FLAGELLAR MEMBRANE ADHESION AND SEXUAL SIGNALING

细胞识别、粘附和随后的细胞反应被认为是 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 是为了了解鞭毛间的性粘附机制 来了解这种粘附信号是如何传递的 该细胞分泌酶，肌动蛋白，制备其质膜 用于细胞融合，并修改其细胞器DNA遗传的模式。 成功的基础是我们成功地获得了高比活性的 通过处理制备交配型Plus（MT+）和MT-凝集素 用12.5 mM EDTA在体内培养细胞。 部分纯化通过以下进行： 蔗糖梯度离心和凝胶过滤;亲和纯化 将使用目前正在生产的单克隆抗体进行 对部分纯化的材料。 凝集素鉴定 将使用4种方法制造对粘附重要的域：1） 将寻求识别MT特异性肽单克隆抗体 决定因素; 2）突变和失活凝集素将寻求在8 非凝集突变株和分离的合子细胞，如果 发现，将与它们的野生型对应物进行比较; 3）体外 将利用两种凝集素类型的中和来检测 对中和重要的肽;和4）可裂解的交联 试剂将用于偶联粘附表面并鉴定相互作用 两种凝集素类型的区域。 内在膜的鉴定 将通过结合研究制备与凝集素结合的蛋白质 和凝集素亲和层析，并制备抗体， 探测其表面定位和凝集素定位，使用 免疫电镜 鞭毛尖端激活， 对粘附的形态学反应，这对于信号 透射，将通过免疫电子显微镜和 生物化学提取和分析的纤维材料，填补了 激活提示。 尝试在无粘连的情况下诱导头端激活， 也可以做。 最后，我们将分析现有的遗传特性， 突变体，并继续分离和表征新的突变体。

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.