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

GJA1-20K是肌动蛋白动力学和心肌细胞-细胞耦合的新调节因子

• 批准号: 9922119
• 负责人: Rachel Baum
• 金额: $ 4.55万
• 依托单位: CEDARS-SINAI MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9922119
• 关键词: Actin-Binding Protein; Actins; Adult; Affect; Arrhythmia; Binding; Binding Sites; Cardiac; Cardiac Myocytes; Cause of Death; Cell Nucleus; Cell membrane; Cells; Cessation of life; Code; Connexin 43; Connexins; Coupling; Cytoskeleton; Data; Exons; Fiber; G Actin; Gap Junctions; Generations; Genes; Goals; Heart; Heart Contractilities; Heart Diseases; Hela Cells; Image; Incubated; Ions; Ischemia; Laboratories; Mediating; Messenger RNA; Methionine; Methods; Microfilaments; Microtubules; Molecular; Muscle Contraction; Mutagenesis; Myocardial; Myocardial Contraction; Myocardial Ischemia; Myocardium; Play; Polymers; Process; Protein Isoforms; Proteins; Publications; Rest; Role; Site; Stress; Tail; Testing; Thick; Translating; United States; Work; base; cellular imaging; domain mapping; heart function; improved; latrunculin A; novel; overexpression; polymerization; pre-doctoral; prevent; trafficking

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.

项目摘要-摘要 心脏病是美国人死亡的主要原因。在内心深处， 连接蛋白43(Cx43)缝隙连接对健康的心脏功能和 通过允许心肌细胞之间的离子通道来同步肌肉收缩。 Cx43由单个编码外显子的GJA1基因编码，但GJA1基因的mRNA包含 6个内部蛋氨酸起始点，编码Cx43的6个小亚型 同样的C-末端尾巴。GJA1-20K是含量最丰富的20 kDa亚型 表示，Shaw实验室发现它是人口贩运所必需的 Cx43从细胞核沿微管进入细胞膜。 我们小组最近的研究表明，肌动蛋白细胞骨架也是 Cx43贩运，无论是在货物停靠站还是为了适当的微管定向到 细胞膜。我们还发现GJA1-20k在HeLa细胞和 心肌细胞导致细胞内肌动蛋白纤维总量增加和增厚 稳定肌动蛋白细丝以对抗Latrunculin A处理，但直接相互作用 GJA1-20K和肌动蛋白之间的关系从未被研究过。 在目标1中，我将利用肌动蛋白动力学的无细胞TIRF成像来揭示其作用 GJA1-20k为肌动蛋白结合蛋白。我会用诱变技术找到结合部位 在GJA1-20K和肌动蛋白之间。在目标2中，我将使用分离的心肌细胞来了解 GJA1-20K介导的肌动蛋白稳定保护GAP的形成和功能 接合处的斑块。这些结果将揭示GJA1-20k是一种新的肌动蛋白结合蛋白 并更好地了解GJA1-20k如何利用其与肌动蛋白的相互作用来 保护心脏缝隙连接斑块的形成。