IDENTIFICATION OF INTERACTING PROTEINS OF SPINDLY

Spindly 相互作用蛋白的鉴定

• 批准号: 8171402
• 负责人: Arshad Desai
• 金额: $ 0.24万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171402
• 关键词: Aneuploidy; Binding; Biology; Characteristics; Chromosome Segregation; Chromosomes; Complex; Computer Retrieval of Information on Scientific Projects Database; Cultured Cells; Dynein ATPase; Ensure; Funding; Goals; Grant; Immunoprecipitation; Institution; Kinetochores; Laboratories; Mass Spectrum Analysis; Microtubules; Mitosis; Mitotic; Mitotic Spindle Apparatus; Nocodazole; Proteins; Recruitment Activity; Research; Research Personnel; Resources; Role; Source; Transgenes; United States National Institutes of Health; Work; daughter cell; dynactin; human tissue; insight; mutant; neoplastic cell; tissue culture

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The kinetochore facilitates chromosome segregation in mitosis by interacting with microtubules of the mitotic spindle apparatus. Proper functioning of the kinetochore is essential to avoid uneven distribution of chromosomes to daughter cells, which leads to aneuploidy, a common characteristic of tumors cells. The dynein/dynactin complex localizes to the kinetochore in mitosis and has been implicated in both kinetochore-microtubule attachment and in the silencing of the spindle checkpoint, which ensures that mitotic exit does not occur in the presence of unattached kinetochores. Work in our laboratory has identified an outer kinetochore component, called Spindly, which has a key role in recruiting the dynein/dynactin complex to unattached kinetochores, but the molecular interactions involved are not known. Our goal is to gain insight into the mechanism of dynein/dynactin recruitment to kinetochores by identifying the dynein/dynactin subunits that interact with Spindly. We will use mass spectrometry after immunoprecipitation of Myc-tagged Spindly transgenes from nocodazole-arrested human tissue culture cells to identify candidate Spindly binding partners. As a negative control, we will use a Spindly point mutant which is unable to recruit dynein/dynactin to kinetochores, but whose own kinetochore recruitment is unaffected. In addition to dynein/dynactin subunits, we expect the mass spectrometric analysis to reveal the components that recruit Spindly itself to kinetochores. Mass spectrometry analysis is therefore crucial to answer fundamental questions in kinetochore biology.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 着丝粒通过与有丝分裂纺锤体的微管相互作用来促进有丝分裂中的染色体分离。 着丝粒的正常功能对于避免子细胞染色体分布不均至关重要，从而导致非整倍性，这是肿瘤细胞的常见特征。 动力蛋白/动力蛋白复合物定位于有丝分裂中的着丝粒，并与着丝粒-微管附着和纺锤体检查点沉默有关，这确保在存在未附着的着丝粒的情况下不会发生有丝分裂退出。 我们实验室的工作已经确定了一种称为 Spindly 的外部着丝粒成分，它在将动力蛋白/动力蛋白复合物募集到未附着的着丝粒方面发挥着关键作用，但所涉及的分子相互作用尚不清楚。 我们的目标是通过识别与 Spindly 相互作用的动力蛋白/动力蛋白亚基，深入了解动力蛋白/动力蛋白招募到着丝粒的机制。 我们将在对来自诺考达唑捕获的人体组织培养细胞的 Myc 标记的 Spindly 转基因进行免疫沉淀后使用质谱法来鉴定候选 Spindly 结合伴侣。 作为阴性对照，我们将使用 Spindly 点突变体，该突变体无法将动力蛋白/动力蛋白募集到动粒，但其自身的动粒募集不受影响。 除了动力蛋白/动力蛋白亚基之外，我们期望质谱分析能够揭示将 Spindly 自身招募到着丝粒的成分。 因此，质谱分析对于回答动粒生物学的基本问题至关重要。