Microtubules, which constitute one of the three known elements of the cytoskeleton, are dynamic structures which change their location and function during the cell cycle. We wish to determine the kinetic parameters for assembly and disassembly of microtubules and learn how these are influenced by solution variable, microtubule-associated proteins and drugs which bind to tubulin. Our long-term goal is to learn how the cytoskeleton is regulated during the cell cycle. Also, microtubules are the target for a number of antitumor drugs and development of better agents for cancer chemotherapy will be advanced by understanding how microtubules are regulated.
The specific aims of the proposed research include determination of the role of GTP hydrolysis in microtubule assembly. We wish to know how GTP is used in the nucleation and elongation processes, as well as when the GTP is hydrolyzed, in relation to the time that tubulin subunits are incorporated into microtubules. Studies of the effects of GDP on microtubule assembly and stability are projected, in order to elucidate the mechanism underlying a puzzling phenomenon: although tubulin-GDP subunits do not readily elongate microtubules, they are able to react so as to maintain microtubules in a metastable steady state. The GDP effects are also of interest since it has recently been found that the kinetics for microtubule assembly and disassembly cannot be accounted for in terms of the relatively simple protein condensation model of Oosawa. The Oosawa model does not consider the effects of GTP hydrolysis and the observed deviations from this theory suggest that the GTP hydrolysis is an important element in regulation of microtubules. We wish to further develop a kinetic model which we have recently derived for microtubule assembly and disassembly, which takes into account the GTP hydrolysis reaction.
