Limits...
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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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.
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fig6: 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.

Mentions: It has been suggested that constitutive or inducible localization of Fas in membrane areas enriched in cholesterol and sphingolipids (lipid rafts) can be necessary for both clustering and activation of Fas by soluble agonists (Grassme et al., 2001a,b; Hueber et al., 2002; Aouad et al., 2004; Muppidi and Siegel, 2004). Accordingly, Fas signaling was abrogated in various studies by pretreatment of cells with the cholesterol depleting compound methyl-β-cyclodextrin (βMCD; Cremesti et al., 2001; Grassme et al., 2001a,b; Hueber et al., 2002; Muppidi and Siegel, 2004). This raised the question of whether the induction of supramolecular Fas clusters by membrane FasL was dependent of lipid rafts. First, we analyzed association of Fas-YFP and YFP-FasL with lipid rafts in HeLa cells using cell fractionation on sucrose gradients. Transiently expressed lipid raft marker proteins, such as Lck-GFP and a palmitylated YFP variant were found as expected in the detergent insoluble factions obtained in our experiments (Fig. 6 A). In contrast, cytosolic proteins such as JNK and predominantly cytosolic proteins as tubulin which are not (JNK) or only barely (tubulin) associated with lipid rafts were found in the detergent soluble fractions (Fig. 6 A). Notably, Fas-YFP and YFP-FasL were almost exclusively detected in the detergent insoluble fraction in HeLa cells indicating a constitutive association with lipid rafts (Fig. 6 A). This strong association with lipid rafts was also maintained when both proteins were incorporated in supramolecular clusters. Interestingly, lipid raft association of Fas-YFP was not dependent on its cytoplasmic domain, because the corresponding Fas deletion mutant was also mainly detected in the detergent insoluble fractions (Fig. 6 A). The constitutive association of Fas with lipid rafts was further confirmed by fluorescence microscopy. HeLa cells expressing Fas-CFP were labeled with rhodamine-conjugated cholera toxin B to visualize ganglioside GM1. After patching with an anti–cholera toxin B antibody, strong colocalization in punctuate structures of Fas and cholera toxin was observed in the majority of cells (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200501048/DC1). The putative requirement of lipid rafts for the formation of FasL-Fas clusters was then analyzed in cells, which were depleted of cholesterol by treatment with βMCD. In these experiments, we pretreated Fas-YFP expressing HeLa cells in serum free medium with 20 mM βMCD for 20 min at 37°C, added freshly harvested CFP-FasL expressing cells and followed cluster formation online. Cell fractionation of cholesterol depleted cells on sucrose gradients confirmed that the vast majority of Fas-YFP or Lck-GFP had shifted into the detergent soluble fraction (Fig. 6 A). Successful cholesterol depletion was also evident from the fact that βMCD-treated cells showed significant morphological changes. FasL-Fas clusters were still efficiently formed, when cells expressing the corresponding molecules came into contact, although the assembly of cluster formation was delayed (Fig. 6, B and C). Moreover, clusters were even formed and maintained in cells that were rounded up and detached from the culture dish.


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)

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.
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Related In: Results  -  Collection

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fig6: 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.
Mentions: It has been suggested that constitutive or inducible localization of Fas in membrane areas enriched in cholesterol and sphingolipids (lipid rafts) can be necessary for both clustering and activation of Fas by soluble agonists (Grassme et al., 2001a,b; Hueber et al., 2002; Aouad et al., 2004; Muppidi and Siegel, 2004). Accordingly, Fas signaling was abrogated in various studies by pretreatment of cells with the cholesterol depleting compound methyl-β-cyclodextrin (βMCD; Cremesti et al., 2001; Grassme et al., 2001a,b; Hueber et al., 2002; Muppidi and Siegel, 2004). This raised the question of whether the induction of supramolecular Fas clusters by membrane FasL was dependent of lipid rafts. First, we analyzed association of Fas-YFP and YFP-FasL with lipid rafts in HeLa cells using cell fractionation on sucrose gradients. Transiently expressed lipid raft marker proteins, such as Lck-GFP and a palmitylated YFP variant were found as expected in the detergent insoluble factions obtained in our experiments (Fig. 6 A). In contrast, cytosolic proteins such as JNK and predominantly cytosolic proteins as tubulin which are not (JNK) or only barely (tubulin) associated with lipid rafts were found in the detergent soluble fractions (Fig. 6 A). Notably, Fas-YFP and YFP-FasL were almost exclusively detected in the detergent insoluble fraction in HeLa cells indicating a constitutive association with lipid rafts (Fig. 6 A). This strong association with lipid rafts was also maintained when both proteins were incorporated in supramolecular clusters. Interestingly, lipid raft association of Fas-YFP was not dependent on its cytoplasmic domain, because the corresponding Fas deletion mutant was also mainly detected in the detergent insoluble fractions (Fig. 6 A). The constitutive association of Fas with lipid rafts was further confirmed by fluorescence microscopy. HeLa cells expressing Fas-CFP were labeled with rhodamine-conjugated cholera toxin B to visualize ganglioside GM1. After patching with an anti–cholera toxin B antibody, strong colocalization in punctuate structures of Fas and cholera toxin was observed in the majority of cells (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200501048/DC1). The putative requirement of lipid rafts for the formation of FasL-Fas clusters was then analyzed in cells, which were depleted of cholesterol by treatment with βMCD. In these experiments, we pretreated Fas-YFP expressing HeLa cells in serum free medium with 20 mM βMCD for 20 min at 37°C, added freshly harvested CFP-FasL expressing cells and followed cluster formation online. Cell fractionation of cholesterol depleted cells on sucrose gradients confirmed that the vast majority of Fas-YFP or Lck-GFP had shifted into the detergent soluble fraction (Fig. 6 A). Successful cholesterol depletion was also evident from the fact that βMCD-treated cells showed significant morphological changes. FasL-Fas clusters were still efficiently formed, when cells expressing the corresponding molecules came into contact, although the assembly of cluster formation was delayed (Fig. 6, B and C). Moreover, clusters were even formed and maintained in cells that were rounded up and detached from the culture dish.

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