Article |
Address correspondence to Gordon C. Shore, 3655 Promenade Sir William Osler, McIntyre Medical Sciences Building (906), Dept. of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6. Tel.: (514) 398-7282. Fax: (514) 398-7384. E-mail: gordon.shore{at}mcgill.ca
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: apoptosis; caspase-8; BID; BAX; Drp1
* Abbreviations used in this paper: crBAP31, caspase-resistant BAP31; cyt.c, cytochrome c; HA, hemagglutinin; OMM, outer mitochondrial membrane; RTA, reverse tet transactivating protein; TG, thapsigargin.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although it is clear that BCL-2 family members govern mitochondrial dysfunction, it remains unclear at what point the functions of these proteins intercede with gross alterations in mitochondrial morphology that occur during apoptosis. Normal mitochondrial morphology can vary dramatically between cell types, but in most cases mitochondria form long "wormlike" tubules that may (Rizzuto et al., 1998) or may not (Collins et al., 2002) make up interconnected networks. The distribution of mitochondria depends on interactions with microtubules whereas mitochondrial size and shape is the result of constant fusion and fission processes (Bereiter-Hahn and Voth, 1994). Little is known about the mechanism of mitochondrial fission and fusion except that it is regulated by a group of evolutionary conserved GTPases; fusion is dependent on Fzo/Mfn, whereas fission relies on a dynamin related protein, Drp1 (Osteryoung, 2001; Shaw and Nunnari, 2002). During apoptosis mitochondria remodel inner membrane cristae (Scorrano et al., 2002), fragment into small punctiform organelles that sometimes cluster in the perinuclear region (Desagher and Martinou, 2000; Frank et al., 2001; Pinton et al., 2001), and eventually undergo matrix swelling leading to OMM rupture (Petit et al., 1998; Mootha et al., 2001). Recently, Frank et al. (2001) demonstrated that fragmentation of the mitochondrial network during apoptosis is caused by large-scale activation of Drp1-dependent mitochondrial fission, and that this event is requisite for the mitochondrial phase of apoptosis. How apoptotic signals converge on the fission machinery, however, is unclear.
In the current paper, we present evidence that caspase cleavage of BAP31 at the ER can trigger the onset of mitochondrial fission. BAP31 is a polytopic integral protein of the ER membrane that forms a large hetero-oligomeric complex with the related BAP29 protein and components of the actomyosin network (Adachi et al., 1996; Ng et al., 1997; Nguyen et al., 2000). After activation of cell surface death receptors, human BAP31 is cleaved at two identical caspase recognition sites in its cytosolic tail, generating a membrane-embedded fragment, called p20, which induces apoptosis when expressed ectopically (Ng et al., 1997; Nguyen et al., 2000). Cleavage of BAP31 seems to be an important event in the Fas pathway because cells expressing a caspase-resistant BAP31 (crBAP31) mutant retain a near normal morphology after stimulation and resist apoptotic membrane blebbing/fragmentation, disruption of the actin network, and irreversible loss of cell growth potential after removal of the Fas stimulus (Nguyen et al., 2000). In addition, crBAP31 prevents mitochondrial remodeling and the release of cyt.c in the face of activated caspases, suggesting that events at the ER can modulate mitochondrial dysfunction in intact cells (Nguyen et al., 2000). To better understand this communication between ER and mitochondria, and how BAP31 contributes to Fas signaling in general, we investigated the role of p20 in apoptotic progression. We find that p20 stimulates ER Ca2+ release, resulting in the activation of Drp1-dependent fission of mitochondria, which ultimately sensitizes this organelle to caspase-8induced cyt.c release.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Prolonged expression of p20 induces apoptosis
Expression of p20 was observed by 10 h post-infection of KB cells with Adp20, and remained stable for over 50 h (Fig. 2 A). 3040 h after infection, Adp20 induced the activation of caspases, measured by the hydrolysis of the caspase substrate DEVD-amc and by processing of procaspase-3, in many cell types including KB, H1299, HeLa, and Rat1 cells (Fig. 2 B; unpublished data). The mechanism of this caspase activation seemed to occur via the classical mitochondrial apoptosome stress pathway. For example, p20 expression resulted in the insertion of BAX into the OMM, homo-oligomerization of BAK, and release of cyt.c from mitochondria in the presence of the pan-caspase inhibitor, zVAD-fmk (Fig. 2 C; unpublished data). In contrast, p20-induced caspase activation was abrogated in APAF-1null cells (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200212059/DC1). Control adenovectors expressing either LacZ or the reverse tet transactivating protein (RTA) did not cause any of the aforementioned apoptotic changes (unpublished data). Inhibition of caspases using zVAD-fmk, or overexpression of BCL-2 or BCL-xL, blocked downstream morphological features of apoptosis including loss of plasma membrane integrity as assessed by trypan blue uptake (Fig. 2 D). In the absence of these inhibitors, cells showed typical signs of apoptosis, including nuclear condensation and fragmentation, membrane blebbing and cell surface exposure of phosphatidylserine (unpublished data).
