Measurement of membrane permeability and permeability transition of mitochondria.
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Animals↗
Biomedical subjects
G Kroemer
Publications and source records attributed to G Kroemer.
The mitochondrion in apoptosis: how Pandora's box opens.
There is widespread agreement that mitochondria have a function in apoptosis, but the mechanisms behind their involvement remain controversial. Here we suggest that opening of a multiprotein complex called the mitochondrial permeability transition pore complex is sufficient (and, usually, necessary) for triggering apoptosis.
Animals↗
Heat-shock protein 70 antagonizes apoptosis-inducing factor.
Heat-shock protein 70 (Hsp70) has been reported to block apoptosis by binding apoptosis protease activating factor-1 (Apaf-1), thereby preventing constitution of the apoptosome, the Apaf-1/cytochrome c/caspase-9 activation complex [1,2]. Here we show that overexpression of Hsp70 protects Apaf-1-/- cells against death induced by serum withdrawal, indicating that Apaf-1 is not the only target of the anti-apoptotic action of Hsp70. We investigated the effect of Hsp70 on apoptosis mediated by the caspase-independent death effector apoptosis inducing factor (AIF), which is a mitochondrial intermembrane flavoprotein [3,4]. In a cell-free system, Hsp70 prevented the AIF-induced chromatin condensation of purified nuclei. Hsp70 specifically interacted with AIF, as shown by ligand blots and co-immunoprecipitation. Cells overexpressing Hsp70 were protected against the apoptogenic effects of AIF targeted to the extramitochondrial compartment. In contrast, an anti-sense Hsp70 complementary DNA, which reduced the expression of endogenous Hsp70, increased sensitivity to the lethal effect of AIF. The ATP-binding domain of Hsp70 seemed to be dispensable for inhibiting cell death induced by serum withdrawal, AIF binding and AIF inhibition, although it was required for Apaf-1 binding. Together, our data indicate that Hsp70 can inhibit apoptosis by interfering with target proteins other than Apaf-1, one of which is AIF.
Animals↗
Organelle-specific initiation of cell death pathways.
Nuclear DNA damage and ligation of plasma-membrane death receptors have long been recognized as initial triggers of apoptosis that induce mitochondrial membrane permeabilization (MMP) and/or the direct activation of caspases. Accumulating evidence suggests that other organelles, including the endoplasmic reticulum (ER), lysosomes and the Golgi apparatus, are also major points of integration of pro-apoptotic signalling or damage sensing. Each organelle possesses sensors that detect specific alterations, locally activates signal transduction pathways and emits signals that ensure inter-organellar cross-talk. The ER senses local stress through chaperones, Ca2+-binding proteins and Ca2+ release channels, which might transmit ER Ca2+ responses to mitochondria. The ER also contains several Bcl-2-binding proteins, and Bcl-2 has been reported to exert part of its cytoprotective effect within the ER. Upon membrane destabilization, lysosomes release cathepsins that are endowed with the capacity of triggering MMP. The Golgi apparatus constitutes a privileged site for the generation of the pro-apoptotic mediator ganglioside GD3, facilitates local caspase-2 activation and might serve as a storage organelle for latent death receptors. Intriguingly, most organelle-specific death responses finally lead to either MMP or caspase activation, both of which might function as central integrators of the death pathway, thereby streamlining lysosome-, Golgi- or ER-elicited responses into a common pathway.
Animals↗
The European Cell Death Group.
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Apoptosis↗
Erythrocytes: death of a mummy.
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Animals↗
Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor.
The complete AIF cDNA comprising the amino-terminal mitochondrial localization sequence (MLS) and the oxidoreductase domain has been fused in its carboxyl terminus to enhanced green fluorescent protein (GFP), thereby engineering an AIF-GFP fusion protein that is selectively targeted to the mitochondrial intermembrane space. Upon induction of apoptosis, the AIF-GFP protein translocates together with cytochrome c (Cyt-c) to the extramitochondrial compartment. Microinjection of recombinant AIF leads to the release of AIF-GFP and Cyt-c-GFP, indicating that ectopic AIF can favor permeabilization of the outer mitochondrial membrane. These mitochondrial effects of AIF are caspase independent, whereas the Cyt-c-microinjection induced translocation of AIF-GFP and Cyt-c-GFP is suppressed by the pan-caspase inhibitor Z-VAD.fmk. Upon prolonged culture, transfection-enforced overexpression of AIF results in spontaneous translocation of AIF-GFP from mitochondria, nuclear chromatin condensation, and cell death. These effects are caspase independent and do not rely on the oxidoreductase function of AIF. Spontaneous AIF-GFP translocation and subsequent nuclear apoptosis can be retarded by overexpression of a Bcl-2 protein selectively targeted to mitochondria, but not by a Bcl-2 protein targeted to the endoplasmic reticulum. Overexpression of a mutant AIF protein in which the MLS has been deleted (AIF Delta 1-100) results in the primary cytosolic accumulation of AIF. AIF Delta 1-100-induced cell death is suppressed by neither Z-VAD.fmk or by Bcl-2. Thus, extramitochondrially targeted AIF is a dominant cell death inducer.
