STRUCTURAL ANALYSIS OF GAP JUNCTION TRAFFICKING

描述（由申请人提供）：缝隙连接在大多数细胞类型中创建直接的细胞-细胞通信。这些膜特化包括来自两个相对细胞的一个质膜和跨越两个膜的数十到数千个十二聚体连接蛋白通道。这些在静止（非有丝分裂）阶段形成离散和可识别的细胞结构。细胞通过调节这些连接蛋白通道的合成、转运、门控和周转来动态调节间隙连接通讯。目前的教科书将间隙连接描述为静态的、独立的结构，现在被连接蛋白、半通道、通道和间隙连接的新范式所取代，它们是非常移动的、动态的和相互作用的组合。蛋白质磷酸化是一种重要的调节机制，通过该机制蛋白质可以控制细胞功能和/或定位，这一过程最近被称为“空间细胞生物学”。间隙连接蛋白connexin43具有高度调控的生命周期，在此过程中，在其c端的几个特定丝氨酸残基上发生了几个分层磷酸化事件。不同的磷酸化事件发生在细胞周期的各个阶段，可以改变哪些蛋白质与connexin43相互作用，connexin43运输、组装、门控和周转的动力学和/或定位，以细胞周期阶段特定的方式影响重要的生物过程，如细胞迁移和增殖。该项目的重点是成像连接蛋白43磷酸化，其细胞定位和细胞周期之间的优雅相互作用。本研究的三个具体目的是：(1)确定某些激酶是否在其生命周期的特定阶段与connexin43形成复合物；(2)将特定丝氨酸残基的磷酸化与其细胞位置单独或串联联系起来；(3)阐明在细胞周期中特定的磷酸化事件是如何与细胞定位相关联的。该建议的重点是使用活细胞成像，相关光和电子断层扫描与蛋白质标签或探针来识别和表征连接蛋白运输结构，以产生细胞中选择性标记的连接蛋白的3D重建。开发和应用多探针相关光和电子显微镜的方法是必不可少的成像贩运中间体的成功。结合野生型和突变型Cx43蛋白的生化和抑制剂分析，总体目标是在3D电子断层扫描分辨率（~40-60 E）下研究这些磷酸化形式，以确定它们在其他细胞成分背景下的形态和位置。从这些研究中，我们将通过先进的成像技术了解连接蛋白磷酸化如何控制静止细胞和细胞周期中间隙连接依赖通讯的功能。这项研究的重要性在于，在癌变过程中，连接蛋白定位和间隙连接通讯的变化是细胞增殖、迁移和生长控制丧失的精细控制的一部分。

DESCRIPTION (provided by applicant): Gap junctions create direct cell-cell communication in most cell types. These membrane specializations contain one plasma membrane from two apposing cells and tens to thousands of dodecameric connexin channels spanning the two membranes. These form discrete and recognizable cellular structures during quiescent (non-mitotic) phases. Cells dynamically modulate gap junctional communication by regulating the synthesis, transport, gating and turnover of these connexin channels. Current textbook descriptions depicting gap junctions as static, stand-alone structures now are replaced with a new paradigm of connexins, hemichannels, channels and gap junctions as very mobile, dynamic and interactive assemblies. Protein phosphorylation is an important regulatory mechanism by which proteins can control cellular function and/or localization in a process newly termed "spatial cell biology". The gap junction protein, connexin43, has a highly regulated life cycle during which several, hierarchical phosphorylation events occur at several specific serine residues in its C-terminus. Different phosphorylation events occur during all stages of the cell cycle and can change which proteins interact with connexin43, the kinetics and/or localization of connexin43 trafficking, assembly, gating, and turnover in a cell cycle stage specific manner that affects important biological processes such as cell migration and proliferation. This project is focused on imaging the elegant interplay between connexin43 phosphorylation, its cellular localization and the cell cycle. The three specific aims of this proposed research are: (1) determine whether certain kinases form complexes with connexin43 at particular stage(s) of its life cycle; (2) to correlate the phosphorylation of specific serine residues with their cellular location singly and in tandem; and (3) to elucidate how specific phosphorylation events are linked to cellular localization during the cell cycle. This proposal focuses on the identification and characterization of connexin trafficking structures using live cell imaging, correlative light and electron tomography with protein tags or probes to produce 3D reconstructions of selectively labeled connexins in cells. Methods for developing and applying multiple probes for correlated light and electron microscopy are essential to the success in imaging trafficking intermediates. In combination with biochemical and inhibitor analyses of wild type and mutant Cx43 proteins, the overall goal is to study these phospho-forms at electron tomographic resolution (~40-60 E) in 3D to determine their morphologies and locations within the context of other cellular components. From these studies, we will gain a mechanistic understanding through advanced imaging how connexin phosphorylation in controls gap junction communication-dependent functions in quiescent cells and during the cell cycle. The importance of this research is driven by the fact that changes in connexin localization and gap junctional communication are part the exquisite control of cellular proliferation, migration and with a loss of growth control during carcinogenesis. PUBLIC HEALTH RELEVANCE: Project Narrative Direct cell-cell communication as mediated by gap junctions has been shown repeatedly to be a necessary component of homeostasis and is highly regulated during the cell cycle, developmental processes and cell proliferation. Connexin diseases result when gap junction proteins mis-traffic or mis-function and loss of gap junction intercellular communication is concomitant with carcinogenesis. We investigate the connexin43 trafficking process using an imaging based approach examining the hierarchy of connexin43 phosphorylation events and where within the cell cycle, connexin43-kinase(s) interactions occurs.