Department of Biology, University of Konstanz, 78467 Konstanz, Germany.
Department of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland.
Eur J Cell Biol. 2015 Nov;94(11):531-45. doi: 10.1016/j.ejcb.2015.07.003. Epub 2015 Jul 31.
Reggies/flotillins are implicated in trafficking of membrane proteins to their target sites and in the regulation of the Rab11a-dependent targeted recycling of E-cadherin to adherens junctions (AJs). Here we demonstrate a function of reggies in focal adhesion (FA) formation and α5- and β1-integrin recycling to FAs. Downregulation of reggie-1 in HeLa and A431 cells by siRNA and shRNA increased the number of FAs, impaired their distribution and modified FA turnover. This was coupled to enhanced focal adhesion kinase (FAK) and Rac1 signaling and gain in plasma membrane motility. Wild type and constitutively-active (CA) Rab11a rescued the phenotype (normal number of FAs) whereas dominant-negative (DN) Rab11a mimicked the loss-of-reggie phenotype in control cells. That reggie-1 affects integrin trafficking emerged from the faster loss of internalized antibody-labeled β1-integrin in reggie-deficient cells. Moreover, live imaging using TIRF microscopy revealed vesicles containing reggie-1 and α5- or β1-integrin, trafficking close to the substrate-near membrane and making kiss-and-run contacts with FAs. Thus, reggie-1 in interaction with Rab11a controls Rac1 and FAK activation and coordinates the targeted recycling of α5- and β1-integrins to FAs to regulate FA formation and membrane dynamics.
Reggies/flotillins 参与膜蛋白向其靶位的运输，以及 Rab11a 依赖性 E-钙黏蛋白靶向回收至黏附连接点（AJs）的调节。在这里，我们证明了 Reggies 在焦点黏附（FA）形成和α5 和β1 整合素向 FA 的循环中的功能。siRNA 和 shRNA 下调 HeLa 和 A431 细胞中的 Reggie-1 增加了 FA 的数量，破坏了它们的分布并改变了 FA 的周转率。这与增强的焦点黏附激酶（FAK）和 Rac1 信号转导以及质膜流动性的增加有关。野生型和组成激活型（CA）Rab11a 挽救了表型（正常数量的 FA），而显性负性（DN）Rab11a 在对照细胞中模拟了 Reggie 缺失表型。Reggie-1 影响整合素运输，这是因为 Reggie 缺陷细胞中内化的抗体标记的β1 整合素更快丢失。此外，使用 TIRF 显微镜进行的实时成像显示了含有 Reggie-1 和α5 或β1 整合素的囊泡，它们在靠近基质的膜附近运输，并与 FA 进行吻接和跑离接触。因此，Reggie-1 通过与 Rab11a 的相互作用控制 Rac1 和 FAK 的激活，并协调α5 和β1 整合素靶向回收至 FA，以调节 FA 的形成和膜动力学。
