University of California San Francisco, Cardiovascular Research Institute, 555 Mission Bay Boulevard South, San Francisco, CA 94158, USA.
Circ Res. 2012 Mar 30;110(7):978-89. doi: 10.1161/CIRCRESAHA.111.257964. Epub 2012 Feb 9.
The intracellular trafficking of connexin 43 (Cx43) hemichannels presents opportunities to regulate cardiomyocyte gap junction coupling. Although it is known that Cx43 hemichannels are transported along microtubules to the plasma membrane, the role of actin in Cx43 forward trafficking is unknown.
We explored whether the actin cytoskeleton is involved in Cx43 forward trafficking.
High-resolution imaging reveals that Cx43 vesicles colocalize with nonsarcomeric actin in adult cardiomyocytes. Live-cell fluorescence imaging reveals Cx43 vesicles as stationary or traveling slowly (average speed 0.09 μm/s) when associated with actin. At any time, the majority (81.7%) of vesicles travel at subkinesin rates, suggesting that actin is important for Cx43 transport. Using Cx43 containing a hemagglutinin tag in the second extracellular loop, we developed an assay to detect transport of de novo Cx43 hemichannels to the plasma membrane after release from Brefeldin A-induced endoplasmic reticulum/Golgi vesicular transport block. Latrunculin A (for specific interference of actin) was used as an intervention after reinitiation of vesicular transport. Disruption of actin inhibits delivery of Cx43 to the cell surface. Moreover, using the assay in primary cardiomyocytes, actin inhibition causes an 82% decrease (P<0.01) in de novo endogenous Cx43 delivery to cell-cell borders. In Langendorff-perfused mouse heart preparations, Cx43/β-actin complexing is disrupted during acute ischemia, and inhibition of actin polymerization is sufficient to reduce levels of Cx43 gap junctions at intercalated discs.
Actin is a necessary component of the cytoskeleton-based forward trafficking apparatus for Cx43. In cardiomyocytes, Cx43 vesicles spend a majority of their time pausing at nonsarcomeric actin rest stops when not undergoing microtubule-based transport to the plasma membrane. Deleterious effects on this interaction between Cx43 and the actin cytoskeleton during acute ischemia contribute to losses in Cx43 localization at intercalated discs.
连接蛋白 43 (Cx43) 半通道的细胞内转运为调节心肌细胞缝隙连接偶联提供了机会。虽然已知 Cx43 半通道沿微管转运到质膜，但肌动蛋白在 Cx43 正向转运中的作用尚不清楚。
我们探讨了肌动蛋白细胞骨架是否参与 Cx43 的正向转运。
高分辨率成像显示，成年心肌细胞中 Cx43 小泡与非肌节肌动蛋白共定位。活细胞荧光成像显示，当 Cx43 小泡与肌动蛋白结合时，小泡是静止的或缓慢移动（平均速度为 0.09 μm/s）。在任何时候，大多数（81.7%）小泡以亚驱动蛋白的速度移动，这表明肌动蛋白对 Cx43 的运输很重要。使用第二细胞外环中含有血凝素标签的 Cx43，我们开发了一种测定法，用于检测从布雷菲德菌素 A 诱导的内质网/高尔基体囊泡转运阻断中释放后新形成的 Cx43 半通道向质膜的转运。Latrunculin A（用于肌动蛋白的特异性干扰）在囊泡转运重新开始后用作干预。肌动蛋白的破坏抑制了 Cx43 向细胞表面的传递。此外，在原代心肌细胞中，该测定法显示肌动蛋白抑制导致新形成的内源性 Cx43 向细胞-细胞边界的传递减少 82%（P<0.01）。在 Langendorff 灌注的小鼠心脏制剂中，急性缺血期间 Cx43/β-肌动蛋白复合物形成被破坏，肌动蛋白聚合的抑制足以降低连接蛋白在闰盘处的水平。
肌动蛋白是 Cx43 正向转运细胞骨架基础装置的必要组成部分。在心肌细胞中，Cx43 小泡在未进行基于微管向质膜转运时，大部分时间都在非肌节肌动蛋白的静止点暂停。在急性缺血期间，Cx43 与肌动蛋白细胞骨架之间的这种相互作用受到有害影响，导致 Cx43 在闰盘处的定位丢失。
