Endocytic Machinery Involved in Gap Junction Plaque Internalization

知识价值和科学影响：缝隙连接蛋白几乎存在于脊椎动物身体的每个细胞中，因此更好地了解细胞如何维持和处理它们的缝隙连接将对许多科学领域产生影响。它们聚集在细胞的质膜上，在细胞与邻近细胞紧密相对的地方，形成高度组织的斑块或专利通道集合（斑块），允许小分子在邻近细胞之间运动。由此产生的通过小分子在相邻细胞之间的“通信”是组织稳态和生理学的一个重要方面。虽然许多研究者已经研究了在这些连接细胞的膜结构的生物发生过程中调节间隙连接蛋白的生物合成、运输和组装的因素，但很少有研究涉及现有间隙连接斑块内化的成分，这是细胞定量调节其细胞表面间隙连接表达的机制。这种内化是一个非常关键但却很难理解的过程。在这项研究中，Murray博士将使用肾上腺细胞作为研究间隙连接内在化的模型系统。本项目的目的是验证缝隙连接斑块内化是由网格蛋白及其相关蛋白介导的假设。Murray博士将研究网格蛋白和网格蛋白相关蛋白的关联（目的一）以及它们在间隙连接斑块和斑块片段内化的调节中的作用（目的二）。通过结合免疫细胞化学、活细胞成像、免疫电子显微镜和结合分析技术，将对网格蛋白、衔接蛋白和间隙连接之间存在的关联进行假设测试。这个提议的一个新颖和创新的组成部分是应用“量子点”可视化技术，在其他实验室开发的，以分析间隙结内在化的调节。Murray博士的方法还将采用一系列不同的程序来消除或减少网格蛋白或网格蛋白相关蛋白与肾上腺细胞的主要连接蛋白连接蛋白43 （Cx43）的相互作用，以评估这种操作对间隙连接内化的影响。Murray博士进一步假设，作用于Cx43 c端的因子可能调节整个间隙连接斑块或斑块碎片进入两个接触细胞的细胞质的内在化。因此，连接蛋白43的细胞内“尾巴”上的几个位点将被消除或破坏，在可以监测对间隙连接膜内化的影响的条件下。将使用三种方法来选择性地减少或消除网格蛋白或相关蛋白与Cx43的相互作用：1)通过一组选择性改变其活性的破坏剂来抑制网格蛋白；2)连接蛋白突变和截断技术将用于修饰c端尾部区域，该区域被认为是与网格蛋白相关蛋白结合的关键区域；3)短干扰RNA （siRNA）技术将用于降低所选网格蛋白相关蛋白的表达，从而降低可与连接蛋白相互作用的蛋白质的细胞含量。在存在或不存在这些不同治疗的情况下，肾上腺细胞内化间隙连接的能力将通过免疫细胞化学、量子点和免疫电镜分析来确定。在一些实验中，荧光标记的Cx43蛋白将被表达，用延时活细胞成像技术分析间隙连接的内化。对间隙连接斑块内化的理解对于理解细胞间通讯以及间隙连接、膜调节和细胞行为之间的关系至关重要。更广泛的影响和基础设施的贡献：该实验室将继续作为一个重要的资源，为本科生和研究生，博士后研究员，少数民族机构的客座教授，以及高中学生和教师（影子计划）的培训。过去的NSF研究经费使该实验室培养了27名本科生、9名研究生、12名医学研究生、4名博士后、5名少数民族院校的客座教授、16名高中生和2名教师。通过在国内和国际上举办的研讨会和讲习班，已经并将继续向科学界成员提供来自若干学科的信息。

Intellectual Merit and Scientific Impact: Gap junction proteins are found in almost every cell of the vertebrate body, and therefore a better understanding of how cells maintain and process their gap junctions would have an impact on a multitude of scientific fields. These assemble at the cell's plasma membrane at sites where the cells are closely apposed to neighboring cells, to form highly organized patches or collections (plaques) of patent channels that allow the movement of small molecules between the neighboring cells. The resulting "communication" via small molecules between neighboring cells is an important aspect of tissue homeostasis and physiology. While many investigators have studied factors that regulate gap junction protein biosynthesis, trafficking and assembly during the biogenesis of these membrane structures that connect cells to one another, very few have studied the components involved in the internalization of existing gap junction plaques, which is a mechanism whereby cells quantitatively regulate gap junctional expression at their cell surfaces. This internalization is a critical but poorly understood process. In this study, Dr. Murray will use adrenal cells as a model system for studying gap junction internalization. The goal of this project is to test the hypothesis that gap junction plaque internalization is mediated by clathrin and its associated proteins. Dr. Murray will investigate the association of clathrin and clathrin-associated proteins (Objective I) and their role in the regulation of gap junction plaque and plaque fragment internalization (Objective II). The hypothesized existence of associations between clathrin, adaptor proteins and gap junctions will be tested through a combination of immunocytochemistry, live cell imaging, immuno-electron microscopy and binding assay techniques. A novel and innovative component of this proposal is the application of the "quantum dot" visualization technique, developed in other laboratories, to the analysis of the regulation of gap junction internalization. Dr. Murray's approach will also employ a battery of different procedures to eliminate or reduce the interactions of clathrin or clathrin-associated proteins with the major connexin of adrenal cells, connexin 43 (Cx43), in order to assess the effect of such manipulations on gap junction internalization. Dr. Murray further hypothesizes that factors acting at the C-terminus of Cx43 may regulate internalization of the entire gap junction plaque or plaque fragments into the cytoplasms of two contacting cells. Therefore, several sites on the intracellular "tail" of the connexin 43 protein will be eliminated or disrupted under conditions where effects on gap junction membrane internalization can be monitored. Three methodologies will be used to selectively decrease or eliminate the interaction of clathrin or associated protein with Cx43: 1) Clathrin will be inhibited by treatment with a panel of disruptive agents that selectively alter its activities; 2) connexin mutation and truncation techniques will be used to modify the C-terminal tail region thought to be critical for binding to clathrin-associated proteins; 3) short-interfering RNA (siRNA) techniques will be used to decrease the selected clathrin associated protein expression, and thus the cellular content of the protein available to interact with connexin. The capacity of adrenal cells to internalize gap junctions in the presence or absence of these various treatments will be determined with immunocytochemical, quantum dot and immunoelectron microscopic analysis. In some experiments, in which fluorescently tagged Cx43 protein will be expressed, the gap junction internalization will be analyzed with time lapse live cell imaging techniques. An understanding of gap junction plaque internalization is critical to an understanding of cell-cell communication in general, as well as to an understanding of the relationship between gap junctions, membrane regulation and cell behaviors. Broader Impact and Infrastructural Contributions:The laboratory will continue to serve as a significant resource for the training of undergraduate and graduate students, postdoctoral fellows, and visiting professors from minority institutions, and for high school students and teachers (shadow program). Past NSF research funding has enabled the laboratory to train twenty seven undergraduates, nine graduate students, twelve medical research students, four postdoctoral fellows, five visiting professors from minority institutions, and sixteen high school students and two teachers. Through seminars and workshops presented both nationally and internationally, information has been, and will continue to be, provided to members of the scientific community from a number of disciplines.