Abstract: BH3 interacting domain death agonist (Bid), a pro-apoptotic member of the Bcl-2 family of proteins, is activated through cleavage by caspase-8. The active C-terminal fragment of Bid (tBid) translocates to the mitochondria where it interacts with cardiolipins at contact sites and induces the release of cytochrome c by a mechanism that is not yet fully understood. It has been shown that the alpha-helices alphaH6 and alphaH7 (which create the hairpin-forming domain of tBid) mediate the insertion of Bid into mitochondrial membranes and are essential for the cytochrome c-releasing activity. In the present study, we focused on the interaction between the alphaH6 and the mitochondrial membrane. By the use of single-cell electropermeabilization associated with flow cytometric analysis of intact cells, we demonstrated that H6 is able to induce cell death when used in the micromolar range. We also studied the interactions of the alphaH6 with artificial monolayers. We showed that the presence of negatively charged cardiolipins greatly enhances the insertion of alphaH6 into the phospholipid monolayer. The modification of two charged amino acid residues in alphaH6 abolished its insertion and also its in vivo effects. Furthermore, the negative values of the excess areas of mixing indicate that attractive interactions between cardiolipins and alphaH6 occur in the mixed monolayers. Fluorescence microscopy observations revealed that alphaH6 significantly disrupts cardiolipin packing and stabilizes the fluid lipid phase. These results suggest that cardiolipins at the contact sites between the two mitochondrial membranes could mediate the binding of tBid via alphaH6.
Abstract: In several neurodegenerative diseases of the CNS, oligodendrocytes are implicated in an inflammatory process associated with altered levels of oxysterols and inflammatory enzymes such as secreted phospholipase A2 (sPLA2). In view of the scarce literature related to this topic, we investigated oxysterol effects on these myelinating glial cells. Natural oxysterol 25-hydroxycholesterol (25-OH; 1 and 10 microM) altered oligodendrocyte cell line (158N) morphology and triggered apoptosis (75% of apoptosis after 72 h). These effects were mimicked by 22(S)-OH (1 and 10 microM) which does not activate liver X receptor (LXR) but not by a synthetic LXR ligand (T0901317). Therefore, oxysterol-induced apoptosis appears to be independent of LXR. Interestingly, sPLA2 type IIA (sPLA2-IIA) over-expression partially rescued 158N cells from oxysterol-induced apoptosis. In fact, 25-OH, 24(S)-OH, and T0901317 stimulated sPLA2-IIA promoter and sPLA2 activity in oligodendrocyte cell line. Accordingly, administration of T0901317 to mice enhanced sPLA2 activity in brain extracts by twofold. Short interfering RNA strategy allowed to establish that stimulation of sPLA2-IIA is mediated by pregnane X receptor (PXR) at high oxysterol concentration (10 microM) and by LXR beta at basal oxysterol concentration. Finally, GC coupled to mass spectrometry established that oligodendrocytes contain oxysterols and express their biosynthetic enzymes, suggesting that they may act through autocrine/paracrine mechanism. Our results show the diversity of oxysterol signalling in the CNS and highlight the positive effects of the LXR/PXR pathway which may open new perspectives in the treatment of demyelinating and neurodegenerative diseases.
Abstract: Bax is activated and translocated onto mitochondria to mediate cytochrome c release and apoptosis. The molecular mechanisms of Bax activation during apoptosis remain a subject of debate. We addressed the question of whether reactive oxygen species could directly activate Bax for its subsequent translocation and apoptosis. Using the SW480 human colon adenocarcinoma cell line stably expressing Bax fused to GFP, we showed that H2O2 induces Bax conformational change, mitochondrial translocation, and subsequent oligomerization at mitochondria. We found that H2O2-induced Bax activation is dependent on the conserved cysteine residue 62 of Bax. Mutation of cysteine 62, but not cysteine 126, to serine or alanine abolished its activation by H2O2 but not other death stimuli, both in SW480 and Bax-deficient HCT116 cells, whereas wild type Bax sensitizes these cells to apoptosis. Cysteines of Bax could chemically react with H2O2. Mutation of Bax BH3 domain in the presence of cysteine 62 also abolished Bax proapoptotic activity. We conclude that reactive oxygen species could be a direct signal for Bax activation by reacting with cysteine residues. Our results identify a critical role of cysteine 62 in oxidative stress-induced Bax activation and subsequent apoptosis.
Abstract: Cardiolipin is a mitochondria-specific phospholipid known to be intimately involved with apoptosis. However, the lack of appropriate cellular models to date restricted analysis of its role in cell death. The maturation of cardiolipin requires the transacylase tafazzin, which is mutated in the human disorder Barth syndrome. Using Barth syndrome patient-derived cells and HeLa cells in which tafazzin was knocked down, we show that cardiolipin is required for apoptosis in the type II mitochondria-dependent response to Fas stimulation. Cardiolipin provides an anchor and activating platform for caspase-8 translocation to, and embedding in, the mitochondrial membrane, where it oligomerizes and is further activated, steps that are necessary for an efficient type II apoptotic response.
Abstract: Cardiolipin (CL) is a mitochondria-specific phospholipid synthesized by CL synthase (CLS). We describe here a human gene for CLS and its analysis via RNAi knockdown on apoptotic progression. Although mitochondrial membrane potential is unchanged in cells containing only 25% of the normal amount of CL, free cytochrome c (cyt. c) is detected in the intermembrane space and the mitochondria exhibit signs of reorganized cristae. However, the release of cyt. c from the mitochondria still requires apoptotic stimulation. Increased sensitivity to apoptotic signals and accelerated rates of apoptosis are observed in CL-deficient cells, followed by elevated levels of secondary necrosis. Apoptosis is thought to progress via binding of truncated Bid (tBid) to mitochondrial CL, followed by CL oxidation which results in cyt. c release. The exaggerated and accelerated apoptosis observed in CL-deficient cells is matched by an accelerated reduction in membrane potential and increased cyt. c release, but not by decreased tBid binding. This study suggests that the CL/cyt. c relationship is important in apoptotic progression and that regulating CL oxidation or/and deacylation could represent a possible therapeutic target.
Abstract: TNFR1/Fas engagement results in the cleavage of cytosolic Bid to truncated Bid (tBid), which translocates to mitochondria. We demonstrate that recombinant tBid induces in vitro immediate destabilization of the mitochondrial bioenergetic homeostasis. These alterations result in mild uncoupling of mitochondrial state-4 respiration, associated with an inhibition the adenosine diphosphate (ADP)-stimulated respiration and phosphorylation rate. tBid disruption of mitochondrial homeostasis was inhibited in mitochondria overexpressing Bcl-2 and Bcl-XL. The inhibition of state-3 respiration is mediated by the reorganization of cardiolipin within the mitochondrial membranes, which indirectly affects the activity of the ADP/ATP translocator. Cardiolipin-deficient yeast mitochondria did not exhibit any respiratory inhibition by tBid, proving the absolute requirement for cardiolipin for tBid binding and activity. In contrast, the wild-type yeast mitochondria underwent a similar inhibition of ADP-stimulated respiration associated with reduced ATP synthesis. These events suggest that mitochondrial lipids rather than proteins are the key determinants of tBid-induced destabilization of mitochondrial bioenergetics.