|
p20 sensitizes mitochondria to caspase-8induced cyt.c release
Given that BAP31 is a caspase-8 substrate, p20 might cooperate with other products generated by caspase-8, such as tBID, to enhance mitochondrial dysfunction. According to this model, immediately after its expression, p20 should activate a signal that is slow to induce cyt.c release on its own, but able to synergize with other apoptotic signals during Fas-mediated apoptosis. Therefore, we investigated whether p20 could enhance caspase-8driven cyt.c release. Death receptordependent caspase-8 activation was mimicked by infecting H1299 cells with adenovector-expressing triplicate copies of Fpk (a mutant of FKBP) fused to the catalytic subunits of caspase-8 (AdMFpk3FLICE; Muzio et al., 1998). After its expression in cells, oligomerization and autoactivation of the caspase-8 fusion protein was induced with the Fpk-dimerizing compound, FK1012Z. This approach has the benefit of delivering synchronized caspase-8 signals to cells without stimulating caspase-8independent pathways activated by death receptors (Schulze-Osthoff et al., 1998; Wang et al., 2001). In Fig. 3, H1299 cells were co-infected with AdRTA (control adenovector) and AdMFpk3FLICE, or with Adp20 and AdMFpk3FLICE. 16 h after infection, a time when Adp20 alone did not induce cyt.c release or caspase activation (Figs. 2 and 3), the cells were exposed to a short treatment (45 or 90 min) with FK1012Z or vehicle (DMSO) alone, and the mitochondrial and post-mitochondrial fractions were isolated. Compared with caspase-8 activation in the presence of the control protein RTA, caspase-8 activation in the presence of ectopic p20 strongly induced release of cyt.c to the cytosol (Fig. 3 A), but it did not affect the amount of caspase-8generated tBID that was recovered in the mitochondrial fraction (Fig. 3 B). In all cases, equivalent amounts of MFpk3FLICE were produced (unpublished data). Therefore, these results suggest that p20-mediated signals from the ER might cooperate with other caspase-8generated signals to increase cyt.c release from mitochondria.
|
|
H1299 cells treated with Adp20 + zVAD-fmk for 24 h and stained with cyt.c displayed dramatically fragmented mitochondria compared with mock-infected cells (Fig. 4 B). Remarkably, in cells pre-treated with TG or expressing b5-BCL-2, the mitochondrial network remained intact and highly interconnected with no signs of mitochondrial fragmentation (Fig. 4 B; unpublished data). Quantification of the two mitochondrial phenotypes revealed that TG and b5-BCL-2 reduced the number of cells showing signs of mitochondrial fragmentation from 52% to 10 and 13%, respectively (Fig. 4 C). Pretreatment of cells with BAPTA or Ru360 also reduced the number of cells manifesting fragmented mitochondria in response to p20 (Fig. 4 C). The expression of p20 was not affected by any of the treatments (unpublished data). TG, b5-BCL-2, BAPTA, and Ru360 also inhibited p20-induced release of cyt.c from mitochondria, which occurred subsequent to fragmentation (unpublished data; see following section). Therefore, inhibition of Ca2+ transport between the ER and mitochondria inhibits the effect of p20 on mitochondrial morphology and redistribution of cyt.c.
