Molecular ordering of the initial signaling events of CD95

Abstract

Binding of either ligand or agonistic antibodies to the death receptor CD95 (APO-1/Fas) induces the formation of the death-inducing signaling complex (DISC). We now show that signal initiation of CD95 in type I cells can be further separated into at least four distinct steps. (i) The first step is ligand-induced formation of CD95 microaggregates at the cell surface. (ii) The second step is recruitment of FADD to form a DISC. This step is dependent on actin filaments. (iii) The third step involves formation of large CD95 surface clusters. This event is positively regulated by DISC-generated caspase 8. (iv) The fourth step is internalization of activated CD95 through an endosomal pathway. The latter step is again dependent on the presence of actin filaments. The data indicate that the signal initiation by CD95 is a complex process actively regulated at various levels, providing a number of new drug targets to specifically modulate CD95 signaling.

FIG. 1.

Ligand-induced DISC formation and clustering of CD95. (A) SKW6.4 cells were incubated with an FITC-conjugated anti-CD95 antibody for 45 min on ice (t = 0). Cells were then washed and warmed to 37°C and analyzed after the indicated times. After incubation, cells were attached to poly-l-lysine-coated slides and fixed, and samples were analyzed by confocal laser scanning microscopy in the following way: 20 z-sections of each sample (0.5 μm apart) were taken and used to generate a 3D image with Zeiss LSM software. Projections of these 3D images are shown. All cells are shown at the same magnification. (B) Analysis of the DISC of SKW6.4 cells stimulated with anti-CD95 for the indicated times. Migration positions of procaspase 8 (CASP-8), the caspase 8 cleavage intermediates p43 and p41, and FADD are shown. (C) SKW6.4 cells were treated with 1 μg of LZ-CD95L per ml for 45 min on ice and then stimulated for 60 min at 37°C. Receptor redistribution was determined by staining fixed cells with anti-CD95L antibody followed by FITC-conjugated goat anti-mouse antibody. (D) SKW6.4 cells were treated with 1 μg of either anti-CD95 (□) or LZ-CD95L (▪) per ml for different times as described for panel A, and cells were scored for clustering. One hundred cells per condition were counted in duplicate, and the mean is shown. (E) SKW6.4 cells were treated with different amounts of anti-CD95 for 60 min, and clustering of CD95 was quantified as in panel D.

FIG. 2.

Clustering of CD95 is a specific and general effect. (A) Living SKW6.4 cells were treated with FITC-conjugated anti-CD95 and incubated for 60 min on ice (time 0) or for 60 min at 37°C (60′). After stimulation, cells were counterstained with propidium iodide (PI) and analyzed by fluorescence microscopy. (B) To establish specificity of the internalization of CD95, K50 cells were incubated with 1 μg of anti-CD95 per ml for 0 or 60 min and after fixation were stained for CD19 with an FITC-conjugated anti-CD19 mouse MAb. (C and D) CD95 clustering on lymphoid (C) and nonlymphoid (D) cells. Receptor clustering of K50, H9, Boe^R, detached L929-Fas, and detached MCF7-Fas cells was performed as follows. Cells were incubated with anti-CD95 followed by FITC-conjugated goat anti-mouse IgG, each for 45 min on ice (t = 0). Cells were then warmed to 37°C and analyzed after the indicated times. After incubation, cells were attached to poly-l-lysine-coated slides and fixed, and samples were analyzed by fluorescence microscopy. Stimulated cells are shown at the same magnification as unstimulated cells. Adherent L929-Fas cells were grown on Polyprep poly-l-lysine slides (Sigma) to 70% confluency and transferred to ice-cold medium. Directly, FITC-conjugated anti-CD95 was added, and cells were incubated for 45 min on ice. After being washed in medium, CD95 clustering was induced by transfer of slides to 37°C medium and incubation for 60 min. After fixation, all cells were analyzed by fluorescence microscopy.

FIG. 3.

Stimulation-dependent internalization of CD95. (A) SKW6.4 cells were incubated with FITC-conjugated anti-CD95 for 45 min on ice followed by incubation at 37°C for the indicated time. After washing, cells were stained with Texas red-conjugated goat anti-mouse IgG (αmIgG) and then transferred onto poly-l-lysine-coated slides, fixed, and analyzed by fluorescence microscopy as described in Materials and Methods. Arrowheads point to cells that did not stain with the secondary antibody and therefore have fully internalized CD95. (B) 2D projections of 3D movies of SKW6.4 cells treated as in panel A. For orientation, the plasma membrane is labeled by a stippled circle. (C) Quantification of CD95 internalization at time point 0 and after stimulation of K50 and SKW6.4 cells for 60 min. The number of cells with 50% or more of CD95 internalized was determined as described in Materials and Methods. The experiment was done in triplicate, and the mean values with standard deviations are shown. The numbers of propidium iodide-positive cells following the incubation at 37°C were 8% for SKW6.4 and 6% for K50, respectively.