Abstract: The apoptotic effector Bid regulates cell death at the level of mitochondria. Under its native state, Bid is a soluble cytosolic protein that undergoes proteolysis and yields a 15 kDa-activated form tBid (truncated Bid). tBid translocates to mitochondria and participates in cytochrome c efflux by a still unclear mechanism, some of them at least mediated by Bax. Using mitochondria isolated from wild-type and cardiolipin (CL)-synthase-less yeast strains, we observed that tBid perturbs mitochondrial bioenergetics by inhibiting state-3 respiration and ATP synthesis and that this effect was strictly dependent on the presence of CL. In a second set of experiments, heterologous coexpression of tBid and Bax in wild-type and CL-less yeast strains showed that (i) tBid binding and the subsequent alteration of mitochondrial bioenergetics increased Bax-induced cytochrome c release and (ii) the absence of CL favors Bax effects independently of the presence of t-Bid. These data support recent views suggesting a dual function of CL in mitochondria-dependent apoptosis.
Abstract: Little is known about endoplasmic reticulum (ER) export signals, particularly those of members of the G-protein-coupled receptor family. We investigated the structural motifs involved in membrane export of the human pituitary vasopressin V1b/V3 receptor. A series of V3 receptors carrying deletions and point mutations were expressed in AtT20 corticotroph cells. We analyzed the export of these receptors by monitoring radioligand binding and by analysis of a V3 receptor tagged with both green fluorescent protein and Myc epitopes by a novel flow cytometry-based method. This novel method allowed us to quantify total and membrane-bound receptor expression. Receptors lacking the C terminus were not expressed at the cell surface, suggesting the presence of an export motif in this domain. The distal C terminus contains two di-acidic (DXE) ER export motifs; however, mutating both these motifs had no effect on the V3 receptor export. The proximal C terminus contains a di-leucine (345)LL(346) motif surrounded by the hydrophobic residues Phe(341), Asn(342), and Leu(350). The mutation of one or more of these five residues abolished up to 100% of the receptor export. In addition, these mutants colocalized with calnexin, demonstrating that they were retained in the ER. Finally, this motif was sufficient to confer export properties on a CD8alpha glycoprotein-V3 receptor chimera. In conclusion, we have identified a novel export motif, FN(X)(2)LL(X)(3)L, in the C terminus of the V3 receptor.
Abstract: Siva-1 is a death domain-containing proapoptotic protein identified as an intracellular ligand of CD27 and of the glucocorticoid-induced TNFR family-related gene, which are two members of the TNFR family expressed on lymphoid cells. Although Siva-1 expression is up-regulated in multiple pathological processes, little is known about the signaling pathway underlying the Siva-induced apoptosis. In this study, we investigated the mechanism of the proapoptotic activity of Siva-1 and an alternative splice form lacking the death domain of Siva-1, Siva-2, in T lymphocytes in which Siva proteins, CD27, and glucocorticoid-induced TNFR family-related gene are primarily expressed. Overexpression of Siva proteins triggers a typical apoptotic process manifested by cell shrinkage and surface exposure of phosphatidylserine, and confirmed by ultrastructural features. Siva-induced apoptosis is related to the CD27-mediated apoptotic pathway and results in activation of both initiator and effector caspases. This pathway involves a mitochondrial step evidenced by activation of Bid and cytochrome c release, and is modulated by overexpression of Bcl-2 or Bcl-x(L). The determinants for Siva-induced apoptosis are not contained within the death domain found in the central part of Siva-1, but rather in both the N-terminal and C-terminal regions shared by both Siva proteins. The N-terminal region also participates in the translocation of both Siva proteins into the nuclear compartment. These results indicate that Siva-1 and Siva-2 mediate apoptosis in T lymphocytes via a caspase-dependent mitochondrial pathway that likely involves both cytoplasmic and nuclear events.
Abstract: Lethal toxin (LT) from Clostridium sordellii (strain IP82) inactivates in glucosylating the small GTPases Ras, Rap, Ral and Rac. In the present study we show that LT-IP82 induces cell death via an intrinsic apoptotic pathway in the myeloid cell-line HL-60. LT-IP82 was found to disrupt mitochondrial homeostasis as characterized by a decrease in mitochondrial transmembrane potential and cardiolipin alterations, associated with the release of cytochrome c in the cytosol. Time-course studies of caspase activation revealed that caspase-9 and caspase-3 were activated before caspase-8. Moreover, although LT-IP82-induced cell death was abrogated by caspase-inhibitors, these inhibitors did not suppress mitochondrial alterations, indicating that caspase activation occurs downstream of mitochondria. Protection of mitochondria by Bcl-2 overexpression prevented mitochondrial changes as well as apoptosis induction. Furthermore, evidence is provided that LT-IP82-induced apoptosis is not a consequence of cortical actin disorganization, suggesting that Rac inactivation does not initiate the apoptotic process. Cell exposure to LT-IP82 leads to a co-localization of the toxin with a mitochondrial marker within 2 h. Therefore, we suggest that LT-IP82 could act at the mitochondrion level independently of its enzymatic effect on small GTPases.
Abstract: Cytochrome c release is thought to play an important role in the initiation of apoptosis. The nature of the control exerted by Bcl-2 and Bcl-XL on such a pathway is not precisely known. We addressed this issue by square-wave pulse electroloading of exogenous cytochrome c into Jurkat cells. Three hours after cytochrome c loading into the cells, characteristic phenotypes of apoptosis were observed. However, a significant drop in the mitochondrial membrane potential (Deltapsim) was also observed, while cytochrome c was generally considered to act downstream from the mitochondria. Related to the Deltapsim drop, there was a release of proapoptotic proteins such as AIF and Smac from the mitochondria. This release, as well as NAD(P)H and cardiolipids oxidation, are linked to previous caspase activation. Cytochrome c-linked caspase activation also led to potassium efflux out of the cell. Overexpression of Bcl-2 and Bcl-XL or N-acetyl-DEVD-aldehyde treatment not only prevented the mitochondrial membrane potential decrease, but also protected cells from the apoptosis directly induced by cytochrome c electroloading. Bcl-2 and Bcl-XL protection is based on the inhibition of the caspase-dependent retroactive pathway affecting the mitochondrial compartment.
