STRUCTURAL ANALYSIS OF GAP JUNCTION TRAFFICKING

缺口连接贩运的结构分析

• 批准号: 8208073
• 负责人: GINA E SOSINSKY
• 金额: $ 32.5万
• 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 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8208073
• 关键词: Address; Affect; Amino Acids; Antibodies; Binding; Biochemical; Biochemistry; Biological; Biological Process; Biology; Brain; CDC2 Protein Kinase; CSNK1A1 gene; Cataract; Cell Communication; Cell Cycle; Cell Cycle Regulation; Cell Cycle Stage; Cell Proliferation; Cell Surface Proteins; Cell membrane; Cell physiology; Cells; Cellular Structures; Cellular biology; Communication; Complex; Connexin 43; Connexins; Cyclin B; Development; Developmental Process; Disease; Electron Microscopy; Electrons; Endothelium; Event; Functional disorder; Gap Junctions; Goals; Growth; Growth and Development function; Heart; Homeostasis; Image; Imagery; Individual; Investigation; Ion Channel; Kinetics; Label; Life; Life Cycle Stages; Light; Link; Location; MAP Kinase Gene; Mediating; Membrane; Membrane Protein Traffic; Methods; Mitosis; Morphology; Mutation; Neuropathy; Optics; Organelles; Organism; Outcome; Phase; Phospho-Specific Antibodies; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiologic pulse; Play; Population; Process; Protein Isoforms; Proteins; Recycling; Research; Resolution; Role; Serine; Signal Transduction; Site; Site-Directed Mutagenesis; Staging; Structure; Testing; Textbooks; Time; Tissues; Work; base; carcinogenesis; cell growth; cell motility; cell type; cellular imaging; deafness; electron tomography; gap junction channel; imaging modality; inhibitor/antagonist; innovation; insight; intercellular communication; light microscopy; loss of function; migration; mutant; novel; programs; public health relevance; reconstruction; research study; skin disorder; spatiotemporal; success; trafficking

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.

描述（由申请人提供）：间隙连接在大多数细胞类型中产生直接的细胞间通讯。这些膜特化包含来自两个并列细胞的一个质膜和跨越两个膜的数十至数千个十二聚体连接蛋白通道。这些在静止期（非有丝分裂）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、门控和转换来动态调节间隙连接通讯。目前的教科书描述描绘间隙连接作为静态的，独立的结构，现在被替换为连接蛋白，半通道，通道和间隙连接作为非常移动的，动态和互动的组件的新的范例。 蛋白质磷酸化是一种重要的调控机制，蛋白质可以通过这种机制来控制细胞功能和/或定位，这一过程被称为“空间细胞生物学”。差距连接蛋白，连接蛋白43，具有高度调节的生命周期，在此期间，在其C-末端的几个特定丝氨酸残基处发生几个分级磷酸化事件。不同的磷酸化事件发生在细胞周期的所有阶段，并且可以改变哪些蛋白质与连接蛋白43相互作用，连接蛋白43以细胞周期阶段特异性方式运输、组装、门控和周转的动力学和/或定位，其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的相互作用。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与连接蛋白43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关联起来;（3）阐明特定磷酸化事件如何与细胞周期中的细胞定位相关联。 该建议的重点是使用活细胞成像，相关的光和电子断层扫描与蛋白质标签或探针，以产生选择性标记的连接蛋白在细胞中的三维重建的连接蛋白运输结构的识别和表征。开发和应用相关的光学和电子显微镜的多个探针的方法是必不可少的成功成像贩运中间体。结合野生型和突变型Cx43蛋白的生物化学和抑制剂分析，总体目标是在3D中以电子断层扫描分辨率（~40-60 E）研究这些磷酸形式，以确定它们在其他细胞组分背景下的形态和位置。从这些研究中，我们将获得一个机制的理解，通过先进的成像如何连接蛋白磷酸化控制间隙连接通讯依赖的功能，在静止细胞和细胞周期。这项研究的重要性是由这样一个事实驱动的，即连接蛋白定位和间隙连接通讯的变化是细胞增殖，迁移和癌发生过程中生长控制丧失的精密控制的一部分。 公共卫生相关性：项目叙述由间隙连接介导的直接细胞间通讯已多次被证明是体内平衡的必要组成部分，并且在细胞周期、发育过程和细胞增殖期间受到高度调节。间隙连接蛋白疾病是由于间隙连接蛋白的错误运输或错误功能以及间隙连接细胞间通讯的丧失伴随着癌症发生而引起的。我们调查的connexin 43运输过程中使用成像为基础的方法检查connexin 43磷酸化事件的层次结构，在细胞周期内，connexin 43激酶（S）的相互作用发生。