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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 在有丝分裂中，动粒通过与有丝分裂纺锤体中的微管相互作用促进染色体分离。 着丝粒的正常功能对于避免染色体向子细胞的不均匀分布是至关重要的，这导致非整倍性，这是肿瘤细胞的共同特征。 动力蛋白/动力肌动蛋白复合物定位于有丝分裂中的动粒，并与动粒-微管附着和纺锤体检查点的沉默有关，这确保了有丝分裂退出在未附着的动粒存在下不会发生。 我们实验室的工作已经确定了一个外部动粒组分，称为纺锤体，它在招募动力蛋白/动力蛋白复合物到独立的动粒中起关键作用，但所涉及的分子相互作用尚不清楚。 我们的目标是通过鉴定与Spindly相互作用的动力蛋白/动力肌动蛋白亚基来深入了解动力蛋白/动力肌动蛋白招募到着丝粒的机制。 我们将在免疫沉淀来自诺考达唑抑制的人类组织培养细胞的Myc标记的Spindly转基因后使用质谱法来鉴定候选Spindly结合伴侣。 作为阴性对照，我们将使用纺锤形点突变体，其不能将动力蛋白/动力肌动蛋白募集到动粒，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，我们希望质谱分析揭示的组件，招聘纺锤体本身的着丝粒。 因此，质谱分析对于回答动粒生物学中的基本问题至关重要。