MONOCLONAL ANTIBODY ANALYSIS OF MDCK MEMBRANE DOMAINS

MDCK 膜域的单克隆抗体分析

• 批准号: 3446047
• 负责人: ZAID D SMITH
• 金额: $ 5.86万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1986-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3446047
• 关键词: affinity chromatography; antibody formation; antibody specificity; apical membrane; autoradiography; basolateral membrane; cell fusion; crosslink; electron microscopy; enzyme linked immunosorbent assay; epithelium; extracellular matrix; gel filtration chromatography; hybridomas; immunofluorescence technique; immunoprecipitation; intercellular connection; ion exchange chromatography; kidney cell; membrane activity; membrane reconstitution /synthesis; monoclonal antibody; pinocytosis; radionuclide double label; spectrometry; tissue /cell culture; vesicle /vacuole

A characteristic feature of cells is their ability to place enzymes, receptors, etc., in the membrane domain appropriate for cellular and organ functioning. This property is expressed especially clearly in epithelial cells where the plasmalemma is divided into two distinct domains, apical and basolateral, with distinct populations of receptors, pumps, and transporters. It is quickly becoming understood how the segregation of membrane proteins into these domains is important for the normal physiology of all epithelial tissues such as is found in the kidney, pancreas, liver, intestine, etc., but the mechanisms whereby the cell is capable of sorting membrane proteins and directing them into the correct domain needs further investigation. Much work has been done on this problem using virally-infected MDCK cells, where preferential budding of viral particles from one domain or the other has been used to examine the biogenesis of membrane polarity. With monoclonal antibodies specific for proteins in the apical or basolateral plasmalemma, and current immunolocalization techniques, it is now possible to examine the formation and maintenance of membrane domains in normal cells using probes for native membrane proteins. Herein it is proposed to investigate normal membrane biogenesis and domain formation in the Madin-Darby canine kidney cell line, using a panel of monoclonal antibodies directed exclusively to either the apical or basolateral plasmalemma domains. Experiments will be done to achieve the following aims: 1) To determine morphologically the topological distribution of biosynthetic vesicles; and where sorting of domain-specific proteins may take place; 2) To study processing kinetics and times to arrival at the plasmalemma of apical and basolateral membrane proteins; 3) To examine the spatial stability of membrane domains, and the ability of proteins to move to other domains in various conditions; 4) To determine whether or not domain-specific membrane proteins are initially inserted into their proper domains, or if they are randomly inserted and subsequently redistributed; 5) To determine the cellular conditions necessary for maintenance of plasmalemmal domains.

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