CELL SURFACE CONTROL OF LYMPHOCYTE PHYSIOLOGY

淋巴细胞生理学的细胞表面控制

• 批准号: 3299850
• 负责人: SUSAN W CRAIG
• 金额: $ 25.28万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-08-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3299850
• 关键词: actins; affinity chromatography; antibody formation; binding proteins; calcium; calmodulin; cell cell interaction; cell growth regulation; cell membrane; cell motility; chickens; electron microscopy; flow cytometry; fluorescence microscopy; immunodiffusion; immunofluorescence technique; intestinal villi; iodine; laboratory mouse; laboratory rabbit; lymphocyte; molecular biology; monoclonal antibody; myosins; radionuclides; radiotracer; smooth muscle; tritium; vinculin

The objective of this research is to learn how actin interacts with plasma membrane. Three examples of actin-membrane linkages are being studied. (1) We are determining the molecular composition of the actin to membrane cross filament in intestinal microvilli. a) We propose to isolate the microvillar 110K protein, a candidate for the cross filament, and to determine whether and how it interacts with actin and the microvillar membrane in vitro. b) We will test the proposed roles of the microvillar core proteins (fimbrin, villin, 110K, actin, and calmodulin) in the structure and membrane association of the core bundle of actin filaments by immunoultrastructural localization of these proteins in situ, using 50 angstroms Fab' fragments of monoclonal antibodies complexed to 50 angstroms gold particles. c) We will screen for microvillar proteins which link actin to membrane by a reconstitution binding assay employing 3H-actin, microvillar membranes, and fractionated microvillar proteins. (2) We are analyzing the association of vinculin-like proteins with the plasma membrane. We have discovered two new proteins in smooth muscle, 150K meta-vinculin, and 300K vinculin-like polypeptide (300K-VLP) that are antigenically related to 130K vinculin, but which unlike vinculin, have solubility properties of amphiphilic membrane proteins and could be direct links between actin filaments and the cell membrane. We propose to analyze the molecular basis for the antigenic similarity and solubility difference between these proteins by peptide mapping, charge shift electrophoresis, biosynthetic labelling and pulse-chase experiments, isolation of native metavinculin and 300K-VLP and characterization of their interaction with actin, plasma membrane and liposomes in vitro. (3) We have defined a cell surface actin (CSA) on some murine lymphocytes by immunofluorescence and flow cytometry. a) We propose to determine by two-color immunofluorescence and flow cytometry if CSA is a marker for new murine and human lymphocyte subpopulations within the known subdivisions of T and B-cells. b) We will assay for biochemical differences between CSA and intracellular actin by determining if CSA is a glycoprotein and if it has a hydrophobic domain. c) We plan to test for the possible function of CSA in mediating adherence of lymphocytes to fibronectin and to cells of the lymphoreticuloendothelial system, and for the possible function of CSA as part of a cell surface growth control complex.

本研究的目的是了解肌动蛋白如何与血浆相互作用 膜的 正在研究肌动蛋白-膜连接的三个例子。 (1)我们正在确定肌动蛋白的分子组成， 小肠微绒毛内有交叉丝。 （a）我们建议将 微绒毛110 K蛋白，交叉丝的候选者，以及 确定它是否以及如何与肌动蛋白和微绒毛相互作用， 膜在体外。 B）我们将测试微绒毛的拟议作用， 核心蛋白（fimplatin，villin，110 K，actin和calmodulin） 肌动蛋白丝的核心束的结构和膜协会， 这些蛋白质的原位免疫超微结构定位，使用50 与50埃复合的单克隆抗体的Fab'片段 黄金颗粒 c）我们将筛选微绒毛蛋白， 通过使用3 H-肌动蛋白的重构结合测定， 微绒毛膜和分级微绒毛蛋白。 (2)我们 分析黏着斑蛋白样蛋白与血浆的结合 膜的 我们在平滑肌中发现了两种新的蛋白质，150 K 后黏着斑蛋白和300 K黏着斑蛋白样多肽（300 K-VLP）， 与130 K黏着斑蛋白抗原相关，但与黏着斑蛋白不同， 两亲性膜蛋白的溶解性，并可以直接 连接肌动蛋白丝和细胞膜。 我们建议分析 抗原相似性和溶解性差异的分子基础 通过肽图谱，电荷位移电泳， 生物合成标记和脉冲追踪实验，分离天然 metavinculin和300 K-VLP的相互作用及其表征 肌动蛋白、质膜和脂质体。 (3)我们定义了一个单元格 免疫荧光法测定小鼠淋巴细胞表面肌动蛋白（CSA）， 流式细胞仪 a）我们建议用双色免疫荧光法测定 如果CSA是新的鼠和人淋巴细胞的标志物， 已知的T和B细胞亚群。 B）我们会 CSA和细胞内肌动蛋白之间的生化差异的测定 确定CSA是否是糖蛋白以及它是否具有疏水结构域。 c）我们计划测试CSA在调解依从性方面的可能功能 纤维连接蛋白和淋巴网状内皮细胞 系统，以及CSA作为细胞表面的一部分的可能功能 生长控制复合体