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化技术来了解脊椎动物细胞中着丝点-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正端上。它们通过着丝点来完成这一过程，着丝点是在有丝分裂染色体的初级收缩处形成的一种大型蛋白质组合。这种连锁可能是复杂的，因为它必须是坚固的，以抵抗染色体双向性的力量，但灵活的，以允许流体生长和缩短结合的MT +端。动着点也必须调节这种连接的强度，因为错误连接的mt必须被释放，而正确连接的mt必须稳定。着丝酶相关的NDC80复合体是真核细胞中产生稳定的着丝酶- mt附着体所必需的，但该复合体如何构建和调节纺锤体mt正端的结合位点仍然是有丝分裂领域最重要的未解之谜之一。本提案旨在回答以下问题：NDC80复合体的哪些结构域构成了与MT +端直接接触的点？这些复合物是否系在一个“套筒”中供MT +端插入？如果是这样，这些复合体是如何连接在一起的？弱相关的NDC80复合体是否沿着MT晶格扩散以促进染色体聚集？或者，着丝点和mt之间的接触点是由高亲和力结合相互作用组成的，需要不断释放和重新结合来驱动染色体运动？Aurora B激酶是否通过NDC80复合物的磷酸化来调节着丝酶- mt结合强度？什么磷酸酶抑制激酶活性以确保着丝酶- mt的稳定？这些问题将通过以下方法得到解答：首先，将在PtK1细胞中开发NDC80复合物组分的基因沉默/拯救策略，以明确评估表达突变NDC80复合物的细胞中的着丝酶- mt附着表型。其次，利用NDC80突变体进行生化和生物物理实验，以了解NDC80复合物如何结合并沿MT易位的机制，以及该复合物的哪些特征负责物理耦合正端MT动力学以迫使染色体运动产生。第三，通过开发着丝点特异性荧光相互作用测定，将首次在体内在着丝点- mt界面上绘制蛋白质-蛋白质相互作用。这些研究将为有丝分裂领域一系列尚未解决的关键问题提供答案，并且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：有丝分裂过程中不正确的着丝酶- mt附着体是非整倍体的主要原因，它与人类肿瘤的发生和发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着丝酶- mt附着体对人类健康至关重要。

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.