STRUCTURE AND ASSEMBLY OF CYTOSKELETAL FILAMENTS

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Our major objective is to understand the mechanisms of "dynamic instability", the well established phenomenon in which microtubules cycle between phases of slow elongation and rapid shortening. Probably the most important unsolved question is the mechanism of "catastrophe," the abrupt transition from elongation to shortening. In the favored current model catastrophe is thought to be the loss of a "GTP cap," which when present, stabilizes the end of the MT and favors elongation. Cap loss exposes the very labile core of GDP tubulin subunits, which then rapidly disassembles. Catastrophe is therefore determined by the dynamics of the GTP cap, which involves at least three stochastic reactions" association and dissociation of GTP tubulin subunits from the end of the MT, and hydrolysis of GTP. At present there are speculations, but little definitive data, on the size of the cap, the rate of GTP hydrolysis, and the mechanism of cap loss. Our first priority in the next grant period will be to extend our study of dynamic instability by video microscopy of single microtubules. We have recently completed an exhaustive study in collaboration with the laboratory of E.D. Salmon. This study measured the kinetics for all phases of dynamic instability, but was limited to a single buffer. We now propose to extend these studies to explore important buffer conditions and ligands. Ongoing studies have already demonstrated that magnesium has a dramatic effect on the rapid shortening reaction, with depolymerization rates up to 8,000 subunits per sec at 7 mM Mg. Calcium, pH, and nucleotides, all potential regulators of physiological importance, will be investigated in this system to determine at which phases(s) of dynamic instability they exert their effects. We propose to complement these studies of individual microtubules by using quench-flow and stopped-flow techniques to measure GTP hydrolysis and cap loss at time intervals down to 5 msec. Our recent experiments, at time intervals of 10 and 1 s, have set upper bounds for the size of the cap, but much faster techniques are needed to resolve the mechanisms of cap dynamics. The quench-flow studies should determine not only the rate of hydrolysis but also the location within the cap at which hydrolysis takes place. The stopped-flow analysis of depolymerization should also determine the size of the cap, by a different approach. By repeating these experiments over a range of assembly conditions we hope to determine the complete mechanism of cap dynamics and catastrophe. The ultimate understanding of tubulin biochemistry will require an atomic structure, from x-ray crystallography. We are in an excellent position to attempt crystallization. The first step will be to determine conditions that keep tubulin as a solution of single protein molecules, stable for weeks, and at the same time poisoned for the two normal associations of tubulin: MT and ring formation. We propose to determine the necessary biochemical conditions and initiate crystallization attempts.