Abstract 16815: GJA1-20k, a Truncated Isoform of Connexin 43, Nucleates De Novo Actin Clusters

Abstract

Introduction: Connexin 43 (Cx43) gap junctions allow rapid communication of signals between cardiomyocytes. Loss of Cx43 gap junction coupling contributes to arrhythmogenesis. GJA1-20k, a truncated internally translated isoform of the Cx43 gene GJA1 , is necessary for full length Cx43 trafficking to intercalated discs for gap junction formation. GJA1-20k stabilizes actin filaments, necessary for the Cx43 delivery highway. The mechanism of GJA1-20k interaction with actin, while essential for gap junction formation, is still not known. Methods: Cell free individual actin dynamics were observed by TIRF microscopy. For real time imaging of actin nucleation and polymerization, glass bottom chamber slides were pre-incubated with 100nM N-ethylmaleimide myosin and loaded with Alexa Fluor 488-conjugated actin (Thermo Fisher) with GJA1-20k in ATP containing buffer. Actin binding assays were performed using a spin-down assay kit (Cytoskeleton, Inc.). Actin polymerization was studied by using pyrene labeled actin (Cytoskeleton, Inc.). Results: Cell free biochemical binding assays show that GJA1-20k binds to G-actin molecules, nucleating de novo actin clusters yet also surprisingly preventing G-actin polymerization. Pyrene actin assays further confirm that GJA1-20k alone does not promote polymerization. Cell free TIRF imaging also reveals that GJA1-20k promotes the formation of de novo G-actin clusters (Figure 1). Once a critical degree of nucleation is present however, polymerization occurs at a rate that promotes the formation of thicker actin fibers. Because GJA1-20k preferentially supports nucleation over polymerization, GJA1-20k introduces novel foci of new actin growth that, when a critical mass is present, rapidly overflows to polymerization. Conclusions: GJA1-20k nucleates de novo actin clusters, and thus is able to seed novel cytoskeleton growth patterns, dictating the direction of gap junction delivery.