Department of Health Sciences, Boston University, Boston, Massachusetts, USA.
Am J Physiol Cell Physiol. 2013 Jul 15;305(2):C215-27. doi: 10.1152/ajpcell.00103.2013. Epub 2013 May 22.
Turnover of focal adhesions (FAs) is known to be critical for cell migration and adhesion of proliferative vascular smooth muscle (VSM) cells. However, it is often assumed that FAs in nonmigratory, differentiated VSM (dVSM) cells embedded in the wall of healthy blood vessels are stable structures. Recent work has demonstrated agonist-induced actin polymerization and Src-dependent FA phosphorylation in dVSM cells, suggesting that agonist-induced FA remodeling occurs. However, the mechanisms and extent of FA remodeling are largely unknown in dVSM. Here we show, for the first time, that a distinct subpopulation of dVSM FA proteins, but not the entire FA, remodels in response to the α-agonist phenylephrine. Vasodilator-stimulated phosphoprotein and zyxin displayed the largest redistributions, while β-integrin and FA kinase showed undetectable redistribution. Vinculin, metavinculin, Src, Crk-associated substrate, and paxillin displayed intermediate degrees of redistribution. Redistributions into membrane fractions were especially prominent, suggesting endosomal mechanisms. Deconvolution microscopy, quantitative colocalization analysis, and Duolink proximity ligation assays revealed that phenylephrine increases the association of FA proteins with early endosomal markers Rab5 and early endosomal antigen 1. Endosomal disruption with the small-molecule inhibitor primaquine inhibits agonist-induced redistribution of FA proteins, confirming endosomal recycling. FA recycling was also inhibited by cytochalasin D, latrunculin B, and colchicine, indicating that the redistribution is actin- and microtubule-dependent. Furthermore, inhibition of endosomal recycling causes a significant inhibition of the rate of development of agonist-induced dVSM contractions. Thus these studies are consistent with the concept that FAs in dVSM cells, embedded in the wall of the aorta, remodel during the action of a vasoconstrictor.
细胞迁移和增殖性血管平滑肌（VSM）细胞黏附的关键是焦点黏附（FA）的周转率。然而，人们通常认为，健康血管壁中嵌入的非迁移性、分化的 VSM（dVSM）细胞中的 FA 是稳定的结构。最近的工作表明，激动剂诱导的 dVSM 细胞中的肌动蛋白聚合和Src 依赖性 FA 磷酸化，表明激动剂诱导的 FA 重塑发生。然而，dVSM 中 FA 重塑的机制和程度在很大程度上是未知的。在这里，我们首次表明，在响应α激动剂苯肾上腺素时，dVSM FA 蛋白的一个独特亚群而不是整个 FA 重塑。血管扩张刺激磷酸蛋白和zyxin 显示出最大的重分布，而β整合素和 FA 激酶显示出无法检测到的重分布。 vinculin、metavinculin、Src、Crk 相关底物和 paxillin 显示出中间程度的重分布。膜部分的重分布尤为明显，表明存在内体机制。去卷积显微镜、定量共定位分析和 Duolink 邻近连接分析显示，苯肾上腺素增加了 FA 蛋白与早期内体标志物 Rab5 和早期内体抗原 1 的关联。小分子抑制剂 primaquine 对内体的破坏抑制了 FA 蛋白激动剂诱导的重分布，证实了内体的再循环。细胞松弛素 D、拉坦环素 B 和秋水仙碱也抑制了 FA 的重分布，表明这种重分布依赖于肌动蛋白和微管。此外，内体再循环的抑制导致激动剂诱导的 dVSM 收缩发展速度显著抑制。因此，这些研究与 FA 在 dVSM 细胞中的概念一致，即嵌入主动脉壁中的 FA 在血管收缩剂的作用下重塑。
