The extracellular domains of FasL and Fas are sufficient for the formation of supramolecular FasL-Fas clusters of high stability

Abstract

Using fluorescent variants of Fas and FasL, we show that membrane FasL and Fas form supramolecular clusters that are of flexible shape, but nevertheless stable and persistent. Membrane FasL-induced Fas clusters were formed in caspase-8- or FADD-deficient cells or when a cytoplasmic deletion mutant of Fas was used suggesting that cluster formation is independent of the assembly of the cytoplasmic Fas signaling complex and downstream activated signaling pathways. In contrast, cross-linked soluble FasL failed to aggregate the cytoplasmic deletion mutant of Fas, but still induced aggregation of signaling competent full-length Fas. Moreover, membrane FasL-induced Fas cluster formation occurred in the presence of the lipid raft destabilizing component methyl-beta-cyclodextrin, whereas Fas aggregation by soluble FasL was blocked. Together, these data suggest that the extracellular domains of Fas and FasL alone are sufficient to drive membrane FasL-induced formation of supramolecular Fas-FasL complexes, whereas soluble FasL-induced Fas aggregation is dependent on lipid rafts and mechanisms associated with the intracellular domain of Fas.

Figure 1.

Membrane FasL and Fas form supramolecular clusters. (A) Scheme of YFP and CFP fusion proteins of Fas and FasL. (B) HeLa cells were transfected with expression plasmids encoding YFP-FasL or CFP-FasL and Fas-YFP or with a mixture of the latter two plasmids in the presence of z-VAD-fmk (20 μM). Images shown were taken after 24 h and are representative for each experimental group. (C) HeLa cells transfected with CFP-FasL and Fas-YFP, respectively, were harvested 24 h after transfection, mixed at a 1:1 ratio, and cocultured for additional 24 h. Images shown are representative for neighboring cells expressing CFP-FasL and Fas-YFP, respectively. (D) HeLa cells were separately transfected with plasmids encoding the indicated proteins and cocultured overnight. After 24 h, at least 100 yellow fluorescent cells that neighbored one or more blue fluorescent cells were selected on randomly chosen sections of the slide and analyzed for cluster formation. The portions of cells that displayed ligand receptor clusters were determined. (E) HeLa cells were transfected with CFP-FasL and were cocultured 24 h after transfection with SKW cells in the presence of 20 μM z-VAD-fmk for 3 h. Cells were fixed and stained with anti-Fas and a CY5-labeled secondary antibody.

Figure 2.

Supramolecular FasL-Fas clusters are highly stable durable structures. HeLa cells were transfected with CFP-FasL and Fas-YFP, respectively, and were cocultured in the presence of 20 μM z-VAD-fmk. The next day, 25 μg/ml CHX were added and after an additional hour a representative pair of transfected cells was analyzed by life imaging fluorescence microscopy. After the indicated times images were taken. (B, left) HeLa cells overexpressing Fas were pretreated with CHX (25 μg/ml) for 30 min and were then challenged with 400 ng/ml of M2-cross-linked Flag-FasL on ice or at 37°C. After 1 h Fas cell surface expression was determined by FACS analysis. The experiment was done in triplicates. (B, right) HeLa cells transfected with Fas-YFP or CFP-FasL were cocultivated. Cells were then pretreated with CHX and fluorescence intensities of nine Fas-YFP expressing cells forming FasL-Fas clusters with neighboring CFP-FasL cells were determined for a 2-h interval by confocal microscopy. (C) HeLa cells were transfected with an expression plasmid encoding Fas-YFP and cultured without z-VAD-fmk. The next day, cells were challenged with a suspension of freshly harvested HEK293 cells, expressing CFP-FasL. Imaging was started when first FasL-Fas clusters became apparent (arrows) and followed until the dying Fas-YFP expressing cell detached from the plastic surface.

Figure 3.

FLIP analysis of free and clustered YFP-FasL and Fas-YFP. HeLa cells transfected with YFP-FasL and Fas-YFP, respectively, were cultured individually or together with HeLa cells expressing CFP fusion proteins of the corresponding receptor or ligand. Images of a representative cell from each experimental group are shown. Bleaching areas covering most of the cell except the analyzed FasL-Fas cluster and/or a control region in the plasma membrane were defined (dotted lines). Cells were initially bleached for 5 min to bleach the majority of the highly diffusible intracellular fraction of the YFP fusion proteins. After this, bleaching was continued, and images were recorded in 1-min intervals. Using these images, loss of fluorescence in nonbleached areas, which contained clusters or control membrane, was determined, as described in Materials and methods. These areas were defined as ROI, and are indicated (dotted line, bleach area; solid line, ROI of clustered or free YFP fusion protein; dashed line, control ROI). Measurements of clustered YFP fusion proteins (Fas-YFP, closed squares top diagram; YFP-FasL, closed squares bottom diagram) were corrected for each time point for the residual fluorescence of the corresponding “free” protein outside the bleach region. Fluorescence intensities of nonclustered Fas-YFP (open squares top diagram) and YFP-FasL (open squares bottom diagram) determined outside the bleach regions were corrected for the rest fluorescence within the bleach region. Corrected fluorescence intensities were normalized against the relative fluorescence intensity obtained after the initial five bleach cycles. This time point of each experiment was defined as t = 0 min. Averaged (n = 12–15) normalized relative fluorescence intensities were shown in the diagrams. The fluorescence loss indicating dissociation was linearized by plotting the natural logarithm of the relative fluorescence intensities over time. The third row in the top part shows a control experiment where besides Fas-YFP membrane CFP were coexpressed to demonstrate that the overall morphology of bleached cells was not affected.

