STRUCTURAL ANALYSIS OF GAP JUNCTION TRAFFICKING

缺口连接贩运的结构分析

• 批准号: 7929432
• 负责人: GINA E SOSINSKY
• 金额: $ 9.47万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7929432
• 关键词: 3-Dimensional; Biological Assay; Canis familiaris; Cataract; Cell Line; Cell membrane; Cells; Cellular Structures; Cellular biology; Charcot-Marie-Tooth Disease; Cholesterol; Communication; Complex; Connexin 43; Connexins; Connexon; Cyclodextrins; Data; Developmental Process; Disease; Docking; Electron Microscopy; Electrons; Fluorescence; Gap Junctions; Genetic; Goals; Hela Cells; Hepatocyte; Homeostasis; Human; Image; Imagery; In Situ; Ion Channel; Kidney; Label; Life; Life Cycle Stages; Ligands; Light; Lipids; Location; Lysosomes; Maintenance; Microscopic; Microscopy; Mitosis; Molecular; Mutation; Neural Conduction; Normal Cell; Optics; Outcome; Pathway interactions; Physiologic pulse; Play; Process; Proteins; Publishing; Qualifying; Rattus; Recycling; Regulation; Resolution; Role; Route; Sensorineural Hearing Loss; Shapes; Signal Transduction; Skin Abnormalities; Structure; Symptoms; System; Techniques; Testing; Time; Tissues; Vertebrates; Vesicle; cell type; electron tomography; follow-up; gap junction channel; hearing impairment; inhibitor/antagonist; intercellular communication; interest; kidney cell; light microscopy; polarized cell; protein transport; reconstruction; tool; trafficking; transmission process; uptake

DESCRIPTION (provided by applicant): Gap junctions (GJ) are defined as clusters of closely packed membrane channels containing the connexin protein, connecting two adjoining cells. The channel is composed of two hexamers from each cell and contains oligomers of one or more connexins. GJ serve important functions in direct intercellular communication in almost all vertebrates cell types. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Normal cell and tissue homeostasis as well as developmental processes are dependent on the proper trafficking of connexins and the fast turnover rate of connexins has been hypothesized to be one mechanism of channel function regulation. Many of the mutations in connexin diseases (sensorineural deafness, Charcot-Marie-Tooth disease, cataracts, for example) result in abnormal trafficking. This proposal focuses on the identification and characterization of connexin trafficking structures using the techniques of tetracysteine genetic tags complexed with biarsenical fluorescent ligands, optical pulse-chase, fluorescence photooxidation, correlative light and electron microscopy and electron tomography to produce 3D reconstructions of selectively labeled connexins in cells. Using these techniques in combination, we aim to study these intermediates at reasonably high electron tomographic resolution (approximately 40-60 Angstroms) in 3D to determine their composition and locations within the context of other cellular components. We have four specific aims for the requested five-year period. Specific Aim 1 follows up on our initial study of Gaietta et al., (2002) by dissecting "unstimulated" or normal connexin trafficking pathways in endogenously expressing connexin cell lines. Initial studies were done in HeLa cells, a cell line known to produce artificially large quantities of connexins. We are expanding our initial study to investigate cell lines that are unpolarized, polarized and primary to see if the mechanism of adding new gap junction channels to the edges of the plaques is a universal one. Specific Aim 2 explores the question of whether hemichannels are a stable part of the plasma membrane or a short-lived trafficking intermediate. Specific Aim 3 is focused on the connexin structures found during mitosis and whether recycling of connexins occurs. Specific Aim 4 investigates investigate the role that cholesterol plays in plaque maintenance by examining the effect of cholesterol depleting agents and the re-uptake of cholesterol temporally examining this process at higher resolution and in 3-D than previously published.

描述（由申请人提供）：间隙连接（GJ）定义为包含连接蛋白的紧密包装膜通道的簇，连接了两个相邻的细胞。该通道由每个细胞的两个六聚体组成，并包含一个或多个连接素的低聚物。 GJ在几乎所有脊椎动物细胞类型的直接细胞间通信中都起着重要的功能。细胞通过调节这些通道的合成，传输和周转来通过GJ动态调节通信。正常的细胞和组织稳态以及发育过程取决于连接素的适当运输，并且连接蛋白的快速周转率已被认为是通道功能调节的一种机制。连接性疾病中的许多突变（例如，感觉性耳聋，肉体 - 马里齿病，白内障）导致贩运异常。 This proposal focuses on the identification and characterization of connexin trafficking structures using the techniques of tetracysteine genetic tags complexed with biarsenical fluorescent ligands, optical pulse-chase, fluorescence photooxidation, correlative light and electron microscopy and electron tomography to produce 3D reconstructions of selectively labeled connexins in cells.使用这些技术结合使用，我们旨在研究3D中相当高的电子断层分辨率（约40-60埃）的这些中间体，以在其他细胞成分的背景下确定它们的组成和位置。我们在要求的五年期间有四个具体目标。特定的目标1通过在内源表达连接蛋白细胞系中解剖“未刺激”或正常连接的连接蛋白运输途径，遵循我们对Gaietta等人（2002）的初步研究。初步研究是在HeLa细胞中进行的，Hela细胞是一种已知人为产生大量连接素的细胞系。我们正在扩大初步研究，以研究不偏振，极化和主要的细胞系，以查看将新的间隙连接通道添加到斑块边缘的机制是否是通用的。具体目标2探讨了半通道是质膜还是短暂贩运中间体的稳定部分的问题。具体的目标3集中在有丝分裂过程中发现的连接蛋白结构以及连接素的回收。具体目标4研究调查了胆固醇通过检查胆固醇耗尽剂的作用以及胆固醇在时间更高的分辨率和3-D中的胆固醇的效果，与先前发表的胆固醇的重新摄入的作用。