p20-induced fragmentation of the mitochondrial network
The observation that p20 caused mitochondrial fragmentation was extended in Fig. 5. p20 induced an early fragmentation of the mitochondrial network into small punctiform organelles in all cell types tested, including H1299, Rat1, and HeLa cells (Figs. 4 and 5; unpublished data). The gross morphological changes in the mitochondrial network could be observed 1516 h after Adp20 infection (i.e., 23 h after the onset of Ca2+ release), a time when p20 sensitized mitochondria to caspase-8induced cyt.c release (Fig. 3). Induction of mitochondrial fragmentation by p20 occurred in the absence of zVAD-fmksensitive caspase activation and cell shrinkage or disruption of microtubules. For example, Fig. 5 A shows that Rat1 fibroblasts expressing p20 + zVAD-fmk for 20 h and costained with anti-tubulin and anti-TOM20 antibodies displayed a normal microtubule distribution despite having fragmented mitochondria. The punctiform mitochondria could be observed in living cells stained with MitoTracker® Red (unpublished data), indicating that fragmented mitochondria maintain membrane potential and were not an artifact of fixation. Co-staining of Rat1 fibroblasts expressing p20 + zVAD-fmk with antibody to cyt.c and antibody selective for the active conformation of BAX (Desagher et al., 1999) revealed that the transition of mitochondria into punctiform organelles preceded cyt.c release and activation of BAX. As exemplified in Fig. 5 B, most cells expressing p20 + zVAD-fmk for 25 h displayed fragmented mitochondria but showed no signs of cyt.c release or BAX conformation-specific immunoreactivity. BAX immunoreactivity could only be observed in apoptotic cells that had released cyt.c from mitochondria, and all cells that had undergone cyt.c release stained positive for BAX. These results suggest that p20 induces early fragmentation of mitochondria, which precedes BAX activation and cyt.c release. Given that disintegration of the mitochondrial network has been demonstrated to contribute to apoptotic progression (Desagher and Martinou, 2000; Frank et al., 2001) but that BAX/BAK activation and cyt.c release are normally stimulated by BH3-only molecules (Korsmeyer et al., 2000), it is likely that p20 mediates its sensitizing effect by inducing early fragmentation of mitochondria.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The importance of BAP31 cleavage during Fas-mediated apoptosis was first highlighted by the observation that expression of crBAP31 strongly inhibited apoptotic membrane blebbing and release of cyt.c from mitochondria (Nguyen et al., 2000), suggesting that ER-mitochondrial signaling played a role in this pathway. When we reexamined photographs of mitochondria in crBAP31 cells undergoing Fas-induced apoptosis, it was apparent that mitochondrial fragmentation was also strongly inhibited (Nguyen et al., 2000). Thus, full-length BAP31 and p20 have opposing functions during Fas-mediated apoptosis, the former inhibiting mitochondrial fission and egress of cyt.c from mitochondria, and the latter stimulating these events. Importantly, however, p20 operates independently of BAP31 and BAP29 because p20 caused apoptosis in Bap31- and Bap29,31-null cells (Fig. 2 E). Therefore, caspase-8 cleavage of BAP31 converts it from an inhibitor to an activator of cell death; a paradigm that has been ascribed to other caspase targets such as BCL-2 (Cheng et al., 1997), BCL-xL (Clem et al., 1998), and RIP (Lin et al., 1999).
Cleavage of BAP31 may contribute to other cell death pathways that signal through caspase-8. For example, we recently reported that BAP31 and BAP29 play a role in the recruitment and activation of procaspase-8L at the ER during E1A-induced apoptosis (Breckenridge et al., 2002). The kinetics of procaspase-8L processing strongly correlated with BAP31 cleavage in response to E1A, suggesting that activated procaspase-8L may hydrolyze BAP31. The ensuing p20-induced Ca2+ release and mitochondrial fission might then enhance cyt.c release by other pro-apoptotic regulators that are activated by E1A, including BIK (Breckenridge and Shore, 2000; Mathai et al., 2002).
Based on studies using pharmacological modulators of Ca2+ signaling and inhibitors of apoptosis and mitochondrial fission, our results suggest that p20 induces an apoptotic pathway between the ER and mitochondria (Fig. 8 A). This is initiated by ER Ca2+ release coupled to mitochondrial Ca2+ uptake. An important caveat, of course, is that such conclusions rely on the specificity of the inhibitors that are widely used to interfere with Ca2+ signaling. Moreover, it cannot be ruled out that additional mechanisms are also involved. Importantly, however, it has been demonstrated that Drp1 recruitment to mitochondria initiates fission (Labrousse et al., 1999; Smirnova et al., 2001). Because either the lowering of ER Ca2+ stores, or chelating cytosolic Ca2+, or preventing mitochondrial Ca2+ uptake all prevented p20-induced fission of mitochondria, it is likely that ER-mitochondrial Ca2+ transmission acts upstream of Drp1 translocation in this context. Drp1 recruitment is likely mediated by an OMM receptor protein(s), and this complex likely cooperates with an inner mitochondrial membrane reorganizing enzyme(s) to mediate organelle fission (Shaw and Nunnari, 2002). Mitochondrial membranes are often in close proximity and privileged Ca2+ exchange between the two organelles has previously been implicated during apoptosis. For example, IP3 receptor and ryanodine receptormediated Ca2+ spikes that modulate mitochondrial metabolism in healthy cells also sensitize mitochondria to pro-apoptotic stimuli during cell death (Szalai et al., 1999; Hajnoczky et al., 2000). Moreover, manipulations that increase [Ca2+]ER also increase agonist-induced Ca2+ spikes and enhance mitochondrial cyt.c release and apoptosis, whereas a lowering of ER Ca2+ stores has the opposite effect (Nakamura et al., 2000; Pinton et al., 2001). Modulation of the frequency, amplitude and spatio-temporal pattern of ER Ca2+ release during apoptosis may determine how mitochondria respond to Ca2+ signals (Berridge et al., 2000; Pacher and Hajnoczky, 2001). Our results suggest that caspase cleavage of BAP31 may be one mechanism to generate such pro-apoptotic ER-mitochondrial Ca2+-dependent crosstalk in the Fas pathway.
