Functional estrogen receptors in the mitochondria of breast cancer cells

Steroid hormones have been reported to indirectly impact mitochondrial functions, attributed to nuclear receptor-induced production of proteins that localize in this cytoplasmic organelle. Here we show high-affinity estrogen receptors in the mitochondria of MCF-7 breast cancer cells and endothelial cells, compatible with classical estrogen receptors ERalpha and ERbeta. We report that in MCF-7, estrogen inhibits UV radiation-induced cytochrome C release, the decrease of the mitochondrial membrane potential, and apoptotic cell death. UV stimulated the formation of mitochondrial reactive oxygen species (mROS), and mROS were essential to inducing mitochondrial events of cell death. mROS mediated the UV activation of c-jun N-terminal kinase (JNK), and protein kinase C (PKC) delta, underlying the subsequent translocation of Bax to the mitochondria where oligomerization was promoted. E2 (estradiol) inhibited all these events, directly acting in mitochondria to inhibit mROS by rapidly up-regulating manganese superoxide dismutase activity. We implicate novel functions of ER in the mitochondria of breast cancer that lead to the survival of the tumor cells.     

Widespread but tissue-specific patterns of interferon-induced transmembrane protein 3 (IFITM3, FRAGILIS,MIL-1) in the mouse gastrula

Interferon-induced transmembrane protein 3 (IFITM3; FRAGILIS; MIL-1) is part of a larger family of important small interferon-induced transmembrane genes and proteins involved in early development, cell adhesion, and cell proliferation, and which also play a major role in response to bacterial and viral infections and, more recently, in pronounced malignancies. IFITM3, together with tissue-nonspecific alkaline phosphatase (TNAP), PRDM1, and STELLA, has been claimed to be a hallmark of segregated primordial germ cells (PGCs) (Saitou et al., 2002). However, whether IFITM3, like STELLA, is part of a broader stem/progenitor pool that builds the posterior region of the mouse conceptus (Mikedis and Downs, 2012) is obscure. To discover the whereabouts of IFITM3 during mouse gastrulation (～E6.5-9.0), systematic immunohistochemical analysis was carried out at closely spaced 2-4-h intervals. Results revealed diverse, yet consistent, profiles of IFITM3 localization throughout the gastrula. Within the putative PGC trajectory and surrounding posterior tissues, IFITM3 localized as a large cytoplasmic spot with or without staining in the plasma membrane. IFITM3, like STELLA, was also found in the ventral ectodermal ridge (VER), a posterior progenitor pool that builds the tailbud. The large cytoplasmic spot with plasma membrane staining was exclusive to the posterior region; the visceral yolk sac, non-posterior tissues, and epithelial tissues exhibited spots of IFITM3 without cell surface staining. Colocalization of the intracellular IFITM3 spot with the endoplasmic reticulum, Golgi apparatus, or endolysosomes was not observed. That relatively high levels of IFITM3 were found throughout the posterior primitive streak and its derivatives is consistent with evidence that IFITM3, like STELLA, is part of a larger stem/progenitor cell pool at the posterior end of the primitive streak that forms the base of the allantois and builds the fetal-umbilical connection, thus further obfuscating practical phenotypic distinctions between so-called PGCs and surrounding soma.     

Alphavirus Restriction by IFITM Proteins

Interferon inducible transmembrane proteins (IFITMs) are broad‐spectrum antiviral factors. In cell culture the entry of many enveloped viruses, including orthomyxo‐, flavi‐, and filoviruses, is inhibited by IFITMs, though the mechanism(s) involved remain unclear and may vary between viruses. We demonstrate that Sindbis and Semliki Forest virus (SFV), which both use endocytosis and acid‐induced membrane fusion in early endosomes to infect cells, are restricted by the early endosomal IFITM3. The late endosomal IFITM2 is less restrictive and the plasma membrane IFITM1 does not inhibit normal infection by either virus. IFITM3 inhibits release of the SFV capsid into the cytosol, without inhibiting binding, internalization, trafficking to endosomes or low pH‐induced conformational changes in the envelope glycoprotein. Infection by SFV fusion at the cell surface was inhibited by IFITM1, but was equally inhibited by IFITM3. Furthermore, an IFITM3 mutant (Y20A) that is localized to the plasma membrane inhibited infection by cell surface fusion more potently than IFITM1. Together, these results indicate that IFITMs, in particular IFITM3, can restrict alphavirus infection by inhibiting viral fusion with cellular membranes. That IFITM3 can restrict SFV infection by fusion at the cell surface equivalently to IFITM1 suggests that IFITM3 has greater antiviral potency against SFV.      

