Correspondence to Harald Wajant: harald.wajant{at}mail.uni-wuerzburg.de
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Formation of signaling competent Fas complexes is accompanied by recruitment of the cytoplasmic death domain-containing adaptor protein Fas-associated death domain (FADD; Peter and Krammer, 2003). FADD recruitment depends on the interaction between the death domains of Fas and FADD. Fas-bound FADD in turn is able to bind procaspase-8. Within this death-inducing signaling complex (DISC), procaspase-8 is activated by dimerization (Boatright et al., 2003; Donepudi et al., 2003). DISC-bound active procaspase-8 dimers are then converted by autoproteolytic processing into the mature and active heterotetrameric form of the enzyme which is released from the Fas signaling complex. Active caspase-8 cleaves a limited set of substrates including caspase-3 and the BH3-only protein Bid. Two types of cells can be defined. In type I cells caspase-8 mediated activation of caspase-3 is sufficient to ensure execution of the final steps of apoptosis (Barnhart et al., 2003; Peter and Krammer, 2003). In contrast, in type II cells caspase-8 activation is less efficient and/or activation of effector caspases is inhibited by members of the inhibitor of apoptosis (IAP) protein family (Barnhart et al., 2003; Peter and Krammer, 2003). In these cells, a caspase-8 generated cleavage product of Bid, named truncated Bid, may contribute to apoptosis by inducing Bax/Bak-dependent release of apoptogenic proteins from mitochondria, especially cytochrome c and further SMAC/Diablo and HtrA2/Omi (Barnhart et al., 2003; Peter and Krammer, 2003). Cytochrome c assembles in the cytoplasm with ATP and the scaffold protein apoptosis promoting factor-1 to form the caspase-9 activating apoptosome (Shi, 2002), which in turn processes and activates caspase-3. Smac/Diablo and HtrA2/Omi block caspase inhibition by members of the IAP protein family (Verhagen and Vaux, 2002). Thus, both mechanisms enhance the effect of initially DISC-activated caspase-8 and facilitate activation of effector caspases, especially caspase-3. A contribution of the mitochondrial pathway to Fas-induced apoptosis has been experimentally defined in vitro by ectopic overexpression of the anti-apoptotic Bcl2 protein (Scaffidi et al., 1998). In type I cells, Fas-induced apoptosis is not affected by the Bcl2-dependent inhibition of Bax/Bak-mediated release of apoptogenic factors. In contrast, in type II cells Bcl2 expression attenuates apoptosis induction by Fas (Barnhart et al., 2003; Peter and Krammer, 2003). In vivo, thymocytes have consistently been recognized as type I cells. Although some in vivo studies using suboptimal doses of agonistic anti-Fas antibodies found a contribution of the mitochondrial pathway in Fas-induced apoptosis of hepatocytes, the conclusions regarding the in vivo relevance of the mitochondrial pathway for Fas-induced apoptosis extracted from these data are discussed differently (Schmitz et al., 1999; Huang et al., 2000).
