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. 2011;6(6):e19783.
doi: 10.1371/journal.pone.0019783. Epub 2011 Jun 13.

Mitochondrial ceramide-rich macrodomains functionalize Bax upon irradiation

Hyunmi Lee  1 , Jimmy A Rotolo, Judith Mesicek, Tuula Penate-Medina, Andreas Rimner, Wen-Chieh Liao, Xianglei Yin, Govind Ragupathi, Desiree Ehleiter, Erich Gulbins, Dayong Zhai, John C Reed, Adriana Haimovitz-Friedman, Zvi Fuks, Richard Kolesnick
Affiliations

Mitochondrial ceramide-rich macrodomains functionalize Bax upon irradiation

Hyunmi Lee et al. PLoS One. 2011.

Erratum in

Abstract

Background: Evidence indicates that Bax functions as a "lipidic" pore to regulate mitochondrial outer membrane permeabilization (MOMP), the apoptosis commitment step, through unknown membrane elements. Here we show mitochondrial ceramide elevation facilitates MOMP-mediated cytochrome c release in HeLa cells by generating a previously-unrecognized mitochondrial ceramide-rich macrodomain (MCRM), which we visualize and isolate, into which Bax integrates.

Methodology/principal findings: MCRMs, virtually non-existent in resting cells, form upon irradiation coupled to ceramide synthase-mediated ceramide elevation, optimizing Bax insertion/oligomerization and MOMP. MCRMs are detected by confocal microscopy in intact HeLa cells and isolated biophysically as a light membrane fraction from HeLa cell lysates. Inhibiting ceramide generation using a well-defined natural ceramide synthase inhibitor, Fumonisin B1, prevented radiation-induced Bax insertion, oligomerization and MOMP. MCRM deconstruction using purified mouse hepatic mitochondria revealed ceramide alone is non-apoptogenic. Rather Bax integrates into MCRMs, oligomerizing therein, conferring 1-2 log enhanced cytochrome c release. Consistent with this mechanism, MCRM Bax isolates as high molecular weight "pore-forming" oligomers, while non-MCRM membrane contains exclusively MOMP-incompatible monomeric Bax.

