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

Henkler F, Behrle E, Dennehy KM, Wicovsky A, Peters N, Warnke C, Pfizenmaier K, Wajant H - J. Cell Biol. (2005)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Internal Medicine, Medical Polyclinic, University of Wuerzburg, 97070 Wuerzburg, Germany.

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.

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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).
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fig5: 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).

Mentions: The fact that the caspase inhibitor z-VAD-fmk does not interfere with the formation of FasL-Fas clusters in our experiments argues against a crucial role of caspase-8 in FasL-induced Fas aggregation. As studies with soluble Fas agonists have reported such a role we further substantiate this finding by analyzing Fas cluster formation in Jurkat clones deficient in the expression of FADD or caspase-8 after cocultivation with CFP-FasL expressing HeLa cells. Jurkat cells were only transfected with low efficiency (0.5 – 3%). Accordingly, there was only a low number (20–50) of neighboring cell pairs that expressed Fas-YFP and CFP-FasL, respectively. However, clusters were observed in the vast majority of such cell pairs, indicating that this process is independent from FADD or caspase-8 (Fig. 4). To exclude a contribution of other Fas interacting signaling components in cluster formation, we analyzed the capacity of a Fas deletion mutant lacking the entire cytoplasmic domain (FasΔcyt) to form clusters (Fig. 5 A). As in case of the Fas-related CD40-CD40L system, a reverse signaling activity of the ligand has been implicated in the formation of supramolecular clusters (Grassme et al., 2002), we also analyzed deletion mutants of FasL lacking the cytoplasmic domain. Although the FasΔcyt mutants were unable to activate the signaling pathways that are associated with Fas and act as dominant-negative variants, the FasL deletion mutants were comparably active as the wild-type ligand (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200501048/DC1). In HeLa cells, which has been transfected with Fas-YFP or FasΔcyt-YFP, cluster induction was initiated by overlaying FasL-CFP expressing 293 cells. After 1 h coculture near to 100% cluster incidence was observed in Fas-YFP as well as in FasΔcyt-YFP expressing cells that were in contact with CFP-FasL expressing cells (Fig. 5 B). This indicates that the extracellular domain of Fas is sufficient to assemble receptor–ligand clusters. However, the cytosolic domain of Fas might exert a modulating effect, as at earlier time points cluster formation was significantly delayed (Fig. 5 B). Notably, FasΔcyt-YFP showed also an altered dissociation kinetic, as compared with full-length Fas. When it was clustered with CFP-FasL or CFP-FasLΔcyt, a significant reduction in t1/2 of ∼50% was observed in both cases (compare Fig. 5 C with Fig. 3). These observations open the possibility that the cytosolic DISC or homotypic interaction of the Fas death domain might further stabilize the ligand–receptor interactions, without having an essential role on its initiation. Similar as the corresponding full-length counterparts, both fluorescent FasL-deletion proteins localized to the plasma membrane. In contrast to the speckled membrane distribution of YFP-FasL (Fig. 1 B), FasLΔcyt showed a rather homogenous distribution comparable with Fas receptor fusion proteins. Despite these slightly different distributions, there were no significant differences in the mobility of free YFP-FasLΔcyt and YFP-FasL in the membrane of transfected cells. Furthermore, both proteins showed comparable dissociation kinetics when they were incorporated in clusters with Fas-CFP (Fig. 5 C). Together these data show that the cytoplasmic domains of Fas and FasL, as well as the associated signaling pathways are dispensable for the formation of supramolecular FasL-Fas clusters. Furthermore, the cytoplasmic domain of Fas, but not FasL, has a modulating effect in this process. However, it is possible that the somewhat reduced stability of FasΔcyt-YFP clusters reflects a clustering promoting effect of Fas signaling or the Fas death domain, which may become relevant when Fas agonists of restricted activity, as antibodies or cross-linked soluble FasL, were used.


