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First published online 29 April 2008
doi: 10.1242/jcs.022640

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Isoform B of myosin II heavy chain mediates actomyosin contractility during TNF-induced apoptosis

Department of Pathology and Cell Biology, Université de Montréal, 2900 Edouard-Montpetit, Montréal, Québec, H3T 1J4, Canada

View larger version (71K): [in this window] [in a new window]   Fig. 1. TtT/GF cell apoptosis following long-term treatment with TNF. TtT/GF cells were incubated with culture medium alone (control) or with medium containing 20 ng/ml TNF (TNF) for increasing periods of time. (A) The phase-contrast micrographs show control TtT/GF cells with a typical morphology of spreading cells with abundant filopodia and lamellipodia. After 48-96 hours in culture, the cells became elongated and lamellipodia became less abundant. Following TNF treatment, the cells progressively lost the membrane extensions, they shrunk and detached. Scale bar: 250 µm. Inset: Differential interference contrast microscopy shows membrane blebbings of detached cells that had been treated with TNF for 96 hours. Scale bar: 25 µm. (B) Representative western blots demonstrating the absence and the presence of active caspase 8 in total-cell lysates from control and TNF-treated cells, respectively. (C) Following treatments, total-cell lysates were obtained and subjected to electrophoresis and western blotting with an antibody that exclusively recognises the active caspase 3 fragment and with anti-actin (loading control). The representative western blots show a 17 kDa band corresponding to the active caspase 3 in cells that had been treated with TNF for 72 hours and 96 hours, but not in control TtT/GF cells. (D) TtT/GF cells were incubated with either control culture medium or culture medium containing TNF (20 ng/ml final concentration) both in the absence or in the presence of the caspase inhibitor Z-VADfmk (5 µM, final concentration) for 96 hours. Following treatments, total cell lysates (T), non-cytoskeleton (N) and cytoskeleton (C) fractions were obtained and subjected to electrophoresis and western blotting with an anti-active caspase 3. Cleaved caspase 3 was recovered in the cytoskeleton fraction and the cleavage was blocked by Z-VAD-fmk. (E) Time-course studies showing the increased chromatin condensation (P<0.001, 72 hours vs 48 hours), nucleosome release (*P<0.0001, 48 hours vs 24 hours) and cell detachment (**P<0.0002, 48 hours vs 24 hours) in TNF-treated TtT/GF cells. Chromatin condensation in apoptotic cells was evaluated by labelling the cells with Hoechst 33342 and propidium iodide. Nucleosome release was measured by quantifying cytoplasmic histone-associated-DNA fragments (mono and oligonucleosomes). Cell detachment was quantified by counting detached cells with a hemocytometer. Data from three independent experiments are expressed as the ratio of treated to control cells ± s.e.m. (F) TNF-induced cell shrinkage and detachment were blocked by caspase inhibition (scale bar: 50 µm). (G) TNF-induced nucleosome release was blocked by the caspase inhibitor Z-VAD-fmk. (H) After incubation with medium alone (control) or containing TNF for increasing periods of time, total cell lysates were subjected to electrophoresis and western blotting with -actinin and spectrin antibodies. The representative western blot shows -actinin downregulation and spectrin cleavage into two fragments of 120 kDa and 150 kDa of molecular mass, respectively (arrows) in TNF-treated cells.

