Kinesin like Motors in Mitotic Movements

University Of Colorado At Boulder, Boulder CO

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Abstract

DESCRIPTION: (provided by applicant) We will study the mechanisms of chromosome motion in the fission yeast, Schizosaccharomyces pombe. S. pombe has several virtues for mitotic research. It contains only three, easily visualized chromosomes. As in many other eukaryotes, each of its centromeres is large, comprising many l0s of kb of DNA, and each binds 2-4 microtubules (MTs). Its mitotic cycle includes prometaphase congression to the spindle equator and both anaphase A and B. Of the 10 MT-associated motors now known in this organism, we have selected 3 kinesin-like proteins (KLPs) for detailed study. One of these (Klp2p) localizes to spots on mitotic chromosomes that are probably kinetochores, and two of them (Klp5p and 6p) are essential for normal prometaphase chromosome motion and spindle length control. Our plans include four specific aims. Aim 1) To characterize the motility and domain structure of these KLPs, using a combination of molecular, biochemical, and cell biological approaches. The results should clarify motor directionality, speed, and enzymology. They should also identify any effects of motor action on MT dynamics, characterize the protein domains responsible for cargo binding and localization, and isolate additional alleles of each motor. Aim 2) To identify genes and their products that interact with these motors, both to bind them to cargo and to control their activity in vivo. Aim 3) To study the structure of kinetochores in fission yeast, using electron microscope tomography and EM immunolocalization of known kinetochore components. Aim 4) To take advantage of a recently developed method for isolating fission yeast chromosomes and characterize kinetochore-MT interaction in vitro. We will employ already-useful systems for the assembly of MTs into organized arrays and use laser tweezers to manipulate the kinetochore-MT connection. Chromosomes prepared from strains lacking specific kinetochore components and/or with chemistry modified in vitro (e.g., through the action of relevant phosphatases or kinases), will be used to identify the molecules necessary for kinetochore-MT interaction and the ways in which mechanical activities at this interface (e.g., tension) modify the physiology of the kinetochore