STUDIES ON THE INTERACTION OF TAXOL WITH CELLULAR MICROTUBULES
MANFREDI, JAMES JOHN
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Taxol is a low molecular weight plant derivative which enhances microtubule assembly in vitro and has the unique ability to promote the formation of discrete microtubule bundles in interphase cells. Here are reported studies in cells on the binding of ('3)H-taxol and the mechanism of the formation of these unusual microtubule arrays.;('3)H-Taxol binds to a macrophage-like cell line in a specific, saturable manner. Scatchard analysis demonstrates a single set of high affinity binding sites. Conditions which depolymerize microtubules in intact and extracted cells inhibit the binding of ('3)H-taxol. These data suggest that taxol binds specifically to cellular microtubules. Extraction with NP-40 or treatment with sodium azide prevents the characteristic taxol-induced bundle formation; the binding of ('3)H-taxol is retained under these conditions.;Taxol induces the formation of parallel arrays of microtubules. Cells which are pretreated with the reversible inhibitor, nocodazole, and then treated with taxol form bundles faster; bundle formation is facilitated by depolymerization of cellular microtubules. Cells which are pretreated with the irreversible inhibitor, colchicine, and then treated with taxol form a single microtubule aster associated with the nucleus and numerous discrete sites of microtubule nucleation scattered throughout the cytoplasm.;Treatment with taxol stabilizes cellular microtubules against depolymerization by calcium. These calcium-stable microtubules can be depolymerized in an intact cell by an appropriate treatment with colchicine. Both colchicine-induced depolymerization and taxol-induced bundle formation are energy dependent and require an intact cell. Depolymerization resulting from calcium is not energy dependent and occurs in an extracted cell.;At low concentrations, vinblastine depolymerizes cellular microtubules; at high concentrations the drug induces the formation of paracrystals. These paracrystals are retained in cytoskeletons prepared by Triton extraction and are stable to treatment with calcium. No effect is seen if vinblastine is added directly to cytoskeletons; vinblastine requires an intact cell to exert its effects.;The following scheme is proposed: Taxol crosses the plasma membrane, probably by passive diffusion. It binds directly to preexisting cytoplasmic microtubules conferring stability to calcium-induced depolymerization. These calcium-stable microtubules are depolymerized in an energy dependent manner by an endogenous colchicine-like activity. Parallel arrays of microtubules form in an organized manner at discrete cytoplasmic sites; this bundle formation is energy dependent.
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