Evidence for Chemokine-mediated Coalescence of Preformed Flotillin Hetero-oligomers in Human T-cells

Baumann, Tommy; Affentranger, Sarah; Niggli, Verena

doi:10.1074/jbc.m112.412742

Cited by 14 publications

(24 citation statements)

References 19 publications

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“…Note that these FRAP experiments were carried out at 20°C, as rapid shape changes and migration occurring at 37°C precluded accurate bleaching, acquisition and evaluation of FRAP data. The T cells were cotransfected with flotillin-1 and -2 tagged with mCherry as uropod markers (singly expressed flotillins do not cap, see Baumann et al, 2012) and bleaching of EGFP was carried out in the region of flotillin caps, if present. As shown in Table 1, 75% of T567D ezrin-EGFP is immobilized in cells not exposed to SDF-1, as compared to only 26% of WT ezrin.…”

Section: Resultsmentioning

confidence: 99%

“…Localized positive feedback loops and inhibitory effects of front signaling pathways on rear signaling and vice versa are thought to reinforce this biochemical and structural cell polarization (Bagorda and Parent, 2008). Specific proteins, including adhesion receptors, cytoskeletal proteins, and membrane-microdomain scaffolding proteins such as flotillin-1 and -2, have been shown to segregate to the uropod of polarizing T-lymphocytes (Sánchez-Madrid and Serrador, 2009; Affentranger et al, 2011; Baumann et al, 2012). Localized Rho activation in the rear results in uropod contraction and suppresses formation of protrusions; localized Rac activation in the front induces formation of F-actin-rich protrusions.…”

Section: Introductionmentioning

confidence: 99%

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

Martinelli¹,

Chen²,

Clarke³

et al. 2013

Front. Immunol.

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Martinelli¹,

Chen²,

Clarke³

et al. 2013

Front. Immunol.

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“…It has been reported that high-order oligomerization and membrane-binding motifs artificially target cytosolic proteins to membrane patches, which accumulate at the lagging edge in polarized cells (48)(49)(50)(51)(52). Oligomerizing membrane microdomain components also enrich at the rear of polarized neutrophils or T cells (53)(54)(55)(56)(57). The WD40 domain enhances the stability and immobility of CynA at the lagging edge, either through oligomerization alone or by interaction with other proteins, as evidenced by the differences between CynA 1-574 and either CynA or the tandem PH domain, CynA 1-204+1-204 .…”

Section: Discussionmentioning

confidence: 99%

Novel protein Callipygian defines the back of migrating cells

Swaney

Borleis

Iglesias

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Asymmetric protein localization is essential for cell polarity and migration. We report a novel protein, Callipygian (CynA), which localizes to the lagging edge before other proteins and becomes more tightly restricted as cells polarize; additionally, it accumulates in the cleavage furrow during cytokinesis. CynA protein that is tightly localized, or "clustered," to the cell rear is immobile, but when polarity is disrupted, it disperses throughout the membrane and responds to uniform chemoattractant stimulation by transiently localizing to the cytosol. These behaviors require a pleckstrin homology-domain membrane tether and a WD40 clustering domain, which can also direct other membrane proteins to the back. Fragments of CynA lacking the pleckstrin homology domain, which are normally found in the cytosol, localize to the lagging edge membrane when coexpressed with full-length protein, showing that CynA clustering is mediated by oligomerization. Cells lacking CynA have aberrant lateral protrusions, altered leading-edge morphology, and decreased directional persistence, whereas those overexpressing the protein display exaggerated features of polarity. Consistently, actin polymerization is inhibited at sites of CynA accumulation, thereby restricting protrusions to the opposite edge. We suggest that the mutual antagonism between CynA and regions of responsiveness creates a positive feedback loop that restricts CynA to the rear and contributes to the establishment of the cell axis.chemotaxis | polarity | asymmetry | Dictyostelium | protein localization

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“…The uropod is a plasma membrane protrusion that contains specific organelles along with cytoskeletal, adhesion and signaling proteins (Serrador et al, 1997; Sánchez-Madrid & Serrador, 2009). Flotillins, membrane microdomain scaffolding proteins, are also enriched in leukocyte uropods and are involved in uropod formation (Rossy et al, 2009; Ludwig et al, 2010; Affentranger et al, 2011; Baumann, Affentranger & Niggli, 2012). Recent in vivo data using inhibition of uropod formation by suppressing Rho-kinase activity suggest that the uropod is especially important for T cell migration through constricted spaces (Soriano et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Determinants of phosphatidylinositol-4-phosphate 5-kinase type Iγ90 uropod location in T-lymphocytes and its role in uropod formation

Mathis

Wernimont

Affentranger

et al. 2013

PeerJ

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We have previously identified phosphatidylinositol-4-phosphate 5-kinase type I (PIPKI)γ90 as a T cell uropod component. However, the molecular determinants and functional consequences of its localization remain unknown. In this report, we seek to better understand the mechanisms involved in PIPKIγ90 uropod targeting and the role that PIPKIγ90 plays in T cell uropod formation. During T cell activation, PIPKIγ90 cocaps with the membrane microdomain-associated proteins flotillin-1 and -2 and accumulates in the uropod. We report that the C-terminal 26 amino acid extension of PIPKIγ90 is required for its localization to the uropod. We further use T cells from PIPKIγ90−/− mice and human T cells expressing a kinase-dead PIPKIγ90 mutant to examine the role of PIPKIγ90 in a T cell uropod formation. We find that PIPKIγ90 deficient T cells have elongated uropods on ICAM-1. Moreover, in human T cells overexpression of PIPKIγ87, a naturally occurring isoform lacking the last 26 amino acids, suppresses uropod formation and impairs capping of uropod proteins such as flotillins. Transfection of human T cells with a dominant-negative mutant of flotillin-2 in turn attenuates capping of PIPKIγ90. Our data contribute to the understanding of the molecular mechanisms that regulate T cell uropod formation.

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Evidence for Chemokine-mediated Coalescence of Preformed Flotillin Hetero-oligomers in Human T-cells

Cited by 14 publications

References 19 publications

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

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

Novel protein Callipygian defines the back of migrating cells

Determinants of phosphatidylinositol-4-phosphate 5-kinase type Iγ90 uropod location in T-lymphocytes and its role in uropod formation

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