It has become increasingly evident that microtubule (MT) networks in cells are highly dynamic structures. While it is generally accepted that the different dynamic behaviors of MTs are almost certainly essential for their cell functions, it is not understood how MT dynamic properties are generated, nor how they are regulated in the cell to suit specific cell functions. It has been suggested, however, that the loss and regain of a """"""""cap"""""""" of tubulin-GTP subunits at a MT end could contribute to MT dynamic behavior and there is considerable evidence consistent with such a model. A primary focus of this proposal is to test this model by generating tubulin mutants with altered GTPase activity and examining what an altered tubulin GTPase means for MT dynamics in vitro and for MT dynamics and functions in cells. Domains in beta-tubulin likely involved with GTP binding and hydrolysis at the E-site have been identified by cross-linking studies and by sequence comparisons with other known GTP-binding proteins. Site-directed mutagenesis of these sites will be carried out using mutagenic oligonucleotides on TUB2, the single copy beta-tubulin gene in the yeast S. cerevisiae. The mutant genes will be introduced into yeast cells by transformation and the effects of these mutations will be examined on yeast cell phenotypes, such as growth rates and benomyl sensitivity. The consequences of these mutations for the MT-based cell functions, nuclear migration and separation, will also be examined. The in vitro biochemical effects of the mutations on tubulin GTPase activity, on GTP binding affinity and specificity, and on MT dynamics, such as treadmilling and dynamic instability, will also be measured. These data will provide a test of: 1) the GTP """"""""cap"""""""" model, 2) models of GTP hydrolysis and 3) the role of MT dynamics in cell function. A search for non-tubulin gene suppressors of bona fide tubulin GTPase mutations will also be undertaken;
the aim will be to identify proteins which regulate the extent, the type and the timing of MT dynamics in the cell via the tubulin GTPase site. Finally, the approach with yeast will also be extended into HeLa cell lines to exploit the superior cytology of the mammalian cell lines; this will make it possible to assess the effects of tubulin GTP-site mutations on spindle MT dynamics and function with far greater resolution than would be possible in yeast.