Role of mitochondrial membrane potential in the regulation of murine erythroleukemia cell differentiation

The level of cytoplasmic calcium ions appears to be important in the control of murine erythroleukemia (MEL) cell differentiation. Our interest in this study focuses on the relationship between the regulation of calcium concentration and differentiation. We used the fluorescent membrane probe DiOC₆ to examine the relationship between MEL cell mitochondria and changes in cytoplasmic calcium levels occurring at the initiation of commitment. Fluorescence microscopy reveals the selective association of DiOC₆ with MEL cell mitochondria, where an enhanced fluorescence is observed. Treatment of cells with dimethylsulfoxide (DMSO) or other inducers causes a decrease in mitochondria-associated fluorescence levels that occurs with the initiation of commitment. A decrease in DiOC₆ fluorescence is caused by agents that reduce mitochondrial membrane potential, but is only slightly affected by agents that alter plasma membrane potential. Amiloride and EGTA, agents that prevent commitment and inhibit calcium uptake, also prevent the decrease in DiOC₆ uptake caused by DMSO. The effect of DMSO on MEL cell mitochondria is mimicked by FCCP, a proton ionophore that dissipates mitochondrial membrane potential. FCCP also caused MEL cell mitochondria to release calcium into the cytoplasm. When MEL cells are treated with DMSO plus FCCP, commitment is initiated without the lag period observed when cells are treated with DMSO alone. These results are consistent with the hypothesis that mitochondrial transmembrane potential is important in the regulation of cytoplasmic calcium levels at the time of commitment of MEL cells to terminal differentiation.     

Mitochondrial expression of the human equilibrative nucleoside transporter 1 (hENT1) results in enhanced mitochondrial toxicity of antiviral drugs

Many antiviral drugs (e.g. fialuridine; FIAU) produce clinically significant mitochondrial toxicity that limits their dose or prevents their use in the clinic. Because the majority of nucleoside drugs is too hydrophilic to cross the highly impermeable mitochondrial membrane, we have hypothesized that they must be transported into the mitochondria to produce their toxicity. To test this hypothesis, we have sought to determine whether the nucleoside transporters, human equilibrative nucleoside transporter 1 (hENT1) or human concentrative nucleoside transporter 1 (hCNT1), when stably expressed in Madin-Darby canine kidney cells as yellow fluorescent fusion protein (YFP), are localized to the mitochondria. By using organelle-selective dyes and confocal microscopy, we have found that hENT1-YFP is localized to the mitochondria as well as the plasma membrane, whereas hCNT1-YFP was found predominantly on the plasma membrane. hENT1-YFP was not localized to the nuclear envelope, endosomes, lysosomes, or Golgi complex. Western blotting confirmed the presence of hENT1-YFP or endogenous hENT1 in mitochondria isolated from hENT1-YFP-expressing cells and human livers, respectively. In agreement with these localization data, [14C]FIAU was efficiently transported into the mitochondria of cells expressing hENT1-YFP but not of cells expressing hCNT1-YFP. The mitochondrial toxicity of FIAU to Madin-Darby canine kidney cells was enhanced by hENT1-YFP, even when hENT1 activity on the plasma membrane was selectively blocked by 10 nm nitrobenzylthioinosine. Moreover, FIAU (50 microm) produced significant mitochondrial toxicity ( approximately 70% decrease in mitochondrial DNA synthesis) when it was directly incubated with mitochondria isolated from hENT1-expressing cells. In conclusion, we have identified for the first time that hENT1 is expressed on the mitochondrial membrane and that this expression enhances the mitochondrial toxicity of nucleoside drugs such as FIAU. Mitochondrial expression of hENTs may explain the clinically significant mitochondrial toxicity caused by the anti-HIV nucleoside drugs such as zidovudine, stavudine, and didanosine.     

Antiviral Role of IFITM Proteins in African Swine Fever Virus Infection

The interferon-induced transmembrane (IFITM) protein family is a group of antiviral restriction factors that impair flexibility and inhibit membrane fusion at the plasma or the endosomal membrane, restricting viral progression at entry. While IFITMs are widely known to inhibit several single-stranded RNA viruses, there are limited reports available regarding their effect in double-stranded DNA viruses. In this work, we have analyzed a possible antiviral function of IFITMs against a double stranded DNA virus, the African swine fever virus (ASFV). Infection with cell-adapted ASFV isolate Ba71V is IFN sensitive and it induces IFITMs expression. Interestingly, high levels of IFITMs caused a collapse of the endosomal pathway to the perinuclear area. Given that ASFV entry is strongly dependent on endocytosis, we investigated whether IFITM expression could impair viral infection. Expression of IFITM1, 2 and 3 reduced virus infectivity in Vero cells, with IFITM2 and IFITM3 having an impact on viral entry/uncoating. The role of IFITM2 in the inhibition of ASFV in Vero cells could be related to impaired endocytosis-mediated viral entry and alterations in the cholesterol efflux, suggesting that IFITM2 is acting at the late endosome, preventing the decapsidation stage of ASFV.     

