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First published online 26 April 2005
doi: 10.1242/jcs.02343

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CD95 capping is ROCK-dependent and dispensable for apoptosis

¹ Department of Biology, Åbo Akademi University, FI-20520, Turku, Finland
² Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, PO Box 123, FI20521, Turku, Finland
³ Department of Human Microbial Ecology, National Public Health Institute, FI-20520, Turku, Finland
⁴ Department of Biology, Laboratory of Animal Physiology, University of Turku, FI-20014, Turku, Finland

View larger version (89K): [in a new window]   Fig. 1. CD95 capping occurs by different mechanisms dependent on cell type. (A) Jurkat and (B) H9 cells were labeled with CD95 for 30 minutes on ice. After washing, the cells were incubated with a fluorescent secondary antibody on ice. The labeled cells were then stimulated by incubation at 37°C for the indicated time (0-10 minutes) to induce CD95 capping. In C,D, Jurkat and H9 cells were preincubated with cytD or zIETD for 1 hour to determine the requirement for actin or caspase-8 activation for CD95 capping. Fixed samples were then analyzed by confocal laser scanning microscopy. Maximum projections were generated from z-sections and representative fields from at least three separate experiments are shown.

View larger version (38K): [in a new window]   Fig. 2. CD95 capping does not correlate with induction of apoptosis. (A) Jurkat and (B) H9 cells were pre-treated for 1 hour with cytD or zIETD and were then incubated with 200 ng/ml CD95 for 2 hours (Jurkat) or 6 hours (H9) to induce apoptosis. Cells were then labeled and analyzed for the presence of activated caspase-3, by flow cytometry. The data are mean values (±s.e.m.) from a minimum of three separate experiments. (C) Nuclear morphology of CytD- and CD95-treated cells was further analyzed by DAPI labeling. Typical apoptotic cells with condensed chromatin can be seen in the CD95 treated samples. CytD treatment did not inhibit the nuclear alterations associated with apoptosis. (D) Cells were treated as in A. and were then assessed for overall cleavage of caspase-8 by immunoblotting. Overall caspase-8 cleavage does not correlate with the capping of CD95 (Fig. 1C,D). Representative immunoblots from two separate experiments are shown.

View larger version (57K): [in a new window]   Fig. 3. Assembly of the CD95 DISC is independent of CD95 capping. (A) Jurkat and H9 cells were assessed for the presence of caspase-8 in the CD95 DISC by immunoprecipitation of CD95 after stimulation with CD95 for the indicated time. (B) Jurkat and H9 cells were pretreated as in Fig. 2A and were then subjected to immunoprecipitation of CD95 after stimulation with CD95 for 10 minutes. Cell lysates and IP samples were analyzed for the presence of caspase-8. The treatments that abolished CD95 capping did not inhibit recruitment of caspase-8 and assembly of the CD95 DISC. Representative immunoblots from two separate experiments are shown.

View larger version (47K): [in a new window]   Fig. 4. Actin colocalizes with CD95 capping, giving the cells a polarized phenotype. (A) Jurkat cells were treated with CD95 for 10 minutes at 37°C to stimulate the cells. Fixed cells were then labeled with Alexa Fluor 488 phalloidin to detect F-actin. Maximum projections from confocal z-sections were generated and representative cells from at least three separate experiments are shown. F-actin rapidly reorganizes to one pole of the cell upon CD95 activation. (B) Jurkat and (C) H9 cells were treated and labeled for CD95 (green) as in Fig. 1. The fixed and permeabilized cells were then further labeled with Alexa Fluor 546 phalloidin (red). Finally, single confocal z-sections were acquired. Overlay pictures of representative cells from at least three separate experiments are shown. CD95 colocalizes with F-actin only when CD95 capping is allowed (yellow).