Although Fas signaling is well understood, the mechanisms that drive the formation of signaling competent Fas complexes are poorly defined. Soluble trimeric FasL interacts with three molecules of Fas but fails to activate the receptor efficiently. In contrast, hexameric soluble FasL or antibody cross-linked soluble FasL are sufficient to induce Fas signaling (Schneider et al., 1998; Holler et al., 2003). Therefore, it has been suggested that Fas activation requires secondary interaction of trimeric FasLFas (FasL3Fas3) complexes. This concept is also in agreement with the observation that agonistic Fas specific antibodies belong to the IgM or IgG3 subclass or require secondary aggregation e.g., by protein A (Kischkel et al., 1995; Huang et al., 1999). Treatment of Fas expressing cells with cross-linked FasL or agonistic anti-Fas antibodies induces higher order clusters ("capping"), which are detectable by immunofluorescent microscopy (von Reyher et al., 1998; Cremesti et al., 2001; Grassme et al., 2001a, b; Algeciras-Schimnich et al., 2002). Although the Fas DISC is rapidly assembled, several lines of evidence suggest that formation of this procaspase-8 converting protein complex is not the first consequence of Fas stimulation but rather a later step in the conversion of preassembled signaling incompetent Fas complexes into "activated" receptor complexes (Grassme et al., 2001a, HREF="#BIB12">b; Cremesti et al., 2001; Algeciras-Schimnich et al., 2002). In particular, formation of Fas microclusters (Kischkel et al., 1995; Kamitani et al., 1997; Ruiz-Ruiz et al., 1999; Varadhachary et al., 2001; Algeciras-Schimnich et al., 2002), actin reorganization (Algeciras-Schimnich et al., 2002), inducible or constitutive association with membrane rafts (Grassme et al., 2001a, HREF="#BIB12">b; Hueber et al., 2002; Aouad et al., 2004, Muppidi and Siegel, 2004) and acid sphingomyelinase mediated ceramide production (Cremesti et al., 2001) have been discussed as important intermediate steps preceding robust DISC formation.
Here, we show that membrane FasL and Fas form supramolecular clusters of high stability, persisting for several hours. In contrast to early, soluble FasL-induced Fas aggregates, recently named signaling protein oligomeric transduction structures (SPOTS), membrane FasL-induced Fas clusters form independently of the intracellular domain of Fas and occur in the absence of FADD or caspase-8 suggesting that the formation of supramolecular FasL-Fas clusters precedes and is independent of recruitment and activation of downstream signaling components.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Using fluorescent fusion proteins of FasL and Fas we describe here that membrane FasL, induces efficient receptor clustering similar to cross-linked sFasL and anti-Fas antibodies. This suggests that the formation of higher order complexes of Fas and FasL is an essential step in membrane FasL-induced Fas activation. Time lapse microscopy and fluorescence loss in photobleaching (FLIP) in living cells revealed that membrane FasL-Fas clusters formed between neighboring cells expressing membrane CFP-FasL and Fas-YFP, respectively, are highly stable and enduring over a period of several hours, Moreover, there was no evidence for internalization of Fas-FasL clusters formed after cellcell contact (Fig. 2). This is an interesting difference from Fas activation with soluble reagents, where internalization has been observed (Algeciras-Schimnich et al., 2002; Eramo et al., 2004). Therefore, it appears possible that Fas internalization is not a major consequence of Fas activation but rather an effect related to soluble agonists. Moreover, membrane FasL-induced Fas cluster formation at the cell membrane resulted in gene induction or was followed by rapid cell death in the absence of the caspase inhibitor zVAD-fmk (Fig. 7 B; Fig. S1). Thus, our data demonstrate that proper Fas signaling is initiated at the membrane and does not require internalization. The latter may only becomes relevant when clusters were formed between membrane FasL and Fas, expressed in the same cell or when cluster-bound membrane FasL is shed. The long persistence of membrane FasL-Fas clusters could be of special relevance when FasL expressing cells stimulate apoptosis in resistant Fas expressing cells that can however become sensitive by regulatory mechanisms.
Membrane FasL-induced higher order complexes with Fas were also formed when Fas was expressed in FADD- or caspase-8deficient cells, in the presence of a broad range caspase inhibitor and when a cytoplasmic Fas deletion mutant was used. Thus, cluster formation occurred under circumstances, where activation of the currently known Fas-induced signaling pathways, in particular caspase-8 activation, was blocked. The concept that Fas induced caspase-8 activation is not an obligate step in robust Fas activation is also in agreement with the signaling capabilities of mutated Fas in lpr-cg mice (Desbarats et al., 2003). These mice show normal Fas-induced ERK activation despite a mutation in the death domain of Fas which prevents caspase-8 activation and apoptosis induction. Moreover, an independent Fas deletion mutant has been described, which interferes with FADD binding and apoptosis induction but still has the capacity to trigger JNK activation (Chang et al., 1999).
