Ledesma Fernandez, ME; (2016) Functional Genomics Characterisation of the Genetic Pathways that Control Kinetochore Function. Doctoral thesis , (UCL) University College London.

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Abstract

Kinetochores serve both a structural role linking chromosomes to the mitotic spindle and a regulatory role, controlling the timing of mitosis via the spindle assembly checkpoint. Defects in kinetochore function can lead to aneuploidy, a hallmark of cancer cells. To understand how cells regulate the segregation of their chromosomes during cell division it is important to understand which genes regulate kinetochores function. I have examined the distribution of the outer kinetochore complex DAM1/DASH complex in the budding yeast Saccharomyces cerevisiae using fluorescence imaging. I modified the endogenous DAD4 and SPC42 alleles to introduce the gene encoding yellow fluorescent protein (YFP) and red fluorescent protein (RFP) respectively. I have used the synthetic genetic array technology to transfer these two alleles into all of the ~6000 non-essential gene deletions and essential temperature sensitive mutants in yeast. Fluorescence microscopy of these mutant strains has allowed me to examine quantitative and qualitative kinetochore phenotypes. First, I assessed the levels of YFP fluorescence intensity at the kinetochore as a surrogate for protein concentration to determine changes in DAM1/DASH complex homeostasis. Then, I annotated abnormal distributions of YFP fluorescence by visualisation. I have identified a number of mutants that alter Dad4 homeostasis. For example, ~3% of the non-essential deletions and ~27% of essential conditional alleles produce either an increase or a decrease in Dad4 intensity. I have also identified mutants that cause a mislocalisation of Dad4, some of which represent mitotic spindle defects. Finally, I have investigated the mechanism underlying the phenotype caused by a kinase from the cell wall integrity pathway, Pkc1, and its connection to the kinetochore via the microtubule and actin cytoskeleton. This study highlights the importance of chromatin remodeling complexes, RNA processing enzymes and a number of protein kinases in maintaining kinetochore homeostasis.

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