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.

Show MeSH

Related in: MedlinePlus

FLIP analysis of free and clustered YFP-FasL and Fas-YFP. HeLa cells transfected with YFP-FasL and Fas-YFP, respectively, were cultured individually or together with HeLa cells expressing CFP fusion proteins of the corresponding receptor or ligand. Images of a representative cell from each experimental group are shown. Bleaching areas covering most of the cell except the analyzed FasL-Fas cluster and/or a control region in the plasma membrane were defined (dotted lines). Cells were initially bleached for 5 min to bleach the majority of the highly diffusible intracellular fraction of the YFP fusion proteins. After this, bleaching was continued, and images were recorded in 1-min intervals. Using these images, loss of fluorescence in nonbleached areas, which contained clusters or control membrane, was determined, as described in Materials and methods. These areas were defined as ROI, and are indicated (dotted line, bleach area; solid line, ROI of clustered or free YFP fusion protein; dashed line, control ROI). Measurements of clustered YFP fusion proteins (Fas-YFP, closed squares top diagram; YFP-FasL, closed squares bottom diagram) were corrected for each time point for the residual fluorescence of the corresponding “free” protein outside the bleach region. Fluorescence intensities of nonclustered Fas-YFP (open squares top diagram) and YFP-FasL (open squares bottom diagram) determined outside the bleach regions were corrected for the rest fluorescence within the bleach region. Corrected fluorescence intensities were normalized against the relative fluorescence intensity obtained after the initial five bleach cycles. This time point of each experiment was defined as t = 0 min. Averaged (n = 12–15) normalized relative fluorescence intensities were shown in the diagrams. The fluorescence loss indicating dissociation was linearized by plotting the natural logarithm of the relative fluorescence intensities over time. The third row in the top part shows a control experiment where besides Fas-YFP membrane CFP were coexpressed to demonstrate that the overall morphology of bleached cells was not affected.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171833&req=5

fig3: FLIP analysis of free and clustered YFP-FasL and Fas-YFP. HeLa cells transfected with YFP-FasL and Fas-YFP, respectively, were cultured individually or together with HeLa cells expressing CFP fusion proteins of the corresponding receptor or ligand. Images of a representative cell from each experimental group are shown. Bleaching areas covering most of the cell except the analyzed FasL-Fas cluster and/or a control region in the plasma membrane were defined (dotted lines). Cells were initially bleached for 5 min to bleach the majority of the highly diffusible intracellular fraction of the YFP fusion proteins. After this, bleaching was continued, and images were recorded in 1-min intervals. Using these images, loss of fluorescence in nonbleached areas, which contained clusters or control membrane, was determined, as described in Materials and methods. These areas were defined as ROI, and are indicated (dotted line, bleach area; solid line, ROI of clustered or free YFP fusion protein; dashed line, control ROI). Measurements of clustered YFP fusion proteins (Fas-YFP, closed squares top diagram; YFP-FasL, closed squares bottom diagram) were corrected for each time point for the residual fluorescence of the corresponding “free” protein outside the bleach region. Fluorescence intensities of nonclustered Fas-YFP (open squares top diagram) and YFP-FasL (open squares bottom diagram) determined outside the bleach regions were corrected for the rest fluorescence within the bleach region. Corrected fluorescence intensities were normalized against the relative fluorescence intensity obtained after the initial five bleach cycles. This time point of each experiment was defined as t = 0 min. Averaged (n = 12–15) normalized relative fluorescence intensities were shown in the diagrams. The fluorescence loss indicating dissociation was linearized by plotting the natural logarithm of the relative fluorescence intensities over time. The third row in the top part shows a control experiment where besides Fas-YFP membrane CFP were coexpressed to demonstrate that the overall morphology of bleached cells was not affected.

Mentions: To analyze the stability of the FasL-Fas clusters further, we determined the exchange of unbound and cluster-bound molecules of YFP-FasL and Fas-YFP. We selected transfected cells with moderate YFP fusion protein levels and measured the loss of fluorescence in the FasL-Fas clusters or in plasma membrane areas of comparable size while continuously bleaching the YFP fusion protein in the remaining area of transfected cells (Fig. 3). This revealed a clear difference of fluorescence loss between “free” and clustered fusion proteins. In case of nonclustered YFP-FasL and Fas-YFP, half of the fluorescence was lost in 7.2 and 3.5 min, respectively. Thus, free membrane Fas exerts a significantly higher mobility than nonclustered FasL. However, when YFP-FasL and Fas-YFP were localized in clusters, loss of fluorescence was comparable for both molecules, but strongly reduced, as compared with their nonclustered counterparts. Decay of fluorescence of the clustered proteins was <15% within 15 min. Based on the assumption that the fluorescence of the clustered fusion proteins declines with exponential kinetics the average retention periods were ∼72 and 59 min.