Abstract: Bcl-2 is a prosurvival factor that reportedly prevents the nonspecific permeabilization of mitochondrial membranes, yet enhances specific ADP/ATP exchange by these organelles. Here, we show that Bcl-2 enhances the ADP/ATP exchange in proteoliposomes containing the purified adenine nucleotide translocase (ANT) in isolated mitochondria and mitoplasts, as well as in intact cells in which mitochondrial matrix ATP was monitored continuously using a specific luciferase-based assay system. Conversely, Bax, which displaces Bcl-2 from ANT in apoptotic cells, inhibits ADP/ATP exchange through a direct action on ANT. The Bax-mediated inhibition of ADP/ATP exchange can be separated from Bax-stimulated formation of nonspecific pores by ANT. Chemotherapy-induced apoptosis caused an inhibition of ANT activity, which preceded the loss of the mitochondrial transmembrane potential and could be prevented by overexpression of Bcl-2. These data are compatible with a model of mitochondrial apoptosis regulation in which ANT interacts with either Bax or Bcl-2, which both influence ANT function in opposing manners. Bcl-2 would maintain the translocase activity at high levels, whereas Bax would inhibit the translocase function of ANT.
Abstract: Leishmania major is a protozoan parasite from one of the most ancient phylogenic branches of unicellular eukaryotes, and containing only one giant mitochondrion. Here we report that staurosporine, that induces apoptosis in all mammalian nucleated cells, also induces in L. major a death process with several cytoplasmic and nuclear features of apoptosis, including cell shrinkage, phosphatidyl serine exposure, maintenance of plasma membrane integrity, mitochondrial transmembrane potential (DeltaPsim) loss and cytochrome c release, nuclear chromatin condensation and fragmentation, and DNA degradation. Nuclear apoptosis-like features were prevented by cysteine proteinase inhibitors, and cell free assays using dying L. major cytoplasmic extracts indicated that the cysteine proteinases involved (i) also induced nuclear apoptosis-like features in isolated mammalian nuclei, and (ii) shared at least two nuclear substrates, but no cleavage site preference, with human effector caspases. Finally, isolated L. major mitochondria released cytochrome c and cysteine proteinases with nuclear pro-apoptotic activity when incubated with human recombinant Bax, even (although much less efficiently) when Bax was deleted of its transmembrane domain required for insertion in mitochondrial outermembranes, implying that L. major mitochondrion may express proteins able to interact with Bax. The recruitment of cysteine proteinases and mitochondria to the cell death machinery may be of very ancient evolutionary origin. Alternately, host/parasite interactions may have exerted selective pressures on the cell death phenotype of kinetoplastid parasites, resulting in the more recent emergence of an apoptotic machinery through a process of convergent evolution.
Abstract: Mitochondria play a pivotal role in apoptosis in multicellular organisms by releasing apoptogenic factors such as cytochrome c that activate the caspases effector pathway, and apoptosis-inducing factor (AIF) that is involved in a caspase-independent cell death pathway. Here we report that cell death in the single-celled organism Dictyostelium discoideum involves early disruption of mitochondrial transmembrane potential (DeltaPsim) that precedes the induction of several apoptosis-like features, including exposure of the phosphatidyl residues at the external surface of the plasma membrane, an intense vacuolization, a fragmentation of DNA into large fragments, an autophagy, and the release of apoptotic corpses that are engulfed by neighboring cells. We have cloned a Dictyostelium homolog of mammalian AIF that is localized into mitochondria and is translocated from the mitochondria to the cytoplasm and the nucleus after the onset of cell death. Cytoplasmic extracts from dying Dictyostelium cells trigger the breakdown of isolated mammalian and Dictyostelium nuclei in a cell-free system, and this process is inhibited by a polyclonal antibody specific for Dictyostelium discoideum apoptosis-inducing factor (DdAIF), suggesting that DdAIF is involved in DNA degradation during Dictyostelium cell death. Our findings indicate that the cell death pathway in Dictyostelium involves mitochondria and an AIF homolog, suggesting the evolutionary conservation of at least part of the cell death pathway in unicellular and multicellular organisms.
Abstract: The multicellular development of the single celled eukaryote Dictyostelium discoideum is induced by starvation and consists of initial aggregation of the isolated amoebae, followed by their differentiation into viable spores and dead stalk cells. These stalk cells retain their structural integrity inside a stalk tube that support the spores in the fruiting body. Terminal differentiation into stalk cells has been shown to share several features with programmed cell death (Cornillon et al. (1994), J. Cell Sci. 107, 2691-2704). Here we report that, in the absence of aggregation and differentiation, D. discoideum can undergo another form of programmed cell death that closely resembles apoptosis of most mammalian cells, involves loss of mitochondrial transmembrane potential, phosphatidylserine surface exposure, and engulfment of dying cells by neighboring D. discoideum cells. This death has been studied by various techniques (light microscopy and scanning or transmission electron microscopy, flow cytometry, DNA electrophoresis), in two different conditions inhibiting D. discoideum multicellular development. The first one, corresponding to an induced unicellular cell death, was obtained by starving the cells in a "conditioned" cell-free buffer, prepared by previous starvation of another D. discoideum cell population in potassium phosphate buffer (pH 6.8). The second one, corresponding to death of D. discoideum after axenic growth in suspension, was obtained by keeping stationary cells in their culture medium. In both cases of these unicellular-specific cell deaths, microscopy revealed morphological features known as hallmarks of apoptosis for higher eukaryotic cells and apoptosis was further corroborated by flow cytometry. The occurrence in D. discoideum of programmed cell death with two different phenotypes, depending on its multicellular or unicellular status, is further discussed.
Abstract: Jurkat T cells showed a major, early decrease in blue autofluorescence in response to Fas/Apo-1/CD95 cross-linking or stimulation with cell-permeant ceramide. This indicates the oxidation/depletion of NADH or NADPH before the onset of apoptosis. Kinetic studies, cytofluorimetric multiparameter analyses and cell sorting experiments indicated a close temporal relationship between NAD(P)H oxidation/depletion and the dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)). In contrast, NAD(P)H depletion was detected well before several other changes associated with late apoptosis, including enhanced superoxide generation, phosphatidylserine exposure on the cell surface, loss of cytosolic K(+), decreased cytoplasmic pH, nuclear DNA fragmentation, cell shrinkage, loss of viability and the appearance of the mitochondrial antigen APO2.7. Full activation of caspase 9 and caspase 3 appeared to be correlated with the appearance of superoxide anions in the mitochondria, and followed the drop in NADPH. Overexpression of the apoptosis-inhibitory proto-oncogene Bcl-2, which encodes an inhibitor of the mitochondrial permeability transition (PT) pore, delayed both the DeltaPsi(m) disruption and the depletion of NAD(P)H. Similar effects were observed with the pharmacological PT pore inhibitors, bongkrekic acid and cyclosporin A. Thus there appears to be a close functional relationship between mitochondrial and cellular redox changes during early apoptosis; events that are inhibited by Bcl-2.
