ANALYSIS OF SPINDLE MICROTUBULE DYNAMICS

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The molecular mechanism responsible for chromosome movement during mitosis remains a major unanswered question in cell biology. The mitotic spindle is composed, in part, of a bipolar arrangement of microtubules (MTs) which radiate from the spindle poles and overlap at the metaphase plate. In an effort to elucidate the role of MTs in chromosome motion, techniques have been developed to introduce fluorochrome labeled tubulin into living cells to monitor tubulin behavior at steady-state. In the experiments presented here, the pathways of tubulin subunit association and dissociation with spindle fiber MTs, in particular the kinetochore fiber MTs, will be examined by measuring the rate and pattern of fluorescence redistribution after photobleaching (FRAP). In these FRAP experiments, tubulin fluorescence in spindle fibers is photobleached in a selected area and recovery of fluorescence, which occurs as unbleached subunits exchange with bleached subunits in MTs, is monitored using video or photometric techniques. The site or sites of spindle MT disassembly at anaphase will be determined by FRAP experiments on large spindles of Haemanthus endosperm or spermatocytes. The dynamic properties of microtubule associated proteins, MAPs, will be measured by preparing fluorescent analogs of several different MAPs and measuring FRAP. The ability of these proteins to modulate the rate of tubulin FRAP will also be determined. The role of cellular ATP in chromosome motion, tubulin and MAP dynamics will be determined. Finally, conditions which maintain the dynamic activity of MTs in vitro will be determined so that a functional model of the mitotic spindle can be obtained. A more complete understanding of the process of mitosis may lead to improvements in the treatment of many diseases where uncontrolled cell proliferation occurs. The ability to specifically control cell division may be realized through analysis of the dynamic properties of the components of the mitotic spindle.