The relevance of lipid rafts, distinct plasma membrane microdomains enriched in cholesterol and sphingolipids, for Fas-induced DISC formation has been intensively studied. Although some studies found Fas constitutively outside of lipid rafts (Ko et al., 1999; Algeciras-Schimnich et al., 2002, Legler et al., 2003), other reports described significant incorporation of Fas and the DISC components caspase-8 and FADD in such membrane areas constitutively or after stimulation (Grassme et al., 2001a,b; Hueber et al., 2002; Aouad et al., 2004). These discrepancies have been partly attributed to different roles of lipid rafts for Fas signaling in type I and type II cells and may also depend on regulatory events not causally linked to Fas activation. For example, it has been recently shown that TCR signaling in activated CD4+ T cells induces relocation of Fas into lipid rafts (Muppidi and Siegel, 2004). Due to the complex and presumably cell type specific role of lipid rafts in Fas activation, we have clarified here the relationship between lipid rafts and the formation of membrane FasL-induced Fas clusters. We found that both Fas-YFP and YFP-FasL were constitutively associated with lipid rafts in HeLa cells (Fig. 6 A). This finding is in agreement with a previous report by Hueber et al. (2002), showing a comparable constitutive raft association of Fas in thymus cells. However, HeLa cells were strongly protected by Bcl2 overexpression against Fas-induced apoptosis (Mandal et al., 1996; unpublished data) and are therefore type II cells, whereas thymocytes are type I cells. Thus, constitutive raft association of Fas does not necessarily correlate with grouping cells into type I and type II. Noteworthy, Fas-YFP remained in association with lipid rafts when it was incorporated into membrane FasL-induced clusters (Fig. 6 A). In our experiments, lipid raft association of Fas did not crucially depend on its cytoplasmic domain suggesting that the Fas lipid raft association is not related to receptor activation in HeLa cells. The lipid raft association of Fas was not necessary for membrane FasL-induced cluster formation and showed only a modulating effect on this process (Fig. 6 B). In contrast to membrane FasL, cross-linked soluble FasL failed to induce SPOTS after dislocation of Fas from lipid rafts (Fig. 7). Moreover, SPOTS formation was also dependent on an intact cytoplasmic Fas domain (Fig. 7). SPOTS induced by soluble FasL and supramolecular clusters induced by membrane FasL are both activation-associated structures composed of Fas aggregates and are possibly formed by related mechanisms. However, due to the differential relevance of lipid raft association and the cytoplasmic domain of Fas for SPOTS and cluster formation, some differences must exist for the formation of these structures. Based on the experiments with the Fascyt mutants, it appears that Fas associated signaling events have a crucial role in Fas aggregation during SPOTS formation, but are not essential for Fas aggregation in membrane FasL-induced cluster formation. In fact, as already discussed above, it has been shown that initial Fas-mediated caspase-8 activation is necessary to trigger a positive feedback loop ensuring robust Fas signaling after stimulation with soluble FasL (or agonistic Fas antibodies; Grassme et al., 2001b; Algeciras-Schimnich et al., 2002). This gives rise to the question of why these Fas signaling dependent mechanisms are less important in membrane FasL-induced cluster formation and Fas activation. Perhaps, the spatial orientation and mobility reduction of membrane FasL in the plasma membrane facilitates Fas aggregation, making cytosolic events dispensable for membrane FasL-induced Fas clustering. The absence of Fas signaling may therefore only result in the observed delay in cluster formation. In contrast, it is tempting to speculate that in case of soluble FasL, the signaling dependent mechanisms of Fas aggregation gain higher relevance or are even essential, as cross-linked soluble FasL can possibly not fully substitute for plasma membrane organized FasL molecules. A similar argumentation may also explain the differential relevance of lipid rafts in membrane FasL-induced Fas clustering and soluble FasL-induced SPOTS formation. However, as first Fas resides constitutively in lipid rafts, also in unstimulated cells, and second Fas
cyt-YFP is still raft associated, enhancement of Fas aggregation by initial Fas signaling is most likely not related to association of Fas with lipid rafts. Thus, lipid raft association of Fas and a positive feedback of events associated with the Fas cytoplasmic domain may represent two independent mechanisms, compensating for the missing plasma membrane enforced spatial orientation of FasL when its soluble variant is used for Fas activation.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Determination of Fas-mediated apoptosis
Cells transfected with the indicated expression plasmids were cultured overnight. The next day, 2.5 µg/ml CHX was added and after 10 h, cell death was analyzed by crystal violet staining. Alternatively, Fas-YFP expressing cells were stimulated with cross-linked soluble Fas for 2 h. In these experiments, apoptotic effects were analyzed mircoscopically. Cells that rounded up and/or detached from the cover slide were defined as dying cell. Dying and normal cells were counted to determine the viability of Fas-YFP expressing cells.
