Endocytic Machinery Involved in Gap Junction Plaque Internalization

University Of Pittsburgh, Pittsburgh PA

Investigators

Abstract

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.