FLAGELLAR MEMBRANE ADHESION AND SEXUAL SIGNALING

细胞-细胞粘附对真核细胞生物学至关重要，其许多最多样化的表现形式发生在受精反应期间的配子之间。 本研究的长期目标是表征性凝集素，其显示在单细胞绿色莱茵衣藻的正配子和负配子的鞭毛膜上，并介导强而特异的粘附以启动交配反应。 凝集素是富含羟脯氨酸的糖蛋白（HRGP）家族的成员，该家族的成员参与高等植物的形态发生、伤口愈合和病原体防御，它们的相互作用刺激鞭毛内腺苷酸环化酶的激活和cAMP介导的途径，导致细胞融合。 编码负凝集素的基因已被克隆并几乎完全测序。 它编码一个长PPSPX重复序列，在C. reinhardtii，其形成膜结合域和长纤维轴，以及形成头部的大球状域。该提案有三个具体目标。 1)鉴定和测序已知在形态上与阴性种属相似的阳性凝集素，其中阳性和阴性蛋白质之间的相似性和差异可能表明性别特异性结构域的位置。 构建绿色荧光蛋白（GFP）标记的凝集素基因，并生成域特异性抗体，以允许通过实时共聚焦显微镜和速冻深蚀刻复制品识别/可视化凝集素。 2)利用凝集素是第一个被克隆的鞭毛膜蛋白的事实，使用GFP标记的野生型和鞭毛马达缺陷型突变体进行一系列实验，以询问鞭毛膜蛋白是否通过与用于运输轴丝组分的系统相同或不同的系统被运输到鞭毛中。 定位鞭毛膜池并研究其在鞭毛生长过程中向鞭毛的募集。 定位在cAMP刺激下聚集到鞭毛膜上的凝集素池，并询问它是否与鞭毛生长池不同。 3)确定凝集素是内源性还是外源性膜蛋白，如果是内源性的，它是否具有转运和/或启动性信号的重要结构域。 研究伴随性凝集和细胞融合时发生的解离，询问它们是相同还是不同的事件以及它们是如何发生的，目标是了解粘附过程本身。

Cell-cell adhesion is vital to eukaryotic cell biology, and many of its most diverse manifestations occur between gametes during fertilization reactions. The long-term objective of this research is to characterize the sexual agglutinins that are displayed on the flagellar membranes of plus and minus gametes of the unicellular green alga Chlamydomonas reinhardtii and that mediate a strong and specific adhesion to initiate the mating reaction. The agglutinins are members of the hydoxyproline-rich glycoprotein (HRGP) family, members of which participate in morphogenesis, wound healing, and pathogen defense in higher plants, and their interaction stimulates an activation of intraflagellar adenylyl cyclase and a cAMP-mediated pathway that results in cell fusion. The gene encoding the minus agglutinin has been cloned and almost completely sequenced. It encodes a long PPSPX repeat, found as well in 2 other HRGPs in C. reinhardtii, that forms a membrane-binding domain and a long fibrous shaft, and a large globular domain that forms a head. The proposal has 3 specific aims. 1) Identify and sequence the plus agglutinin, known to be morphologically similar to the minus species, where the similarities and differences between the plus and minus proteins may suggest the location of sex-specific domains. Construct green fluorescent protein (GFP)-tagged agglutinin genes and generate domain-specific antibodies to allow agglutinins to be identified/visualized by real-time confocal microscopy and in quick-freeze deep-etch replicas. 2) Taking advantage of the fact that the agglutinin is the first flagellar membrane protein to be cloned, perform a series of experiments using GFP-tagged wild-type and flagellar-motor-defective mutants to ask whether flagellar membrane protein is transported into the flagellum via the same or different systems than those used to transport axonemal components. Localize the flagellar membrane pool and study its recruitment to the flagellum during flagellar outgrowth. Localize the pool of agglutinin that is recruited to the flagellar membrane with cAMP stimulation and ask whether it differs from the flagellar-outgrowth pool. 3) Determine whether the agglutinin is an intrinsic or extrinsic membrane protein and whether, if intrinsic, it possesses domains important for transport and/or for initiating sexual signaling. Study the disadhesion that accompanies sexual agglutination and occurs at the time of cell fusion, asking whether they are the same or different events and how they occur, the goal being to gain understanding of the adhesion process itself.