Microscopic analysis of living cells or fixed samples
An inverted confocal microscope (model DM IRBE; Leica) with a 63x objective (HCX PL APO 63x/1.32 oil) was used for online analysis of living cells. The microscope carried a preconditioned chamber (37°C, 5% CO2) on its stage and was operated by confocal software TCS SL (v. 2.5.1227, serial no. 194043; Leica). Living cells were grown on glass bottom culture dishes, observed over 20 min up to several hours and imaged in regular intervals. Fixed and mounted cells were analyzed using a DM RE confocal microscope with a 100x objective (HCX PL APO 100x/1.40 oil; Leica). This microscope was operated by confocal software TCS SP2 (v. 2.5.1227; Leica). In all microscopic experiments excitation of CFP fusion proteins was performed at 456 nm, the emission was detected between 470 and 500 nm and visualized blue. Excitation of YFP proteins was done at 514 nm, the emission signal was detected above 600 nm and visualized yellow. Analysis of fluorescent samples, containing both CFP and YFP proteins were done by sequential screening. The absence of nonspecific signals in both selected emission ranges was verified. Overlays of different staining patterns in living cells were directly obtained as snapshots from Leica experimental files. Photographs of fixed cells, showing CFP/YFP costaining were derived by overlay of individual images in Adobe photoshop version 7.0.
FLIP analysis
These experiments were performed using cocultures of HeLa cells that expressed complementary YFP/CFP variants of Fas and FasL. Neighboring cells showing suitable receptorligand aggregates were analyzed. In all photo-bleaching experiments the excitation was performed at 514 nm (YFP channel) and the laser energy was constantly maintained at 80% of maximum. The bleached area included most of the cell that expressed the YFP-tagged fusion protein, but excluded receptorligand clusters or a small part of the plasma membrane. The analysis took further advantage of the time lapse function, provided by the Leica confocal software. Cells were sequentially imaged in both YFP and CFP channels in 1-min time intervals. Between these times, a maximal excitation applied to the selected bleached areas. Images of each experiment were merged into a single Leica experimental series, as recommended in the software manual. This facilitated the determination of fluorescence intensities in corresponding sections of all merged images. Receptorligand clusters, plasma membrane areas and control regions were selected as region of interests (ROIs) and ROI fluorescence was quantified, using an integrated function of the Leica Confocal Software. Importantly, we have manually verified that the selected ROIs corresponded in all cases to the analyzed structures in each single image, because we did occasionally observe movements of the cells or the microscopic stage. The cluster specific YFP fluorescence was obtained by correction for the fluorescence of the corresponding "free" compound in a comparable ROI. Dependent on the morphology of the clusters and the shape of the ROIs fluorescence of "free" YFP-FasL or Fas-YFP was between 50 and 80% of the fluorescence of the clustered molecules. However, after five bleach cycles the fluorescence intensities of the nonclustered fusion proteins outside the bleach region was regularly below 5% of the fluorescence of clustered molecules. This time point was therefore defined as t = 0. In FLIP experiments with nonclustered Fas-YFP and YFP-FasL, the corresponding fluorescence was corrected for the bleach dynamics in the bleach region itself. The corrected fluorescence intensities were normalized against the corrected relative fluorescence intensity obtained after the initial 5 min. Normalized relative fluorescence intensities of corresponding time points of 1215 FLIP experiments were averaged and plotted over time. To calculate the time (t1/2) when 50% of the fluorescence of the YFP fusion proteins was bleached, the curves obtained were linearized by plotting the natural logarithm of the relative fluorescence intensities over time.