Abstract: Hypericin (HY) is a powerful photo-inducer of apoptosis in Jurkat cells as measured by caspase-3 activation, cell shrinkage, phosphatidylserine (PS) exposure and the appearance of hypoploid DNA. These processes are preceded by rapid Bcl-2-independent mitochondrial transmembrane depolarization and a drop in cytoplasmic pH. Pre-incubation of cells with inhibitors of the mitochondrial permeability transition pore, such as cyclosporin A or bongkrekic acid, does not protect cells from mitochondrial membrane potential (deltapsim) decrease. However, monitoring of mitochondrial entrapped calcein by confocal fluorescence imaging gives clear evidence of HY photo-induced mitochondrial permeability. This should be considered as the result of a non-specific alteration of mitochondrial membrane integrity brought about by lipid peroxidation. Nevertheless, synthesis of the anti-apoptotic protein Bcl-2 appears to delay the subsequent time course of PS exposure and to reduce caspase-3 activation and the fraction of cells which become hypoploid. We interpret this partially protective effect as the consequence of a direct interaction of Bcl-2 with cytosolic cytochrome c previously released from mitochondria upon deltapsim decrease and/or of Bcl-2 inhibition of the deleterious retro-effect of caspase-3 on the mitochondrial permeability transition pore and/or the mitochondrial membrane components.
Abstract: The molecular mode of action of lonidamine, a therapeutic agent employed in cancer chemotherapy, has been elusive. Here we provide evidence that lonidamine (LND) acts on mitochondria to induce apoptosis. LND provokes a disruption of the mitochondrial transmembrane potential which precedes signs of nuclear apoptosis and cytolysis. The mitochondrial and cytocidal effects of LND are not prevented by inhibitors of caspases or of mRNA or protein synthesis. However, they are prevented by transfection-enforced overexpression of Bcl-2, an oncoprotein which inhibits apoptosis by stabilizing the mitochondrial membrane barrier function. Accordingly, the cell death-inducing effect of LND is amplified by simultaneous addition of PK11195, an isoquinoline ligand of the peripheral benzodiazepine receptor which antagonizes the cytoprotective effect of Bcl-2. When added to isolated nuclei, LND fails to provoke DNA degradation unless mitochondria are added simultaneously. In isolated mitochondria, LND causes the dissipation of the mitochondrial inner transmembrane potential and the release of apoptogenic factors capable of inducing nuclear apoptosis in vitro. Thus the mitochondrion is the subcellular target of LND. All effects of LND on isolated mitochondria are counteracted by cyclosporin A, an inhibitor of the mitochondrial PT pore. We therefore tested the effect of LND on the purified PT pore reconstituted into liposomes. LND permeabilizes liposomal membranes containing the PT pore. This effect is prevented by addition of recombinant Bcl-2 protein but not by a mutant Bcl-2 protein that has lost its apoptosis-inhibitory function. Altogether these data indicate that LND represents a novel type of anti-cancer agent which induces apoptosis via a direct effect on the mitochondrial PT pore.
Abstract: We have previously shown that fibroblasts from ultra-violet (UV) hypersensitive xeroderma pigmentosum patients (XP) are markedly deficient in catalase activity resulting in high intracellular levels of hydrogen peroxide (H2O2) following UV irradiation. No direct correlation between catalase activity and repair ability was found since XP variant cells which are proficient in nucleotide excision repair (NER) showed activities as low as those found in NER deficient classical XP groups A and D. However, in contrast to the skin cancer prone XP patients, another NER deficient syndrome, trichothiodystrophy (TTD), which does not exhibit any cancer predisposition, was found to present normal catalase activity. Moreover, it was found that a variety of SV40 transformed human cell lines also showed decreased catalase activities. Our previous data showed that a molecular analysis of the normal, XP, TTD or transformed human fibroblast cell lines did not reveal any differences in levels of catalase transcription or amount of catalase protein subunits. These results incited us to examine the structure/function relationship of the tetrameric active enzyme form of catalase (which is the only one able to carry out H2O2 dismutation) with its cofactor NADPH. In the present study, we have measured the effects on catalase activity after adding NADPH either to acellular extracts or during cell culture of the different cell types. The NADPH levels were also quantified directly in intact cells using flow cytometry. Our results show a clear relationship between low catalase activity and striking decrease in intracellular NADPH levels.
Abstract: Mitochondrial intermembrane proteins including cytochrome c are known to activate caspases. Accordingly, a disruption of the mitochondrial membrane barrier function with release of cytochrome into the cytosol has been shown to precede caspase activation in a number of different models of apoptosis. Here, we addressed the question of whether caspases themselves can affect mitochondrial membrane function. Recombinant caspases were added to purified mitochondria and were found to affect the permeability of both mitochondrial membranes. Thus, caspases cause a dissipation of the mitochondrial inner transmembrane potential. In addition, caspases cause intermembrane proteins including cytochrome c and AIF (apoptosis-inducing factor) to be released through the outer mitochondrial membrane. These observations suggest that caspases and mitochondria can engage in a circular self-amplification loop. An increase in mitochondrial membrane permeability would cause the release of caspase activators, and caspases, once activated, would in turn increase the mitochondrial membrane permeability. Such a self-amplifying system could accelerate the apoptotic process and/or coordinate the apoptotic response between different mitochondria within the same cell.
Abstract: Upon induction of permeability transition with different agents (Ca2+, tert-butyl hydroperoxide, atractyloside), mouse hepatocyte mitochondria manifest a disruption of outer membrane integrity leading to the release of cytochrome c and apoptosis-inducing factor (AIF), two proteins which are involved in programmed cell death (apoptosis). Chelation of Ca2+ shortly (within 2 min) after its addition to isolated mitochondria reestablished the mitochondrial transmembrane potential (deltapsi(m)), prevented induction of large amplitude swelling and release of both cytochrome c and AIF. In contrast, late Ca2+ chelation (10 min after addition of Ca2+) failed to affect these parameters. Cytochrome c appears to be released through a mechanically damaged outer mitochondrial membrane rather than via a specific release mechanism. These findings clarify the mechanisms through which irreversible permeability transition occurs with subsequent large amplitude swelling culminating in the release of intermembrane proteins from mitochondria. Moreover, they confirm the hypothesis formulated by Skulachev [FEBS Lett. 397 (1996) 7-10 and Q. Rev. Biophys. 29 (1996) 169-2021 linking permeability transition to activation of the apoptogenic catabolic enzymes.