|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture, virus infection, and transfection
KB epithelial cells and H1299 lung carcinoma cells were maintained in MEM- supplemented with 10% FBS and 100 U/ml streptomycin and penicillin. Rat1 fibroblast, CHO, and HeLa cells were grown in DME supplemented as above. KB cells stably expressing HA-Bcl-2 and HA-Bcl-XL have been described previously (Nguyen et al., 1998; Ruffolo et al., 2000). H1299 b5-BCL-2 cells were created by transfecting H1299 cells with pcDNA3 vector encoding human HA-Bcl-2 with amino acids 215239 swapped with the transmembrane sequence of human cytochrome B5 (amino acids 107134) and selecting for resistance to geneticin. Bap31- and Bap29-null mouse embryonic stem cells were maintained as described previously (Breckenridge et al., 2002).
For the construction of Adp20, AdMFpk3FLICE, and AdRTA, cDNAs encoding p20-HA, MFpk3FLICE (Muzio et al., 1998) and RTA were subcloned into a variant of pCA14 containing the T-RExTM promoter (Invitrogen), which functioned as a shuttle to produce the adenoviral vectors as described previously (Bett et al., 1994; Mathai et al., 2002). All adenoviral infections were conducted at a multiplicity of infection of 100 pfu/cell as described previously (Ng et al., 1997), except for in Fig. 3, where the viruses were mixed to generate a total of 100 pfu/cell. LipofectAMINETM Plus (Invitrogen) was used for all transfections using the manufacturer's protocols. In experiments where Adp20 infection followed transfection of CFP-Drp1 or CFP-Drp1K38E, the transfection medium was removed 3 h after transfection and medium containing serum and Adp20 virus was added back.
Apoptotic assays
DEVDase activity was measured from 25 µg of cell lysate protein according to the manufacturer's protocol (Upstate Biotechnology). For statistical analysis of mitochondrial fission and cyt.c release, cells were stained for TOM20 or cyt.c, and the distribution of cyt.c and the morphology of mitochondria were analyzed by conventional immunofluorescence microscopy. In all cases, at least five independent experiments were conducted, where three counts of 150 randomly selected cells was done per experiment. In Fig. 7 B, only cells showing CFP expression were assessed for cyt.c release. Biochemical isolation of the heavy membrane fraction enriched in mitochondria or post-mitochondrial supernatant for measurement of cyt.c release and Drp1 recruitment was done as described previously (Ruffolo et al., 2000). In Fig. 3, the intensity of each cyt.c and tBID Western blot signal was quantified using ImageQuantTM (Amersham Biosciences) software and compared with the intensity of a loading control signal in the same lane (actin or TOM20, respectively) after the subtraction of background. The relative values were expressed as arbitrary units.
Fluorescence microscopy
Cells were typically seeded at 50% confluency on glass coverslips and mock infected or infected with Adp20, always in the presence of zVAD-fmk to prevent apoptosis and cell detachment. At the indicated times after infection, cells were washed in PBS, and were then fixed in PFA solution (4% PFA, 23 mM NaH2PO4, and 77 mM Na2HPO4, pH 7.3). Cells were briefly permeabilized in PBS/0.2% Triton X-100, then blocked in blocking solution (PBS containing 10% FCS and 0.1% Triton X-100). Primary and secondary antibody incubations were done in blocking solution for 1 h at RT using the indicated antibodies and goat antimouse IgG or goat antirabbit IgG secondary antibody coupled to Alexa® 488 (green) or Alexa® 594 (red; Molecular Probes, Inc.). Cells were visualized by confocal microscopy or by conventional fluorescence microscopy on an inverted microscope (TE-FM Epi-fl; Nikon).