FIG. 4.

Colocalization of CD95 with the endosomal marker TfR. (A) Electron microscopic analysis of MCF7-Fas cells stimulated for 0 or 20 min with anti-CD95 and goat anti-mouse IgG covalently labeled with 20-nm-diameter colloidal gold particles. PM, plasma membrane. Gold particles are indicated with arrows. The bar represents 200 nm. (B and C) Detached MCF7-Fas (B) and SKW6.4 (C) cells were treated with anti-CD95 and stimulated for the indicated times at 37°C as described in Fig. 2C. CD95 was detected with an FITC-conjugated goat anti-mouse antibody (green). To detect TfR after CD95 staining and fixation, cells were stained with an anti-TfR antibody followed by Texas red-conjugated goat anti-mouse IgG1 antibody (red). Stainings were analyzed by confocal microscopy. The extent of internalization of MCF7-Fas cells shown in panel B was 80%.

FIG. 5.

Actin filaments are required for CD95 DISC formation and receptor internalization. (A) SKW6.4 cells were left untreated or pretreated with 2.5 μM Ltn A or 100 μM MDC for 1 h at 37°C. Cells were then treated with anti-CD95 and left unstimulated (time 0) or stimulated for 60 min (60′) at 37°C as described in Fig. 2C. Samples were analyzed by fluorescence microscopy. (B) 2D projection of a 3D analysis of an Ltn A-treated SKW6.4 cell stimulated for 1 h at 37°C as described for panel A. Cells were analyzed as described in Fig. 3B. The yellow color indicates that CD95 is at the cell surface. For orientation, the outline of the cell is indicated by a stippled circle. (C) Quantification of inhibition of CD95 internalization on SKW6.4 (S) and K50 (K) cells by 5 μM Ltn A. The number of cells with 50% or more of CD95 internalized was determined as described in Materials and Methods. The experiment was done in triplicate, and the mean values with standard deviations are shown. (D) SKW6.4 or H9 cells were pretreated with Ltn A (2.5 or 5.0 μM) for 1 h at 37°C. CD95 was immunoprecipitated from either 10⁷ untreated or anti-CD95-treated (5 min) SKW6.4 or H9 cells. Immunoprecipitates were subjected to SDS-PAGE (12% polyacrylamide) and immunoblotted with anti-FADD MAb and anti-CD95 C20. Migration positions for each protein are indicated. Cell lysates equivalent to 40 μg of protein were subjected to SDS-PAGE (12% polyacrylamide) and immunoblotted with anti-FADD antibody. (E) SKW6.4 and H9 cells were treated with 1 μg of anti-CD95 per ml for different periods of time in the absence (•) or presence of LtnA (2.5 μM [▪] or 5.0 μM [□]). Caspase 8 activity was analyzed by cleavage of the fluorogenic substrate IETD-AFC. This result was confirmed by a Western blot analysis that demonstrated reduced activation of caspase 8 in Ltn A-treated cells (data not shown). FU, fluorescence units. (F) SKW6.4 (circles) and H9 (squares) cells were preincubated with the indicated concentrations of Ltn A for 1 h and then incubated for 16 h with 1 μg of anti-CD95 (open symbols) or LZ-CD95L (solid symbols) per ml. After incubation, cells were harvested and analyzed by flow cytometry for DNA fragmentation with nuclear staining with propidium iodide. Nontoxic concentrations of Ltn A were chosen after titration of Ltn A to toxic levels. The data represent the percentage of decrease of apoptosis in the presence of Ltn A. The percentages of specific apoptosis in the absence of Ltn A were 58% (56%) and 70% (80%) for SKW6.4 and H9 cells treated with anti-CD95 (LZ-CD95L), respectively. The experiment is representative of three independent experiments.

FIG. 6.