Active Transport, Cell Nucleus↗
Resveratrol, a tumor-suppressive compound from grapes, induces apoptosis via a novel mitochondrial pathway controlled by Bcl-2.
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Anticarcinogenic Agents↗
[Mitochondrial control of apoptosis].
The dysregulation of programmed cell death (apoptosis) is involved in different pathologies including cancer, which is frequently associated with an increase resistance to apoptosis induction. We discovered in 1994 the implication of a specific organelle, the mitochondrion, in apoptosis. Our result have demonstrated that mitochondrial membrane permeabilization (MMP) constitutes a decisive step of the apoptotic process. MMP is regulated by numerous effectors, including the proteins from the Bcl-2/Bax family (oncogenes or tumor suppressor genes which modulate apoptosis), which interact with sessile proteins of mitochondria. MMP can be induced by a large number of pro-apoptotic second messengers, as well as by some experimental anti-cancer agents, suggesting that MMP constitutes a point of integration of the apoptotic response. As a result of MMP, several apoptogenic proteins normally confined to mitochondria are released in the extra-mitochondrial space and participate in the suicidal dismantling of the cell. We have identified several mitochondrial apoptogenic proteins, one of which, the apoptosis inducing factor (AIF) has been cloned. AIF appears to be one of the principal effectors of the apoptotic machinery. Genetic inactivation of AIF abolishes the first wave of apoptosis indispensable for early embryonic morphogenesis. In contrast, its presence in the extra-mitochondrial compartment suffices to kill cells. Altogether, these results allow for the development of new strategies aiming at inducing apoptosis in cancer cells.
Apoptosis↗
Bid acts on the permeability transition pore complex to induce apoptosis.
Similar to most if not all pro-apoptotic members of the Bcl-2 family, Bid (and its truncated product t-Bid) triggers cell death via mitochondrial membrane permeabilization (MMP). This effect can be monitored in intact cells, upon microinjection of recombinant Bid protein into the cytoplasm, as well as in purified mitochondria, upon addition of Bid protein. Here we show that Bid-induced MMP can be inhibited, both in cells and in the cell-free system, by three pharmacological inhibitors of the permeability transition pore complex (PTPC), namely cyclosporin A, N-methyl-4-Val-cyclosporin A, and bongkrekic acid (a ligand of the adenine nucleotide translocase, ANT, one of the PTPC components). Bid effects on synthetic membranes were studied either in proteoliposomes or in synthetic bilayers subjected to electrophysiological measurements. Full length Bid preferentially permeabilizes membranes and induces the formation of large conductance channels at neutral pH, when added to liposomes or bilayers containing both purified ANT and Bax, yet has no or little effect combined with ANT or Bax alone. t-Bid acts on membranes containing ANT alone with the same efficiency as on those containing both ANT and Bax. These results suggest that the proapoptotic effects of Bid are mediated, at least in part, by its functional interaction with ANT, one of the major components of PTPC.
Animals↗
Apoptosis of syncytia induced by the HIV-1-envelope glycoprotein complex: influence of cell shape and size.
Cells stably transfected with a lymphotropic HIV-1 Env gene form syncytia when cocultured with CD4(+)CXCR4(+) cells. Heterokaryons then spontaneously undergo apoptosis, while manifesting signs of mitochondrial membrane pemeabilization as well as nuclear chromatin condensation. Modulation of cellular geometry was achieved by growing syncytia on self-assembled monolayers of terminally substituted alkanethiolates designed to control the adhesive properties of the substrates. Spreading of syncytia, induced by culturing them on small circular adhesive islets (diameter 5 microm), placed at a distance that cells can bridge (10 microm), inhibited spontaneous and staurosporin-induced signs of apoptosis, both at the mitochondrial and at the nuclear levels, and allowed for the generation of larger syncytia. Transient cell spreading conferred a memory of apoptosis inhibition which was conserved upon adoption of a conventional cell shape. Limiting syncytium size by culturing them on square-shaped planar adhesive islands of defined size (400 to 2500 microm(2)), separated by nonadhesive regions, enhanced the rate of apoptotic cell death, as indicated by an accelerated permeabilization of the outer mitochondrial membrane, loss of the mitochondrial inner transmembrane potential, and an increased frequency of nuclear apoptosis. In conclusion, external constraints on syncytial size and shape strongly modulate their propensity to undergo apoptosis.