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

Henkler F, Behrle E, Dennehy KM, Wicovsky A, Peters N, Warnke C, Pfizenmaier K, Wajant H - J. Cell Biol. (2005)

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).
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fig5: 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).
Mentions: The fact that the caspase inhibitor z-VAD-fmk does not interfere with the formation of FasL-Fas clusters in our experiments argues against a crucial role of caspase-8 in FasL-induced Fas aggregation. As studies with soluble Fas agonists have reported such a role we further substantiate this finding by analyzing Fas cluster formation in Jurkat clones deficient in the expression of FADD or caspase-8 after cocultivation with CFP-FasL expressing HeLa cells. Jurkat cells were only transfected with low efficiency (0.5 – 3%). Accordingly, there was only a low number (20–50) of neighboring cell pairs that expressed Fas-YFP and CFP-FasL, respectively. However, clusters were observed in the vast majority of such cell pairs, indicating that this process is independent from FADD or caspase-8 (Fig. 4). To exclude a contribution of other Fas interacting signaling components in cluster formation, we analyzed the capacity of a Fas deletion mutant lacking the entire cytoplasmic domain (FasΔcyt) to form clusters (Fig. 5 A). As in case of the Fas-related CD40-CD40L system, a reverse signaling activity of the ligand has been implicated in the formation of supramolecular clusters (Grassme et al., 2002), we also analyzed deletion mutants of FasL lacking the cytoplasmic domain. Although the FasΔcyt mutants were unable to activate the signaling pathways that are associated with Fas and act as dominant-negative variants, the FasL deletion mutants were comparably active as the wild-type ligand (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200501048/DC1). In HeLa cells, which has been transfected with Fas-YFP or FasΔcyt-YFP, cluster induction was initiated by overlaying FasL-CFP expressing 293 cells. After 1 h coculture near to 100% cluster incidence was observed in Fas-YFP as well as in FasΔcyt-YFP expressing cells that were in contact with CFP-FasL expressing cells (Fig. 5 B). This indicates that the extracellular domain of Fas is sufficient to assemble receptor–ligand clusters. However, the cytosolic domain of Fas might exert a modulating effect, as at earlier time points cluster formation was significantly delayed (Fig. 5 B). Notably, FasΔcyt-YFP showed also an altered dissociation kinetic, as compared with full-length Fas. When it was clustered with CFP-FasL or CFP-FasLΔcyt, a significant reduction in t1/2 of ∼50% was observed in both cases (compare Fig. 5 C with Fig. 3). These observations open the possibility that the cytosolic DISC or homotypic interaction of the Fas death domain might further stabilize the ligand–receptor interactions, without having an essential role on its initiation. Similar as the corresponding full-length counterparts, both fluorescent FasL-deletion proteins localized to the plasma membrane. In contrast to the speckled membrane distribution of YFP-FasL (Fig. 1 B), FasLΔcyt showed a rather homogenous distribution comparable with Fas receptor fusion proteins. Despite these slightly different distributions, there were no significant differences in the mobility of free YFP-FasLΔcyt and YFP-FasL in the membrane of transfected cells. Furthermore, both proteins showed comparable dissociation kinetics when they were incorporated in clusters with Fas-CFP (Fig. 5 C). Together these data show that the cytoplasmic domains of Fas and FasL, as well as the associated signaling pathways are dispensable for the formation of supramolecular FasL-Fas clusters. Furthermore, the cytoplasmic domain of Fas, but not FasL, has a modulating effect in this process. However, it is possible that the somewhat reduced stability of FasΔcyt-YFP clusters reflects a clustering promoting effect of Fas signaling or the Fas death domain, which may become relevant when Fas agonists of restricted activity, as antibodies or cross-linked soluble FasL, were used.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Internal Medicine, Medical Polyclinic, University of Wuerzburg, 97070 Wuerzburg, Germany.

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.

Show MeSH
Related in: MedlinePlus