Kinetochore Function in Vertebrate Cells

脊椎动物细胞的动粒功能

• 批准号: 8054166
• 负责人: Jennifer G DeLuca
• 金额: $ 32.15万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8054166
• 关键词: Affect; Affinity; Amino Acids; Aneuploid Cells; Aneuploidy; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Cells; Centromere; Charge; Chromosomes; Complex; Congenital Abnormality; Coupling; Cultured Cells; Development; Diffuse; Diffusion; Ensure; Eukaryotic Cell; Fluorescence; Fluorescence Resonance Energy Transfer; Gene Silencing; Goals; Growth; Health; Human; Kinetochores; Length; Link; Liquid substance; Maps; Measures; Mediating; Microtubule Stabilization; Microtubules; Mitosis; Mitotic; Mitotic Chromosome; Molecular; N-terminal; Nature; Pathway interactions; Phenotype; Phospho-Specific Antibodies; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Plus End of the Microtubule; Process; Production; Protein-Protein Interaction Map; Proteins; Research; Role; Site; Tail; Techniques; Testing; Time; aurora B kinase; base; chromosome movement; constriction; daughter cell; design; flexibility; in vitro Assay; in vivo; molecular recognition; mutant; prevent; protein protein interaction; public health relevance; research study; tumor; tumor progression

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.

描述(申请人提供)：这项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动粒-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+末端上。它们通过着丝粒完成这一过程，这是一种建立在有丝分裂染色体初级收缩处的大型蛋白质组合。这种连锁可能很复杂，因为它必须强大地抵抗染色体双向的力量，但又必须灵活，以允许液体生长和缩短结合的MT+末端。动圈还必须调节该附件的强度，因为错误连接的MTS必须松开，正确连接的MTS必须稳定。动粒相关的NDC80复合体是在真核细胞中产生稳定的动粒-MT连接所必需的，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中最重要的悬而未决的问题之一。这项建议旨在回答以下问题：NDC80复合体的哪些结构域构成与MT+末端的直接接触点？复合体是否被套在一起，以便MT加端插入？如果是这样，这些复合体是如何捆绑在一起的？弱相关的NDC80复合体是否沿MT晶格扩散以促进染色体聚集？或者，动点和MT之间的接触点是由高亲和力的结合相互作用组成的，需要持续释放和重新结合来驱动染色体运动吗？动粒-MT结合强度是通过极光B激酶对NDC80复合体的磷酸化来调节的吗？是什么磷酸酶抑制了激酶活性，以确保动粒-MT的稳定？这些问题将通过以下方法得到解答：首先，将在PtK1细胞中开发一种针对NDC80复杂成分的基因沉默/挽救策略，以明确地评估表达突变的NDC80复杂成分的细胞中的动粒-MT附着表型。其次，将使用NDC80突变体进行生化和生物物理实验，以从力学上了解NDC80复合体如何与MT结合并沿MT移位，以及该复合体的哪些特征负责物理耦合正端MT动力学以迫使生产用于染色体运动。第三，通过开发动粒特异的荧光相互作用分析方法，将首次在体内绘制动粒-MT界面上的蛋白质-蛋白质相互作用图谱。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进一步研究有丝分裂蛋白和过程。相关性：有丝分裂的进展与不正确的动粒-MT连接是非整倍体的主要原因，非整倍体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动粒-MT连接对人类健康至关重要。 公共卫生相关性：在有丝分裂期间，染色体必须正确分离，以防止非整倍体细胞的形成，因为非整倍体细胞含有错误的染色体数量。这对人类健康至关重要，因为非整倍体是众所周知的导致出生缺陷的原因，并被认为是肿瘤发生和发展的致病因素。了解细胞用来正确地将其染色体等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是至关重要的。