Modulation of Innate Immune Signalling by Lipid-Mediated MAVS Transmembrane Domain Oligomerization

RIG-I-like receptors detect viral RNA in infected cells and promote oligomerization of the outer mitochondrial membrane protein MAVS to induce innate immunity to viral infection through type I interferon production. Mitochondrial reactive oxygen species (mROS) have been shown to enhance anti-viral MAVS signalling, but the mechanisms have remained obscure. Using a biochemical oligomerization-reporter fused to the transmembrane domain of MAVS, we found that mROS inducers promoted lipid-dependent MAVS transmembrane domain oligomerization in the plane of the outer mitochondrial membrane. These events were mirrored by Sendai virus infection, which similarly induced lipid peroxidation and promoted lipid-dependent MAVS transmembrane domain oligomerization. Our observations point to a role for mROS-induced changes in lipid bilayer properties in modulating antiviral innate signalling by favouring the oligomerization of MAVS transmembrane domain in the outer-mitochondrial membrane.     

Prohibitin involvement in the generation of mitochondrial superoxide at complex I in human sperm

Prohibitin (PHB), a major mitochondrial membrane protein, has been shown earlier in our laboratoryto regulate sperm motility via an alteration in mitochondrial membrane potential (MMP) in infertile men with poor sperm quality. To test if PHB expression is associated with sperm mitochondrial superoxide (mROS) levels, here we examined sperm mROS levels, high MMP and lipid peroxidation in infertile men with poor sperm motility (asthenospermia, A) and/or low sperm concentrations (oligoasthenospermia, OA). The diaphorase‐type activity of sperm mitochondrial complex I (MCI) and PHB expression were also determined. We demonstrate that mROS and lipid peroxidation levels are significantly higher in sperm from A and OA subjects than in normospermic subjects, whereas high MMP and PHB expression are significantly lower. A positive correlation between mROS and lipid peroxidation and a negative correlation of mROS with PHB expression, high MMP, and sperm motility were found in these subjects. The finding of similar diaphorase‐type activity levels of sperm MCI in the three groups studied suggests that the catalytic subunits of MCI in the matrix arm may produce mROS on its own. There may be a dysfunction of electron transport at MCI associated with decreased expression of PHB in sperm with poor quality. We conclude that mROS level is increased and associated with decreased PHB expression, and it may regulate sperm motility via increases in low MMP and lipid peroxidation. This is the first report on the involvement of PHB in human sperm motility loss associated with increased generation of mROS at MCI.     

Mitochondrial ascorbic acid transport is mediated by a low-affinity form of the sodium-coupled ascorbic acid transporter-2

Despite the fundamental importance of the redox metabolism of mitochondria under normal and pathological conditions, our knowledge regarding the transport of vitamin C across mitochondrial membranes remains far from complete. We report here that human HEK-293 cells express a mitochondrial low-affinity ascorbic acid transporter that molecularly corresponds to SVCT2, a member of the sodium-coupled ascorbic acid transporter family 2. The transporter SVCT1 is absent from HEK-293 cells. Confocal colocalization experiments with anti-SVCT2 and anti-organelle protein markers revealed that most of the SVCT2 immunoreactivity was associated with mitochondria, with minor colocalization at the endoplasmic reticulum and very low immunoreactivity at the plasma membrane. Immunoblotting of proteins extracted from highly purified mitochondrial fractions confirmed that SVCT2 protein was associated with mitochondria, and transport analysis revealed a sigmoidal ascorbic acid concentration curve with an apparent ascorbic acid transport K_m of 0.6 mM. Use of SVCT2 siRNA for silencing SVCT2 expression produced a major decrease in mitochondrial SVCT2 immunoreactivity, and immunoblotting revealed decreased SVCT2 protein expression by approximately 75%. Most importantly, the decreased protein expression was accompanied by a concomitant decrease in the mitochondrial ascorbic acid transport rate. Further studies using HEK-293 cells overexpressing SVCT2 at the plasma membrane revealed that the altered kinetic properties of mitochondrial SVCT2 are due to the ionic intracellular microenvironment (low in sodium and high in potassium), with potassium acting as a concentration-dependent inhibitor of SVCT2. We discarded the participation of two glucose transporters previously described as mitochondrial dehydroascorbic acid transporters; GLUT1 is absent from mitochondria and GLUT10 is not expressed in HEK-293 cells. Overall, our data indicate that intracellular SVCT2 is localized in mitochondria, is sensitive to an intracellular microenvironment low in sodium and high in potassium, and functions as a low-affinity ascorbic acid transporter. We propose that the mitochondrial localization of SVCT2 is a property shared across cells, tissues, and species.     

