The long-term objective is to define the molecular mechanisms utilized to achieve sexual adhesion, extracellular matrix (ECM) assembly, and sexual signalling in Chlamydomonas reinhardtii and related species, the rationale being that Chlamydomonas is likely to be closely related to the eukaryotic unicells that """"""""invented"""""""" such cellular processes, and their understanding will therefore increase our understanding of metazoan animals and plants. The sexual agglutinins and the ECM components are homologous hydroxyproline-rich glycoprotein (HRGP's) whose modes of recognition and assembly are likely to follow similar molecular rules. To probe these mechanisms, experiments are proposed to identify structural and immunoreactive domains that are conserved in Volvocacean HRGP's, these likely to be essential for recognition/assembly; to identify structural and immunological differences between interacting HRGP's (e.g. the plus and minus sexual agglutinins) that may confer recognition specificity; to evaluate the role of disulfides in establishing the globular domains present on these fibrous proteins; to identify peptides important for recognition/assembly; and to evaluate the role of side-to-side interactions between fibrous domains in the interaction of HRGP's. In vitro adhesion and ECM assembly systems have recently been developed; these will be used to visualize adhesion/ disadhesion and assembly/disassembly by quick-freeze deep-etch TEM; to analyze the biochemical basis for agglutinin inactivation during mating; to understand how the ECM layers are laid down and how some of them are rendered insoluble; and to ask whether heterologous proteins (mixtures of Chlamydomonas and Volvox HRGP's, for example) will co-assemble. Finally, experiments are proposed to identify and prepare antisera against the intrinsic membrane protein to which agglutinin binds, and to ask how cross- linking of this protein elicits sexual signals. We will evaluate the hypothesis that the signals are mediated by cAMP by seeking a flagellar-localized adenylate cyclase that is responsive to receptor cross-linking, and will evaluate the hypothesis that the signals are mediate by Ca++ by following Ca+ levels in mating cells via Quin2 fluorescence. A flagellar adenylate cyclase has been identified; flagellar guanylate cyclase and cAMP/cGMP phosphodiesterase activities will be sought and the role of such enzymes in signal generation evaluated. The ability of cyclic nucleotides to generate sexual signals in the absence of adhesion vill be explored and the role(s) played by Ca++ and PI lipid metabolism in signal generation and/or response will be assessed.
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