FLAGELLAR MEMBRANE ADHESION AND SEXUAL SIGNALING

细胞-细胞粘附在真核细胞生物学中是至关重要的，它的许多最多样化的表现发生在受精反应中的配子之间。本研究的长期目标是表征单细胞绿藻莱茵衣藻正配子和负配子鞭毛膜上显示的性凝集素，并介导强烈和特异性的粘附以启动交配反应。凝集素是富含羟脯氨酸糖蛋白（HRGP）家族的成员，在高等植物中参与形态发生、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介导的细胞融合途径。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的其他2个hrgp中也发现，形成一个膜结合结构域和一个长纤维轴，以及一个形成头部的大球状结构域。该提案有三个具体目标。1)鉴定和测序正凝集素，已知在形态上与负物种相似，其中正和负蛋白质之间的相似性和差异可能提示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，并产生区域特异性抗体，使凝集素能够通过实时共聚焦显微镜和速冻深蚀刻复制品进行识别/可视化。2)利用凝集素是第一个被克隆的鞭毛膜蛋白这一事实，利用gfp标记的野生型和鞭毛运动缺陷突变体进行一系列实验，了解鞭毛膜蛋白是否通过与轴突成分运输相同或不同的系统运输到鞭毛。定位鞭毛膜池并研究其在鞭毛生长过程中向鞭毛的吸收。用cAMP刺激定位被招募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，它是否具有对运输和/或启动性信号传导重要的结构域。研究伴随性凝集并在细胞融合时发生的分离，询问它们是相同的还是不同的事件，以及它们是如何发生的，目的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.