Determination of FasL-Fas cluster incidence
5 x 106 HeLa cells were transfected with 20 µg of plasmid DNA encoding Fas-YFP or Fascyt-YFP by electroporation and cultured in 6 well plates, containing one 18 x 18 cm glass cover slide per well. In parallel, 2 x 107 HEK293 cells were transiently transfected with CFP-FasL and cultured overnight. The next day, the medium was removed from the 6 well plates and 2 ml of a 4°C cold cell suspension, containing 2 x 106 transfected HEK293 cells was added into each well. Plates were centrifuged at 1,200 rpm for 5 min at 4°C and the cover glasses were then incubated at 37°C for the indicated times. Cells were fixed and investigated by fluorescence microscopy. A field of
150 x 150 µm of was randomly selected. All Fas-YFP or Fas
cyt-YFP expressing cells found within this field were analyzed of whether they were in contact with one or more CFP-FasL expressing HEK293 cell. Isolated Fas-YFP or Fas
cyt-YFP expressing HeLa cells that were not neighbored or overlaid by CFP-FasL expressing HEK293 cells were excluded from further analysis. Fas-YFP or Fas
cyt-YFP expressing HeLa cells that were in apparent contact with CFP-FasL transfected cells were investigated for cluster formation. Clusters were defined by their spot-like or aggregated morphology, as well as by strong codetection of blue and yellow fluorescence. This analysis was continued in equivalent fields until 100 cell pairs were analyzed. The percentage of cell pairs with clusters of Fas-YFP/Fas
cyt-YFP and CFP-FasL are indicated. Cluster incidence of Fas-YFP in Jurkat cells was determined by online fluorescence microscopy. HeLa cells expressing CFP-FasL were cultured in Matek glass bottom dishes and overlaid with 5 x106 cells of the indicated Jurkat cell line, which had been transfected with 50 µg Fas-YFP cells by electroporation. After 3 h, all Fas-YFP expressing cells in the dish which were in apparent contact with CFP-FasL expressing HeLa cells were investigated for cluster formation and the cluster incidence was calculated.
Preparation of detergent insoluble raft membrane microdomain fractions
5 x 107 cells, expressing the proteins of interest were harvested with a rubber policemen, pelleted, resuspended in 200 µl serum free RPMI medium, and kept on ice. Cells were then lysed in 200 µl ice-cold Brij-96 lysis buffer [0.1% Brij-96 in TNE (25 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM Pefabloc, 5 mM iodoacetamide; 1 mM Na3VO4,1 mM NaF)]. The lysates were mixed with 80% sucrose in TNE buffer, transferred into an ultra centrifugation tube and overlaid with 2.8 ml 30% sucrose in TNE, followed by 400 µl TNE. Samples were centrifuged at 50,000 rpm in a TST 60.4 rotor (Sorvall) for 22 h at 4°C. Proteins in the four collected fractions were precipitated by addition of 1.5 ml ice-cold acetone. Precipitates were resolved in SDS sample buffer and analyzed by Western blotting, using the following antibodies: anti-GFP (mAb; Roche); anti-JNK (rabbit pAb; Cell Signaling) and anti-tubulin (NeoMarkers). Based on localization of Lck-GFP (Janes et al., 1999) and palmitylated YFP (Zacharias et al., 2002) the top fraction (1) was identified as detergent insoluble raft membrane microdomain fraction. The bottom fraction (4), which contained the vast majority of total protein was correspondingly identified as detergent soluble. To dissolve lipid rafts, cells were treated with 20 mM of the cholesterol-depleting drug ßMCD in serum-free medium at 37°C for 20 min. Cells were then harvested for analysis or analyzed further by online fluorescence microscopy (37°C, 5% CO2) for up to one more hour.
