STRUCTURAL ANALYSIS OF GAP JUNCTION TRAFFICKING

描述（由申请人提供）：间隙连接（GJ）定义为含有连接蛋白的紧密包装的膜通道簇，连接两个相邻细胞。通道由来自每个细胞的两个六聚体组成，并且含有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的直接细胞间通讯中起重要作用。细胞通过调节这些通道的合成、运输和周转来动态地调节通过GJ的通信。正常细胞和组织的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已被假设为通道功能调节的机制之一。连接蛋白疾病（例如感觉神经性耳聋、腓骨肌萎缩症、白内障）中的许多突变导致异常运输。该建议的重点是识别和表征的连接蛋白的运输结构，使用技术的四半胱氨酸基因标签与双砷荧光配体，光学脉冲追逐，荧光光氧化，相关的光和电子显微镜和电子断层扫描，以产生选择性标记的连接蛋白在细胞中的三维重建。结合使用这些技术，我们的目标是在3D中以合理的高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组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。具体目标1跟进了我们对Gaietta等人的初步研究，（2002）通过剖析内源性表达连接蛋白细胞系中的“未刺激的”或正常连接蛋白运输途径。最初的研究是在HeLa细胞中进行的，HeLa细胞是一种已知能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，以调查非极化，极化和主要的细胞系，看看是否增加新的缝隙连接通道的斑块边缘的机制是一个普遍的。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集中在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。具体目标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，以比先前发表的更高的分辨率和3-D暂时检查这一过程。

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.