Chromosome Movement in Prometaphase

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DESCRIPTION (APPLICANT'S ABSTRACT): The goals of this project are to understand how mitotic chromosomes move, how they align at metaphase, and how microtubule attachment and physical tension at the kinetochores of chromosomes controls the timing of chromosome segregation. Defects in the regulation of chromosome movement and segregation lead to chromosome instability, the gain or loss of chromosomes during cell division. Chromosome instability in the formation of human gametes leads to birth defects such as Down's syndrome. Chromosome instability in the division of somatic cells is an important contributing factor to malignancy in cancer. Paradoxically, chromosome instability may also be critical for the effectiveness of certain drugs used in cancer chemotherapy. Important steps of cell division are regulated by the conjugation of the protein ubiquitin to other proteins. Many of the component enzymes involved in ubiquitin conjugation have been identified but the molecular pathways by which they are temporally regulated in the cell cycle or spatially regulated within different parts of the cell are unknown. The aims of this project are to identify new regulator proteins of these systems and to determine how temporal and spatial control of the ubiquitin conjugation machinery takes place within cell division. These studies use a combination of molecular, biochemical, and advanced microscopic techniques to track the biochemical changes and protein interactions of the ubiquitin conjugation machinery that are important in the regulation of mitosis in mammalian cells. In vitro model systems of several types using cell extracts, detergent-lysed cells or synthesized components are also used. These model systems partially reproduce events within the living cells but provide for better opportunities to control and manipulate the molecular environment. One set of objectives seeks to map the steps by which chromosome movements themselves are controlled by the ubiquitin conjugation machinery during prometaphase in early mitosis. Another set is pointed toward understanding the regulation of the cell cycle checkpoint pathway that functions in mitosis in controlling ubiquitin conjugation to prevent premature chromosome segregation and chromosome instability.