The functions that microtubules serve in cells require explanations for the mechanisms that determine the initiation, growth, directionality and spatial localization of these structures. The principal objective of this proposal is to elucidate the molecular mechanism of the in vitro self-assembly of microtubules in terms of the specific chemical steps that occur between the depolymerized state and the fully assembled structure. A kinetic analysis of assembly and disassembly using turbidity to measure the amount of material in the polymeric state is proposed. It is intended to determine the range of conditions under which the polymerization is a multi-step process with nucleation by intermediate subassemblies. The identification of these nuclei by the use of overshoot kinetics, analytical ultracentrifugation and electron microscopy is planned. The roles of ligands in formation and stabilization of reaction intermediates will be examined using the above methods plus acid-base titrations. BIBLIOGRAPHIC REFERENCES: Borisy, G.G., Marcum, J.M., Olmsted, J.B., Murphy, D. and Johnson, K. Purification of tubulin and associated high molecular weight proteins from porcine brain and characterization of microtubule assembly in vitro. New York Acad. Sci. Symp. on Microtubules 253:107-132 (1975). Olmsted, J.B. and Borisy, G.G. Ionic and nucleotide requirements for microtubule polymerization in vitro. Biochemistry 14:2996-3004 (1975).