GJA1-20K is a new regulator of actin dynamics and myocardial cell-cell coupling

项目摘要-摘要 心脏病是美国人死亡的主要原因。在心中， 连接蛋白43（Cx43）间隙连接是健康心脏功能不可或缺的， 通过允许心肌细胞之间的离子通道来同步肌肉收缩。 Cx43由GJA 1基因在单个编码外显子中编码，但GJA 1 mRNA含有 六个内部甲硫氨酸起始位点，编码六种小的Cx43亚型， 相同的C末端GJA 1 - 20 k，20 kDa的同种型，是最丰富的 表示，肖实验室已经发现，这是必要的贩运 Cx43沿着微管从细胞核到细胞膜。 我们小组最近的工作表明，肌动蛋白细胞骨架也是 Cx43运输，既作为货物休息站，也用于适当的微管定向到 细胞膜我们还表明，GJA 1 - 20 k在HeLa细胞和 心肌细胞导致细胞中总肌动蛋白纤维和更厚的肌动蛋白纤维的增加， 稳定肌动蛋白丝对Latrunculin A治疗，但直接相互作用 GJA 1 - 20 k和肌动蛋白之间的关系从未被研究。 在目标1中，我将利用肌动蛋白动力学的无细胞TIRF成像来揭示 GJA 1 - 20 k作为肌动蛋白结合蛋白。我会用诱变技术找到结合位点 GJA 1 - 20 k和肌动蛋白之间的关系。在目标2中，我将使用分离的心肌细胞来观察 GJA 1 - 20 k介导的肌动蛋白稳定化保护gap的形成和功能 连接斑块。这些结果将揭示GJA 1 - 20 k作为一种新的肌动蛋白结合蛋白 并提供更好的理解GJA 1 - 20 k如何利用其与肌动蛋白的相互作用， 保护心脏缝隙连接斑块的形成。

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.