Abstract: Purified nuclei exposed to apoptogenic factors in vitro undergo morphological and biochemical changes in chromatin organization. Most cell-free models of nuclear apoptosis are based on the quantitation of endonuclease-mediated DNA fragmentation on agarose gels or on the changes of nuclear morphology revealed by the DNA-intercalating fluorochrome 4'-6-diamidino-2-phenylindole dihydrochloride. In this work we develop a cytofluorometric system for the accurate quantitation of nuclear DNA loss. This system has been used to determine the conditions of nuclear apoptosis induced by apoptosis-inducing factor (AIF) contained in the supernatant of mitochondria induced to undergo permeability transition. AIF can provoke significant nuclear DNA loss in < or = 5 min, acts over a wide pH range (pH 6 to 9), and resists cysteine protease inhibitors such as iodoacetamide and N-ethylmaleimide. Moreover, we applied this system to the question of how the proapoptotic second messenger ceramide would induce apoptosis in vitro: via a direct effect on nuclei, a direct effect on mitochondria, or via indirect mechanisms? Our data indicate that ceramide has to activate yet unknown cytosolic effectors that, in the presence of mitochondria, can induce nuclear apoptosis in vitro.
Abstract: Both physiological cell death (apoptosis) and at least some cases of accidental cell death (necrosis) involve a two-step-process. At first level, numerous physiological or pathological stimuli can trigger mitochondrial permeability transition which constitutes a rate-limiting event and initiates the common phase of the death process. Mitochondrial permeability transition (PT) involves the formation of proteaceous, regulated pores, probably by apposition of inner and outer mitochondrial membrane proteins which cooperate to form the mitochondrial PT pore complex. Inhibition of PT by pharmacological intervention on mitochondrial structures or mitochondrial expression of the apoptosis-inhibitory oncoprotein Bcl-2 thus can prevent cell death. At a second level, the consequences of mitochondrial dysfunction (collapse of the mitochondrial transmembrane potential, uncoupling of the respiratory chain, hyperproduction of superoxide anions, disruption of mitochondrial biogenesis, outflow of matrix calcium and glutathione, and release of soluble intermembrane proteins) can entail a biogenetic catastrophe culminating in the disruption of plasma membrane integrity (necrosis) and/or the activation and action of apoptogenic proteases with secondary endonuclease activation and consequent oligonucleosomal DNA fragmentation (apoptosis). The acquisition of the biochemical and ultrastructural features of apoptosis critically relies on the liberation of apoptogenic proteases or protease activators from the mitochondrial intermembrane space. This scenario applies to very different models of cell death. The notion that mitochondrial events control cell death has major implications for the development of death-inhibitory drugs.
Abstract: The induction phase of programmed cell death (PCD) or apoptosis is characterized by an extreme heterogeneity of potential PCD-triggering signal transduction pathways. During the subsequent effector phase, the numerous PCD-inducing stimuli converge into a few stereotypical pathways and cells pass a 'point of no return', thus becoming irreversibly committed to death. Evidence is accumulating that cytoplasmic structures, including mitochondria, participate in the critical effector stage and that alterations usually considered to define apoptosis, as nuclear chromatolysis and cytolysis, have to be ascribed to the late degradation phase. We and others have recently shown that nuclear features of apoptosis are preceded by alterations in mitochondrial function and structure. The importance of these alterations for the apoptotic process and also the possible link between, these observations, the permeability transition pore and the programmed cell death, are discussed.
Abstract: Paromomycin is an aminocyclitol aminoglycoside antibiotic used for the treatment of leishmaniasis. In view of the central role of mitochondria in cellular energetics and metabolism, its effect on in vivo mitochondrial activities of Leishmania donovani promastigotes-the parasite flagellate form-was investigated. The approach used flow cytometry, amperometric measure of O2 consumption, and, as a global estimate of mitochondrial dehydrogenases, thiazolyl blue reduction (MTT test); some in vitro controls were also made. When added to promastigote cultures for 24-72 h at 150-200 microM (= LC50), paromomycin doubled the generation time, inhibited respiration, and lowered its associated electric potential difference across mitochondrial membranes, as measured by rhodamine 123 fluorescence. The chemical analogue neomycin was ineffective. Furthermore, the in vivo mitochondrial dehydrogenase activities were lower, seemingly because of the shortage of respiratory substrates. Indeed, succinate addition to paromomycin-treated cultures partly restored mitochondrial membrane potential. However, no immediate effect of paromomycin on respiration was observed, neither inhibition of redox chain nor increase of membrane permeability (uncoupling). It is proposed that paromomycin acts at a metabolic level upstream of the respiratory chain itself. This would have the observed delayed consequence because the cell energy supply would progressively decline since it depends upon the proton gradient-viz., membrane potential-generated by respiration. In conclusion, paromomycin is an antibiotic affecting the cell's energetic metabolism; the respiratory dysfunction it induces may be a crucial aspect of its action against Leishmania and possibly other cells.
Abstract: When tested on HeLa cells, bis-pyridinium oximes (BPO), a family of newly synthesized molecules whose charged pyridinium moieties are linked by a linear polymethylene chain of variable length (N = 3 to 12) have been shown to possess an inhibitory effect on cell growth and finally to provoke cell death. BPO-affected cells displayed reduced mitochondrial oxygen consumption and ATP stores and were blocked in the G1 phase of the cell cycle. Mitochondrial membrane potential, as assayed with the dye 3,3'-diexyloxacarbocyanine iodide [DiOC6(3)], increased in BPO-treated cells with time of exposure. Cell growth inhibition as well mitochondrial dysfunction were observed only with derivatives having a long polymethylene linking chain (N > or = 6). Furthermore, the concentration of the compound eliciting such effects was inversely related to the number of methylene groups in the linking chain. None of the BPO with N = 6 to 12 modified the mitochondrial DNA content, relative to the nuclear DNA content. In BPO (N = 8 and N = 12)-treated cells, chromatin fragmentation and internucleosomal DNA cleavage occurred massively, indicating that the death mode induced by these compounds is apoptosis. The possible pathway of action and the potential pharmacological interest of these compounds are discussed.
Abstract: Programmed cell death, or apoptosis, has in the past few years undoubtedly become one of the most intensively investigated biological processes. However, fundamental questions concerning the molecular and biochemical mechanisms remain to be elucidated. The central question concerns the biochemical steps shared by the numerous death induction pathways elicited by different stimuli. Heterogeneous death signals precede a common effector phase during which cells pass a threshold of 'no return' and are engaged in a degradation phase where they acquire the typical onset of late apoptosis. Alterations in mitochondrial permeability transition linked to membrane potential disruption precede nuclear and plasma membrane changes. In vitro induction of permeability transition in isolated mitochondria provokes the release of a protein factor capable of inducing nuclear chromatin condensation and fragmentation. This permeability transition is regulated by multiple endogenous effectors, including members of the bcl-2 gene family. Inhibition of these effects prevents apoptosis.