CD95 receptor clustering and internalization require caspase 8 activation. (A) Induction of CD95 clustering on BJAB vector transfectants or BJAB cells expressing FADD-DN as described in Fig. 2C. (B) Induction of CD95 clustering of SKW6 vector-transfected cells or SKW6 cells stably expressing Bcl-2 as described in Fig. 2C. (C) SKW6-vec (•) or SKW6-Bcl-2 (○) transfectants were treated with 1 μg of anti-CD95 per ml for different periods of time. δΨ_m was determined as described in Materials and Methods. (D) MCF7-Fas cells were treated with either 20 or 50 μM caspase inhibitor zVAD-fmk (polycaspase inhibitor), zDEVD-fmk (caspase 3 or 7), or zIETD-fmk (caspase 8). After treatment, cells were incubated with anti-CD95 and kept on ice (time 0) or stimulated for 60 min (60′) at 37°C. Receptor localization was determined as described in Fig. 2C.

FIG. 7.

CD95 signaling on SKW6.4 cells cannot be inhibited by destruction of lipid rafts (A) SKW6.4 cells were left untreated or were pretreated with 10 μg of nystatin per ml, 1 μg of filipin per ml, or 2 mM methyl-β-cyclodextrin (β-CD) for 1 h at 37°C. Cells were then treated with anti-CD95 and left unstimulated (time 0) or were stimulated for 60 min (60′) at 37°C as described in Fig. 1A. Samples were analyzed by fluorescence microscopy. (B) Analysis of the DISC of 10⁷ SKW6.4 cells treated for different times with the same reagents as in panel A was performed as described in Fig. 1B. (C) Titration of anti-CD95 onto SKW6.4 cells in the presence of the indicated reagents used at the same concentrations as in panel A. Cells were treated in either serum-free medium for 9 h or serum-containing medium for 16 h, and DNA fragmentation was quantified as described in Materials and Methods. Similar data were obtained when H9 cells were tested in the same way (data not shown).

FIG. 8.

Formation of SDS-stable CD95 microaggregates is independent of actin and caspase activity. (A) CD95 was immunoprecipitated from either 10⁷ unstimulated (U) or anti-CD95 stimulated (5 min) SKW6.4 cells that were left untreated (S) or that had been pretreated with 50 μM zVAD-fmk (Z). Immunoprecipitates were subjected to SDS-PAGE (12% polyacrylamide) and immunoblotted with anti-caspase 8 and anti-FADD antibodies. p43 and p41 are cleavage intermediates of activated caspase 8. DISC formation was also unaffected by zVAD-fmk when cells were stimulated with anti-CD95 for 60 min (data not shown). (B) SKW6.4 cells were incubated with anti-CD95 and left unstimulated (time 0) or stimulated for 60 min (60′) at 37°C as described in Fig. 2C after pretreatment with 50 μM zVAD-fmk. 0, mock treatment with 0.5% dimethyl sulfoxide (DMSO) alone. Samples were analyzed by fluorescence microscopy. (C) Ligand-induced internalization of SKW6.4 cells or K50 cells untreated or pretreated with zIETD-fmk for 60 min was quantified as described in Fig. 3C. (D) A total of 10⁶ SKW6.4 and H9 cells stimulated with anti-CD95 (20 min) were left untreated (U) or had been pretreated with 5 μM Ltn A (L) or 50 μM zVAD-fmk (Z). Unstimulated cells were analyzed as a control (C). Cell lysates were subjected to SDS-PAGE (10% polyacrylamide) and immunoblotted with anti-CD95 (C20) antibody. CD95^hi corresponds to SDS-stable CD95 microaggregates migrating at around 220 kDa during SDS-PAGE.

FIG. 9.

The membrane-proximal events of CD95 signaling. The signal initiation phase of CD95 can be subdivided into five distinct steps. (I) Ligand-independent receptor preassociation (37). (II) Formation of microaggregates. These submicroscopic microaggregates can be detected as SDS-stable high-molecular-weight CD95 bands in SDS-PAGE (Fig. 8C). (III) Formation of the DISC. This step is dependent on actin filaments, since it can be inhibited by Ltn A (Fig. 5D). DISC components are FADD/Mort1, caspase 8, caspase 10 (unpublished data), and c-FLIP (not shown) (34). (IV) Receptor clustering. CD95 clusters can be seen by fluorescence microscopy. This step depends on generation of active caspase 8 by the DISC and can be efficiently prevented by preincubating cells with either zVAD-fmk or zIETD-fmk (Fig. 6D). Caspase 8 activation is therefore part of a positive feedback loop. (V) Internalization of the DISC. This step again is dependent on actin, since treatment of cells with Ltn A prevents internalization of the clustered receptor (Fig. 5C). Ltn A does not affect type II cells (data not shown), indicating that the actin-dependent steps are reduced or absent and explaining why type II cells form very little DISC. Blue domains, DED; red domain, DD. The N-terminal PLAD in CD95 is shown in a different green tone.