Microtubules are essential to cell growth, morphology and motility. They are filaments that the cell organizes into quite different structures depending upon which of those functions they are performing. The filaments themselves are primarily polymers of subunits of tubulin heterodimers. The heterodimers in turn are composed of one alpha-tubulin and one beta-tubulin polypeptide. The free tubulin polypeptides can occur when the heterodimer dissociates or when it does not form properly. At each of these levels - organization of the microtubules, polymerization of the subunits, and formation of the heterodimer - there are cellular regulatory mechanisms. Our work focuses on the lowest of these levels - the formation of the heterodimer and its regulation. Our interest in this issue arises from several observations in yeast cells. First, several genetic tests of microtubule function performed by our lab and others identify genes that affect this process. Second, we have shown that even small amounts of beta-tubulin polypeptide that is not incorporated into heterodimer and therefore is free, are toxic to the cell. There are genes which function to protect the cell against beta-tubulin toxicity. Third, we have recently identified a cellular mechanism that decreases expression of tubulin, perhaps in response to the formation of free tubulin polypeptides. These results provide evidence for regulation of very early steps in microtubule morphogenesis. Our long-range objective is to understand the mechanism of this regulation and its biological implications. This proposal will identify the molecular trigger of this regulatory process as well as the downstream targets of that trigger. In this way, it will identify the molecular mechanism of action. It will also investigate why free beta-tubulin polypeptides are toxic, and so identify the molecular process disrupted by that protein. Based on these results, the focus will shift to determining if similar mechanisms exist in animal cells. The results will provide basic information about the control of early steps in microtubule morphogenesis as part of the normal cell cycle and provide insight into response to those anti-cancer drugs that act by poisoning microtubule assembly.
