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）被定义为包含连接蛋白的紧密堆积的膜通道簇，连接两个相邻的细胞。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蛋白的寡聚体。 GJ 在几乎所有脊椎动物细胞类型中的直接细胞间通讯中发挥着重要作用。细胞通过 GJ 调节这些通道的合成、运输和周转来动态调节通讯。正常细胞和组织稳态以及发育过程依赖于连接蛋白的适当运输，并且连接蛋白的快速周转率被假设为通道功能调节的一种机制。连接蛋白疾病（例如感音神经性耳聋、腓骨肌萎缩症、白内障）的许多突变都会导致异常运输。该提案的重点是使用四半胱氨酸遗传标签与双砷荧光配体复合、光学脉冲追踪、荧光光氧化、相关光学和电子显微镜以及电子断层扫描技术来识别和表征连接蛋白运输结构，以产生选择性标记的连接蛋白的 3D 重建。细胞。结合使用这些技术，我们的目标是在 3D 中以相当高的电子断层扫描分辨率（约 40-60 埃）研究这些中间体，以确定它们在其他细胞成分中的组成和位置。我们在所要求的五年期间有四个具体目标。具体目标 1 是我们对 Gaietta 等人 (2002) 的初步研究的后续，剖析了内源表达连接蛋白细胞系中的“未刺激”或正常连接蛋白运输途径。最初的研究是在海拉细胞中进行的，海拉细胞是一种已知能人为产生大量连接蛋白的细胞系。我们正在扩大我们的初步研究，以调查非极化、极化和原代细胞系，看看在斑块边缘添加新间隙连接通道的机制是否是通用的。具体目标 2 探讨了半通道是质膜的稳定部分还是短暂的运输中间体的问题。具体目标 3 重点关注有丝分裂过程中发现的连接蛋白结构以及是否发生连接蛋白的回收。具体目标 4 通过检查胆固醇消耗剂的作用和胆固醇的再摄取，以比之前发表的更高的分辨率和 3D 形式暂时检查这一过程，从而研究胆固醇在斑块维持中的作用。