Endocytic Machinery Involved in Gap Junction Plaque Internalization

参与间隙连接斑块内化的内吞机制

• 批准号: 0647748
• 负责人: Sandra Murray
• 金额: $ 40.64万
• 依托单位: University of Pittsburgh
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0647748&HistoricalAwards=false
• 关键词: Endocytic; Machinery; Involved; Gap; Junction

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.

知识价值和科学影响： 间隙连接蛋白几乎存在于脊椎动物体内的每一个细胞中，因此更好地了解细胞如何维持和处理它们的间隙连接将对许多科学领域产生影响。 这些细胞聚集在细胞质膜上，在细胞与相邻细胞紧密贴壁的部位，形成高度组织化的斑块或专利通道的集合（斑块），允许小分子在相邻细胞之间移动。 通过相邻细胞之间的小分子产生的"通信"是组织稳态和生理学的重要方面。 虽然许多研究人员已经研究了在这些将细胞彼此连接的膜结构的生物发生期间调节间隙连接蛋白的生物合成、运输和组装的因素，但是很少有人研究了参与现有间隙连接斑块的内化的组分，这是细胞定量调节其细胞表面的间隙连接表达的机制。 这种内在化是一个关键的过程，但人们对其了解甚少。 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相关性（目标I）及其在间隙连接斑块和斑块片段内化调节中的作用（目标II）。 网格蛋白，衔接蛋白和间隙连接之间的关联的假设存在将通过免疫细胞化学，活细胞成像，免疫电子显微镜和结合试验技术的组合进行测试。 这一建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，间隙连接内化的监管分析。 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末端的因子可能调节整个间隙连接斑块或斑块片段内化到两个接触细胞的细胞间隙中。因此，在可以监测对间隙连接膜内化的影响的条件下，连接蛋白43蛋白的细胞内"尾"上的几个位点将被消除或破坏。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）网格蛋白将通过用一组选择性改变其活性的破坏性试剂处理来抑制; 2）连接蛋白突变和截短技术将用于修饰被认为对于结合网格蛋白相关蛋白至关重要的C末端尾区; 3）短干扰RNA（siRNA）技术将用于降低所选网格蛋白相关蛋白质的表达，并因此降低可用于与连接蛋白相互作用的蛋白质的细胞含量。将用免疫细胞化学、量子点和免疫电子显微镜分析来确定肾上腺细胞在存在或不存在这些各种处理的情况下内化间隙连接的能力。在其中将表达荧光标记的Cx43蛋白的一些实验中，将用时间推移活细胞成像技术分析差距连接内化。理解间隙连接斑块内化对于理解细胞-细胞通信以及理解间隙连接、膜调节和细胞行为之间的关系至关重要。更广泛的影响和结构性贡献：该实验室将继续作为一个重要的资源，用于培训本科生和研究生，博士后研究员，来自少数民族机构的访问教授，以及高中学生和教师（影子计划）。过去的NSF研究资金使实验室能够培养27名本科生，9名研究生，12名医学研究生，4名博士后研究员，5名来自少数民族机构的客座教授，16名高中生和2名教师。通过在国家和国际一级举办的研讨会和讲习班，已经并将继续向来自若干学科的科学界成员提供信息。