View larger version (68K): [in this window] [in a new window]   Fig. 2. Effect of long-term TNF treatment on MLC phosphorylation state in TtT/GF cells. TtT/GF cells were incubated with medium alone or with medium containing 20 ng/ml TNF for 96 hours. Following the treatments, cells were scraped off and total-cell lysate (T), non-cytoskeleton (N) and cytoskeleton (C) fractions were prepared and subjected to electrophoresis and western blotting with different antibodies. (A) Specific antibody against MLC phosphorylated at Ser19 [labelled P-(Ser19)-MLC] detected phosphorylated MLC in the cytoskeleton-enriched (C) fraction of control and treated cells. TNF increased MLC-Ser19-P levels. The increase was blocked by the caspase inhibitor Z-VADfmk. The same membrane was stripped and reprobed for MLC. Most MLC (arrow) was recovered in the cytoskeleton fraction. Cytoskeleton-associated MLC was slightly increased by TNF, whereas non-cytoskeleton-associated MLC decreased in TNF-treated cells. The decrease was partly abolished by the caspase inhibitor Z-VAD-fmk. The actin immunoreactive bands correspond to the loading control. (B) Incubation of TtT/GF cells with TNF for 96 hours induced ROCK1 cleavage and translocation to the cytoskeleton fraction of the full-length enzyme. Cleaved, active ROCK1 was recovered in the cytoskeleton-enriched fraction (arrowhead). The caspase inhibitor Z-VAD-fmk blocked TNF-induced ROCK1 cleavage. The actin immunoreactive bands correspond to the loading controls. (C) Representative phase-contrast micrographs of TtT/GF cells incubated with culture medium alone or with medium containing TNF both in the absence (control) or presence of the ROCK inhibitor Y-27632 (5 µM). ROCK inhibition in control cells caused cell-body retraction and increased lamellipodia and cellular processes. TNF-induced cell shrinkage and detachment were abolished by Y-27632. Scale bar: 100 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 3. TNF treatment decreased MHCIIA levels but did not affect MHCIIB levels. (A) TtT/GF cells were treated with culture medium alone or with medium containing 20 ng/ml TNF both in the absence or in the presence of 5 µM Z-VAD-fmk for 96 hours. Next, non-cytoskeleton (N) and cytoskeleton (C) fractions were obtained and subjected to electrophoresis and western blotting with antibodies against MHCIIA. The membrane was then stripped and reprobed with anti-MHCIIB. The actin immunoreactive bands correspond to the loading control. Z-VAD-fmk slightly decreased MHCIIA levels in the cytoskeleton fraction. TNF reduced MHCIIA levels in both subcellular fractions. Inhibition of caspase activity restored control non-cytoskeleton MHCIIA levels but the recovery was not complete in the cytoskeleton fraction. MHCIIB levels in the cytoskeleton fraction were slightly decreased by Z-VAD-fmk but were not affected by TNF. (B) TtT/GF and NIH 3T3 cells were treated with different combinations of TNF (20 ng/ml), cycloheximide (CHX, 5 µg/ml) and Z-VAD-fmk (5 µM). Following a 24-hour treatment, MHCIIA and MHCIIB expression levels were analysed by western blotting in whole-cell lysates. Short-term incubation of the two cell lines with TNF in combination with CHX drastically reduced MHCIIA, whereas MHCIIB expression remained almost unchanged in TtT/GF cells and was slightly reduced in NIH 3T3 fibroblasts. The TNF+CHX-induced drop of MHCIIA levels was abolished by the caspase inhibitor Z-VAD-fmk in both cell lines. (C) TtT/GF cells were incubated with the protein synthesis inhibitor cycloheximide (CHX, 50 µg/ml) for increasing periods of time. Following treatments, total cell proteins were subjected to SDS-PAGE followed by immunoblotting with anti-MHCIIA. Next, the membrane was stripped and incubated with anti-MHCIIB. The actin immunoreactive bands correspond to the loading control. The figure shows representative immunoblots. The bands were scanned and the mean intensity values for each myosin isoform were plotted. Values shown are the mean ± s.e.m. of three independent experiments. (D) TtT/GF cells were incubated with the translation inhibitor actinomycin D (ActD, 1 µg/ml) for increasing periods of time. Blockade of protein or mRNA synthesis affected MHCIIA more rapidly than MHCIIB.

View larger version (43K): [in this window] [in a new window]   Fig. 4. Effect of TNF+CHX treatment on MHCIIA and MHCIIB levels in wild-type and MHCIIB–/– embryonic fibroblasts. (A) Wild-type and MHCIIB–/– embryonic fibroblasts were treated with different combinations of TNF (20 ng/ml), CHX (5 µg/ml), Z-VAD-fmk (5 µM) and Y-27632 (20 µM) for 24 hours. Following treatments, total cell lysates were subjected to western blotting with antibodies against MHCIIA, MHCIIB, spectrin, actin and active caspase 3. TNF+CHX treatment reduced MHCIIA levels and caused the cleavage of spectrin and caspase 3 in both wild-type and MHCIIB–/– cells. Control conditions for each protein were restored by the caspase inhibitor Z-VAD-fmk but not by Y-27632. MHCIIB expression in wild-type cells was not affected by the treatments. The figure shows representative western blots. (B) Quantification of MHCIIA levels in wild type and MHCIIB–/– fibroblasts. The intensity of the MHCIIA immunoreactive bands was measured for each experimental condition described in A and normalised to control cell levels. Values shown are the mean ± s.e.m. of three independent experiments. The graph shows that TNF+CHX treatment had a similar effect on MHCIIA levels in both cell lines. *P<0.01: wild-type fibroblasts, TNF vs TNF+CHX; **P<0.0002: MHCIIB–/– fibroblasts, TNF vs TNF+CHX.