FACS analysis
To analyze the effects of FasL stimulation on Fas cell surface expression Fas overexpressing HeLa cells were challenged with anti-Flag mAb M2 (1 µg/ml) cross-linked soluble Flag-FasL (400 ng/ml) for 1 h on ice or at 37°C and analyzed afterwards by FACS. For this purpose cells were stained with antiFas-FITC mAb FAB128 (R&D Systems) which does not compete with Flag-FasL for Fas binding for 1 h on ice. To avoid interference of protein resynthesis 25 µg/ml CHX was added 30 min before stimulation. Finally cells were analyzed by FACS.
Online supplemental material
Figs. S1 and S2 show that a YFP/CFP tag does not interfere with the functional properties of Fas, FasL, Fascyt and FasL
cyt. Fig. S3 shows colocalization of Fas-CFP and cholera toxin Brhodamine in anticholera toxin B patched cells. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200501048/DC1.
![]() |
Acknowledgments |
---|
Submitted: 10 January 2005
Accepted: 16 February 2005
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Algeciras-Schimnich, A., L. Shen, B.C. Barnhart, A.E. Murmann, J.K. Burkhardt, and M.E. Peter. 2002. Molecular ordering of the initial signaling events of CD95. Mol. Cell. Biol. 22:207220.
Aouad, S.M., L.Y. Cohen, E. Sharif-Askari, E.K. Haddad, A. Alam, and R.P. Sekaly. 2004. Caspase-3 is a component of Fas death-inducing signaling complex in lipid rafts and its activity is required for complete caspase-8 activation during Fas-mediated cell death. J. Immunol. 172:23162323.
Barnhart, B.C., E.C. Alappat, and M.E. Peter. 2003. The CD95 type I/type II model. Semin. Immunol. 15:185193.[CrossRef]
Boatright, K.M., M. Renatus, F.L. Scott, S. Sperandio, H. Shin, I.M. Pedersen, J.E. Ricci, W.A. Edris, D.P. Sutherlin, D.R. Green, and G.S. Salvesen. 2003. A unified model for apical caspase activation. Mol. Cell. 11:529541.[CrossRef][Medline]
Chang, H.Y., X. Yang, and D. Baltimore. 1999. Dissecting Fas signaling with an altered-specificity death-domain mutant: requirement of FADD binding for apoptosis but not Jun N-terminal kinase activation. Proc. Natl. Acad. Sci. USA. 96:12521256.
Cremesti, A., F. Paris, H. Grassme, N. Holler, J. Tschopp, Z. Fuks, E. Gulbins, and R. Kolesnick. 2001. Ceramide enables fas to cap and kill. J. Biol. Chem. 276:2395423961.
Desbarats, J., R.B. Birge, M. Mimouni-Rongy, D.E. Weinstein, J.S. Palerme, and M.K. Newell. 2003. Fas engagement induces neurite growth through ERK activation and p35 upregulation. Nat. Cell Biol. 5:118125.[CrossRef][Medline]
Donepudi, M., A. Mac Sweeney, C. Briand, and M.G. Grutter. 2003. Insights into the regulatory mechanism for caspase-8 activation. Mol. Cell. 11:543549.[Medline]
Eramo, A., M. Sargiacomo, L. Ricci-Vitiani, M. Todaro, G. Stassi, C.G. Messina, I. Parolini, F. Lotti, G. Sette, C. Peschle, and R. De Maria. 2004. CD95 death-inducing signaling complex formation and internalization occur in lipid rafts of type I and type II cells. Eur. J. Immunol. 34:19301940.[CrossRef][Medline]
Fesik, S.W. 2000. Insights into programmed cell death through structural biology. Cell. 103:273282.[Medline]
Grassme, H., A. Jekle, A. Riehle, H. Schwarz, J. Berger, K. Sandhoff, R. Kolesnick, and E. Gulbins. 2001a. CD95 signaling via ceramide-rich membrane rafts. J. Biol. Chem. 276:2058920596.