Conclusions/significance: Our recent studies in the C. elegans germline indicate that mitochondrial ceramide generation is obligate for radiation-induced apoptosis, although a mechanism for ceramide action was not delineated. Here we demonstrate that ceramide, generated in the mitochondrial outer membrane of mammalian cells upon irradiation, forms a platform into which Bax inserts, oligomerizes and functionalizes as a pore. We posit conceptualization of ceramide as a membrane-based stress calibrator, driving membrane macrodomain organization, which in mitochondria regulates intensity of Bax-induced MOMP, and is pharmacologically tractable in vitro and in vivo.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Ionizing radiation-induced ceramide elevation is confined to mitochondria.
(A) Time- and dose-dependent induction of apoptosis in response to ionizing radiation. Morphologic changes of nuclear apoptosis were detected using the DNA-specific fluorochrome bis-benzimide. Data (mean±SEM) are collated from 3 experiments analyzing 500 cells per point. (B) Time-dependent cytochrome c (Cyt. c) release after 10 Gy. Cytosolic fractions of HeLa cells, collected at the indicated times post-irradiation were analyzed by immunoblotting using α-Cyt. c. Data are from 1 representative of 3 studies. (C) Increased CS activity in isolated mitochondria (MT) after 20 Gy. CS activity was measured at 28 h post-irradiation. Data are from 2 experiments. (D) Radiation increases mitochondrial ceramide content. At 33 h post-irradiation, ceramide was quantified by the diacylglycerol kinase assay in mitochondrial-enriched and ER-enriched fractions from HeLa cells. Inset: The ER-enriched fraction was devoid of mitochondrial contamination based on western blotting with anti-COXII (mitochondrial marker), while the mitochondrial fraction was 10.9±1.7% (mean±SE) ER based on anti-Climp63 (ER marker) blotting. Data (mean±SE) are from 4 experiments performed in triplicate. *, p<0.05 vs. control. (E) Ionizing radiation increases CS activity in MAM and ceramide levels in MAM-free mitochondria (MT). HeLa cells were harvested 33 h post-10 Gy and organelles isolated as in Materials and Methods. ER (P100) was fractionated by differential centrifugation and mitochondria and MAM within the heavy membrane fraction (P10) were further separated from each other by 30% Percoll gradient. Upper panel; CS activity was measured in each fraction using sphinganine and palmitoyl-CoA as substrates as in Materials and Methods. Middle panel: purity of ER, Mitochondria and MAM fractions was analyzed by immunoblotting with antibodies to Calnexin (ER marker) and COXII (MT marker). Based on anti-Calnexin blotting, Percoll-purified mitochondria were 3–4% contaminated with ER. Lower panel; Ceramide levels were quantified by diacylglycerol kinase assay as in Materials and Methods. (F) Co-localization of mitochondrial ceramide and COXI after 20 Gy. At 33 h post-irradiation, HeLa cells were stained with anti-ceramide IgM (red) and anti-COXI IgG (green). Images were acquired with a Leica TCS AOBS SP2 confocal microscope equipped with a 63×1.4NA OIL DIC D objective combined with 4× scan zoom, and co-localization (yellow) was analyzed with MetaMorph 7.5 software. Scale bar; 10 µm. Inset images (rectangles) represent 4× magnification of left upper (0 Gy) and left lower (20 Gy) regions for observation of co-localization. Data are from 1 of 5 experiments.
Figure 2
Figure 2. FB1 prevents radiation-induced MOMP in HeLa cells.
(A) FB1 blocks mitochondrial ceramide generation. HeLa cells were irradiated (IR) and treated with 15 µM FB1 20 h post-irradiation. Ceramide in isolated mitochondria was quantified by diacylglycerol kinase assay at 36 h post-irradiation. Data (mean±SEM) are from 2 experiments performed in triplicate. *, p<0.05 vs. control; **, p<0.01 vs. irradiated. (B) FB1 prevents radiation-induced Bax insertion into the MOM. Alkali-resistant mitochondrial fractions containing inserted Bax were isolated after 34 h from HeLa cells irradiated with 20 Gy and treated with 25 µM FB1 or 75 nM ISP-1 at 20 h post-irradiation. COXII was used as mitochondrial loading control. Data are from 1 of 4 studies. (C) FB1 blocks radiation-induced Bax oligomerization. At 34 h post-irradiation, mitochondrial proteins from HeLa cells treated as in (B) were separated by gel filtration. Data are from 2 studies. (D) FB1 attenuates radiation-induced cytochrome c release in HeLa cells and BAEC. HeLa cells were irradiated with 10 Gy, and 15 µM FB1 was added 20 h post-irradiation. BAEC cells were treated with 25 µM FB1 1 h before irradiation with 5 Gy. 36 h (HeLa) and 12 h (BAEC) post-irradiation, cytosolic fractions were analyzed by immunoblotting using mouse monoclonal anti-Cyt. c and mouse monoclonal anti-tubulin antibodies. Data are from 1 of 2 studies in HeLa and BAEC each. (E) FB1 attenuates radiation-induced caspase activity. FB1 was added to cells 20 h after 10 Gy and caspase activity measured at 36 h post-irradiation using the fluorogenic caspase substrate Z-DEVD-AFC. Data (mean±SEM) are from 1 of 2 investigations performed in triplicate.
Figure 3
Figure 3. Effect of C16-ceramide on MOMP in isolated HeLa mitochondria.
(A) Ceramide induces cytochrome c release from isolated HeLa mitochondria. C16-ceramide (0–1 µM) was incubated with HeLa mitochondria (1 µg/µl) in MSB buffer. After 1 h at 30°C, samples were centrifuged at 14,000×g for 5 min at 4°C to separate released (supernatant) and retained (pellet) mitochondrial proteins, and analyzed for cytochrome c release by immunoblotting using anti-Cyt.c and anti-COXII as loading control. Data are from 1 of 4 investigations. (B) Ceramide induces insertion of endogenous Bax into HeLa mitochondrial membranes. Isolated mitochondria were incubated with 1 µM C16-ceramide and mitochondrial pellets were collected after incubation as in (A). Attached and inserted Bax were separated by alkali extraction of mitochondrial pellets as in Figure 2B and analyzed by Western blot with anti-Bax and anti-COXII as loading control. Data are from 1 of 4 investigations. (C) FB1 inhibits tBid-induced cytochrome c release. Isolated HeLa mitochondria were incubated with 0.25–12.5 ng of caspase-8 cleaved human Bid for 30 min and cytochrome c release was analyzed as in (A). Data represent one of three similar studies. (D) FB1 inhibits BaxΔC-induced cytochrome c release. HeLa mitochondria, replete (control) or depleted of ceramide (from 35 µM FB1-pretreated cells), were incubated with BaxΔC (0–1 µM) for 30 min and cytochrome c release was analyzed as in (A). The left panel shows the cytochrome c content of mitochondria isolated from FB1-pretreated cells was not different than that from untreated HeLa cells. Data are from 1 of 5 investigations.