Measurement of ER Ca2+ content
The ER Ca2+ store was measured as the sudden increase in [Ca2+]c on addition of TG. [Ca2+]c was measured by the cell permeable fluorescent indicator Fura2-AM. In brief, 2 x 106 cells were washed in Ca2+-free buffer (20 mM Hepes, pH 7.4, 143 mM NaCl, 6 mM KCL, 1 mM MgSO4, 0.1% glucose, and 250 µM sulfinpyrazone), then loaded with 3 mM Fura2-AM for 30 min at 37°C in Ca2+-free buffer containing 0.02% pluronic acid and 0.1% BSA. After a final wash, cells were resuspended in Ca2+-free buffer and [Ca2+]c was measured as 340/380 nm excitation wavelength ratio at 510 nm wavelength emission (340/380 ratio) in a luminescence spectrophotometer (model LS 50B; PerkinElmer). The ER calcium content was measured as the difference between the baseline 340/380 ratio before TG addition and the peak 340/380 ratio after TG addition. This value was arbitrarily set at 100% for untreated cells.
Measurement of [Ca2+]m
5 x 105 cells were collected, washed once in PBS, then resuspended in 1 ml Earl's balanced salt solution and loaded with 2 µM Rhod2-AM in the presence of 0.02% pluronic acid for 20 min at RT. Cells were washed twice in the same buffer and Rhod2 fluorescence (F) was measured as above at 550/580 excitation/emission wavelengths. Minimum and maximum fluorescence values (Fmax and Fmin, respectively) were then obtained on the sequential addition of EGTA and saturating amounts of CaCl2 in the presence of detergent. [Ca2+]m was determined by the equation
[Ca2+] = Kd (F - Fmin)/(Fmax - F) where Kd is the dissociation constant of Rhod2. Rhod2 was judged to be localized to mitochondria based on analysis by immunofluorescence microscopy and by the fact that fluorescence was reduced to basal levels on the addition of the mitochondrial uncoupler, CCCP.
Online supplemental material
Fig. S1 documents that Adp20-induced release of cyt.c from mitochondria, procaspase-3 processing, and caspase activation are blocked in APAF-1null MEFs (Yoshida et al., 1998; a gift from T. Mak, Ontario Cancer Institute, Toronto, Canada). Fig. S2 shows that Adp20 infection does not affect ER-Golgi trafficking of temperature-sensitive VSV-G-EGFP. Fig. S3 documents that TG and b5-BCL-2 effectively lowered resting ER Ca2+ stores. Immunofluorescence microscopy confirmed that b5-BCL-2 was located exclusively at the ER. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200212059/DC1.
![]() |
Acknowledgments |
---|
D.G. Breckenridge is a recipient of a Canadian Institutes of Health Research Doctoral award. This work was supported by grants to G.C. Shore from the National Cancer Institute of Canada, and the Canadian Institutes of Health Research.
Submitted: 10 December 2002
Revised: 6 February 2003
Accepted: 11 February 2003
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adachi, T., W.W. Schamel, K.M. Kim, T. Watanabe, B. Becker, P.J. Nielsen, and M. Reth. 1996. The specificity of association of the IgD molecule with the accessory proteins BAP31/BAP29 lies in the IgD transmembrane sequence. EMBO J. 15:15341541.[Abstract]
Bereiter-Hahn, J., and M. Voth. 1994. Dynamics of mitochondria in living cells: shape changes, dislocations, fusion, and fission of mitochondria. Microsc. Res. Tech. 27:198219.[Medline]
Berridge, M.J., P. Lipp, and M.D. Bootman. 2000. The versatility and universality of calcium signalling. Nat. Rev. Mol. Cell Biol. 1:1121.[CrossRef][Medline]
Bett, A.J., W. Haddara, L. Prevec, and F.L. Graham. 1994. An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc. Natl. Acad. Sci. USA. 91:88028806.[Abstract]
Bleazard, W., J.M. McCaffery, E.J. King, S. Bale, A. Mozdy, Q. Tieu, J. Nunnari, and J.M. Shaw. 1999. The dynamin-related GTPase Dnm1 regulates mitochondrial fission in yeast. Nat. Cell Biol. 1:298304.[CrossRef][Medline]
Breckenridge, D.G., and G.C. Shore. 2000. Regulation of apoptosis by E1A and Myc oncoproteins. Crit. Rev. Eukaryot. Gene Exp. 10:273280.[Medline]
Breckenridge, D.G., and G.C. Shore. 2002. The endoplasmic reticulum and apoptosis. Genetics of Apoptosis. S. Grimm, editor. BIOS Scientific Publishers Inc., Oxford. 95113.
