Cells communicate with one another through pores in the membrane called gap junction channels. Movement of molecules through the gap junction channels has been implicated in the development, differentiation and function of most cells of the body. The assembly and clustering of channels into functional gap junction plaques as well as the removal of these gap junction plaques from the cell membrane are thought to be critical to cell behavior. Gap junction plaque removal occurs by a unique internalization process in which the two opposing membranes are incorporated into the cytoplasm of one of two contacting cells, to form annular gap junction vesicles. There is strong evidence that clathrin (a protein known to be involved in endocytosis of membrane protein) and protein kinases are involved in this internalization process. In this research study, the hypothesis is that protein kinase-mediated phosphorylation of the gap junction channel protein (Connexin 43) control the recruitment of clathrin to the gap junction plaque and thus regulate gap junction plaque internalization. To test this hypothesis a combination of morphological methodologies (immunocytochemistry, live cell imaging, electron microscopy) and molecular alterations (truncations and mutations to modify critical kinase binding and/or phosphorylation sites on the gap junction protein) will be used in adrenal, uterine and ovarian cell cultures. In addition, protein kinase activity will be altered by chemical treatments, knockdown (siRNA) protocols, micro-chromophore-assisted laser inactivation of SNAP-tag proteins and infection with replication-defective adenovirus vectors. Information resulting from this study will increase our understanding of gap junction protein trafficking as well as of membrane trafficking in general. Furthermore, elucidating the mechanism of gap junction internalization will impact our capacity for targeting gap junctions for treatments to control and/or correct cell behavior.
Broader Impact The laboratory will continue to serve as a resource for the training of high school, undergraduate and graduate students, as well as visiting professors from minority institutions. Trainees will be involved in all aspects of this project and they will be encouraged to write and present their findings in the research laboratory, both to the scientific community and to the general public. In addition to obtaining instructions and experience with the tools of cell and molecular biology, students will receive training in public speaking, grant writing, manuscript preparation, laboratory operations, and career management. Through publications, seminars, lectures and workshops, presented both nationally and internationally, the research team is expected to make an impact on the scientific community. Novel methods, such as instillation art, for demonstrating processes in cell biology and for engaging and educating the general public. will continue to be developed and implemented.
