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Mitochondrial ceramide-rich macrodomains functionalize Bax upon irradiation.

Lee H, Rotolo JA, Mesicek J, Penate-Medina T, Rimner A, Liao WC, Yin X, Ragupathi G, Ehleiter D, Gulbins E, Zhai D, Reed JC, Haimovitz-Friedman A, Fuks Z, Kolesnick R - PLoS ONE (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.

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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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.
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pone-0019783-g003: 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.

Mentions: To provide direct evidence that ceramide effects Bax-mediated MOMP, isolated HeLa mitochondria were treated with natural C16-ceramide (0.05–1 µM), resulting in dose-dependent cytochrome c release (Figure 3A). In contrast, treatment with anti-apoptotic sphingosine-1-phosphate (0.05–1 µM) failed to induce cytochrome c release under the same conditions (not shown). 1 µM C16-ceramide also rendered the endogenous Bax known to co-purify with HeLa mitochondria (termed attached Bax; [37], [46]) resistant to alkali extraction (Figure 3B), indicating attached Bax had inserted into the MOM. The ED50 for cytochrome c release of approximately 0.2 µM C16-ceramide (Figure 3A) and peak response at 0.5 µM (Figure 3A) were right-shifted from the ED50 for Bax insertion (approximately 0.05 µM C16-ceramide with maximal insertion at 0.12 µM) (Figure S4).


Mitochondrial ceramide-rich macrodomains functionalize Bax upon irradiation.

Lee H, Rotolo JA, Mesicek J, Penate-Medina T, Rimner A, Liao WC, Yin X, Ragupathi G, Ehleiter D, Gulbins E, Zhai D, Reed JC, Haimovitz-Friedman A, Fuks Z, Kolesnick R - PLoS ONE (2011)

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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113798&req=5

pone-0019783-g003: 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.
Mentions: To provide direct evidence that ceramide effects Bax-mediated MOMP, isolated HeLa mitochondria were treated with natural C16-ceramide (0.05–1 µM), resulting in dose-dependent cytochrome c release (Figure 3A). In contrast, treatment with anti-apoptotic sphingosine-1-phosphate (0.05–1 µM) failed to induce cytochrome c release under the same conditions (not shown). 1 µM C16-ceramide also rendered the endogenous Bax known to co-purify with HeLa mitochondria (termed attached Bax; [37], [46]) resistant to alkali extraction (Figure 3B), indicating attached Bax had inserted into the MOM. The ED50 for cytochrome c release of approximately 0.2 µM C16-ceramide (Figure 3A) and peak response at 0.5 µM (Figure 3A) were right-shifted from the ED50 for Bax insertion (approximately 0.05 µM C16-ceramide with maximal insertion at 0.12 µM) (Figure S4).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.

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.

Show MeSH