Recycling: An Alternative Method for Rapid Gap Junction Plaque Assembly

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Gap junctions are cellular channels that are critical for cell-to-cell communication. The main component of gap junctions are the connexin proteins. This Project is expected to describe a new mechanism by which gap junctions assemble at the surface of the cell. This Project will determine the extent that connexins can be recycled and re-used in the formation of new functional gap junction channels. It will also identify the molecular mechanisms that regulate connexin recycling versus degradation. Together, this will provide a better understanding of the turnover and trafficking of connexins. This knowledge will be useful for understanding the numerous cellular phenomena in which cell-cell communication is critical. Experimentally collected data will also be used to develop software that will mathematically predict how the modification of connexins determines their biological functions, including movement to different cytoplasmic compartments. The Broader Impacts of the Project are three-fold, broadening participation in science, creating novel science-oriented art exhibits, and providing research training. Participation in science will be strengthened by providing an opportunity for individuals from the University of Pittsburgh, smaller colleges from extended rural areas surrounding Pittsburgh, and minority serving institutions to participate in innovative research opportunities, to which they would not otherwise have access. Training will be provided to researchers at all levels, from undergraduates and graduate students to postdoctoral fellows and visiting professors. They will gain hands on training in the use of cutting edge imaging and mathematical modeling tools to study protein dynamics, recycling and degradation. Scientific and computational workshops, where students will actively use modeling software to analyze experimental morphological data, will lead to novel insights, as well as interesting training and career paths. In addition, students will be directed in the production of installation art depicting protein dynamics and regulated cell behavior. These art exhibits will be presented at workshops, in the community, and at local art museums. These exhibits are intended to encourage career paths in science, technology, engineering, and math (STEM). In addition, they will introduce and familiarize the public with the world of research, highlighting the beauty and intricacy of living cells. The major protein subunits of gap junctions are the connexin proteins, the most abundant of which is connexin 43. The Project will test the hypothesis that recycling of connexin 43 contributes to the formation of new gap junction channels. Specifically, the first Aim of this proposal will determine the dynamics of connexin 43 after endocytosis in multiple cell types. In the second Aim, the hypothesis will be tested that endocytosis and recycling of connexins can be regulated by multiple kinases and phosphatases. To test these hypotheses, the PI will use biochemistry, confocal microscopy, morphological, and cell biological methods, in combination with phosphor-mimetics and pharmacological activators. Aim 3 will use the dynamic parameters for endocytosis, recycling and degradation to generate a differential rate-equation model that will characterize and predict details of connexin trafficking. Information gained from this study and the training provided will advance the science of cell-cell communication that is ultimately needed to understand complex physiological processes, and thus contribute to public welfare.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.