Flotillins are involved in the polarization of primitive and mature hematopoietic cells

Abstract

Background: Migration of mature and immature leukocytes in response to chemokines is not only essential during inflammation and host defense, but also during development of the hematopoietic system. Many molecules implicated in migratory polarity show uniform cellular distribution under non-activated conditions, but acquire a polarized localization upon exposure to migratory cues.

Methodology/principal findings: Here, we present evidence that raft-associated endocytic proteins (flotillins) are pre-assembled in lymphoid, myeloid and primitive hematopoietic cells and accumulate in the uropod during migration. Furthermore, flotillins display a polarized distribution during immunological synapse formation. Employing the membrane lipid-order sensitive probe Laurdan, we show that flotillin accumulation in the immunological synapse is concomittant with membrane ordering in these regions.

Conclusions: Together with the observation that flotillin polarization does not occur in other polarized cell types such as polarized epithelial cells, our results suggest a specific role for flotillins in hematopoietic cell polarization. Based on our results, we propose that in hematopoietic cells, flotillins provide intrinsic cues that govern segregation of certain microdomain-associated molecules during immune cell polarization.

Figure 1. Pre-assembled platforms of flotillins polarize to the uropods upon chemokinesis and the spatial segregation of flotillins and actin cytoskeleton during polarization.

a) T lymphoblasts were treated with SDF-1α and allowed to migrate on fibronectin. The control cells were just plated on fibronectin without any chemoattractant. The cells were then fixed and stained with β-tubulin (red) and DAPI (blue) to stain the microtubules and the nucleus, respectively. Note that during chemokinesis, the microtubules and the MTOCs reorient themselves to the rear end of the cell. b) Untreated T lymphoblasts show uniform actin distribution but very polarized flotillin-1 staining (green). But chemoattracted (SDF-1α or RANTES) treated cells show polarized morphology accumulating actin (red) at the leading edge and flotillins (green) at the uropod. The images represent several sets of experiments and images collected. c) Magnified image of an SDF-1α treated lymphocyte displaying a clear spatial distribution of flotillins and actin cytoskeleton. d) Note that, even before a morphologic polarization took place, flotillins and actin spatially distribute themselves as early as 5 min after stimulation.

Figure 2. Flotillins co-localize with uropod markers upon chemokinesis in T lymphoblasts.

a) SDF-1α or RANTES treated lymphoblasts were stained with anti-flotillin-2 (green) and anti-ERM (red) antibodies. DIC images show morphological polarization during migration. b) ERM (red) and flotillin-1 (green) stained RANTES treated lymphoblast show that both the molecules concentrate at the uropod during migration. Note that ERM staining is also found at the leading edge due to the fact that the antibody also recognizes ezrin, which is localized at the leading edge. c) Peripheral T lymphoblasts were treated with RANTES, fixed and stained for flotillins (green) and ERM (red) proteins. Series of Z-stack images show that flotillins are more contained to the uropods. d) Chemokine stimulated cells were allowed to migrate on fibronectin and stained with anti-CD43 (upper panel, red), CD44 (lower panel, red) and anti-flotillin antibodies (green). The DIC images show the contrast image of the polarized lymphoblasts.

Figure 3. Flotillin polarization to the uropods in cultivated CD34⁺ cells.

In morphological polarized CD34⁺ cells flotillin-1 (a) and flotillin-2 (b) are highly concentrated in the tip of the uropod while moesin, CD43 and CD44 are located in the whole uropod. Sometimes an additional domain of flotillin-2 is found at the base of the uropod (arrow). Actin marks the leading edge. (c) Cyto B panel shows the morphology and the staining of actin and flotillin-1 in cytochalasin B treated cells.

Figure 4. Spatial distribution of flotillins to the uropods and actin to the leading edge during GM-CSF induced monocyte polarization.

GM-CSF untreated (upper panel) and treated (lower panel) macrophages were stained with anti-actin (red) and anti-flotillin-1 (green).

Figure 5. SDF-1α treatment induces reorganization of GM1- and flotillin-microdomains but does not affect DRM localization of flotillins in T cells.

a) Unstimulated (upper panel) and SDF-1a treated (lower panel) cells were stained with mouse anti-flotillin-1 (green) to detect flotillin-1. To detect the ganglioside, GM1, cells were stained first with Cholera toxin – B (CTX-B) subunit and then with rabbit anti- CTX-B antibodies (red). b) Unstimulated (upper panel) and SDF-1a treated (lower panel) cells were stained with mouse anti-flotillin-2 (green) to detect flotillin-2. GM1, on the other hand is detected by staining the cells first with Cholera toxin – B (CTX-B) subunit and then with rabbit anti- CTX-B antibodies (red). c) DRM fractions isolated from control (left) and SDF-1α treated (right) T cells were blotted for flotillin-1 (upper panel), flotillin-2 (middle panel) and PI3K (lower panel). Note that microdomain residency of flotillins is not affected during the treatment but PI3K specifically translocates to DRMs only upon chemokine stimulation. d) Whole cell lysates of SDF-1α treated cells for various times as indicated were boiled, electrophoresed and transferred onto a nitrocellulose membrane. Upper panel: The membrane was then probed with anti-phospho-ERK1/2 antibodies. Increased ERK phosphorylation accompanied chemokine treatment at 5 min and decreases with time. Lower panel: Blotting with anti-ERK antibody shows the total ERK content.

Figure 6. Flotillin polarization to the immunological synapse is concomitant with membrane condensation.

a) Jurkat cells stably expressing Flotillin-2-GFP were incubated with either unpulsed or SEE-pulsed EBV-B cells for 20 min. Confocal images were taken with identical settings. b) Quantitation of the conjugate formation in non-pulsed and SEE-pulsed conditions. c) Jurkat cells were labeled with Laurdan, conjugated with anti-CD3 antibody- or anti-TfR antibody-coated beads on ice and activated for 10 min at 37°C. After fixation, cells were immunostained for flotillin-1, adhered to poly-L-lysine-coated coverslips, mounted and imaged. The confocal image of flotillin-1 is recorded at the identical focal depth as the Laurdan images that are converted into GP images as described in Methods. GP images are pseudocolored with high GP (ordered membranes) in yellow and low GP (fluid membranes) in green.

Figure 7. Flotillins are not polarized in MDCK cells.

Filter-grown MDCK cells were immunostained for apical marker protein, GP135 (green) and flotillin-1 (blue) or with Phalloidin to stain actin (red) and flotillin-2 (blue). Note that GP135 marks exclusively the apical membrane while flotillin-1 and -2 are mainly associated with intracellular vesicles. Actin, on the other hand marks both apical and basolateral membranes.