The structural protein tubulin is involved in a variety of cellular processes important in morphogenesis, including cell division and flagellar assembly. In many cases, tubulin involved in different processes is found in structurally different microtubule arrays. The regulation of tubulin assembly into microtubule arrays with different functions may utilize discrete structural domains of the tubulin molecule and may be mediated by interactions between tubulin and different sets of microtubule associated proteins. The goal of our research is to determine how tubulin structure regulates tubulin function in vivo and to identify gene products which interact with tubulin during its several functions and which may regulate its assembly. As a model system, we propose a genetic, biochemical, and ultrastructural analysis of the function of tubulin and the molecules with which it interacts during spermatogenesis in Drosophila. We propose to identify genes encoding proteins which interact with the testis-specific Beta tubulin (Beta2) by isolation and characterization of mutations which fail to complement mutant alleles of Beta2 tubulin but which map to other sites on the genome. We have already identified two such """"""""second-site"""""""" recessive male sterile mutations. We plan to identify the mutant gene products by 2D gel analysis or recombinant DNA techniques. We will investigate the function of the mutant gene products in spermatogenesis and the nature of their interaction with Beta2 tubulin by light and electron microscopic analysis of defects in spermatogenesis caused by """"""""second-site"""""""" mutations both as homozygotes and in heterogyzous combination with Beta2 tubulin mutants. We also propose to analyze changes in tubulin structure caused by mutations in the Beta2 tubulin gene which affect some, but not all, of the functions of Beta2 tubulin during spermatogenesis. Changes in mutant tubulin structure will be assayed using monoclonal antibodies specific for different determinants on the tubulin molecule as probes of molecular structure. Structural changes will be correlated with changes in function of mutant tubulins in vivo and in vitro to determine if discrete structural regions of the tubulin molecule function uniquely in different kinds of microtubule dependent processes.
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