Aneuploidy, the gain or loss of one or more chromosomes from a diploid genome, is the most frequent manifestation of genomic instability in human cancer cells. Progress made in our laboratory and elsewhere dunng the past five years has brought us much closer to understanding the molecular basis of mitosis and chromosome segregation in eukaryotic cells during normal and neoplastic cell conditions. Our proposed research will continue to focus on the centromere-kinetochore complex of mitotic chromosomes with aims designed to advance our knowledge on the structure and regulation of this vital locus.
The specific aims will be to (1) determine the higher-order structure and folding of chromatin within centromere domains of human chromosomes, (2) investigate the molecular dynamics and structural relationships of centromeric chromatin and associated proteins using live cell imaging and quantitative photobleaching, (3) define and characterize the molecular determinants (genetic/epigenetic) that specify the site for assembly of the centromerekinetochore complex on mammalian chromosomes, and (4) to analyze centromere-kinetochore components utilizing conventional EM and FlAsH epitope tagging and detection in both living and fixed cells by light and electron microscopy. The experimental plan utilizing both human and non-human mammalian cells is designed to better characterize kinetochore-forniing chromatin and the higher-order structure of the centromere-kinetochore complex. The proposed research plan will represent new experiments along with a continuation of innovative studies ongoing in the Brinkley laboratory. Additional experiments and novel methodology will be introduced such as Fluorescence Recovery After Photobleaching (FRAP) to track the movement and dynamics of bioluminescent proteins in live cells and new FIAsH-epitope technology that enables the extension of experiments from light microscopy to the EM level in the same cells with uncompromised ultrastructure. This project will provide vital information on the centromere-kinetochore complex required for understanding the molecular basis for normal chromosome segregation and defects in chromosome partitioning that lead to aneuploidy as seen in most human tumors.
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