The long-term objective of the research described is to increase our understanding of the regulation of microtubule assembly and of the mechanism of action of certain anti-tumor drugs directed against tubulin, the structural subunit of microtubules. We intend to investigate the binding site of the anti-tumor drug maytansine to see its relationship to tau, one of the microtubule-associated proteins (MAPs). We also will study the dynamics of purified reconstituted microtubules consisting of tubulin and one MAP, either tau or MAP2. Such microtubules are likely to be more meaningful analogues of intracellular microtubules than the recycled microtubules used by many investigators. We will examine treadmilling and dynamic instability in these microtubules to get an idea of the role of each MAP in regulating microtubule dynamic behavior. We then will see how these parameters are perturbed by maytansine, phosphorylation (both cyclic AMP-dependent and calcium/calmodulin-dependent) of the MAPs, limited proteolysis of MAP2, and the MAP-directed anti-tumor drug estramustine phosphate. These experiments are expected to illuminate the roles of MAPs in general and tau and MAP2 in particular, in microtubule-related processes, such as transport, secretion and perhaps mitosis; they also may elucidate the mechanisms of action of the anti-tumor drugs maytansine and estramustine phosphate. We recently have described two new anti-tubulin compounds, Bis(1,8-anilinonaphthalenesulfonate) (FisANS) and 2,6-anilinoaphthalenesulfonate (2,6ANS). BisANS and 2,6ANS are, respectively, a potent inhibitor and a potent enhacer of microtubule assembly. We intend to examine the effects of BisANS and 2,6ANS on microtubule dynamics and on living cultured cells as well as on permeabilized cells. We hope to understand the in vitro and in vivo behavior of these novel anti-tubulin compounds. It is possible that these compounds, perhaps after some structural modification, could become therapeutically useful anti-tumor drugs.
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