DRUG INTERACTIONS WITH BRAIN MICROTUBULE PROTEINS

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Microtubules are important components of the cytoskeleton of eukaryotic cells, and participate in the development and maintenance of cell shape and in many kinds of intracellular movement. They are prominent in the central nervous system where they are especially important in the organization and function of axonal and dendritic processes of neurons. Microtubules exhibit a wide variety of polymerization behaviors in vitro and in cells, and there is strong reason to believe that the various polymerization behaviors are important in microtubule function. Microtubule functions such as those related to the organization and development of axons and dendrites may be determined and regulated by modulation of the assembly and disassemble reactions at microtubule ends. The role of polymerization dynamics in the function of microtubules at a mechanistic level is not understood. With a focus on brain microtubule systems, the main strategy of this proposal is to investigate the dynamics of tubulin addition and loss at microtubule ends in vitro, with the goals of understanding the mechanisms that govern tubulin addition and loss and identifying and elucidating mechanistically the actions of microtubule- associated proteins that control assembly and disassembly dynamics in neuronal cells. A combination of procedures will be employed that can distinguish tubulin addition and loss events at the individual microtubule ends. The dynamics of tubulin addition and loss at the ends of microtubules composed of unmodified and posttranslationally-modified brain tubulins (detyrosinated, glutamylated, ADP-ribosylated) and specific unmodified and modified (phosphorylated) neuronal MAPs will be examined. The work will focus on the actions of MAP-2 and Tau, two prominent neuronal microtubule-associated proteins that are important in the development, organization, and functions of dendrites and axons, respectively. The work will also focus on the role of GTP hydrolysis in microtubule polymerization. The objective is to characterize the flux and dynamic instability parameters of the microtubules, and to determine to what extent and how specific microtubule-associated proteins, specific tubulin isotypes, posttranslational modifications of tubulin and microtubule- associated proteins, and the drug colchicine, modulate and regulate microtubule polymerization.