Breckenridge, D.G., M. Nguyen, S. Kuppig, M. Reth, and G.C. Shore. 2002. The procaspase-8 isoform, procaspase-8L, recruited to the BAP31 complex at the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA. 99:43314336.
Budihardjo, I., H. Oliver, M. Lutter, X. Luo, and X. Wang. 1999. Biochemical pathways of caspase activation during apoptosis. Annu. Rev. Cell Dev. Biol. 15:269290.[CrossRef][Medline]
Byrne, A.M., J.J. Lemasters, and A.L. Nieminen. 1999. Contribution of increased mitochondrial free Ca2+ to the mitochondrial permeability transition induced by tert-butylhydroperoxide in rat hepatocytes. Hepatology. 29:15231531.[Medline]
Cheng, E.H., D.G. Kirsch, R.J. Clem, R. Ravi, M.B. Kastan, A. Bedi, K. Ueno, and J.M. Hardwick. 1997. Conversion of Bcl-2 to a Bax-like death effector by caspases. Science. 278:19661968.
Clem, R.J., E.H. Cheng, C.L. Karp, D.G. Kirsch, K. Ueno, A. Takahashi, M.B. Kastan, D.E. Griffin, W.C. Earnshaw, M.A. Veliuona, and J.M. Hardwick. 1998. Modulation of cell death by Bcl-XL through caspase interaction. Proc. Natl. Acad. Sci. USA. 95:554559.
Collins, T.J., M.J. Berridge, P. Lipp, and M.D. Bootman. 2002. Mitochondria are morphologically and functionally heterogeneous within cells. EMBO J. 21:16161627.
Cory, S., and J.M. Adams. 2002. The Bcl2 family: regulators of the cellular life-or-death switch. Nat. Rev. Cancer. 2:647656.[CrossRef][Medline]
Deng, Y., Y. Lin, and X. Wu. 2002. TRAIL-induced apoptosis requires Bax-dependent mitochondrial release of Smac/DIABLO. Genes Dev. 16:3345.
Desagher, S., and J.C. Martinou. 2000. Mitochondria as the central control point of apoptosis. Trends Cell Biol. 10:369377.[CrossRef][Medline]
Desagher, S., A. Osen-Sand, A. Nichols, R. Eskes, S. Montessuit, S. Lauper, K. Maundrell, B. Antonsson, and J.C. Martinou. 1999. Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J. Cell Biol. 144:891901.
Foyouzi-Youssefi, R., S. Arnaudeau, C. Borner, W.L. Kelley, J. Tschopp, D.P. Lew, N. Demaurex, and K.H. Krause. 2000. Bcl-2 decreases the free Ca2+ concentration within the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA. 97:57235728.
Frank, S., B. Gaume, E.S. Bergmann-Leitner, W.W. Leitner, E.G. Robert, F. Catez, C.L. Smith, and R.J. Youle. 2001. The role of dynamin-related protein 1, a mediator of mitochondrial fission, in apoptosis. Dev. Cell. 1:515525.[Medline]
Fulda, S., W. Wick, M. Weller, and K.M. Debatin. 2002. Smac agonists sensitize for Apo2L/TRAIL- or anticancer drug-induced apoptosis and induce regression of malignant glioma in vivo. Nat. Med. 8:808815.[Medline]
Goping, I.S., D.G. Millar, and G.C. Shore. 1995. Identification of the human mitochondrial protein import receptor, huMas20p. Complementation of delta mas20 in yeast. FEBS Lett. 373:4550.[CrossRef][Medline]
Green, D.R., and J.C. Reed. 1998. Mitochondria and apoptosis. Science. 281:13091312.