Apoptosis↗
HIV induces lymphocyte apoptosis by a p53-initiated, mitochondrial-mediated mechanism.
HIV-1 induces apoptosis and leads to CD4+ T-lymphocyte depletion in humans. It is still unclear whether HIV-1 kills infected cells directly or indirectly. To elucidate the mechanisms of HIV-1-induced apoptosis, we infected human CD4+ T cells with HIV-1. Enzymatic analysis with fluorometric substrates showed that caspase 2, 3, and 9 were activated in CD4+ T cells with peak levels 48 h after infection. Immunoblotting analysis confirmed the cleavage of pro-caspase 3 and 9, and of specific caspase substrates. Release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria was observed in HIV-infected cells. The cytochrome c and AIF release preceded the reduction of the mitochondrial transmembrane potential and nuclear chromatin condensation. H IV infection led to phosphorylation of p53 at the Ser15 residue, detectable as early as 24 h after infection. The p53 phosphorylation was followed by increased mRNA and protein expression of p21, Bax, HDM2, and p53. Up-regulation of surface FasL expression, accompanied by a down-regulation of Fas-associated proteins (FADD, DAXX, and RIP), was observed 72 h after infection. Our results suggest that HIV activates the p53 pathway, leading to cytochrome c and AIF release with ensuing caspase activation.
Apoptosis↗
Apoptosis control in syncytia induced by the HIV type 1-envelope glycoprotein complex: role of mitochondria and caspases.
Syncytia arising from the fusion of cells expressing a lymphotropic HIV type 1-encoded envelope glycoprotein complex (Env) with cells expressing the CD4/CXC chemokine receptor 4 complex spontaneously undergo cell death. Here we show that this process is accompanied by caspase activation and signs of mitochondrial membrane permeabilization (MMP), including the release of intermembrane proteins such as cytochrome c (Cyt-c) and apoptosis-inducing factor (AIF) from mitochondria. In Env-induced syncytia, caspase inhibition did not suppress AIF- and Cyt-c translocation, yet it prevented all signs of nuclear apoptosis. Translocation of Bax to mitochondria led to MMP, which was inhibited by microinjected Bcl-2 protein or bcl-2 transfection. Bcl-2 also prevented the subsequent nuclear chromatin condensation and DNA fragmentation. The release of AIF occurred before that of Cyt-c and before caspase activation. Microinjection of AIF into syncytia sufficed to trigger rapid, caspase-independent Cyt-c release. Neutralization of endogenous AIF by injection of an antibody prevented all signs of spontaneous apoptosis occurring in syncytia, including the Cyt-c release and nuclear apoptosis. In contrast, Cyt-c neutralization only prevented nuclear apoptosis, and did not affect AIF release. Our results establish that the following molecular sequence governs apoptosis of Env-induced syncytia: Bax-mediated/Bcl-2-inhibited MMP --> AIF release --> Cyt-c release --> caspase activation --> nuclear apoptosis.
Apoptosis↗
Involvement of apoptosis-inducing factor during dolichyl monophosphate-induced apoptosis in U937 cells.
Dolichyl monophosphate (Dol-P) has been found to induce apoptosis in human leukemia U937 cells. During this apoptotic execution, the increase of plasma membrane fluidity (5-20 min), caspase-3-like protease activation (2-4 h), chromatin condensation and DNA ladder formation (3-4 h) were observed successively. Here, we report that reduction in mitochondrial transmembrane potential and translocation of apoptosis-inducing factor (AIF) are early events (1-3 h) in the apoptotic process induced by Dol-P in U937 cells. The AIF was concentrated around nuclei and partly translocated to the nuclei, which was confirmed by immunocytochemistry using specific anti-AIF antibody. Both caspase-8 and caspase-3 inhibitors blocked only DNA fragmentation but not mitochondrial processes, AIF migration and chromatin condensation. These results indicate that mitochondrial changes are an early step in the apoptosis induced by Dol-P and AIF is one of the important factors which induce chromatin condensation in nuclei.
Apoptosis↗
Two distinct pathways leading to nuclear apoptosis.
