IDENTIFICATION OF INTERACTING PROTEINS OF SPINDLY

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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.