Grassme, H., H. Schwarz, and E. Gulbins. 2001b. Molecular mechanisms of ceramide-mediated CD95 clustering. Biochem. Biophys. Res. Commun. 284:10161030.[CrossRef][Medline]
Grassme, H., J. Bock, J. Kun, and E. Gulbins. 2002. Clustering of CD40 ligand is required to form a functional contact with CD40. J. Biol. Chem. 277:3028930299.
Holler, N., A. Tardivel, M. Kovacsovics-Bankowski, S. Hertig, O. Gaide, F. Martinon, A. Tinel, D. Deperthes, S. Calderara, T. Schulthess, et al. 2003. Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex. Mol. Cell. Biol. 23:14281440.
Huang, D.C., M. Hahne, M. Schroeter, K. Frei, A. Fontana, A. Villunger, K. Newton, J. Tschopp, and A. Strasser. 1999. Activation of Fas by FasL induces apoptosis by a mechanism that cannot be blocked by Bcl-2 or Bcl-x(L). Proc. Natl. Acad. Sci. USA. 96:1487114876.
Huang, D.C., J. Tschopp, and A. Strasser. 2000. Bcl-2 does not inhibit cell death induced by the physiological Fas ligand: implications for the existence of type I and type II cells. Cell Death Differ. 7:754755.[CrossRef][Medline]
Hueber, A.O., A.M. Bernard, Z. Herincs, A. Couzinet, and H.T. He. 2002. An essential role for membrane rafts in the initiation of Fas/CD95-triggered cell death in mouse thymocytes. EMBO Rep. 3:190196.
Janes, P.W., S.C. Ley, and A.I. Magee. 1999. Aggregation of lipid rafts accompanies signaling via the T cell antigen receptor. J. Cell Biol. 147:447461.
Juo, P., C.J. Kuo, J. Yuan, and J. Blenis. 1998. Essential requirement for caspase-8/FLICE in the initiation of the Fas-induced apoptotic cascade. Curr. Biol. 8:10011008.[Medline]
Juo, P., M.S. Woo, C.J. Kuo, P. Signorelli, H.P. Biemann, Y.A. Hannun, and J. Blenis. 1999. FADD is required for multiple signaling events downstream of the receptor Fas. Cell Growth Differ. 10:797804.
Kamitani, T., H.P. Nguyen, and E.T. Yeh. 1997. Activation-induced aggregation and processing of the human Fas antigen. Detection with cytoplasmic domain-specific antibodies. J. Biol. Chem. 272:2230722314.
Kischkel, F.C., S. Hellbardt, I. Behrmann, M. Germer, M. Pawlita, P.H. Krammer, and M.E. Peter. 1995. Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO J. 14:55795588.[Abstract]
Ko, Y.G., J.S. Lee, Y.S. Kang, J.H. Ahn, and J.S. Seo. 1999. TNF-alpha-mediated apoptosis is initiated in caveolae-like domains. J. Immunol. 162:72177223.
Kreuz, S., D. Siegmund, J.-J. Rumpf, D. Samel, M. Leverkus, O. Janssen, G. Hacker, O. Dittrich-Breiholz, M. Kracht, P. Scheurich, and H. Wajant. 2004. NFkappaB activation by Fas is mediated through FADD, caspase-8, and RIP and is inhibited by FLIP. J. Cell Biol. 166:369380.
Lee, Y., and E. Shacter. 2001. Fas aggregation does not correlate with Fas-mediated apoptosis. J. Immunol. 167:8289.
Legembre, P., M. Beneteau, S. Daburon, J.F. Moreau, and J.L. Taupin. 2003. Cutting edge: SDS-stable Fas microaggregates: an early event of Fas activation occurring with agonistic anti-Fas antibody but not with Fas ligand. J. Immunol. 171:56595662.
