Flotillins Interact with PSGL-1 in Neutrophils and, upon Stimulation, Rapidly Organize into Membrane Domains Subsequently Accumulating in the Uropod

BACKGROUND: Neutrophils polarize and migrate in response to chemokines. Different types of membrane microdomains (rafts) have been postulated to be present in rear and front of polarized leukocytes and disruption of rafts by cholesterol sequestration prevents leukocyte polarization. Reggie/flotillin-1 and -2 are two highly homologous proteins that are ubiquitously enriched in detergent resistant membranes and are thought to shape membrane microdomains by forming homo- and hetero-oligomers. It was the goal of this study to investigate dynamic membrane microdomain reorganization during neutrophil activation. METHODOLOGY/PRINCIPAL FINDINGS: We show now, using immunofluorescence staining and co-immunoprecipitation, that endogenous flotillin-1 and -2 colocalize and associate in resting spherical and polarized primary neutrophils. Flotillins redistribute very early after chemoattractant stimulation, and form distinct caps in more than 90% of the neutrophils. At later time points flotillins accumulate in the uropod of polarized cells. Chemotactic peptide-induced redistribution and capping of flotillins requires integrity and dynamics of the actin cytoskeleton, but does not involve Rho-kinase dependent signaling related to formation of the uropod. Both flotillin isoforms are involved in the formation of this membrane domain, as uropod location of exogenously expressed flotillins is dramatically enhanced by co-overexpression of tagged flotillin-1 and -2 in differentiated HL-60 cells as compared to cells expressing only one tagged isoform. Flotillin-1 and -2 associate with P-selectin glycoprotein ligand 1 (PSGL-1) in resting and in stimulated neutrophils as shown by colocalization and co-immunoprecipitation. Neutrophils isolated from PSGL-1-deficient mice exhibit flotillin caps to the same extent as cells isolated from wild type animals, implying that PSGL-1 is not required for the formation of the flotillin caps. Finally we show that stimulus-dependent redistribution of other uropod-located proteins, CD43 and ezrin/radixin/moesin, occurs much slower than that of flotillins and PSGL-1. CONCLUSIONS/SIGNIFICANCE: These results suggest that flotillin-rich actin-dependent membrane microdomains are importantly involved in neutrophil uropod formation and/or stabilization and organize uropod localization of PSGL-1

Semi-groupe de Lie associé à un cône symétrique

Volcanic arcs are the surface expression of magmatic systems that result from the subduction of mostly oceanic lithosphere at convergent plate boundaries. Arcs with a submarine component include intraoceanic arcs and island arcs that span almost 22,000 km on Earth\u27s surface, the vast majority of which are located in the Pacific region. Hydrothermal systems hosted by submarine arc volcanoes commonly contain a large component of magmatic fluid. This magmatic-hydrothermal signature, coupled with the shallow water depths of arc volcanoes and their high volatile contents, strongly influences the chemistry of the fluids and resulting mineralization and likely has important consequences for the biota associated with these systems. The high metal contents and very acidic fluids in these hydrothermal systems are thought to be important analogs to numerous porphyry copper and epithermal gold deposits mined today on land

Specific roles of Rac1 and Rac2 in motile functions of HT1080 fibrosarcoma cells

Rho family proteins are constitutively activated in the highly invasive human fibrosarcoma HT1080 cells. We now investigated the specific roles of Rac1 and Rac2 in regulating morphology, F-actin organization, adhesion, migration, and chemotaxis of HT1080 cells. Downregulation of Rac1 using specific siRNA probes resulted in cell rounding, markedly decreased spreading, adhesion, and chemotaxis of HT1080 cells. 2D migration on laminin-coated surfaces in contrast was not markedly affected. Selective Rac2 depletion did not affect cell morphology, cell adhesion, and 2D migration, but significantly reduced chemotaxis. Downregulation of both Rac1 and Rac2 resulted in an even more marked reduction, but not complete abolishment, of chemotaxis indicating distinct as well as overlapping roles of both proteins in chemotaxis. Rac1 thus is selectively required for HT1080 cell spreading and adhesion whereas Rac1 and Rac2 are both required for efficient chemotaxis

Ezrin/moesin in motile Walker 256 carcinosarcoma cells: signal-dependent relocalization and role in migration

Rat Walker 256 carcinosarcoma cells spontaneously develop front-tail polarity and migrate in the absence of added stimuli. Constitutive activation of phosphatidylinositol-3 kinase (PI 3-kinase), Rac, Rho and Rho kinase are essential for these processes. Ezrin and moesin are putative targets of these signaling pathways leading to spontaneous migration. To test this hypothesis, we used specific siRNA probes that resulted in a downregulation of ezrin and moesin by about 70% and in a similar reduction in the fraction of migrating cells. Spontaneous polarization however was not affected, indicating a more subtle role of ezrin and moesin in migration. We provide furthermore evidence that endogenous ezrin and moesin colocalize with F-actin at the contracted tail of polarized cells, similar to ectopically expressed green fluorescent protein-tagged ezrin. Our results suggest that myosin light chain and ezrin are markers of front and tail, respectively, even in the absence of morphological polarization. We further show that endogenous ezrin and moesin are phosphorylated and that activities of PI-3 kinase, Rho and Rac, but not of Rho-kinase, are required for this C-terminal phosphorylation. Activation of protein kinase C in contrast suppressed phosphorylation of ezrin and moesin. Inhibition of ezrin phosphorylation prevented its membrane association

Schematic representation of reorganization of flotillins, PSGL-1, P-ERM and CD43 during neutrophil activation.

