The investigation proposed in this application is aimed towards a molecular understanding of the process of flagellar shedding, also called flagellar autotomy, in the unicellular eucaryote Chlamydomonas reinhardtii. Autotomy occurs at a specific site - the cell-distal end of the flagellar transition region - and it results in the release of the flagellum without compromising the integrity of the cell. At first acquaintance, autotomy seems to be a peculiar process with little general cell biological relevance. In fact, just the opposite is true: autotomy is a property of ciliated cells in general, and it involves a number of familiar and fundamental processes, including signalling and membrane fusion, as well as at least one unfamiliar and therefore quite remarkable process -microtubule severing. The experiments proposed in this application make use of recent advances in Chlamydomonas molecular genetic technology to test specific hypotheses about the structural and biochemical nature of autotomy. And, because we can readily assay for autotomy in vitro using detergent extracted cell models or isolated nucleo-basal body apparatuses, the prospect exists for a direct biochemical test of any model(s) favored by the genetic and molecular data. The proposed project has three major foci. The first focus is on the calcium binding protein centrin and its role in autotomy. The gene encoding centrin has been cloned and sequenced, and we have already obtained and analyzed a number of mutations in the gene. We will construct a centrin gene disruption and isolate new centrin mutants, both by selection and by in vitro mutagenesis, to identify those with specific autotomy defects. By examining these mutants at the biochemical functional and structural levels, we hope to acquire a molecular understanding of centrin's role'(s) in autotomy. The second focus is on proteins that interact with centrin; we will attempt to identify centrin binding proteins, and the genes that encode them, using a screen for lambda gt11 clones that encode centrin-binding fusion proteins. The third focus is on genes identified by mutations with autotomydefective phenotypes. We will clone, sequence and analyze a known gene,fa-1, that is required for microtubule severing, and we will identify - and when appropriate isolate, clone and sequence -additional autotomy genes. In all of these cases, once genes of interest have been isolated, we will prepare specific antibodies to the proteins that they encode and use them as probes for protein localization and function.
