The goal of this proposal is to understand the mechanism of anaphase chromosome separation at a molecular and structural level. We use two model organisms; diatoms, where it is possible to isolate highly ordered, spindles that are capable of undergoing spindle elongation in vitro, and fission yeast, a genetically tractable organism that uses both chromosome-to-pole movement (anaphase A) and spindle elongation (anaphase B) to segregate its chromosomes. We focus on the role of kinesin-related proteins (KRPs) during mitosis since these mechanochemical enzymes help generate the forces responsible for moving chromosomes. In diatoms, we identified a KRP, DSK1, that is involved in spindle elongation. We will characterize accessory proteins that may modify DSK1 function and will determine whether other KRPs are also involved in anaphase B in diatoms. In Schizosaccharomyces pombe we have characterized a KRP, pk11, that is involved in spindle organization. Our top priority will be to define the function of pk11 in the spindle and identify what proteins it interacts with. We will systematically identify other KRPs and analyze their contribution to chromosome segregation. To aid in the characterization of mutants, we will describe the kinetics of chromosome movement in living cells, using high resolution DIC and Green Fluorescent Protein labeled spindle proteins. Since pk11 may be functionally redundant with other KRPs we will explore this possibility cytologically, biochemically, and genetically with a synthetic lethal screen. Finally, we will use a genetic screen based on minichromosome segregation to obtain new mutants that affect spindle function, focusing on mutants that may be deficient in chromosome-to-pole movement. We will also develop an in vitro assay for studying anaphase A that can be used to help characterize these new mutants. The principles learned studying mitosis in these organisms will apply to all eukaryotic cells since the organization and function of the spindle is highly conserved. Regulation of mitosis is a topic of major medical interest since uncontrolled cell division is at the heart of the cancer problem and inaccurate chromosomal segregation (aneuploidy) is causal in several congenital malformations and a major cause of premature termination of pregnancy. An improved understanding of mitosis should eventually lead to new approaches to cancer chemotherapy and to control of abnormal chromosome segregation.
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