FLAGELLAR MEMBRANE ADHESION AND SEXUAL SIGNALING

长期目标是确定分子机制 用于实现性粘附，细胞外基质（ECM） 组装和性信号在莱茵衣藻和 相关物种，其基本原理是衣原体可能是 与“发明”的真核单细胞密切相关 这样的细胞过程，他们的理解将因此 增加我们对后生动物和植物的了解。 的 性凝集素和ECM成分是同源的 富含羟脯氨酸的糖蛋白（HRGP），其 识别和组装可能遵循类似分子 规则 为了探索这些机制，提出了实验， 鉴定保守的结构和免疫反应性结构域 在团藻类HRGP中，这些可能是必不可少的 识别/组装;识别结构和免疫学 相互作用的HRGP之间的差异（例如， 性凝集素），可赋予识别特异性; 评估二硫化物在建立球状 结构域存在于这些纤维状蛋白质上;以鉴定肽 重要的承认/组装;并评估的作用 纤维域之间的侧向相互作用 HRGP的互动。 体外粘附和ECM组装系统 最近开发的;这些将用于可视化 粘附/脱粘和速冻组装/拆卸 深蚀刻TEM;分析凝集素的生化基础 在交配过程中失活;了解ECM层是如何 他们中的一些人是如何被安置的，以及他们是如何被安置的。 是否异源蛋白（衣原体和团藻的混合物 例如，HRGP）将共同组装。 最后，实验 提出鉴定和制备抗内源性 膜蛋白，凝集素结合，并问如何交叉- 这种蛋白质的连接激发了性信号。 我们将评估 假设信号是由cAMP介导的， 鞭毛定位的腺苷酸环化酶，其响应于 受体交联，并将评估的假设， 信号是由Ca++介导的，通过跟随交配细胞中的Ca+水平 通过Quin 2荧光。 一种鞭毛腺苷酸环化酶， 鉴定;鞭毛鸟苷酸环化酶和cAMP/cGMP 将寻求磷酸二酯酶活性，并且这种磷酸二酯酶活性的作用将被确定。 酶在信号产生中的作用。 循环能力 在没有粘附的情况下产生性信号的核苷酸 将被探索和Ca++和PI脂质发挥的作用 将评估信号产生和/或响应中的代谢。

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.