Application of recently developed immunocytochemical methods for observing the cellular networks of microtubules (MTs) has revealed that MTs are unexpectedly dynamic. Many MTs in vivo appear to undergo continuous cycles of growth, depolymerization and regrowth, termed """"""""Dynamic Instability"""""""". It has been postulated that this dynamic instability behavior of MTs is involved in cell-shape morphogenesis, cell motility, mitotic spindle morphogenesis and chromosome movement. The mechanistic basis of MT dynamic instability may derive from the gain and loss of a """"""""cap"""""""" of tubulin dimers at a MT end, which have GTP exchangeable bound at the dimer E-site. The GTP binding and hydrolysis activities of the tubulin dimer E-site may therefore play a central role in regulating MT stability and function in the cell. Domains in beta-tubulin most likely involved with GTP-binding at the E-site have been identified by analogy with other GTP-binding proteins and by cross-linking studies. We propose to use mutagenic oligonucleotides directed to these domains to introduce mutations into the unique beta-tubulin gene, TUB2, of S. cerevisiae. Altered copies of TUB2 will be introduced into wild-type cells, using a transplacement vector we have constructed. Transformant phenotypes will be evaluated for cell viability, for MT content (by anti- tubulin immunofluorescence) and for propensity for chromosome loss (by a colony sectoring assay). Tubulin isolated from TUB2 mutants will be assayed in vitro for GTP-binding affinity, for GTPase activity and for dynamic instability using dark-field video- microscopy. The biochemical properties of the tubulins will be correlated with the phenotypes of the corresponding cells from which the tubulin was isolated. This will allow an in vivo test of current models for MT dynamics and for mitotic spindle morphogenesis and function. The goal is to understand the role of GTP binding and hydrolysis at the tubulin dimer E-site in MT dynamics and function in the cell.
