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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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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.
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fig1: 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.

Mentions: The mechanisms leading to Fas clustering and Fas activation have been predominantly analyzed with cross-linked variants of soluble trimeric FasL or agonistic Fas-specific antibodies. However, the prime in vivo activator of Fas is the transmembrane form of FasL. To analyze membrane FasL-induced Fas clustering in living cells by confocal microscopy we have used YFP and CFP fusion proteins of FasL and Fas (Fig. 1 A). Analysis of apoptosis induction, IL8 up-regulation and NFκB activation showed that the various YFP and CFP fusion proteins were functional with respect to apoptotic and nonapoptotic Fas signaling (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200501048/DC1). The confocal images of HeLa cells individually transfected with either CFP-FasL or Fas-YFP in Fig. 1 B show the typical membrane expression pattern of these fusion proteins. As expected, both proteins appear as proteins enriched at the plasma membrane and at intracellular membranes and vesicles, en route to the cell membrane. Although Fas-YFP was found to be homogeneously distributed in the plasma membrane, YFP-FasL (Fig. 1 B) and CFP-FasL (not depicted) showed a speckled distribution. Notably, the detection pattern of FasL did not depend on the individual expression levels of analyzed cells. It is possible that FasL has an intrinsic tendency to form microscopically distinct aggregates. It cannot be formally ruled out that these small aggregates occur partly due to clustering with endogenous Fas. However, the same speckled appearance of YFP-FasL was also observed in cells expressing high amounts of the FasL fusion protein, thus in cells, where the expression level of YFP-FasL is many fold higher than that of endogenous Fas. When CFP-FasL and Fas-YFP were coexpressed in the same cell, both molecules did efficiently colocalize in patches, demonstrating that FasL and Fas can also interact on the same cell (unpublished data). In contrast, patching did not occur when CFP-FasL was cotransfected with CD40-YFP. In coculture assays of cells separately transfected with CFP-FasL and Fas-YFP (or YFP-FasL and Fas-CFP) FasL-Fas clustering between neighboring cells was found with very high incidence (Fig. 1, C and D). There were no effects on the distribution of CFP-FasL and YFP-Fas when these transfectants were cocultured with CD40-YFP and CFP-CD40L transfected cells, respectively (unpublished data). FasL-Fas clusters at sites of cell-to-cell contacts were formed when Fas-YFP was in type I (SV80, SKW) or type II cells (HeLa, Jurkat), as well as after expression in adherent (SV80, HeLa) and suspended cells (Jurkat, SKW) suggesting that the capability of membrane FasL to induce Fas clustering is not a cell-type specific property (unpublished data). Furthermore, in SKW cells, endogenously expressed Fas molecules were also efficiently incorporated in clusters when the cells were cocultured with CFP-FasL expressing cells (Fig. 1 E). Form and size of FasL-Fas clusters between neighboring cells were highly variable. Small “contact plates”, bigger dot-like clusters, and larger membrane stretches of Fas-FasL positive cell–cell contacts were regularly observed. As the majority of the fusion proteins were frequently trapped almost completely within supramolecular FasL-Fas clusters, overall cellular morphology was always ascertained by light microscopy. To prevent induction of apoptosis, FasL-Fas clustering was analyzed in the presence of the pan-caspase inhibitor z-VAD-fmk. This indicates that caspase-8 activation is not necessary for cluster formation.


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171833&req=5

fig1: 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.
Mentions: The mechanisms leading to Fas clustering and Fas activation have been predominantly analyzed with cross-linked variants of soluble trimeric FasL or agonistic Fas-specific antibodies. However, the prime in vivo activator of Fas is the transmembrane form of FasL. To analyze membrane FasL-induced Fas clustering in living cells by confocal microscopy we have used YFP and CFP fusion proteins of FasL and Fas (Fig. 1 A). Analysis of apoptosis induction, IL8 up-regulation and NFκB activation showed that the various YFP and CFP fusion proteins were functional with respect to apoptotic and nonapoptotic Fas signaling (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200501048/DC1). The confocal images of HeLa cells individually transfected with either CFP-FasL or Fas-YFP in Fig. 1 B show the typical membrane expression pattern of these fusion proteins. As expected, both proteins appear as proteins enriched at the plasma membrane and at intracellular membranes and vesicles, en route to the cell membrane. Although Fas-YFP was found to be homogeneously distributed in the plasma membrane, YFP-FasL (Fig. 1 B) and CFP-FasL (not depicted) showed a speckled distribution. Notably, the detection pattern of FasL did not depend on the individual expression levels of analyzed cells. It is possible that FasL has an intrinsic tendency to form microscopically distinct aggregates. It cannot be formally ruled out that these small aggregates occur partly due to clustering with endogenous Fas. However, the same speckled appearance of YFP-FasL was also observed in cells expressing high amounts of the FasL fusion protein, thus in cells, where the expression level of YFP-FasL is many fold higher than that of endogenous Fas. When CFP-FasL and Fas-YFP were coexpressed in the same cell, both molecules did efficiently colocalize in patches, demonstrating that FasL and Fas can also interact on the same cell (unpublished data). In contrast, patching did not occur when CFP-FasL was cotransfected with CD40-YFP. In coculture assays of cells separately transfected with CFP-FasL and Fas-YFP (or YFP-FasL and Fas-CFP) FasL-Fas clustering between neighboring cells was found with very high incidence (Fig. 1, C and D). There were no effects on the distribution of CFP-FasL and YFP-Fas when these transfectants were cocultured with CD40-YFP and CFP-CD40L transfected cells, respectively (unpublished data). FasL-Fas clusters at sites of cell-to-cell contacts were formed when Fas-YFP was in type I (SV80, SKW) or type II cells (HeLa, Jurkat), as well as after expression in adherent (SV80, HeLa) and suspended cells (Jurkat, SKW) suggesting that the capability of membrane FasL to induce Fas clustering is not a cell-type specific property (unpublished data). Furthermore, in SKW cells, endogenously expressed Fas molecules were also efficiently incorporated in clusters when the cells were cocultured with CFP-FasL expressing cells (Fig. 1 E). Form and size of FasL-Fas clusters between neighboring cells were highly variable. Small “contact plates”, bigger dot-like clusters, and larger membrane stretches of Fas-FasL positive cell–cell contacts were regularly observed. As the majority of the fusion proteins were frequently trapped almost completely within supramolecular FasL-Fas clusters, overall cellular morphology was always ascertained by light microscopy. To prevent induction of apoptosis, FasL-Fas clustering was analyzed in the presence of the pan-caspase inhibitor z-VAD-fmk. This indicates that caspase-8 activation is not necessary for cluster formation.

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