Figure 1.
Flotillin-1 and -2 interact and redistribute to the uropod in stimulated human neutrophils.
(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).
Figure 2.
Fast capping and redistribution of flotillin-2 to the uropod in stimulated human neutrophils.
(A) Cells were either incubated in Gey's medium for 25 min at 37°C (ctrl) or were stimulated with 1 nM fNLPNTL for the indicated times. Incubation was stopped by fixation with 10% TCA. Fixed cells were double-labeled for flotillin-2 (flot2; rabbit Ab) and actin (mouse Ab). Bar, 10 µm. (B) The percentage of cells treated as described in panel (A) displaying a flotillin-2 cap was determined by visual counting. 100 cells were inspected per experiment (mean±s.e.m. of 4 independent experiments). Significant differences compared to unstimulated controls are marked: * P<0.001, ** P<0.0001.
Figure 3.
Co-expression of flotillin-1-mCherry and flotillin-2-EGFP in dHL-60 cells promotes uropod enrichment of tagged flotillins.
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.
Figure 4.
Impact of agents that affect F-actin, myosin II or microtubules on flotillin-2 capping.
Human neutrophils were either incubated in Gey's medium for 30 min at 37°C (ctrl) or were preincubated for 20 min without or with (A,C) latrunculin A (lat; 1 µM) or (A) jasplakinolide (jasp; 5 µM) or (B) blebbistatin (ble; 100 µM), or ML-7 (ML; 10 µM), or (B,C) Y-27632 (Y; 10 µM) respectively followed by stimulation with 1 nM fNLPNTL (HP) for 10 min (A,B), or were incubated for 30 min with 10 µM colchicine as indicated (C). Cells were then fixed with 10% TCA and double-labeled for flotillin-2 (rabbit Ab) and actin (mouse Ab). Bar, 10 µm.
Figure 5.
Flotillin-2 capping precedes that of CD43 or P-ERM during polarization of human neutrophils.
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.
Figure 6.
Coordinated redistribution and capping of flotillin-2 and PSGL-1 during polarization of human neutrophils.
(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.
Figure 7.
Co-immunoprecipitation of flotillin-2 by a PSGL-1 antibody.
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.
Figure 8.
Capping of flotillin-2 in PSGL-1 deficient bone marrow neutrophils.
After isolation and 24 h maturation with 25 ng/ml G-CSF, murine bone marrow neutrophils derived from wild type or PSGL-1−/− mice were resuspended in HBSS media, pre-incubated for 15 min at 37°C and incubated for 3–5 min with 1 µM fNLPNTL. Incubation was stopped by fixation with 10% TCA and fixed cells were double-labeled for flotillin-2 (flot2; rabbit Ab) and PSGL-1 (rat Ab). Bar, 10 µm.
Figure 9.
Schematic representation of reorganization of flotillins, PSGL-1, P-ERM and CD43 during neutrophil activation.
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.