Understanding kinetochore dependency pathways using vertebrate conditional knockout cell lines and quantitative proteomics
Wood, Laura Charlotte
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When cells divide, a series of events must proceed in a timely and co-ordinated manner to ensure that all DNA is replicated and partitioned equally between the two daughter cells. A central component of this process is the kinetochore, a large proteinaceous complex (>100 proteins) found within the centromere of all chromosomes. During the dynamic process of cell division, this machinery must be able to capture microtubules, promote chromosome movements towards the spindle midzone and ensure that segregration only occurs once this alignment has been successfully completed. This requires intricate mechanical and regulatory co-ordination between components and it is therefore no surprise that the structures responsible are structurally and functionally varied. It has, however, become clear that many kinetochore proteins assemble into distinct sub-complexes and despite the fact that their specific contributions are well studied, the way the many unique sub-assemblies come together to form a fully operational kinetochore is still poorly understood. Here, chromosome isolation techniques from chicken DT40 cells combined with mass spectrometry employing Stable Isotope Labeling by Amino acids in Cell culture (SILAC), is used to compare the proteome of mitotic chromosomes from different conditional kinetochore knockout (KO) cell lines. This includes components of the inner kinetochore; CENP-C, CENP-T and CENP-W, and a sub-unit of the Ndc80 complex that is important for microtubule attachment. With these large data sets I have focused on the impact these depletions have on the architecture of the holo-kinetochore by measuring the SILAC ratios of individual proteins. From these measurements I can define whether specific components are decreased, increased or unchanged in terms of their abundance on chromosomes in response to the various deletions. I have found that proteins within the same complex typically behave in a similar manner across the different KO conditions. By integrating all of the data sets, dependency networks are revealed, as well as highlighting potential novel kinetochore proteins worthy of further study.