STRUCTURAL ANALYSIS OF GAP JUNCTION TRAFFICKING

描述(申请人提供)：缝隙连接(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。该通道由来自每个细胞的两个六聚体组成，并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细胞间直接通讯中发挥重要作用。细胞通过调节这些通道的合成、运输和周转来动态地调节GJ的通讯。正常的细胞和组织的动态平衡以及发育过程依赖于连接蛋白的正确运输，连接蛋白的快速周转被认为是通道功能调节的机制之一。连接蛋白疾病中的许多突变(例如，感觉神经性耳聋、夏科-玛丽-图斯病、白内障)会导致不正常的贩运。本研究的重点是利用四半胱氨酸基因标签与生物砷荧光配体络合、光脉冲追逐、荧光光氧化、相关光电子显微镜和电子断层扫描等技术来鉴定和表征连接蛋白转运结构，以产生细胞内选择性标记的连接蛋白的三维重建。结合使用这些技术，我们的目标是以合理的高电子断层扫描分辨率(大约40-60埃)在3D中研究这些中间体，以确定它们的组成和在其他细胞成分的背景下的位置。对于所要求的五年期间，我们有四个具体目标。具体目标1是我们对Gaietta等人(2002)最初研究的后续工作，通过解剖内源性表达连接蛋白的细胞系中的“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，这是一种已知能产生大量人工连接蛋白的细胞系。我们正在扩大我们的初步研究，以研究未极化、极化和原发的细胞系，以了解在斑块边缘添加新的缝隙连接通道的机制是否具有普遍性。具体目标2探讨半沟膜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是否发生循环。具体目的4研究胆固醇在斑块维持中的作用，通过检测胆固醇去除剂的效果和胆固醇的重新摄取，以比先前发表的更高的分辨率和3-D的时间检测这一过程。

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.