Long-term objectives of the proposed research are to understand the relationships between GTP hydrolysis by tubulin, binding and function of microtubule-associated proteines (MAPs), and the geometry and kinetics of assembly of microtubules. Five interrelated specific projects are proposed. (1) Understanding of the binding of GTP and GDP to tubulin subunits will be extended to an investigation of the molecular basis of the Mg2+-dependence of assemby and disassembly of microtubules. (2) The kinetics of the transition, during assembly of MAP-free tubulin, from ribbon-like intermediates to microtubules will be investigated. The roles of GTP hydrolysis, MAPs, and polymerization nuclei in producing cylindrical shape in tubulin aggregates will be studied. (3) The noncovalent complexes formed by isolated but unfractionated MAPs, probably composed of more than one type of MAP and tubulin , will be characterized as to their size, composition, and the stoichiometric ratios and exchangeability of their proteins. (4) Potential differences between binding of MAPs to the main lattice of GDP-tubulin in the center of the microtubule and to the GTP-tubulin in the """"""""cap"""""""" at the end will be examined. Differences in both kind and amount of bound protein will be sought. (5) Spontaneous formation by microtubules in solution of regions of nearly parallel alignment will be investigated to learn how well the concentration- and length-dependence of the process fit existing theory. Proposed methods are chiefly biophysical and biochemical. GTP hydrolysis will be assessed by radioisotope measurements, and assembly of microtubules and sheetlike forms by measurement of turbidity and small-angle light-scattering. MAP complexes will be isolated and their protein content obtained by chromatography and electrophoresis. Their size and shape will be measured by gel exclusion chromatography and analytical ultracentrifugation. Exchange of tubulin and MAPs will be assessed by rapid gel-filtration and centrifugation (Airfuge) aided by radioisotope and fluorescent labeling. The work will provide fundamental insight into the mechanisms that allow microtubules to be capable both of dynamic assembly/disassembly and of relative stability, and will help provide a basis for studies of their malfunction in pathological conditions, e.g., interruption of axonal transport in peripheral neuropathies, failures of intracellular movement, and disruption of control of mitosis, as in cancer.
Showing the most recent 10 out of 33 publications