Figure 4
Figure 4. Ceramide is non-apoptogenic by itself and facilitates Bax-induced MOMP.
(A) Ceramide facilitates Bax-induced cytochrome c release from isolated mouse liver mitochondria ex vivo. Recombinant BaxΔC (0–1 µM, upper panel), or C16-ceramide (0–50 µM) plus 0.05 µM BaxΔC (lower panel) were incubated with isolated mouse liver mitochondria (1 µg/µl) in KCl buffer for 5 min at 37°C, supernatants and pellets were separated by centrifugation at 14,000×g for 5 min at 4°C, and cytochrome c release was measured as in Figure 3A. The outer mitochondrial membrane protein VDAC was used as loading control. Data are from 3 studies. (B) Ceramide induces insertion of recombinant BaxΔC into isolated mouse liver mitochondria. Mitochondria were treated as in (A) and attached and inserted BaxΔC analyzed as in Figure 3B. Data are from 2 independent studies. (C) Full-length (FL) Bax co-localizes with MCRMs induced by exogenous C16-ceramide. Mouse liver mitochondria were incubated in MSB-based medium in the presence or absence of 50 nM full-length Bax for 5 min at 30°C. Thereafter, 0 or 1 mM C16-ceramide [1% final solvent concentration (ethanol∶dodecane, 98∶2 v/v)] was added to the mixture for an additional 10 min. Mitochondria were fixed and stained with MitoTracker (blue), and ceramide and Bax were localized using anti-ceramide IgM (red) or anti-Bax IgG (green), respectively. Images, acquired with a Leica TCS AOBS SP2 confocal microscope equipped with a 100×1.4NA OIL DIC D objective combined with 2× scan zoom, were analyzed with MetaMorph 7.5 software. Control IgM and IgG did not yield detectable signals (not shown). Scale bar; 1 µm. Data represent 1 of 3 similar studies. (D) BaxΔC co-localizes with MCRMs induced by exogenous C16-ceramide. Experiments were performed as in (C) using 5 mM C16-ceramide [1% final solvent concentration (ethanol∶dodecane, 98∶2 v/v)] in the presence or absence of 50 nM recombinant BaxΔC. Scale bar; 1 µm. Data represent 1 of 4 similar studies. (E) Biophysical isolation of mouse liver MCRMs. After incubation with 50 nM BaxΔC and 5 µM C16-ceramide, mitochondria (3.3 mg/ml), pelleted as in Figure 4A were resuspended in cold MBS buffer containing 0.05% Triton X-100. After 30 min on ice, mitochondria were homogenized with 20 strokes of a loose-fitting dounce homogenizer. The mitochondrial homogenate was adjusted to 40% final sucrose concentration and subjected to 5–30% continuous sucrose density gradient as in Materials and Methods. 400 ml of each 1 ml fraction were concentrated by 20% TCA precipitation, and proteins were resolved on a 15% SDS-PAGE gel and identified by immunoblot analysis using antibodies to the indicated proteins. Ceramide was measured using 400 µl aliquots by diacylglycerol kinase assay. Data are from 3 independent studies.
Figure 5
Figure 5. Ceramide induces formation of a mitochondrial ceramide-rich macrodomain (MCRM).
(A) Ionizing radiation (10 Gy) induces co-localization of endogenous Bax with MCRMs in HeLa cells. Mitochondria were isolated from HeLa cells 34 h after irradiation and immunostained as described in Supporting Information Text S1. Data represent typical stainings from 1 of 4 similar studies in which 2000 mitochondria were analyzed each. (B) Addition of exogenous C16-ceramide induces co-localization of endogenous full-length Bax with MCRMs in HeLa cells. Mitochondria were isolated from HeLa cells using percoll gradient and treated with ceramide as Figure 3A. After 30 min incubation, mitochondria were fixed and stained with MitoTracker (blue), while ceramide and Bax were localized using anti-ceramide IgM (red) or anti-Bax IgG (green), respectively. Control IgM and IgG did not yield detectable signals (not shown). These data represent 1 of 3 similar studies. (C) Bax translocates into a radiation-generated HeLa MCRM. Upper panel: 34 h post-irradiation, HeLa mitochondria were isolated as in Materials and Methods and incubated with 0.15% Triton X-100 in MBS buffer for 30 min on ice. 40 µl mitochondrial homogenate (3.3 µg/µl) were subjected to 5–30% mini-discontinuous sucrose density gradient centrifugation as described in Materials and Methods. 20 µl aliquots of 80 µl fractions were analyzed by immunoblotting using the indicated antibodies. The protein level of each fraction was assessed using the Bio-Rad Dc protein assay kit (PE, Pellet). Data are from 1 of 4 studies, consisting of 2 independent gradients per study. The gradient shown displays our clearest example of Bax translocation into light membranes. Lower panel: Bax in each fraction, revealed by immunoblotting and quantified using NIH Image software, was normalized to protein content for all 8 gradients. (D) MCRM Bax exists as high molecular weight oligomers. Mitochondria from 10 Gy-irradiated HeLa cells, disrupted by either (a) 1% CHAPS and sonication or (b) dounce homogenization in 0.15% Triton X-100, were subjected to 5–30% discontinuous sucrose gradient for MCRM isolation as in Experimental Procedures. Light (MCRM; fractions 6,7) and heavy fractions (solubilized proteins; fractions 11,12) were analyzed by gel filtration on Sephacryl S-200 column as in Figure 2C. 500 µl of each eluted fraction were concentrated by 20% TCA precipitation for immunoblotting. Data are from 3 independent studies.
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