Hajnoczky, G., G. Csordas, M. Madesh, and P. Pacher. 2000. Control of apoptosis by IP(3) and ryanodine receptor driven calcium signals. Cell Calcium. 28:349363.[CrossRef][Medline]
Korsmeyer, S.J., M.C. Wei, M. Saito, S. Weiler, K.J. Oh, and P.H. Schlesinger. 2000. Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c. Cell Death Differ. 7:11661173.[CrossRef][Medline]
Krammer, P.H. 2000. CD95's deadly mission in the immune system. Nature. 407:789795.[CrossRef][Medline]
Labrousse, A.M., M.D. Zappaterra, D.A. Rube, and A.M. van der Bliek. 1999. C. elegans dynamin-related protein DRP-1 controls severing of the mitochondrial outer membrane. Mol. Cell. 4:815826.[Medline]
Li, H., H. Zhu, C.J. Xu, and J. Yuan. 1998. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell. 94:491501.[Medline]
Li, S., Y. Zhao, X. He, T.H. Kim, D.K. Kuharsky, H. Rabinowich, J. Chen, C. Du, and X.M. Yin. 2002. Relief of extrinsic pathway inhibition by the Bid-dependent mitochondrial release of Smac in Fas-mediated hepatocyte apoptosis. J. Biol. Chem. 277:2691226920.
Lin, Y., A. Devin, Y. Rodriguez, and Z.G. Liu. 1999. Cleavage of the death domain kinase RIP by caspase-8 prompts TNF-induced apoptosis. Genes Dev. 13:25142526.
Luo, X., I. Budihardjo, H. Zou, C. Slaughter, and X. Wang. 1998. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell. 94:481490.[Medline]
Mathai, J.P., M. Germain, R.C. Marcellus, and G.C. Shore. 2002. Induction and endoplasmic reticulum location of BIK/NBK in response to apoptotic signaling by E1A and p53. Oncogene. 21:25342544.[CrossRef][Medline]
Matlib, M.A., Z. Zhou, S. Knight, S. Ahmed, K.M. Choi, J. Krause-Bauer, R. Phillips, R. Altschuld, Y. Katsube, N. Sperelakis, and D.M. Bers. 1998. Oxygen-bridged dinuclear ruthenium amine complex specifically inhibits Ca2+ uptake into mitochondria in vitro and in situ in single cardiac myocytes. J. Biol. Chem. 273:1022310231.
Mootha, V.K., M.C. Wei, K.F. Buttle, L. Scorrano, V. Panoutsakopoulou, C.A. Mannella, and S.J. Korsmeyer. 2001. A reversible component of mitochondrial respiratory dysfunction in apoptosis can be rescued by exogenous cytochrome c. EMBO J. 20:661671.
Muzio, M., B.R. Stockwell, H.R. Stennicke, G.S. Salvesen, and V.M. Dixit. 1998. An induced proximity model for caspase-8 activation. J. Biol. Chem. 273:29262930.
Nakamura, K., E. Bossy-Wetzel, K. Burns, M.P. Fadel, M. Lozyk, I.S. Goping, M. Opas, R.C. Bleackley, D.R. Green, and M. Michalak. 2000. Changes in endoplasmic reticulum luminal environment affect cell sensitivity to apoptosis. J. Cell Biol. 150:731740.
Ng, F.W., M. Nguyen, T. Kwan, P.E. Branton, D.W. Nicholson, J.A. Cromlish, and G.C. Shore. 1997. p28 Bap31, a Bcl-2/Bcl-XL- and procaspase-8-associated protein in the endoplasmic reticulum. J. Cell Biol. 139:327338.
Nguyen, M., P.E. Branton, S. Roy, D.W. Nicholson, E.S. Alnemri, W.C. Yeh, T.W. Mak, and G.C. Shore. 1998. E1A-induced processing of procaspase-8 can occur independently of FADD and is inhibited by Bcl-2. J. Biol. Chem. 273:3309933102.
Nguyen, M., D.G. Breckenridge, A. Ducret, and G.C. Shore. 2000. Caspase-resistant BAP31 inhibits fas-mediated apoptotic membrane fragmentation and release of cytochrome c from mitochondria. Mol. Cell. Biol. 20:67316740.
Osteryoung, K.W. 2001. Organelle fission in eukaryotes. Curr. Opin. Microbiol. 4:639646.[CrossRef][Medline]
Pacher, P., and G. Hajnoczky. 2001. Propagation of the apoptotic signal by mitochondrial waves. EMBO J. 20:41074021.
Petit, P.X., M. Goubern, P. Diolez, S.A. Susin, N. Zamzami, and G. Kroemer. 1998. Disruption of the outer mitochondrial membrane as a result of large amplitude swelling: the impact of irreversible permeability transition. FEBS Lett. 426:111116.[CrossRef][Medline]
Pinton, P., D. Ferrari, P. Magalhaes, K. Schulze-Osthoff, F. Di Virgilio, T. Pozzan, and R. Rizzuto. 2000. Reduced loading of intracellular Ca2+ stores and downregulation of capacitative Ca2+ influx in Bcl-2-overexpressing cells. J. Cell Biol. 148:857862.
