Microtubules participate in diverse cellular events such as the changes in cell shape during development and differentiation, the formation of mitotic and meiotic spindles for chromosomal segregation, and ciliary and flagellar motility. Microtubule dysfunction is implicated in a number of human hereditary disorders such as the immotile cilia syndromes. Despite the diversity and importance of microtubule functions, little is known about the mechanisms that regulate the maintenance and establishment of programmed modifications of these elements during cellular morphogenesis. The long range goal of this proposal is to elucidate the regulatory signals that control the synthesis of tubulins, the major constituents of microtubules. We now know that the synthesis of tubulins is controlled by an apparent autoregulatory pathway in which the intracellular pool of free tubulin determines the rate of new synthesis. This autoregulatory response can be demonstrated by treatment of cultured cells with colchicine, a drug that induces microtubule depolymerization and leads to an increased pool size of free tubulin subunits. I propose to determine the genetic region that confers regulation to tubulin synthesis. The approach will be the introduction and expression of cloned tubulin sequences in cultured cells. Systematic alterations will be introduced to the cloned sequences and the effects of these alterations on colchicine induced modulation of the cloned tubulin gene sequences will be examined. This approach represents a direct way to determine the genetic region(s) that encodes the regulatory signal in tubulin biosynthesis. Additionally, this approach can be used to determine the different regions of the tubulin sequence that are important to the formation of functionally diverse microtubules. Examination of tubulin regulation and function on molecular level is essential to the further understanding of the participation of microtubules in cellular morphogenesis.
