aurora B kinase? What phosphatase counter-acts the kinase activity to ensure kinetochore-MT stabilization? These questions will be answered using the following approaches: First, a gene silence/rescue strategy for NDC80 complex components will be developed in PtK1 cells to unambiguously assess kinetochore-MT attachment phenotypes in cells expressing mutant NDC80 complexes. Second, biochemical and biophysical experiments using NDC80 mutants will be carried out to understand mechanistically how NDC80 complexes bind to and translocate along MTs, and which features of the complex are responsible for physically coupling plus-end MT dynamics to force production for chromosome movement. Third, protein-protein interactions will be mapped at the kinetochore-MT interface for the first time in vivo through the development of kinetochore-specific fluorescence interaction assays. These studies will provide answers to a critical set of unresolved questions in the mitosis field, and the developed techniques will be applicable to further study of mitotic proteins and processes. Relevance: Progression through mitosis with incorrect kinetochore-MT attachments is a major cause of aneuploidy, which has been linked to the initiation and progression of human tumors and also to the formation of birth defects. Thus, understanding how cells generate and regulate kinetochore-MT attachments is of critical importance to human health. PUBLIC HEALTH RELEVANCE: During mitosis, chromosomes must segregate correctly in order to prevent the formation of aneuploid cells, which contain an incorrect number of chromosomes. This is critical for human health, as aneuploidy is well-known for causing birth defects and has been implicated as a causative factor in the initiation and progression of tumors. Understanding the mechanisms that cells use to correctly divide their chromosomes equally into two daughter cells is essential to understand the pathways leading to the emergence of aneuploid cells.