KINESIN LIKE MOTORS IN MITOTIC MOVEMENTS

University Of Colorado At Boulder, Boulder CO

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Abstract

We are working to understand the mechanisms of chromosome motion during mitosis. Several motor enzymes that interact with microtubules (MTs) have recently been implicated in spindle action. It is now clear that a single cell contains many different MT-dependent motors, but the specific role of any one motor in mitosis is still largely unknown. This project will focus on a few individual kinesin-like motor proteins. Through a combination of biochemistry, cell and molecular biology, and genetics we hope to develop an understanding of their roles in chromosome movement. We have selected the fission yeast, Schizosaccharomyces pombe as the most suitable micro-organism for this work because of its good genetics, its acceptable cytology and biochemistry, the wealth of relevant molecular methods, and the similarity of its cell and mitotic cycles to those of higher eukaryotes. We will also work with cultured mammalian cells in order to take advantage of their more convenient cell biology, their suitability for many kinds of molecular studies, and their obvious relevance to problems of human health. Initially, we will characterize a mammalian, mitotic kinesin-like protein recently discovered, cloned and sequenced in our lab; the molecular probes already available will be used to characterize this protein's action both in vivo and in vitro. Analogous motor enzymes will be sought in S. pombe. Here we will combine cellular and molecular approaches with genetic methods to identify and analyze novel mitotic motors and their roles in chromosome motion. Yeast mitotic mutants will be used to help identify novel mammalian mitotic motor molecules by transformation rescue of mutant yeasts with mammalian cDNA. Such genes will then be studied both in yeasts and in mammalian cells to characterize the role their product plays in mitotic motions. Each motor studied will also be characterized with our in vitro system for making chromosomes move in association with MT disassembly. An optical trap will be used to study the relationship between tubulin assembly and motor force, helping to elucidate the relationship between mitotic forces and spindle structure. Through this combination of studies we hope to identify the mitotic roles of several MT-dependent motor enzymes and learn how the enzymes interact with one another and with MT assembly to effect orderly chromosome motion.