Pinton, P., D. Ferrari, E. Rapizzi, F.D. Di Virgilio, T. Pozzan, and R. Rizzuto. 2001. The Ca2+ concentration of the endoplasmic reticulum is a key determinant of ceramide-induced apoptosis: significance for the molecular mechanism of Bcl-2. EMBO J. 20:26902701.
Rizzuto, R., P. Pinton, W. Carrington, F.S. Fay, K.E. Fogarty, L.M. Lifshitz, R.A. Tuft, and T. Pozzan. 1998. Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses. Science. 280:17631766.
Ruffolo, S.C., D.G. Breckenridge, M. Nguyen, I.S. Goping, A. Gross, S.J. Korsmeyer, H. Li, J. Yuan, and G.C. Shore. 2000. BID-dependent and BID-independent pathways for BAX insertion into mitochondria. Cell Death Differ. 7:11011108.[CrossRef][Medline]
Scaffidi, C., S. Fulda, A. Srinivasan, C. Friesen, F. Li, K.J. Tomaselli, K.M. Debatin, P.H. Krammer, and M.E. Peter. 1998. Two CD95 (APO-1/Fas) signaling pathways. EMBO J. 17:16751687.
Schulze-Osthoff, K., D. Ferrari, M. Los, S. Wesselborg, and M.E. Peter. 1998. Apoptosis signaling by death receptors. Eur. J. Biochem. 254:439459.[Abstract]
Scorrano, L., M. Ashiya, K. Buttle, S. Weiler, S.A. Oakes, C.A. Mannella, and S.J. Korsmeyer. 2002. A distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis. Dev. Cell. 2:5567.[CrossRef][Medline]
Sharma, V.K., V. Ramesh, C. Franzini-Armstrong, and S.S. Sheu. 2000. Transport of Ca2+ from sarcoplasmic reticulum to mitochondria in rat ventricular myocytes. J. Bioenerg. Biomembr. 32:97104.[CrossRef][Medline]
Shaw, J.M., and J. Nunnari. 2002. Mitochondrial dynamics and division in budding yeast. Trends Cell Biol. 12:178184.[CrossRef][Medline]
Smirnova, E., L. Griparic, D.L. Shurland, and A.M. van der Bliek. 2001. Dynamin-related protein Drp1 is required for mitochondrial division in mammalian cells. Mol. Biol. Cell. 12:22452256.
Smirnova, E., D.L. Shurland, S.N. Ryazantsev, and A.M. van der Bliek. 1998. A human dynamin-related protein controls the distribution of mitochondria. J. Cell Biol. 143:351358.
Stegh, A.H., H. Herrmann, S. Lampel, D. Weisenberger, K. Andra, M. Seper, G. Wiche, P.H. Krammer, and M.E. Peter. 2000. Identification of the cytolinker plectin as a major early in vivo substrate for caspase 8 during CD95- and tumor necrosis factor receptor-mediated apoptosis. Mol. Cell. Biol. 20:56655679.
Szalai, G., R. Krishnamurthy, and G. Hajnoczky. 1999. Apoptosis driven by IP(3)-linked mitochondrial calcium signals. EMBO J. 18:63496361.
Wang, B., M. Nguyen, D.G. Breckenridge, M. Stojanovic, P.A. Clemons, S. Kuppig, and G.C. Shore. 2003. Uncleaved BAP31 in association with A4 protein at the endoplasmic reticulum is an inhibitor of Fas-initiated release of cytochrome c from mitochondria. J. Biol. Chem. In press.
Wang, J., H.J. Chun, W. Wong, D.M. Spencer, and M.J. Lenardo. 2001. Caspase-10 is an initiator caspase in death receptor signaling. Proc. Natl. Acad. Sci. USA. 98:1388413888.
Yin, X.M., K. Wang, A. Gross, Y. Zhao, S. Zinkel, B. Klocke, K.A. Roth, and S.J. Korsmeyer. 1999. Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis. Nature. 400:886891.[CrossRef][Medline]
Yoshida, H., Y.Y. Kong, R. Yoshida, A.J. Elia, A. Hakem, R. Hakem, J.M. Penninger, and T.W. Mak. 1998. Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell. 94:739750.[Medline]
Zhu, W., A. Cowie, G.W. Wasfy, L.Z. Penn, B. Leber, and D.W. Andrews. 1996. Bcl-2 mutants with restricted subcellular location reveal spatially distinct pathways for apoptosis in different cell types. EMBO J. 15:41304141.[Abstract]
Related Article