PROJECT SUMMARY – ABSTRACT Heart disease is the leading cause of the death in the United States. In the heart, Connexin 43 (Cx43) gap junctions are integral to healthy heart function and synchronized muscle contraction by allowing ion passage between cardiomyocytes. Cx43 is encoded by the GJA1 gene in a single coding exon, but GJA1 mRNA contains six internal methionine start sites, which encode six small isoforms of Cx43 that retain the same C-terminus tail. GJA1-20k, the 20 kDa isoform, is the most abundantly expressed, and the Shaw laboratory has discovered that it is necessary for trafficking Cx43 along microtubules from the nucleus to the cell membrane. Recent work from our group shows that the actin cytoskeleton is also required for Cx43 trafficking, both as cargo rest stops and for proper microtubule orientation to the cell membrane. We have also shown that GJA1-20k expression in both HeLa cells and cardiomyocytes results in an increase in total and thicker actin fibers in the cell and stabilizes actin filaments against Latrunculin A treatment, but a direct interaction between GJA1-20k and actin has never been studied. In Aim 1, I will utilize cell free TIRF imaging of actin dynamics to uncover the role of GJA1-20k as an actin binding protein. I will use mutagenesis to find the binding site between GJA1-20k and actin. In Aim 2, I will use isolated cardiomyocytes to see how GJA1-20k mediated actin stabilization protects the formation and function of gap junction plaques. These results will uncover GJA1-20k as a novel actin binding protein and provide better understanding of how GJA1-20k utilizes its interaction with actin to protect gap junction plaque formation in the heart.