IDENTIFICATION OF INTERACTING PROTEINS OF SPINDLY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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。动粒的正常功能对于避免染色体不均匀地分布到子代细胞是至关重要的，这会导致非整倍体，这是肿瘤细胞的共同特征。在有丝分裂中，动力蛋白/动力蛋白复合体定位于动粒，并参与动粒-微管连接和纺锤体检查点的沉默，这确保了有丝分裂退出时不会出现未附着的动粒。我们实验室的工作已经发现了一种外部动粒成分，称为SPINDLY，它在将动力蛋白/动力蛋白复合体招募到独立的动粒中起关键作用，但其中涉及的分子相互作用尚不清楚。我们的目标是通过鉴定与Spin相互作用的dynein/dynactin亚基，深入了解dynein/dynactin募集到运动中枢的机制。我们将使用免疫沉淀后的质谱仪，从诺康唑抑制的人类组织培养细胞中提取Myc标记的纺锤形转基因，以确定候选的纺锤形结合伙伴。作为阴性对照，我们将使用一个旋转点突变体，它不能将dynein/dynactin招募到动粒，但其自身的动粒招募不受影响。除了动力蛋白/动力蛋白亚基，我们预计质谱分析将揭示纺锤状自我招募到动粒的成分。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。

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.