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)

FLIP analysis of free and clustered YFP-FasL and Fas-YFP. HeLa cells transfected with YFP-FasL and Fas-YFP, respectively, were cultured individually or together with HeLa cells expressing CFP fusion proteins of the corresponding receptor or ligand. Images of a representative cell from each experimental group are shown. Bleaching areas covering most of the cell except the analyzed FasL-Fas cluster and/or a control region in the plasma membrane were defined (dotted lines). Cells were initially bleached for 5 min to bleach the majority of the highly diffusible intracellular fraction of the YFP fusion proteins. After this, bleaching was continued, and images were recorded in 1-min intervals. Using these images, loss of fluorescence in nonbleached areas, which contained clusters or control membrane, was determined, as described in Materials and methods. These areas were defined as ROI, and are indicated (dotted line, bleach area; solid line, ROI of clustered or free YFP fusion protein; dashed line, control ROI). Measurements of clustered YFP fusion proteins (Fas-YFP, closed squares top diagram; YFP-FasL, closed squares bottom diagram) were corrected for each time point for the residual fluorescence of the corresponding “free” protein outside the bleach region. Fluorescence intensities of nonclustered Fas-YFP (open squares top diagram) and YFP-FasL (open squares bottom diagram) determined outside the bleach regions were corrected for the rest fluorescence within the bleach region. Corrected fluorescence intensities were normalized against the relative fluorescence intensity obtained after the initial five bleach cycles. This time point of each experiment was defined as t = 0 min. Averaged (n = 12–15) normalized relative fluorescence intensities were shown in the diagrams. The fluorescence loss indicating dissociation was linearized by plotting the natural logarithm of the relative fluorescence intensities over time. The third row in the top part shows a control experiment where besides Fas-YFP membrane CFP were coexpressed to demonstrate that the overall morphology of bleached cells was not affected.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171833&req=5

fig3: FLIP analysis of free and clustered YFP-FasL and Fas-YFP. HeLa cells transfected with YFP-FasL and Fas-YFP, respectively, were cultured individually or together with HeLa cells expressing CFP fusion proteins of the corresponding receptor or ligand. Images of a representative cell from each experimental group are shown. Bleaching areas covering most of the cell except the analyzed FasL-Fas cluster and/or a control region in the plasma membrane were defined (dotted lines). Cells were initially bleached for 5 min to bleach the majority of the highly diffusible intracellular fraction of the YFP fusion proteins. After this, bleaching was continued, and images were recorded in 1-min intervals. Using these images, loss of fluorescence in nonbleached areas, which contained clusters or control membrane, was determined, as described in Materials and methods. These areas were defined as ROI, and are indicated (dotted line, bleach area; solid line, ROI of clustered or free YFP fusion protein; dashed line, control ROI). Measurements of clustered YFP fusion proteins (Fas-YFP, closed squares top diagram; YFP-FasL, closed squares bottom diagram) were corrected for each time point for the residual fluorescence of the corresponding “free” protein outside the bleach region. Fluorescence intensities of nonclustered Fas-YFP (open squares top diagram) and YFP-FasL (open squares bottom diagram) determined outside the bleach regions were corrected for the rest fluorescence within the bleach region. Corrected fluorescence intensities were normalized against the relative fluorescence intensity obtained after the initial five bleach cycles. This time point of each experiment was defined as t = 0 min. Averaged (n = 12–15) normalized relative fluorescence intensities were shown in the diagrams. The fluorescence loss indicating dissociation was linearized by plotting the natural logarithm of the relative fluorescence intensities over time. The third row in the top part shows a control experiment where besides Fas-YFP membrane CFP were coexpressed to demonstrate that the overall morphology of bleached cells was not affected.
Mentions: To analyze the stability of the FasL-Fas clusters further, we determined the exchange of unbound and cluster-bound molecules of YFP-FasL and Fas-YFP. We selected transfected cells with moderate YFP fusion protein levels and measured the loss of fluorescence in the FasL-Fas clusters or in plasma membrane areas of comparable size while continuously bleaching the YFP fusion protein in the remaining area of transfected cells (Fig. 3). This revealed a clear difference of fluorescence loss between “free” and clustered fusion proteins. In case of nonclustered YFP-FasL and Fas-YFP, half of the fluorescence was lost in 7.2 and 3.5 min, respectively. Thus, free membrane Fas exerts a significantly higher mobility than nonclustered FasL. However, when YFP-FasL and Fas-YFP were localized in clusters, loss of fluorescence was comparable for both molecules, but strongly reduced, as compared with their nonclustered counterparts. Decay of fluorescence of the clustered proteins was <15% within 15 min. Based on the assumption that the fluorescence of the clustered fusion proteins declines with exponential kinetics the average retention periods were ∼72 and 59 min.

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