View larger version (68K): [in a new window]   Fig. 5. CD95 stimulation induces rapid aggregation of lipid rafts. (A) Lipid rafts (GM1) in Jurkat cells were labeled with Alexa Fluor 555 CTxB and the cells were then treated with CD95 for 10 minutes at 37°C. The fluorescence images show that CD95 stimulation rapidly induced aggregation of the lipid rafts to one pole of the cells. (B) Jurkat and (C) H9 cells treated as in Fig. 3 were labeled with Alexa Fluor 555 CTxB (red) and CD95 (green). Cells were kept on ice (0') or transferred to 37°C for 10 minutes (10') to induce capping. CD95 colocalizes with lipid rafts only when CD95 capping is allowed (yellow). Single confocal z-sections were acquired and overlay images of representative cells from three separate experiments are shown.

View larger version (53K): [in a new window]   Fig. 6. CD95 capping but not apoptosis is ROCK dependent. (A) Jurkat and (B) H9 cells pre-treated for 1 hour with the ROCK inhibitor Y-27632 were labeled for CD95 alone (first panel; green) or together with F-actin (second panel; red) as in Fig. 1 and Fig. 4B,C. The cells that were pre-treated with Y-27632 did not show any capping of CD95. Maximum projections (left panels) or single overlay z-sections (right panels) from confocal images are shown. Inhibition of ROCK abolishes CD95 capping in both cell types. (C) Jurkat and H9 cells were pre-treated as in A and were then incubated with 200 ng/ml CD95 for 2 hours (Jurkat) or 4 hours (H9) to induce apoptosis. Cells were then analyzed for activation of caspase-3 as in Fig. 2A,B. Inhibition of ROCK did not inhibit CD95-mediated apoptosis. The data represent mean values (mean±s.e.m.) from a minimum of three separate experiments. (D) The nuclear morphology of samples from B were analyzed by DAPI labeling to confirm that the cells were apoptotic. Y-27632 did not inhibit nuclear alterations associated with apoptosis.

View larger version (25K): [in a new window]   Fig. 7. CD95 activates RhoA only in the type II cell Jurkat. Cells treated with CD95 for the indicated times were analyzed for activation of RhoA by Rho-GTP pull-down assay. Rho-GTP could be detected only in the CD95-treated Jurkat cells, whereas it was absent in the CD95-treated H9 cells. The level of total RhoA in the lysates did not change upon CD95 engagement. Representative immunoblots from two separate experiments with similar results are shown.

View larger version (23K): [in a new window]   Fig. 8. (A) CD95 on the T-cell can polarize towards a CD95L-expressing cell. Raji cells transiently transfected with CD95L-GFP (green) were incubated with Jurkat cells. The mixture was then settled on polylysine-coated coverslips and was finally labeled for CD95 (red). Note that CD95 is polarized towards the CD95L-expressing cell. (B) CD95 capping could regulate the availability of CD95 at immunological synapses. In this model, CD95 triggering by CD95L expressed on an encountering cell leads to ROCK-dependent receptor capping with concomitant aggregation of lipid rafts and F-actin by a mechanism leading to polarization of the cell. When CD95 is polarized towards the synapse, the target cell would eventually die by apoptosis (1). It is also possible that CD95 would cap at the distal pole and the cell would live, unaffected by the CD95L expressed on the surface of the other cell (2). CD95 capping could also be mediated independently of its ligand by signals from the T cell receptor, costimulatory ligands, or anti-tumor drugs that would affect the sensitivity to cell-mediated killing. (C) CD95 capping is ROCK-dependent and uncoupled from apoptosis signaling. ROCK signaling promotes cytoskeletal reorganization and lipid raft aggregation and capping of CD95. In type I cells, ROCK activation could be mediated by caspase cleavage, which is required for both CD95 capping and apoptosis. In the type II cell CD95 engagement leads to RhoA activation, which in turn activates ROCK, actin reorganization, lipid raft aggregation and CD95 capping. The apoptotic pathway, involving caspase activation is separated from the CD95 capping pathway in the type II cell. However, also in these cells ROCK signaling is only required for CD95 capping and not apoptosis. In addition, ROCK signaling suppresses apoptosis in a feedback loop.

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