Apaf-1(-/-) or caspase-3(-/-) cells treated with a variety of apoptosis inducers manifest apoptosis-associated alterations including the translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, large scale DNA fragmentation, and initial chromatin condensation (stage I). However, when compared with normal control cells, Apaf-1(-/-) or caspase-3(-/-) cells fail to exhibit oligonucleosomal chromatin digestion and a more advanced pattern of chromatin condensation (stage II). Microinjection of such cells with recombinant AIF only causes peripheral chromatin condensation (stage I), whereas microinjection with activated caspase-3 or its downstream target caspase-activated DNAse (CAD) causes a more pronounced type of chromatin condensation (stage II). Similarly, when added to purified HeLa nuclei, AIF causes stage I chromatin condensation and large-scale DNA fragmentation, whereas CAD induces stage II chromatin condensation and oligonucleosomal DNA degradation. Furthermore, in a cell-free system, concomitant neutralization of AIF and CAD is required to suppress the nuclear DNA loss caused by cytoplasmic extracts from apoptotic wild-type cells. In contrast, AIF depletion alone suffices to suppress the nuclear DNA loss contained in extracts from apoptotic Apaf-1(-/-) or caspase-3(-/-) cells. As a result, at least two redundant parallel pathways may lead to chromatin processing during apoptosis. One of these pathways involves Apaf-1 and caspases, as well as CAD, and leads to oligonucleosomal DNA fragmentation and advanced chromatin condensation. The other pathway, which is caspase-independent, involves AIF and leads to large-scale DNA fragmentation and peripheral chromatin condensation.
Animals↗
Apoptosis. Mitochondria--the death signal integrators.
Many of the intricate pathways of apoptosis that instruct a cell to kill itself involve the convergence of key proteins on the membranes of mitochondria. Such proteins induce the permeabilization of mitochondrial membranes and the release of caspase enzymes and nuclease activators that set in motion the final stages of programmed cell death. Now, as Brenner and Kroemer discuss in their Perspective, a proapoptotic transcription factor called TR3 has been found to move from its normal location in the nucleus to the mitochondria and to promote release of cytochrome c, a key event in apoptosis (Li et al.)
Animals↗
Caspase-independent commitment phase to apoptosis in activated blood T lymphocytes: reversibility at low apoptotic insult.
Little is known about the mechanisms of programmed death triggered in T lymphocytes by stimuli that can bypass caspase activation. Anti-CD2 monoclonal antibody and staurosporine are such apoptosis inducers because they operate in the presence of broad-spectrum caspase inhibitors BOC-D.fmk and Z-VAD.fmk. A system was devised, based on the isolation according to density of activated blood T cells progressively engaged in the apoptotic process. This allowed definition of a sequence of caspase-dependent and caspase-independent apoptogenic events that are triggered by anti-CD2 and staurosporine. Thus, a commitment phase to apoptosis was defined that is entirely caspase independent and that is characterized by cell volume loss, partial chromatin condensation, and release into the cytosol and the nucleus of mitochondrial "apoptosis-inducing factor " (AIF). Committed cells were viable, displayed a high mitochondrial inner transmembrane potential (triangle upPsim), and lacked large-scale and oligonucleosomal DNA fragmentation. Mitochondrial release of AIF was selective because cytochrome c was retained in mitochondria of the very same cells. Mitochondrial release of cytochrome c occurred later, at the onset of the execution phase of apoptosis, concurrently with triangle upPsim collapse, poly (ADP-ribose) polymerase cleavage, and DNA fragmentation. The apoptogenic events of this commitment phase are reversible if the strength of the stimulus is low and of short duration.
Apoptosis↗
Apoptosis-inducing factor (AIF): a ubiquitous mitochondrial oxidoreductase involved in apoptosis.
Apoptosis-inducing factor (AIF) is encoded by one single gene located on the X chromosome. AIF is ubiquitously expressed, both in normal tissues and in a variety of cancer cell lines. The AIF precursor is synthesized in the cytosol and is imported into mitochondria. The mature AIF protein, a flavoprotein (prosthetic group: flavine adenine dinucleotide) with significant homology to plant ascorbate reductases and bacterial NADH oxidases, is normally confined to the mitochondrial intermembrane space. In a variety of different apoptosis-inducing conditions, AIF translocates through the outer mitochondrial membrane to the cytosol and to the nucleus. Ectopic (extra-mitochondrial) AIF induces nuclear chromatin condensation, as well as large scale ( approximately 50 kb) DNA fragmentation. Thus, similar to cytochrome c, AIF is a phylogenetically old, bifunctional protein with an electron acceptor/donor (oxidoreductase) function and a second apoptogenic function. In contrast to cytochrome c, however, AIF acts in a caspase-independent fashion. The molecular mechanisms via which AIF induces apoptosis are discussed.
Animals↗