FLAGELLAR MEMBRANE ADHESION AND SEXUAL SIGNALING

细胞识别、黏附和随后的细胞反应被认为 对胚胎发育和生长控制至关重要 多细胞生物。拟议研究的长期目标 是为了了解鞭毛之间的性粘连的机制 莱茵衣藻的细胞膜和了解这种黏附信号 细胞分泌酶，聚合肌动蛋白，准备质膜 用于细胞融合，并改变其细胞器DNA遗传模式。 实验是建立在我们成功获得高比活性的基础上的 交配型增强型(mt+)和mt-凝集素的制备 12.5 mM EDTA在体内培养细胞。部分纯化是通过以下方式实现的 蔗糖梯度离心和凝胶过滤；亲和纯化 将使用目前正在产生的克隆抗体进行 与部分提纯的物质进行对比。凝集素的鉴定 将使用4种方法制作对粘合很重要的区域：1) 将寻求识别mt特异性多肽的单抗。 决定因素；2)突变和失活凝集素将在8 非凝集突变株和非粘连合子细胞中，如果 发现，将与它们的野生型同行进行比较；3)体外 将利用这两种凝集素类型的中和来检测 对中和很重要的多肽；4)可裂解的交联物 试剂将用于偶联粘连表面并识别相互作用 这两种凝集素类型的区域。固有膜的鉴定 凝集素与之相关的蛋白质将通过结合研究而制成 和凝集素亲和层析，并将制备抗体 探测它们的表面定位和凝集素定位，使用 免疫电子显微镜。鞭毛尖端激活，一个明显的 对粘连的形态反应，这似乎对信号很重要 传播，将通过免疫电子显微镜和 填充物中纤维状物质的生化提取与分析 激活的提示。尝试在没有粘连的情况下诱导尖端激活将 也会被制造出来。最后，我们将分析现有生物的遗传特性 并继续分离和鉴定新的突变体。

Cell recognition, adhesion, and the ensuing cellular responses are believed to be critical to embryological development and growth control in multicellular organisms. The long-term objective of the proposed research is to understand the mechanisms of sexual adhesion between flagellar membranes of Chlamydomonas reinhardi and to learn how such adhesion signals the cell to secrete enzymes, polymerize actin, prepare its plasma membranes for cell fusion, and modify the pattern of its organelle DNA inheritance. Exerpiments are based on our success in obtaining high-specific-activity preparations of mating-type plus (mt+) and mt- agglutinins by treating cells in vivo with 12.5 mM EDTA. Partial purification is effected by sucrose gradient centrifugation and gel filtration; affinity purification will be undertaken using monoclonal antibodies presently being generated against the partially purified material. Identification of agglutinin domains important for adhesion will be made using 4 approaches: 1) monoclonal antibodies will be sought that recognize mt-specific peptide determinants; 2) mutant and inactive agglutinins will be sought in 8 nonagglutinating mutant strains and in disadhered zygotic cells and, if found, will be compared with their wild-type counterparts; 3) an in vitro neutralization of the two agglutinin types will be exploited to detect peptides important for neutralization; and 4) cleavable crosslinking reagents will be used to couple adhering surfaces and identify interacting regions of the two agglutinin types. Identification of intrinsic membrane proteins with which agglutinins associate will be made via binding studies and agglutinin affinity chromatography, and antibodies will be prepared to probe their surface localization, and agglutinin location, using immunoelectron microscopy. Flagellar tip activation, a pronounced morphological response to adhesion which appears important for signal transmission, will be probed by immunoelectron microscopy and by biochemical extraction and analysis of the fibrous material that fills the activated tips. Attempts to induce tip activation without adhesion will also be made. Finally, we will analyze the genetic properties of existing mutants and continue to isolate and characterize new mutants.