View larger version (35K): [in this window] [in a new window]   Fig. 5. Time course studies on the effect of TNF+CHX treatment on caspase 3 activation and MHCIIA degradation in wild type and MHCIIB–/– embryonic fibroblasts. Wild type and MHCIIB–/– cells were incubated in the absence (0 hour) or in the presence of TNF+CHX for increasing periods of time (2-24 hours). (A) Total cell lysates were subjected to western blotting analyses: the same membrane was incubated with antibodies against active caspase 3, MHCIIA and actin. The figure shows representative western blots. The time course of caspase 3 cleavage and of MHCIIA disappearance was similar in the two cell lines. (B) Following treatments, cells were processed for immunofluorescence microscopy with anti-active caspase 3 and Rhodamine-phalloidin. Total adherent cells and adherent cells positive for active caspase 3 were counted for each time point. TNF+CHX treatment induced an increase in the percentage of cells positive for active caspase 3 with the same time course in both cell lines. The values shown are the mean ± s.e.m. of three independent experiments and more than 300 cells were observed per experimental condition.

View larger version (65K): [in this window] [in a new window]   Fig. 6. Differential morphological responses of wild-type and MHCIIB–/– embryonic fibroblasts during TNF+CHX-induced apoptosis. Wild-type and MHCIIB–/– cells were incubated in the absence (control) or in the presence of TNF+CHX for 24 hours. (A) Representative bright-field micrographs of control and treated cells. TNF+CHX induced cell the shrinkage and detachment of wild-type and MHCIIB–/– embryonic fibroblasts; however, more MHCIIB–/– than wild type cells remained attached. Scale bar: 250 µm. (B) Adherent cells in ten different areas per experimental condition were counted. Quantification of the responses demonstrated a larger number of adherent MHCIIB–/– than wild-type cells after the treatment. Data shown are the mean ± s.e.m. of six independent experiments. *P<0.0001, treated MHCIIB–/– vs treated wild-type fibroblasts. (C) Wild-type and MHCIIB–/– embryonic fibroblasts were incubated for increasing periods of time with TNF+CHX. Following treatments, the cells were processed for immunofluorescence microscopy with anti-active caspase 3 to identify the apoptotic cells and with Rhodamine-phalloidin to facilitate cell visualisation. Adherent, flat cells that were positive for active caspase 3 were counted and the percentage of these cells with respect to the total adherent cell population was calculated. The percentage of apoptotic cells that were flat and adherent was higher in MHCIIB–/– than in wild-type fibroblasts from 16 hours onwards. More than 300 cells per experimental condition were counted. The values shown are the mean ± s.e.m. of three independent experiments. **P<0.00001, wild-type cells vs MHCIIB–/– cells 16 and 24 hours treatment.

View larger version (37K): [in this window] [in a new window]   Fig. 7. Fluorescence microscopy of F-actin distribution and caspase 3 activation in control and TNF+CHX-treated TtT/GF cells, and wild-type and MHCIIB–/– embryonic fibroblasts. The cells were incubated with culture medium alone (control) or containing TNF+CHX for 16 hours. Next, the cells were processed for fluorescence microscopy with Rhodamine-phalloidin and anti-active caspase 3. Control cells were flat and showed cytoplasmic extensions and cortical and cytoplasmic actin filaments. Control cells showed low levels of caspase 3 activation. Following TNF+CHX treatment, the few TtT/GF cells and wild-type fibroblasts that remained attached were round and displayed caspase 3 activation. Some treated TtT/GF cells that remained flat showed no evidence of caspase 3 activation (arrows). Treated MHCIIB–/– cells that remained attached were flat and possessed actin fibers. Moreover, caspase 3 was active in these adherent, TNF+CHX-treated MHCIIB–/– cells. Scale bar: 50 µm.

