Department of Pathology, University of Bern Bern, Switzerland.
Front Immunol. 2013 Apr 8;4:84. doi: 10.3389/fimmu.2013.00084. eCollection 2013.
T cell uropods are enriched in specific proteins including adhesion receptors such as P-selectin glycoprotein ligand-1 (PSGL-1), lipid raft-associated proteins such as flotillins and ezrin/radixin/moesin (ERM) proteins which associate with cholesterol-rich raft domains and anchor adhesion receptors to the actin cytoskeleton. Using dominant mutants and siRNA technology we have tested the interactions among these proteins and their role in shaping the T cell uropod. Expression of wild type (WT) ezrin-EGFP failed to affect the morphology of human T cells or chemokine-induced uropod recruitment of PSGL-1 and flotillin-1 and -2. In contrast, expression of constitutively active T567D ezrin-EGFP induced a motile, polarized phenotype in some of the transfected T cells, even in the absence of chemokine. These cells featured F-actin-rich ruffles in the front and uropod enrichment of PSGL-1 and flotillins. T567D ezrin-EGFP was itself strongly enriched in the rear of the polarized T cells. Uropod formation induced by T567D ezrin-EGFP was actin-dependent as it was attenuated by inhibition of Rho-kinase or myosin II, and abolished by disruption of actin filaments. While expression of constitutively active ezrin enhanced cell polarity, expression of a dominant-negative deletion mutant of ezrin, 1-310 ezrin-EGFP, markedly reduced uropod formation induced by the chemokine SDF-1, T cell front-tail polarity, and capping of PSGL-1 and flotillins. Transfection of T cells with WT or T567D ezrin did not affect chemokine-mediated chemotaxis whereas 1-310 ezrin significantly impaired spontaneous 2D migration and chemotaxis. siRNA-mediated downregulation of flotillins in murine T cells attenuated moesin capping and uropod formation, indicating that ERM proteins and flotillins cooperate in uropod formation. In summary, our results indicate that activated ERM proteins function together with flotillins to promote efficient chemotaxis of T cells by structuring the uropod of migrating T cells.
T 细胞足突富含特定蛋白质，包括黏附受体，如 P 选择素糖蛋白配体-1（PSGL-1），脂质筏相关蛋白，如小窝蛋白和 ezrin/radixin/moesin（ERM）蛋白，它们与富含胆固醇的筏域结合，并将黏附受体锚定在肌动蛋白细胞骨架上。我们使用显性突变体和 siRNA 技术测试了这些蛋白质之间的相互作用及其在塑造 T 细胞足突中的作用。野生型（WT）ezrin-EGFP 的表达未能影响人 T 细胞的形态或趋化因子诱导的 PSGL-1 和小窝蛋白-1 和 -2 的足突募集。相比之下，组成型激活的 T567D ezrin-EGFP 在一些转染的 T 细胞中诱导出一种运动的、极化的表型，即使没有趋化因子也是如此。这些细胞在前部有富含 F-肌动蛋白的皱褶，并且足突富含 PSGL-1 和小窝蛋白。T567D ezrin-EGFP 本身在极化 T 细胞的后部强烈富集。T567D ezrin-EGFP 诱导的足突形成依赖于肌动蛋白，因为它被 Rho 激酶或肌球蛋白 II 的抑制所减弱，并被肌动蛋白丝的破坏所消除。虽然组成型激活的 ezrin 增强了细胞极性，但 ezrin 的显性负突变体 1-310 ezrin-EGFP 的表达显著减少了趋化因子 SDF-1 诱导的足突形成、T 细胞前后极性和 PSGL-1 和小窝蛋白的帽化。WT 或 T567D ezrin 的转染对趋化因子介导的趋化作用没有影响，而 1-310 ezrin 则显著损害了自发的 2D 迁移和趋化作用。在小鼠 T 细胞中用 siRNA 下调小窝蛋白会减弱 moesin 帽化和足突形成，表明 ERM 蛋白和小窝蛋白在足突形成中合作。总之，我们的结果表明，激活的 ERM 蛋白与小窝蛋白一起作用，通过构建迁移 T 细胞的足突来促进 T 细胞的有效趋化。
