FLAGELLAR MEMBRANE ADHESION AND SEXUAL SIGNALING

长期的目标是定义分子机制。 用于实现性黏附的细胞外基质(ECM) 莱茵衣藻和衣藻的组装和性信号 相关物种，其原理是衣藻很可能 与“发明”的真核单细胞密切相关 这种细胞过程，以及他们对这一过程的理解 增加我们对后生动物和植物的了解。这个 性凝集素和细胞外基质成分是同源的 富含羟脯氨酸的糖蛋白(HRGP‘s) 识别和组装很可能遵循相似的分子 规矩。为了探索这些机制，建议进行以下实验 识别保守的结构和免疫活性结构域 在沃尔沃斯动物的HRGP中，这些可能是必不可少的 识别/组装；鉴定结构和免疫学 相互作用的HRGP之间的差异(例如，加号和减号 性凝集素)，可赋予识别特异性； 评估二硫化物在建立球状结构中的作用 存在于这些纤维蛋白上的结构域；用于鉴定多肽 对识别/组装很重要；并评估 纤维结构域之间的侧到侧相互作用 HRGP、体外黏附和细胞外基质组装系统的相互作用 是最近开发的；这些将被用来可视化 用速冻粘合/解粘和组装/拆卸 深蚀电子显微镜分析凝集素的生化基础 在交配期间失活；了解ECM层是如何 以及其中一些是如何变得无法溶解的；并询问 异源蛋白(衣藻和Volvox的混合物 例如，HRGP)将共同组装。最后，实验是 建议鉴定和制备针对固有病毒的抗血清 凝集素结合的膜蛋白，并询问如何交叉- 这种蛋白质的连接引出性信号。我们将评估 假设信号是由cAMP通过寻求一种 鞭毛定位的腺苷环化酶对 受体的交联性，并将评估假设 钙通过交配细胞中的钙水平介导信号的传递 通过Quin2荧光。一种鞭毛状腺苷环化酶 鞭毛型鸟苷环化酶和cAMP/cGMP 将寻求磷酸二酯酶活性和这种作用 评估了信号产生中的酶。循环能力 在没有黏附的情况下产生性信号的核苷酸 钙、磷脂的作用有待探讨(S) 将评估信号生成和/或响应中的新陈代谢。

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.