Abstract: Living yeast cells can be selectively stained with the lipophilic cationic cyanine dye DiOC6(3) in a mitochondrial membrane potential-dependent manner. Our study extends the use of flow cytometric analysis and sorting to DiOC6(3)-stained yeast cells. Experimental conditions were developed that prevented the toxic side effect of the probe and gave a quantitative correlation between fluorescence and mitochondrial membrane potential, without any staining of other membranes. The localization of the fluorochrome was checked by confocal microscopy and image cytometry. The mitochondrial membrane alterations were also tested through cardiolipin staining with nonyl acridine orange. Differences in light scattering and in fluorescence were detected in mutants (rho-, rho degrees, mit-, or pet-) and wild-type (rho+mit+) populations of yeast. The dye uptake of respiratory-deficient yeast strains was significantly reduced as compared to that of the wild-type. Application of an uncoupler (mCICCP), which collapsed the mitochondrial membrane potential (alphapsi(m)), led to a drastic reduction of the dye uptake. It was observed that a decrease in deltapsi(m), was usually correlated with a decrease in cardiolipin stainability by nonyl acridine orange (NAO). Quantitative flow cytometry is a fast and reproducible technique for rapid screening of yeast strains that might be suspected of respiratory dysfunction and/or mitochondrial structural changes. We give evidence that it is an adequate method to characterize and isolate respiratory mutants through sorting procedure, with selective enrichment of the population studied in respiring or non-respiring yeast cells. Confocal microscopy and image cytometry corroborate the flow cytometry results.
Abstract: In this paper we used a multiparametric approach to analyze extensively the events occurring during apoptotic cell death of thymocytes, and furthermore, we asked whether alterations in mitochondrial structure and function are occurring in early stages of apoptosis. A multiparametric quantitative analysis was performed on normal or apoptotic thymocytes emerging from a few-hour culture performed in culture medium or in the presence of dexamethasone. Simultaneous detection of light scattering properties, integrity of plasma membrane (trypan blue exclusion), chromatin condensation (AO/EB staining of entire cells or PI staining of nuclei), and DNA fragmentation (in situ nick-translation in apoptotic cells) allowed a precise analysis of the preapoptotic and apoptotic stages. Moreover a thorough study of mitochondrial transmembrane potential (delta psi m) assessed following in a time course study the uptake by apoptotic cells of the cationic lipophilic dye DiOC6(3) or the J-aggregate-forming cation JC-1, indicates that a drop in delta psi m occurs very early in thymocyte apoptosis, before DNA fragmentation. This is associated with alteration in mitochondrial structure assessed by cytofluorimetric study of NAO uptake in apoptotic cells. Finally these dramatic alterations in mitochondrial structure and function occurring in early stages of apoptosis were confirmed by confocal and electron microscopy analysis.
Abstract: In a number of experimental systems in which lymphocyte depletion was induced by apoptosis-inducing manipulations, no apoptotic morphology and ladder-type DNA fragmentation were detected among freshly isolated peripheral lymphocytes ex vivo. Here we report that one alteration that can be detected among splenocytes stimulated with lymphocyte-depleting doses of dexamethasone (DEX) in vivo is a reduced uptake of 3,3'dihexyloxacarbocyanine iodide (DiOC6[3]), a fluorochrome which incorporates into cells dependent upon their mitochondrial transmembrane potential (delta psi m). In contrast, ex vivo isolated splenocytes still lacked established signs of programmed cell death (PCD):DNA degradation into high or low molecular weight fragments, ultrastructural changes of chromatin arrangement and endoplasmatic reticulum, loss in viability, or accumulation of intracellular peroxides. Moreover, no changes in cell membrane potential could be detected. A reduced delta psi m has been observed in response to different agents inducing lymphoid cell depletion in vivo (superantigen and glucocorticoids [GC]), in mature T and B lymphocytes, as well as their precursors. DEX treatment in vivo, followed by cytofluorometric purification of viable delta psi mlow splenic T cells ex vivo, revealed that this fraction of cells is irreversibly committed to undergoing DNA fragmentation. Immediately after purification neither delta psi mlow, nor delta psi mhigh cells, exhibit detectable DNA fragmentation. However, after short-term culture (37 degrees C, 1 h) delta psi mlow cells show endonucleolysis, followed by cytolysis several hours later. Incubation of delta psi mlow cells in the presence of excess amount of the GC receptor antagonist RU38486 (which displaces DEX from the GC receptor), cytokines that inhibit DEX-induced cell death, or cycloheximide fails to prevent cytolysis. The antioxidant, N-acetylcysteine, as well as linomide, an agent that effectively inhibits DEX or superantigen-induced lymphocyte depletion in vivo, also stabilize the DiOC6(3) uptake. In contrast, the endonuclease inhibitor, aurintricarboxylic acid acts at later stages of apoptosis and only retards the transition from the viable delta psi mlow to the nonviable fraction. Altogether, these data suggest a sequence of PCD-associated events in which a reduction in delta psi m constitutes an obligate irreversible step of ongoing lymphocyte death, preceding other alterations of cellular physiology, and thus allowing for the ex vivo assessment of PCD.
Abstract: Programmed cell death (PCD) is involved in the removal of superfluous and damaged cells in most organ systems. The induction phase of PCD or apoptosis is characterized by an extreme heterogeneity of potential PCD-triggering signal transduction pathways. During the subsequent effector phase, the numerous PCD-inducing stimuli converge into a few stereotypical pathways and cells pass a point of no return, thus becoming irreversibly committed to death. It is only during the successive degradation phase that vital structures and functions are destroyed, giving rise to the full-blown phenotype of PCD. Evidence is accumulating that cytoplasmic structures, including mitochondria, participate in the critical effector stage and that alterations commonly considered to define PCD (apoptotic morphology of the nucleus and regular, oligonucleosomal chromatin fragmentation) have to be ascribed to the late degradation phase. The decision as to whether a cell will undergo PCD or not may be expected to be regulated by "switches" that, once activated, trigger self-amplificatory metabolic pathways. One of these switches may reside in a perturbation of mitochondrial function. Thus, a decrease in mitochondrial transmembrane potential, followed by mitochondrial uncoupling and generation of reactive oxygen species, precedes nuclear alterations. It appears that molecules that participate in apoptotic decision-making also exert functions that are vital for normal cell proliferation and intermediate metabolism.
