Ezrin/Radixin/Moesin Proteins and Flotillins Cooperate to Promote Uropod Formation in T Cells

Abstract

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.

Physical view on migration modes

“…It has been proposed that asymmetries in the strength of membrane-cortex attachment seems to be responsible in targeting the blebs to the cell's leading edge. In line with this, the expression level of the actin-membrane coupler ezrin (belongs to the ezrin-radixin-moesin (ERM) family) is elevated at the back of the Walker carcinosarcoma cells, which is consistent with the hypothesis that the membrane-to-cortex attachment is reduced at the cell's leading edge, supporting the bleb formation in this region (Martinelli et al, 2013;Rossy et al, 2007;Niggli and Rossy, 2008). It has turned out that an increase in the expression level or the activity of ERMs is correlated with reduced blebbing in zebrafish germ cells (Goudarzi et al, 2012), in A375 human melanoma cells (Lorentzen et al, 2011) and in mast cells (Yanase et al, 2011).…”

O-glycans direct selectin ligands to lipid rafts on leukocytes

“…Surprisingly, these glycoproteins also moved to uropods of chemokine-stimulated C1galt1 −/− neutrophils, even though, before stimulation, they did not copatch with GM1 in lipid rafts, and after stimulation, they remained in higher-density, detergent-soluble "nonraft" fractions. Uropods form through membrane interactions with flotillins 1 and 2 and with the actin cytoskeleton (54,55), in part through binding of ERM adaptors to the cytoplasmic domains of membrane glycoproteins (56). PSGL-1 associates with flotillins as measured by coimmunoprecipitation in detergent extracts and by a proximity-ligation assay in intact cells (31,57).…”

The prion protein inhibits monocytic cell migration by stimulating β1 integrin adhesion and uropod formation

“…ERM proteins link membrane proteins to the actin cytoskeletal cortex (Fehon et al, 2010) and active phospho-ERM proteins localize to and regulate formation of the uropod in migrating lymphoblasts (Lee et al, 2004;Martinelli et al, 2013). We observed that the intensity of phospho-ERM labeling appeared to be reduced in shPrP-U937 cells (Fig.…”

“…ERM proteins are activated by phosphorylation of a conserved C-terminal threonine residue, which enables them to crosslink membrane receptors with the underlying actin cytoskeleton (Fehon et al, 2010). ERM protein activation is crucial for uropod formation (Ivetic and Ridley, 2004;Lee et al, 2004;Martinelli et al, 2013) and for β1-integrin-dependent T cell adhesion and polarization Liu et al, 2002). Therefore, the inhibition of ERM phosphorylation in the absence of PrP likely contributes to both the lack of uropods and the reduced β1 integrin adhesion observed for PrP-deficient monocytes.…”

“…Both β1 integrins and phosphorylated ERM ( phospho-ERM) proteins localize to the uropod of migrating leukocytes (Lee et al, 2004;Martinelli et al, 2013). Similarly, phospho-ERM proteins accumulated in the uropod and partially colocalized with β1 integrins in shCtrl-U937 cells migrating on immobilized VCAM-1 (Fig.…”