Neighboring cells need to communicate with one another in order to function properly, maintain proper organization of tissues, and make appropriate responses to changes in their environments. One method of communication involves the direct flow of information through membrane channels that are clustered on the surface of adjacent cells. These clusters of channels are called gap junction plaques and they are composed of proteins called connexin. The removal of gap junction plaques, from the cell surface, is critical to maintaining cell-to-cell communication and impacts numerous cellular functions, including proliferation, cell migration and differentiation. This project will elucidate the needed steps in gap junction plaque removal and determine the underlying molecular mechanisms. Increased information on annular gap junction processing and degradation could facilitate the development of new techniques to influence and control cell behavior, including proliferation and hormonal response. The PI will also continue to serve as a resource for the training of undergraduates, graduate students, and visiting professors from minority institutions. She used past NSF research funding to train forty-five undergraduates, eight graduate students, three postdoctoral fellows, seven visiting professors from minority institutions, five high school students, and ten medical students. Most of the students participating in this research over the years have been African-Americans, a severely under-represented group in science. She will also continue her broader outreach through publications, seminars, lectures and workshops for the scientific community and a unique outreach to the general public through art displays and lectures at a local art museum.
This project will investigate the mechanisms regulating annular gap junction processing. The PI hypothesizes that protein kinases via phosphorylation of sites on the C-terminal tail of the gap junction channel protein, connexin 43 (Cx43), play a pivotal role in annular gap junction vesicle processing needed for degradation. Such processing involves annular gap junction fissions as well as the formation of autophagasomes and multivesicular endosomes. Adrenal cortical cell populations will be used to elucidate the role of kinase-mediated Cx43 phosphorylation in annular gap junction processing and degradation. The approach will be to manipulate three kinases [protein kinase C (PKC), Src, cAMP-dependent protein kinase (PKA)] known to phosphorylate Cx43 and thought to be involved in annular gap junction degradation. In Objective I, To Measure the Annular Gap Junction Vesicle Degradative Process in Cell Populations in Which PKA, PKC, and Src Activities Have Been Altered. Three complementary methodologies will be used to selectively decrease or increase protein kinase activity: 1) chemical treatments with a panel of agents; 2) knockdown (siRNA) protocols; and 3) infection with replication-defective adenovirus vectors which result in constitutively activated kinases. In Objective II To Measure Annular Gap Junction Vesicle Degradative Process in Cells in Which the Cx43 Phosphorylation Sites Have Been Altered. Connexin mutation and truncation techniques will be used to modify the region of the intracellular "tail" of the Cx43 protein thought to be critical for kinase binding and/or phosphorylation. Throughout this study, annular gap junction processing (fission, formation of autophagasomes and multivesicular endosomes and fusion with other organelles (lysosomes, endosomes and plasma membranes [possible recycling of Cx43 to the plasma membrane]) will be investigated with immunocytochemical, quantum dot immuno-electron microscopy, western blot, and time lapse image analysis. Phospho-specific antibodies against the targeted sites are available and will be used to image the spacial and temporal distribution as well as protein amounts of the differing Cx43phosphoforms.The laboratory will continue to serve as a resource for the training of undergraduates, graduate students, and visiting professors from minority institutions. Students will be given a specific project and they will be encouraged to write and present their findings, both to the scientific community and to the general public. The PI will continue to make an impact on the scientific community through publications, seminars, lectures and workshops presented both nationally and internationally. In addition, lectures and installation art displays created, by the PI and displayed at a local art museum, will enable the PI to teach the general public about dynamic cellular events. It is hoped that information gained from this study and the training provided will serve to advance science and thus contribute to public welfare.