View larger version (41K): [in this window] [in a new window]   Fig. 8. Effect of TNF+CHX on PKC cleavage. (A) TtT/GF cells were incubated with TNF+ CHX for increasing periods of time. Next, total cell lysates were prepared and subjected to western blotting with antibodies to PKC and active caspase 3. The membrane was striped and incubated with anti-actin as loading control. The representative western blots shows PKC cleavage in cell incubated with TNF+CHX for more than 16 hours. The cleavage was coincident with caspase 3 activation. (B) TtT/GF cells were incubated culture medium alone or containing TNF+CHX either in the absence or presence of the caspase inhibitor Z-VAD-fmk for 16 hours. Next, the cells were homogenised and non-cytoskeleton (N)- and cytoskeleton (C)-enriched fractions were prepared. The subcellular fractions were subjected to western blot analyses with anti-PKC. The figure shows representative western blots. PKC is present in both subcellular fractions in control and treated cells. TNF+CHX treatment induced the translocation of PKC from the non-cytoskeleton (N) to the cytoskeleton (C) fraction. The PKC cleavage product was only found in the cytoskeleton (C)fraction of TNF+CHX-treated cells. The caspase inhibitor Z-VAD-fmk blocked TNF+CHX-induced PKC cleavage but not its translocation. (C) TtT/GF cells, NIH 3T3 fibroblasts, wild-type and MHCIIB–/– embryonic fibroblasts were treated with TNF+CHX for increasing periods of time. Next, cell lysates were prepared and 10 µg protein per sample were subjected to electrophoresis and western blotting with anti-PKC. The representative western blots show the appearance of a 48 kDa immunoreactive band that corresponds to one of the cleavage products of PKC after TNF+CHX treatment in all cell lines.

View larger version (35K): [in this window] [in a new window]   Fig. 9. Co-immunoprecipitation studies on the effect of TNF+CHX on PKC-MHCIIB interaction. (A) TtT/GF cell lysates were prepared from untreated cells (L). Precleared cell lysates were incubated with proteinA-Sepharose4B beads that had been preincubated with either buffer alone (B) or anti-vimentin (Vm) or anti-MHCIIB (MHCIIB). The pellets were subjected to SDS-PAGE followed by immunoblotting with anti-PKC antibody. A representative membrane shows the presence of a 70 kDa immunoreactive band in the cell lysate and in the MHCIIB immunoprecipitate. (B) Untreated TtT/GF cell lysates (L) were prepared. Precleared cell lysates were incubated with proteinA-Sepharose4B beads that had been preincubated with either buffer alone (B), anti-vimentin (Vm) or anti-PKC (PKC) antibodies. The pellets were subjected to SDS-PAGE followed by immunoblotting with anti-MHCIIB. A representative membrane shows the presence of a 210 kDa immunoreactive band in the cell lysate and in the PKC immunoprecipitate. (C) Control and TtT/GF cells treated for 24 hours with TNF+CHX were subjected to immunoprecipitation with PKC antibody. The pellets were recovered and subjected to western blotting with MHCIIB antibodies. This representative membrane shows presence of a 70 kDa immunoreactive band in the immunoprecipitates of both untreated and treated cells.

View larger version (72K): [in this window] [in a new window]   Fig. 10. Effect of PKC inhibition on TNF+CHX-induced cell shrinkage and detachment. TtT/GF cells, wild-type and MHCIIB–/– embryonic fibroblasts were incubated with culture medium either alone or with medium containing TNF+CHX both in the absence or presence of the specific PKC inhibitor Myr-PKC pseudosubstrate (10 µM, final concentration) for 16 hours. Some cell preparations were double labelled with anti-active caspase 3 to identify apoptotic cells and with Rhodamine-phalloidin to label F-actin. Next, the total numbers of adherent cells, of flat cells positive for active caspase 3 cells, and cells positive for active caspase 3 cells were recorded for each experimental condition. The representative phase-contrast micrographs on the left of the figure show flat and elongated control TtT/GF, wild-type and MHCIIB–/– cells. PKC-inhibited cells remained attached and showed elongated cytoplasmic processes. TNF+CHX treatment induced cell shrinkage and detachment in TtT/GF cells and wild-type fibroblasts, but MHCIIB–/– cells stayed adherent and elongated. PKC inhibition reduced TtT/GF cell and wild-type embryonic fibroblast detachment induced by TNF+CHX. Scale bar, 100 µm. The histograms on the right of the figure show the quantification of these results. PKC inhibition did not affect cell-death rate in any of the cell lines tested; however, it increased the percentage of flat apoptotic TtT/GF cells and wild-type fibroblasts but was without effect on MHCIIB–/– fibroblasts. The values shown are the mean ± s.e.m. of three independent experiments; more than 300 cells per experimental condition were counted. *P<0.03: TNF+CHX+PKC- vs TNF+CHX-treated wild-type fibroblasts; **P<0.005: TNF+CHX+PKC- vs TNF+CHX-treated TtT/GF cells.

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