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产生直接的细胞间通信。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。这些细胞在静止(非有丝分裂)阶段形成离散的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、运输、门控和周转来动态地调节缝隙连接通讯。目前的教科书描述将缝隙连接描述为静态的、独立的结构，现在被连接蛋白、半通道、通道和缝隙连接的新范例所取代，这些连接连接蛋白、半通道和缝隙连接是非常机动、动态和交互的组件。蛋白质磷酸化是蛋白质控制细胞功能和/或定位的重要调节机制，被称为空间细胞生物学。缝隙连接蛋白Cx43有一个高度调控的生命周期，在这个生命周期中，在其C末端的几个特定的丝氨酸残基上会发生几个分级的磷酸化事件。不同的磷酸化事件发生在细胞周期的所有阶段，并可以改变哪些蛋白与连接蛋白43相互作用，以细胞周期阶段特有的方式改变连接蛋白43的运输、组装、门控和周转的动力学和/或定位，从而影响重要的生物过程，如细胞迁移和增殖。该项目致力于成像连接蛋白43磷酸化、其细胞定位和细胞周期之间的优雅相互作用。这项研究的三个具体目标是：(1)确定某些激酶是否在其生命周期的特定阶段(S)与连接蛋白43形成复合体；(2)将特定丝氨酸残基的磷酸化与其单个和连续的细胞位置相关联；以及(3)阐明特定的磷酸化事件如何与细胞周期中的细胞定位有关。这项建议侧重于使用活细胞成像、相关的光和电子断层扫描和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三维重建。开发和应用用于相关光学和电子显微镜的多种探针的方法是成功成像贩运中间体的关键。结合对野生型和突变型Cx43蛋白的生化和抑制分析，总体目标是在电子断层扫描分辨率(~40-60E)的3D下研究这些磷酸形式，以确定它们在其他细胞成分的背景下的形态和位置。通过这些研究，我们将通过先进的成像技术来了解连接蛋白的磷酸化如何控制静止细胞和细胞周期中缝隙连接通讯依赖的功能。这项研究的重要性是因为连接蛋白定位和缝隙连接通讯的变化是细胞增殖、迁移和在癌变过程中失去生长控制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.