<p>Resting cells exhibit randomly dispersed small rafts at the plasma membrane containing flotillins and/or PSGL-1 and/or P-ERM and/or CD43. 20 s after addition of chemotactic stimulus, flotillins and PSGL-1 co-assemble into large caps at the site of the future uropod, whereas P-ERM and CD43 still are randomly distributed at the plasma membrane. ERM phosphorylation is decreased. These flotillin/PSGL-1 caps persist and may mark the site of the future uropod. 5 min after the onset of activation, flotillins, PSGL-1, CD43 and P-ERM all co-cap in the uropod of polarized cells.</p

Flotillin-1 and -2 interact and redistribute to the uropod in stimulated human neutrophils.

<p>(A) Cells were incubated either in Gey's medium for 25 min at 37°C (ctrl) or were stimulated with 1 nM fNLPNTL (HP) for 5 min after a 20 min preincubation. Cells were then fixed with 10% TCA and double-labeled for flotillin-1 (flot1; mouse Ab) and flotillin-2 (flot2; rabbit Ab). Dic; differential interference contrast. Bar, 10 µm. (B) Cells were treated as in (A), lysed and subjected to immunoprecipitation (IP), using a flotillin-2 rabbit antibody. Immunoblots (IB) of the immunoprecipitated material were probed for flotillin-1 (mouse Ab) or, after stripping of the blot, for flotillin-2 (mouse Ab). Comparable amounts of both proteins were present in immunoprecipitates of resting and stimulated cells (upper panels). Three percent input lysates were also analyzed for the presence of flotillin-1 and -2 showing that equal amounts of these proteins were present in the lysates of resting and stimulated cells before immunoprecipitation (lower panels).</p

Co-immunoprecipitation of flotillin-2 by a PSGL-1 antibody.

<p>Cells were incubated either in Gey's medium for 30 min at 37°C (ctrl) or were stimulated with 1 nM fNLPNTL (HP) for 10 min after a 20 min preincubation. Cells were then lysed and subjected to immunoprecipitation (IP), using a murine PSGL-1 antibody or a murine IgG as control, as indicated. Upper parts of the immunoblots (IB) were probed for PSGL-1 (A) (murine Ab) and lower parts of the blots for flotillin-2 (B) (flot2, murine Ab; left upper and lower panels in B). The band of approximately 50 kD detectable in the immunoprecipitates just above flotillin (upper panels in B) corresponds to the heavy chain of IgG, as it also appeared when the blot was stripped and decorated only with the secondary anti-murine IgG antibody (upper right panel in B). Three percent input lysates were in addition analyzed for the presence of PSGL-1 (A) and flotillin-2 (B) showing that equal amounts of these proteins were present in the lysates of resting and stimulated cells before immunoprecipitation (lower panels in A and B). Data representative of 3 independent experiments are shown.</p

Co-expression of flotillin-1-mCherry and flotillin-2-EGFP in dHL-60 cells promotes uropod enrichment of tagged flotillins.

<p>dHL-60 cells were transfected with vectors encoding either flotillin-1-mCherry (flot1-mCherry) or flotillin-2-EGFP (flot2-EGFP) alone (A) or were cotransfected with both plasmids or with pmCherry and pEGFP (B), as indicated and stimulated with 10 nM fNLPNTL for 10 min. Living cells were then placed on the stage of a microscope heated at 37°C and photographed. Bar, 10 µm.</p

Coordinated redistribution and capping of flotillin-2 and PSGL-1 during polarization of human neutrophils.

<p>(A) Cells were either incubated in Gey's medium for 30 min at 37°C (ctrl) or were stimulated with 1 nM fNLPNTL (HP) for 40 s, 2 or 5 min. (B) Cells were preincubated for 20 min with 10 µM ML-7 (ML) followed by stimulation with 1 nM fNLPNTL (HP) for 10 min. (A,B) Incubation was stopped by fixation with 10% TCA and fixed cells were double-labeled (A,B) for flotillin-2 (flot2; rabbit Ab) and PSGL-1 (murine Ab) or (B) for flotillin-2 (murine Ab) and P-ERM (PERM; rabbit Ab). Bar, 10 µm.</p

Flotillin-2 capping precedes that of CD43 or P-ERM during polarization of human neutrophils.

<p>Cells were either incubated in Gey's medium for 25 min at 37°C (ctrl) or were stimulated with 1 nM fNLPNTL for 40 s, 2 or 5 min. Incubation was stopped by fixation with 10% TCA. Fixed cells were double-labeled (A) for flotillin-2 (flot2; rabbit Ab) and CD43 (mouse Ab) or (B) for flotillin-2 (flot2; murine Ab) and P-ERM (rabbit Ab). Bar, 10 µm.</p