Microtubules, cylindrical organelles which play crucial roles in mitosis, are composed of tubulin, which is a dimer of two subunits: alpha and beta, both of which occur as multiple isotypes exhibiting great differences in their tissue distribution. Because of its role in mitosis, tubulin is the target for a variety of anti-tumor drugs. The long-term objective of the proposed research is to increase our understanding of microtubule dynamics and of the interaction of tubulin with anti-tumor drugs. The role of tubulin isotypes in modulating both the dynamic behavior of microtubules and the mechanisms of action of certain drugs shall be explored.
In Specific Aim 1 specific peptides will be used as immunogens to prepare monoclonal antibodies specific for the individual isotypes of alpha- tubulin.
In Specific Aim 2, these monoclonal antibodies, together with antibodies specific for the beta-tubulin isotypes, will be used as tools to prepare isotypically pure tubulin dimers by immunoaffinity chromatography.
In Specific Aim 3, the antibodies will be used to quantitate the different isotypes in various tissues and cultured cells, as well as to localize them within the brain.
In Specific Aim 4, the isotypically purified tubulin dimers will be induced to polymerize into microtubules in vitro and their dynamic properties will be studied. Different hypotheses to account for microtubule dynamic behavior will be tested.
In Specific Aim 5, the effects of anti-tumor drugs on the dynamic behavior of isotypically purified microtubules will be examined. The drugs which will be tested include vinblastine, maytansine and phomopsin A. Maytansine, unlike vinblastine, does not stabilize tubulin against decay, whereas phomopsin A stabilizes tubulin even more than does vinblastine. In fact, in the presence of colchicine, phomopsin A stabilizes tubulin to the point where there is almost no discernible decay. The experiments described in this specific aim should greatly increase our knowledge of the mechanism of action of these drugs and may also identify certain tissues or cells whose microtubules may be more susceptible to particular drugs.
In Specific Aim 6, the isotypically purified tubulin will be incubated with phomopsin A and colchicine under various conditions to determine which is the best way to obtain tubulin crystals. If tubulin crystals could be obtained and subjected to X-ray diffraction, the three-dimensional structure of this critical protein could be determined, which will not only be a major advance in cell biology and biochemistry, but could permit rational design of anti-tumor drugs.
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