Waxse Bennett J, Sengupta Prabuddha, Hesketh Geoffrey G, Lippincott-Schwartz Jennifer, Buss Folma
Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland 20892, USA.
Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, UK.
J Cell Sci. 2017 Mar 1;130(5):827-840. doi: 10.1242/jcs.199083. Epub 2017 Jan 17.
In this study, we demonstrate myosin VI enrichment at Cx43 (also known as GJA1)-containing gap junctions (GJs) in heart tissue, primary cardiomyocytes and cell culture models. In primary cardiac tissue and in fibroblasts from the myosin VI-null mouse as well as in tissue culture cells transfected with siRNA against myosin VI, we observe reduced GJ plaque size with a concomitant reduction in intercellular communication, as shown by fluorescence recovery after photobleaching (FRAP) and a new method of selective calcein administration. Analysis of the molecular role of myosin VI in Cx43 trafficking indicates that myosin VI is dispensable for the delivery of Cx43 to the cell surface and connexon movement in the plasma membrane. Furthermore, we cannot corroborate clathrin or Dab2 localization at gap junctions and we do not observe a function for the myosin-VI-Dab2 complex in clathrin-dependent endocytosis of annular gap junctions. Instead, we found that myosin VI was localized at the edge of Cx43 plaques by using total internal reflection fluorescence (TIRF) microscopy and use FRAP to identify a plaque accretion defect as the primary manifestation of myosin VI loss in Cx43 homeostasis. A fuller understanding of this derangement may explain the cardiomyopathy or gliosis associated with the loss of myosin VI.
在本研究中，我们证明了肌球蛋白VI在心脏组织、原代心肌细胞和细胞培养模型中富含连接蛋白43（也称为GJA1）的间隙连接（GJ）处的富集情况。在原代心脏组织、肌球蛋白VI基因敲除小鼠的成纤维细胞以及用针对肌球蛋白VI的小干扰RNA转染的组织培养细胞中，我们观察到间隙连接斑块大小减小，同时细胞间通讯减少，这通过光漂白后的荧光恢复（FRAP）和一种新的选择性钙黄绿素给药方法得以证明。对肌球蛋白VI在连接蛋白43运输中的分子作用分析表明，肌球蛋白VI对于连接蛋白43向细胞表面的递送以及质膜中连接子的移动并非必需。此外，我们无法证实网格蛋白或Dab2在间隙连接处的定位，并且在环形间隙连接的网格蛋白依赖性内吞作用中未观察到肌球蛋白VI - Dab2复合物的功能。相反，我们发现通过全内反射荧光（TIRF）显微镜观察到肌球蛋白VI定位于连接蛋白43斑块的边缘，并使用FRAP来确定斑块积聚缺陷是肌球蛋白VI缺失在连接蛋白43稳态中的主要表现。对这种紊乱的更全面理解可能解释与肌球蛋白VI缺失相关的心肌病或胶质增生。
