ASSEMBLY AND DISASSEMBLY OF CYTOPLASMIC MICROTUBULES

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Microtubules, ubiquitous components of eukaryotic cells, act as organizers of the cytoplasm and as substrates and guides for intracellular transport. In the course of these activities, they repeatedly dissociate into their constituent proteins and are then reassembled from those proteins. This dynamic behavior, subject to regulation by a variety of factors, affects cellular differentiation, intracellular transport and determination of cell shape. To understand and manipulate it, a fundamental grasp of the affinities of the microtubule's molecules for each other is needed, as well as knowledge of the rates at which those molecules associate and dissociate. This project's overall goal is to get that grasp and to integrate the individual rate constants and equilibrium constants into a mechanistic description of microtubule assembly and disassembly. Its particular aims are four. First, to make quantitative use of video-enhanced (VE) differential-interference-contrast (DIC) microscopy to measure and understand the newly discovered variability of rates of assembly/disassembly of microtubules. This variability must reveal fundamental functional properties of the growing end. Second, to investigate by video-enhanced DIC and fluorescence microscopy how microtubule-associated proteins and molecular motors affect the stability of microtubules. Relatively little is known about the actual mechanism by which this important means of cellular cytoskeletal regulation acts. Third, to characterize the slow but meaningful exchange of tubulin subunits between the wall of the microtubule and the solution. Fourth, to apply folding catalysts, of the cyclophilin and "molecular chaperone" classes, in an attempt to bring about folding of tubulin's polypeptide chains in vitro. Successful refolding would .open a path between molecular biological and functional studies of this important part of the cytoskeleton.