Abstract: Programmed cell death (PCD) is a physiological process commonly defined by alterations in nuclear morphology (apoptosis) and/or characteristic stepwise degradation of chromosomal DNA occurring before cytolysis. However, determined characteristics of PCD such as loss in mitochondrial reductase activity or cytolysis can be induced in enucleated cells, indicating cytoplasmic PCD control. Here we report a sequential disregulation of mitochondrial function that precedes cell shrinkage and nuclear fragmentation. A first cyclosporin A-inhibitable step of ongoing PCD is characterized by a reduction of mitochondrial transmembrane potential, as determined by specific fluorochromes (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine++ + iodide; 3,3'dihexyloxacarbocyanine iodide). Cytofluorometrically purified cells with reduced mitochondrial transmembrane potential are initially incapable of oxidizing hydroethidine (HE) into ethidium. Upon short-term in vitro culture, such cells acquire the capacity of HE oxidation, thus revealing a second step of PCD marked by mitochondrial generation of reactive oxygen species (ROS). This step can be selectively inhibited by rotenone and ruthenium red yet is not affected by cyclosporin A. Finally, cells reduce their volume, a step that is delayed by radical scavengers, indicating the implication of ROS in the apoptotic process. This sequence of alterations accompanying early PCD is found in very different models of apoptosis induction: glucocorticoid-induced death of lymphocytes, activation-induced PCD of T cell hybridomas, and tumor necrosis factor-induced death of U937 cells. Transfection with the antiapoptotic protooncogene Bcl-2 simultaneously inhibits mitochondrial alterations and apoptotic cell death triggered by steroids or ceramide. In vivo injection of fluorochromes such as 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide; 3,3'dihexyloxacarbocyanine iodide; or HE allows for the detection of cells that are programmed for death but still lack nuclear DNA fragmentation. In particular, assessment of mitochondrial ROS generation provides an accurate picture of PCD-mediated lymphocyte depletion. In conclusion, alterations of mitochondrial function constitute an important feature of early PCD.
Abstract: The uncoupling protein (UCP) is uniquely expressed in brown adipose tissue, which is a thermogenic organ of mammals. The UCP uncouples mitochondrial respiration from ATP production by introducing a proton conducting pathway through the mitochondrial inner membrane. The activity of the UCP is regulated: nucleotide binding to the UCP inhibits proton conductance whereas free fatty acids increase it. The similarities between the UCP, the ADP/ATP carrier and the DNA recognition element found in the DNA binding domain of the estrogen receptor suggested that these proteins could share common features in their respective interactions with free nucleotides or DNA, and thus defined a putative 'nucleotide recognition element' in the UCP. This article provides demonstration of the validity of this hypothesis. The putative nucleotide recognition element corresponding to the amino acids 261-269 of the UCP was gradually destroyed, and these mutant proteins were expressed in yeast. Flow cytometry, measuring the mitochondrial membrane potential in vivo, showed increased uncoupling activities of these mutant proteins, and was corroborated with studies with isolated mitochondria. The deletion of the three amino acids Phe267, Lys268 and Gly269, resulted in a mutant where proton leak could be activated by fatty acids but not inhibited by nucleotides.
Abstract: Rodent embryo cells immortalized with temperature-sensitive mutants of simian virus 40 large tumor (T) antigen have a proliferative potential that depends on temperature. At the restrictive temperature, heat-inactivation of large T antigen causes p53 release, growth arrest, and cell death. Morphological and molecular analysis indicate that the induced cell death corresponds to apoptosis. Flow cytometric analysis using a combination of forward light scatter and side scatter allows a discrimination of cells committed to apoptosis within the whole population. These cells display a reduction in cell size and a higher cellular density, confirming the apoptotic nature of the cell death. When cells exhibiting the morphological features of apoptosis were stained with a fluorescent probe of the mitochondrial membrane potential, a decreased accumulation of the dye was recorded. Measures of cellular respiration, performed with whole-cell populations, showed that the lower mitochondrial membrane potential (delta psi m) correlates, as expected, with an uncoupling of electron transport from ATP production and is linked to the induction of apoptosis. We also show that this decrease in delta psi m is associated with a decrease in the rate of mitochondrial translation. These events are detected at early stages of the apoptotic process, when most of the cells are not irreversibly committed to death, suggesting that mitochondria could be a primary target during apoptosis.
Abstract: To better understand quinolone-related arthropathy, we conceived an experimental ex vivo model using cell cultures of articular chondrocytes issued from pretreated New Zealand White rabbits (NZW). Juvenile (4- to 5-week-old) NZW were orally dosed with ofloxacin or pefloxacin (300 mg/kg of body weight for 1 day) or with pefloxacin (300 mg/kg for 7 days). Adult (5-month-old) NZW were treated with pefloxacin (300 mg/kg for 1 day). Chondrocytes were enzymatically recovered from cartilage and were analyzed by cytofluorometry using 2',7'-dichlorofluorescein diacetate (DCFH-DA) and dihydrorhodamine 123 (DHR), reflecting cellular respiratory-burst activity, and rhodamine 123 (Rh123) and 10-N-nonyl-acridine orange (NAO), specific for the mitochondrial activity and mass, respectively. A significant increase in the respiratory burst was detected by DCFH-DA and DHR in all treated groups of young animals, compared with untreated control groups. No significant increase of respiratory burst was noted in older treated rabbits. The 7-day treatment resulted in a decrease in mitochondrial uptake of Rh123 and an increase in NAO uptake. Fluoroquinolone arthrotoxicity seems to involve in its early phase the respiratory burst of immature articular chondrocytes.
Abstract: The viability of rat embryo cells immortalized by thermosensitive mutants of SV40 or polyoma Large T antigen is impaired at the non-permissive temperature thus demonstrating that the immortal phenotype is dominantly maintained by Large T antigens. We have observed that exposing these cells to the restrictive temperature not only induces growth arrest but also causes apoptotic cell death. We present evidence supporting the model that polyomaviruses may indeed establish immortality by antagonizing the lethal effects of tumor suppressor genes via physical interactions between their products and Large T antigens. In the case of SV40-immortalized cells REtsAF, shift-up to 39.5 degrees C dissociates Large T antigen/p53 complexes releasing wild-type p53 molecules capable of inducing apoptotic cell death. In polyomavirus-immortalized cells, apoptosis may result from an alternative pathway mediated by other unidentified negatively acting molecules.
Abstract: The urf13TW gene, which is derived from the mitochondrial T-urf13 gene responsible for Texas cytoplasmic male sterility in maize, was expressed in Saccharomyces cerevisiae by targeting its translation product into mitochondria. Analysis by oxygraphy at the population level revealed that in the presence of methomyl the oxygen uptake of intact yeast cells carrying the targeted protein is strongly stimulated only with ethanol as respiratory substrate and not with glycerol, lactate, pyruvate, or acetate. When malate is the substrate oxidized by isolated mitochondria, interaction between the targeted protein and methomyl results in significant inhibition of oxygen uptake. This inhibition is eliminated and oxygen uptake is stimulated by subsequent addition of NAD+. Using 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] as probe, interactive laser scanning and flow cytometry, which permit analysis at the individual cell level, demonstrated that specific staining of the mitochondrial compartment is obtained and that DiOC6(3) fluorescence serves as a measure of the membrane potential. Finally, it was shown that, as in T cytoplasm maize mitochondria, HmT toxin and methomyl dissipate the membrane potential of yeast mitochondria that carry the foreign protein. Furthermore, the results suggest that the HmT toxin and methomyl response is related to the plasmid copy number per cell and that the deleterious effect induced by HmT toxin is stronger than that of methomyl.