Legler, D.F., O. Micheau, M.A. Doucey, J. Tschopp, and C. Bron. 2003. Recruitment of TNF receptor 1 to lipid rafts is essential for TNFalpha-mediated NF-kappaB activation. Immunity. 18:655664.[Medline]
Mandal, M., S.B. Maggirwar, N. Sharma, S.H. Kaufmann, S.C. Sun, and R. Kumar. 1996. Bcl-2 prevents CD95 (Fas/APO-1)-induced degradation of lamin B and poly(ADP-ribose) polymerase and restores the NF-kappaB signaling pathway. J. Biol. Chem. 271:3035430359.
Muppidi, J.R., and R.M. Siegel. 2004. Fas ligand-independent redistribution of Fas (CD95) into lipid rafts mediates clonotypic T cell death. Nat. Immunol. 5:182189.[CrossRef][Medline]
Peter, M.E., and P.H. Krammer. 2003. The CD95(APO-1/Fas) DISC and beyond. Cell Death Differ. 10:2635.[CrossRef][Medline]
Ruiz-Ruiz, C., G. Robledo, J. Font, M. Izquierdo, and A. Lopez-Rivas. 1999. Protein kinase C inhibits CD95 (Fas/APO-1)-mediated apoptosis by at least two different mechanisms in Jurkat T cells. J. Immunol. 163:47374746.
Scaffidi, C., S. Fulda, A. Srinivasan, C. Friesen, F. Li, K.J. Tomaselli, K.M. Debatin, P.H. Krammer, and M.E. Peter. 1998. Two CD95 (APO-1/Fas) signaling pathways. EMBO J. 17:16751687.
Schmitz, I., H. Walczak, P.H. Krammer, and M.E. Peter. 1999. Differences between CD95 type I and II cells detected with the CD95 ligand. Cell Death Differ. 6:821822.[CrossRef][Medline]
Schneider, P., N. Holler, J.L. Bodmer, M. Hahne, K. Frei, A. Fontana, and J. Tschopp. 1998. Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity. J. Exp. Med. 187:12051213.
Shi, Y. 2002. Apoptosome: the cellular engine for the activation of caspase-9. Structure (Camb). 10:285288.[Medline]
Siegel, R.M., J.K. Frederiksen, D.A. Zacharias, F.K. Chan, M. Johnson, D. Lynch, R.Y. Tsien, and M.J. Lenardo. 2000. Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations. Science. 288:23542357.
Siegel, R.M., J.R. Muppidi, M. Sarker, A. Lobito, M. Jen, D. Martin, S.E. Straus, and M.J. Lenardo. 2004. SPOTS: signaling protein oligomeric transduction structures are early mediators of death receptor-induced apoptosis at the plasma membrane. J. Cell Biol. 167:735744.
Suda, T., H. Hashimoto, M. Tanaka, T. Ochi, and S. Nagata. 1997. Membrane Fas ligand kills human peripheral blood T lymphocytes, and soluble Fas ligand blocks the killing. J. Exp. Med. 186:20452050.
Varadhachary, A.S., M. Edidin, A.M. Hanlon, M.E. Peter, P.H. Krammer, and P. Salgame. 2001. Phosphatidylinositol 3'-kinase blocks CD95 aggregation and caspase-8 cleavage at the death-inducing signaling complex by modulating lateral diffusion of CD95. J. Immunol. 166:65646569.
Verhagen, A.M., and D.L. Vaux. 2002. Cell death regulation by the mammalian IAP antagonist Diablo/Smac. Apoptosis. 7:163166.[CrossRef][Medline]
von Reyher, U., J. Strater, W. Kittstein, M. Gschwendt, P.H. Krammer, and P. Moller. 1998. Colon carcinoma cells use different mechanisms to escape CD95-mediated apoptosis. Cancer Res. 58:526534.[Abstract]
Wajant, H., K. Pfizenmaier, and P. Scheurich. 2003. Non-apoptotic Fas signaling. Cytokine Growth Factor Rev. 14:5366.[CrossRef][Medline]
Zacharias, D.A., J.D. Violin, A.C. Newton, and R.Y. Tsien. 2002. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science. 296:913916.