Figure 4.

The cytoplasmic domains of Fas and FasL and caspase-8 and FADD are dispensable for cluster formation. Parental Jurkat cells and derived clones deficient in FADD or caspase-8 expression were electroporated with Fas-YFP. After 1 d of recovery transfected Jurkat cells were cocultured with CFP-FasL transfected HeLa cells in the presence of 20 μM z-VAD-fmk. After 3 h Fas-YFP expressing Jurkat cells that overlaid or neighbored CFP-FasL expressing HeLa cells were analyzed for occurrence of FasL-Fas clusters. Total numbers and percentage of cluster positive cell pairs are indicated for one representative experiment. Pictures show examples of isolated Fas-YFP expressing Jurkat cells and Jurkat-HeLa cell pairs having Fas-YFP CFP-FasL clusters (arrows).

Figure 5.

Cytoplasmic deletion mutants of FasL and Fas are able to form stable clusters. (A) Scheme of YFP and CFP fusion proteins of Fas and FasL variants lacking the cytoplasmic domains. (B) HeLa cells were either transfected with Fas-YFP or FasΔcyt-YFP, grown overnight on cover glasses in 6 well plates and overlaid by centrifugation with FasL-CFP expressing HEK293 cells. Cells were then cocultured at 37°C and fixed after the indicated time intervals. 90 yellow fluorescent cells which were in contact with blue fluorescent cells were analyzed for cluster incidence. The portions of Fas-YFP and FasΔcyt-YFP expressing cells, which formed clusters are indicated. (C) FLIP analysis of free and clustered YFP fusion proteins of FasL and Fas variants lacking the cytoplasmic domains. HeLa cells were individually transfected with the indicated YFP and CFP fusion proteins and cocultured in the indicated combinations. FLIP experiments were then performed as described in Fig. 3. Again, dotted lines indicate the bleach areas; solid lines, the ROI of clustered or free YFP fusion proteins; and the dashed lines identify the control ROI. A further control with a cell cotransfected with FasΔcyt and membrane CFP is shown to demonstrate maintenance of the overall morphology of bleached cells during bleaching (top, third row).

Figure 6.

Fas-YFP and CFP-FasL are constitutively associated with lipid rafts. (A) HeLa cells were transiently transfected with the indicated fusion proteins and were grown individually or as cocultures. The next day, cells were analyzed by cell fractionation on sucrose gradients as described in Materials and methods. Four fractions were obtained which are shown here from top (1) to bottom (4). Fraction 1, the top fraction corresponded to the lowest sucrose density. This detergent insoluble fraction contained microdomains and lipid rafts. Fraction 4 contained the vast majority of total protein and represented the detergent soluble fraction. The detected proteins and the antibodies used in Western blot analysis are indicated. In experiments shown on the bottom panel, cells had been treated (+) or not (−) with the cholesterol depleting drug βMCD for 20 min before fractionation in order to resolve lipid rafts. (B) HeLa cells expressing Fas-YFP were treated with βMCD for 20 min (gray bars) or left untreated (black bars) and then overlaid with CFP-FasL expressing HEK293 cells by centrifugation. Cells were fixed after the indicated time intervals and the cluster incidence of neighboring cell pairs expressing Fas-YFP and CFP-FasL was determined as described in Materials and methods. The experiment was done in triplicates. (C) HeLa cells were transfected with CFP-FasL and Fas-YFP and grown separately. The next day, Fas-YFP transfectants were depleted for cholesterol (20 mM βMCD, 20 min) in serum-free medium and cocultured with CFP-FasL expressing cells. Online imaging was started when cells came into contact.

Figure 7.

Induction of Fas SPOTS by soluble cross-linked FasL depends on the cytoplasmic domain of Fas and association with lipid rafts. (A, left) HeLa cells were transfected with Fas-YFP, FasΔcyt-YFP, or membrane-YFP and stimulated 18 h after transfection with the indicated combinations of cross-linked soluble Flag-FasL (200 ng/ml) and z-VAD-fmk (20 μM). The proportion of cells that showed SPOTS was determined after 2 h. (Middle) HeLa cells expressing either Fas-YFP (black bars) or FasΔcyt-YFP (gray bars) were cocultured with HEK293 cells, transiently transfected with CFP-FasL. After 1 h of coculture, neighbored blue and yellow fluorescent cells were analyzed and cluster incidence was determined as described in Materials and methods. (Right) HeLa cells expressing Fas-YFP were treated for 20 min with 20 mM βMCD, stimulated for with M2-Flag cross-linked soluble Flag-FasL (200 ng/ml), or cocultured with CFP-FasL expressing HEK293 cells. SPOTS incidence and cluster incidence were determined after 1 h. (B) Microscopic images of cells expressing Fas-YFP or FasΔcyt-YFP before (0 h) and after stimulation with the indicated reagents (2 h). The image showing Flag-FasL/M2 challenged cells was already taken after 1 h.