STRUCTURAL ANALYSIS OF GAP JUNCTION TRAFFICKING

缺口连接贩运的结构分析

• 批准号: 7174774
• 负责人: GINA E SOSINSKY
• 金额: $ 25.64万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7174774
• 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; Green Fluorescent Proteins; 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; Pulse taking; Qualifying; Rate; 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; intracellular protein transport; kidney cell; light microscopy; polarized cell; protein transport; reconstruction; size; 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排列的膜通道簇，连接两个相邻的细胞。该通道由来自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低聚物。GJ在几乎所有脊椎动物细胞类型的直接细胞间通讯中起重要作用。细胞通过调节GJ通道的合成、转运和周转，动态调节GJ通道的通讯。正常细胞和组织的动态平衡以及发育过程都依赖于连接蛋白的适当运输，连接蛋白的快速周转率被认为是通道功能调节的机制之一。连接蛋白疾病（如感音神经性耳聋、沙克-玛丽-图斯病、白内障）的许多突变导致异常的转运。本研究的重点是利用四胱氨酸遗传标签与双砷荧光配体、光脉冲追踪、荧光光氧化、相关光电子显微镜和电子断层扫描等技术鉴定和表征连接蛋白的运输结构，以产生细胞中选择性标记的连接蛋白的三维重建。结合使用这些技术，我们的目标是在3D中以相当高的电子层析分辨率（大约40-60埃）研究这些中间体，以确定它们在其他细胞成分背景下的组成和位置。我们在所要求的五年期间有四个具体目标。Specific Aim 1在我们对Gaietta等人（2002）的初步研究的基础上，剖析了内源性表达连接蛋白细胞系中“未受刺激的”或正常的连接蛋白运输途径。最初的研究是在HeLa细胞中进行的，这是一种已知可以人工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，以研究未极化、极化和初级的细胞系，看看在斑块边缘添加新的间隙连接通道的机制是否具有普遍性。特异性目标2探讨了半通道是质膜的稳定部分还是短期运输中间体的问题。特异性Aim 3关注有丝分裂过程中发现的连接蛋白结构，以及连接蛋白的再循环是否发生。Specific Aim 4研究了胆固醇在斑块维持中的作用，通过检查胆固醇消耗剂的作用和胆固醇的再摄取，暂时以比先前发表的更高分辨率和3d方式检查这一过程。