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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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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.
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fig7: 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.

Mentions: It has been shown in a recent study that cross-linked soluble FasL induce microscopically visible punctuate receptor oligomers in an early stage of Fas-induced apoptosis (Siegel et al., 2004). These structures were termed SPOTS and are possibly related to the supramolecular Fas clusters described in our study. Therefore, we have compared effects of membrane FasL and cross-linked soluble FasL on Fas-YFP aggregation. Although both FasL forms efficiently triggered Fas aggregation (SPOTS or clusters, respectively), some notable differences were also observed. In accordance with the data reported by Siegel et al. (2004), we found that soluble FasL was crucially dependent on the cytoplasmic domain of Fas to induce the formation of SPOTS (Fig. 7, A and B). In contrast, as already shown in Fig. 5, deletion of the cytoplasmic domain of Fas only caused a delay in the kinetics of membrane FasL-induced cluster formation. Furthermore, Fas-YFP SPOTS formation by soluble FasL was completely abrogated in cholesterol depleted cells, whereas membrane FasL-induced cluster formation was only delayed (Fig. 6 B and Fig. 7 A).


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)

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.
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fig7: 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.
Mentions: It has been shown in a recent study that cross-linked soluble FasL induce microscopically visible punctuate receptor oligomers in an early stage of Fas-induced apoptosis (Siegel et al., 2004). These structures were termed SPOTS and are possibly related to the supramolecular Fas clusters described in our study. Therefore, we have compared effects of membrane FasL and cross-linked soluble FasL on Fas-YFP aggregation. Although both FasL forms efficiently triggered Fas aggregation (SPOTS or clusters, respectively), some notable differences were also observed. In accordance with the data reported by Siegel et al. (2004), we found that soluble FasL was crucially dependent on the cytoplasmic domain of Fas to induce the formation of SPOTS (Fig. 7, A and B). In contrast, as already shown in Fig. 5, deletion of the cytoplasmic domain of Fas only caused a delay in the kinetics of membrane FasL-induced cluster formation. Furthermore, Fas-YFP SPOTS formation by soluble FasL was completely abrogated in cholesterol depleted cells, whereas membrane FasL-induced cluster formation was only delayed (Fig. 6 B and Fig. 7 A).

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