Abstract: Highly purified mitochondria from potato (Solanum tuberosum L. cv. Bintje) tubers were subfractionated into a matrix fraction, an inner membrane fraction and an outer membrane fraction with minimal cross-contamination. When the matrix and inner membrane fractions were incubated with [gamma-32P]ATP only one and three prominent phosphoproteins were detected after SDS-PAGE and autoradiography, respectively. In contrast, more than 20 phosphoproteins could be labelled in the outer membrane fraction, the main ones at 12, 18, 26, 43, 58, 60, 65, 74 and 110 kDa. Only one band, at 18 kDa, was detectable when the labelling was done in the presence of EGTA. We conclude that the outer membrane of plant mitochondria contains at least one Ca(2+)-dependent protein kinase and more than 20 endogenous substrates.
Abstract: Intact spinach (Spinacia oleracea) chloroplasts, thylakoid membranes, and inside-out or right-side-out thylakoid vesicles have been characterized by flow cytometry with respect to forward angle light scatter, right angle light scatter, and chlorophyll fluorescence. Analysis of intact chloroplasts with respect to forward light scatter and the chlorophyll fluorescence parameter revealed the presence of truly "intact" and "disrupted" chloroplasts. The forward light scatter parameter, normally considered to reflect object size, was instead found to reflect the particle density. One essential advantage of flow cytometry is that additional parameters such as Ricinus communis agglutinin (linked to fluorescein isothiocyanate) fluorescence can be determined through logical conditions placed on bit-maps, amounting to an analytical purification procedure. In the present case, chloroplast subpopulations with fully preserved envelopes, thylakoid membrane, and inside-out or right-side-out thylakoid membranes vesicles can be distinguished. Flow cytometry is also a useful tool to address the question of availability of glycosyl moities on the membrane surfaces if one keeps in mind that organelle-to-organelle interactions could be partially mediated through a recognition process. A high specific binding of R. communis agglutinin and peanut lectin to the chloroplast envelope was detected. This showed that galactose residues were exposed and accessible to specific lectins on the chloroplast surface. No exposed glucose, fucose, or mannose residues could be detected by the appropriate lectins. Ricin binding to the intact chloroplasts caused a strong aggregation. Disruption of these aggregates by resuspension or during passage in the flow cytometer induced partial breakage of the chloroplasts. Only minor binding of R. communis agglutinin and peanut lectin to the purified thylakoid membranes was detected; the binding was found to be low for both inside-out and right-side-out vesicles of the thylakoid membranes.
Abstract: The fluorescent dye rhodamine 123, which selectively accumulates in mitochondria based on the membrane potential, was used with flow cytometry to evaluate variations in activity of mitochondria isolated from plant tissues. In the presence of succinate and ATP, potato (Solanum tuberosum L.) tuber mitochondrial activity was affected by metabolic inhibitors and compounds that modify the membrane potential. The more uniform the mitochondrial population, the higher the observed membrane potential. The reactive population corresponds to the proportion of intact mitochondria (94-97%) defined by classic methods. Changes in the light-scattering properties are more related to internal modifications affecting the inner membrane-matrix system of the mitochondria during metabolic modulation than to specific volume change or outer membrane surface modifications. We tested our approach using an Arum maculatum preparation that contains three different types of mitochondria and demonstrated the validity of the light-scatter measurements to distinguish the alpha, beta, and [ill] mitochondria and to measure their ability to built up a membrane potential in the presence of succinate. These results demonstrate clearly that flow cytometric techniques using rhodamine 123 can be employed to study the activity in isolated plant mitochondria.
Abstract: Washed and purified rat- or mouse-liver mitochondria exhibiting high membrane integrity and metabolic activity were studied by flow cytometry. The electrophoretic accumulation/redistribution of cationic lipophilic probes, rhodamine 123, safranine O and a cyanine derivative, 3,3'-dihexyloxadicarbocyanine iodide, during the energization process was studied and was consistent with the generation of a negative internal membrane potential. An exception to this was nonylacridine orange which spontaneously bound to the mitochondrial membrane by hydrophobic interactions via its hydrocarbon chain. Energized purified mitochondria stained with potentiometric dyes exhibited both higher fluorescence and population homogeneity than the non-energized or deenergized (nigericin plus valinomycin) mitochondria. By contrast, under non-energized or deenergized conditions, the mitochondrial population exhibited fluorescence intensity heterogeneity related to the residual membrane potential; two subpopulations were evident, one of low fluorescence which may be related to the autofluorescence of the mitochondria (plus non-specific dye binding) and a second population which exhibited high fluorescence. Flow cytometry of the unpurified, simply washed, rat-liver mitochondria stained with rhodamine 123, a classically used dye, provided evidence of their heterogeneity in terms of light-scattering properties and membrane-potential-related fluorescence. One third of the washed mitochondria were found to be non-functional by such assays. The fluorescence of purified rat-liver mitochondria due to the membrane potential built up by endogenous substrates indicates heterogeneity of the mitochondrial population with respect to levels of endogenous substrates. The low-angle light scattering increases upon energization and provides some original information about the shape and modification of the inner mitochondrial conformation accompanying the energization. The heterogeneity of the rat liver mitochondrial population, from a structural, metabolic (existence of endogenous substrates) and functional (active and non-active mitochondrial population dispersion) point of view could thus be demonstrated by flow-cytometry analysis. Two animal models were examined with regard to the alteration of the mitochondrial membrane potential under the effects of drugs (rat-liver mitochondria), and the effects of ammonium toxicity (mouse-liver mitochondria). These results are promising and open new perspectives in the study of mitochondriopathies.
Abstract: Purified mitochondria from potato (Solanum tuberosum L. cv Bintje) tubers were incubated with [gamma-32P]ATP. Total 32P incorporation into proteins saturated after about 2 min and showed a Km (ATP) of 0.2 mM and a broad pH optimum of 6.5-8. About 30 polypeptides were labelled as shown by SDS-PAGE and autoradiography. The major labelled polypeptides were at 11, 14, 16 22-23, 40, 42 (the alpha-subunit of the pyruvate dehydrogenase complex), 45-46, 60, 62, 69, 84-86 and 97 kDa. By the use of atractylate, EGTA and trypsin the major phosphoproteins of 40 and 42 kDa and possibly some minor phosphoproteins in the range 26-33 kDa were localized to the matrix or the inner surface of the inner membrane. All other labelled polypeptides as well as (at least) two kinases (one Ca2(+)-dependent, the other Ca2(+)-independent) are outside the inner membrane.
