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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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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.
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fig2: 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.

Mentions: In z-VAD-fmk protected cells time-lapse microscopy revealed that the FasL-Fas clusters, although undergoing slow dynamic morphologic changes, are stable over hours even in the presence of high concentrations of cycloheximide (25 μg/ml), thus under conditions were synthesis of new proteins was completely inhibited (Fig. 2, A and B). This indicated that under conditions of Fas activation, there was no or only a very limited internalization, an interesting difference to what has been reported for Fas stimulation with soluble reagents (Algeciras-Schimnich et al., 2002; Eramo et al., 2004). To verify whether soluble FasL is capable to induce changes in Fas cell surface expression in the cellular system used here, we comparatively analyzed cell surface expression of soluble FasL-stimulated and nonstimulated Fas transfected HeLa cells. In accordance with soluble FasL-induced internalization, Fas cell surface expression was lowered in the FasL-treated cells by 20–30% 2 h after stimulation (Fig. 2 B). The extent of reduction in Fas cell surface expression was comparable to that observed in the cited studies. To analyze the relationship of cluster formation and apoptosis induction, freshly harvested CFP-FasL expressing cells were added to adherent Fas-YFP transfected cells and cluster formation was monitored online when first signs of cell to cell contact were obvious. Within 1–4 h after initial cluster formation the majority of cycloheximide sensitized (not depicted) as well as a significant portion of nonsensitized (Fig. 2 C) Fas-YFP expressing cells rounded up and detached from the tissue culture plates indicating apoptosis of Fas-YFP expressing cells. Notably, clusters between CFP-FasL expressing cells and dying Fas-YFP transfectants remained stable and intact till detachment and even longer.


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)

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.
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Related In: Results  -  Collection

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fig2: 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.
Mentions: In z-VAD-fmk protected cells time-lapse microscopy revealed that the FasL-Fas clusters, although undergoing slow dynamic morphologic changes, are stable over hours even in the presence of high concentrations of cycloheximide (25 μg/ml), thus under conditions were synthesis of new proteins was completely inhibited (Fig. 2, A and B). This indicated that under conditions of Fas activation, there was no or only a very limited internalization, an interesting difference to what has been reported for Fas stimulation with soluble reagents (Algeciras-Schimnich et al., 2002; Eramo et al., 2004). To verify whether soluble FasL is capable to induce changes in Fas cell surface expression in the cellular system used here, we comparatively analyzed cell surface expression of soluble FasL-stimulated and nonstimulated Fas transfected HeLa cells. In accordance with soluble FasL-induced internalization, Fas cell surface expression was lowered in the FasL-treated cells by 20–30% 2 h after stimulation (Fig. 2 B). The extent of reduction in Fas cell surface expression was comparable to that observed in the cited studies. To analyze the relationship of cluster formation and apoptosis induction, freshly harvested CFP-FasL expressing cells were added to adherent Fas-YFP transfected cells and cluster formation was monitored online when first signs of cell to cell contact were obvious. Within 1–4 h after initial cluster formation the majority of cycloheximide sensitized (not depicted) as well as a significant portion of nonsensitized (Fig. 2 C) Fas-YFP expressing cells rounded up and detached from the tissue culture plates indicating apoptosis of Fas-YFP expressing cells. Notably, clusters between CFP-FasL expressing cells and dying Fas-YFP transfectants remained stable and intact till detachment and even longer.

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