Adaptations of Energy Metabolism Associated with Increased Levels of Mitochondrial Cholesterol in Niemann-Pick Type C1-deficient Cells

Niemann-Pick type C1 (NPC1) is a late endosomal transmembrane protein, which, together with NPC2 in the endosome lumen, mediates the transport of endosomal cholesterol to the plasma membrane and endoplasmic reticulum. Loss of function of NPC1 or NPC2 leads to cholesterol accumulation in late endosomes and causes neuronal dysfunction and neurodegeneration. Recent studies indicate that cholesterol also accumulates in mitochondria of NPC1-deficient cells and brain tissue and that NPC1 deficiency leads to alterations in mitochondrial function and energy metabolism. Here, we have investigated the effects of increased mitochondrial cholesterol levels on energy metabolism, using RNA interference to deplete Chinese hamster ovary cells of NPC1 alone or in combination with MLN64, which mediates endosomal cholesterol transport to mitochondria. Mitochondrial cholesterol levels were also altered by depletion of NPC2 in combination with the expression of NPC2 mutants. We found that the depletion of NPC1 increased lactate secretion, decreased glutamine-dependent mitochondrial respiration, and decreased ATP transport across mitochondrial membranes. These metabolic alterations did not occur when transport of endosomal cholesterol to mitochondria was blocked. In addition, the elevated mitochondrial cholesterol levels in NPC1-depleted cells and in NPC2-depleted cells expressing mutant NPC2 that allows endosomal cholesterol trafficking to mitochondria were associated with increased expression of the antioxidant response factor Nrf2. Antioxidant treatment not only prevented the increase in Nrf2 mRNA levels but also prevented the increased lactate secretion in NPC1-depleted cells. These results suggest that mitochondrial cholesterol accumulation can increase oxidative stress and in turn cause increased glycolysis to lactate and other metabolic alterations.     

The C-terminal transmembrane domain of Bcl-xL mediates changes in mitochondrial morphology

We investigate the effect of mitochondrial localization and the Bcl-x_L C-terminal transmembrane (TM) domain on mitochondrial morphology and subcellular light scattering. CSM 14.1 cell lines stably expressed yellow fluorescent protein (YFP), YFP-Bcl-x_L, YFP-Bcl-x_L-ΔTM, containing the remainder of Bcl-x_L after deletion of the last 21 amino acids corresponding to the TM domain, or YFP-TM, consisting of YFP fused at its C-terminal to the last 21 amino acids of Bcl-x_L. YFP-Bcl-x_L and YFP-TM localized to the mitochondria. Their expression decreased the intensity ratio of wide-to-narrow angle forward scatter by subcellular organelles, and correlated with an increase in the proportion of mitochondria with an expanded matrix having greatly reduced intracristal spaces as observed by electron microscopy. Cells expressing YFP-TM also exhibited significant autophagy. In contrast, YFP-Bcl-x_L-ΔTM was diffusely distributed in the cells, and its expression did not alter light scattering or mitochondrial morphology compared with parental cells. Expression of YFP-Bcl-x_L or YFP-Bcl-x_L-ΔTM provided significant resistance to staurosporine-induced apoptosis. Surprisingly however, YFP-TM expression also conferred a moderate level of cell death resistance in response to staurosporine. Taken together, our results suggest the existence of a secondary Bcl-x_L function that is mediated by the transmembrane domain, alters mitochondrial morphology, and is distinct from BH3 domain sequestration.     

Localization of mRNAs encoding human mitochondrial oxidative phosphorylation proteins

The mitochondrial oxidative phosphorylation (OXPHOS) proteins are encoded by both nuclear and mitochondrial DNA. The nuclear-encoded OXPHOS mRNAs have specific subcellular localizations, but little is known about which localize near mitochondria. Here, we compared mRNAs in mitochondria-bound polysome fractions with those in cytosolic, free polysome fractions. mRNAs encoding hydrophobic OXPHOS proteins, which insert into the inner membrane, were localized near mitochondria. Conversely, OXPHOS gene which mRNAs were predominantly localized in cytosol had less than one transmembrane domain. The RNA-binding protein Y-box binding protein-1 is localized at the mitochondrial outer membrane and bound to the OXPHOS mRNAs. Our findings offer new insight into mitochondrial co-translational import in human cells.     

A novel potassium channel in lymphocyte mitochondria

The margatoxin-sensitive Kv1.3 is the major potassium channel in the plasma membrane of T lymphocytes. Electron microscopy, patch clamp, and immunological studies identified the potassium channel Kv1.3, thought to be localized exclusively in the cell membrane, in the inner mitochondrial membrane of T lymphocytes. Patch clamp of mitoplasts and mitochondrial membrane potential measurements disclose the functional expression of a mitochondrial margatoxin-sensitive potassium channel. To identify unambiguously the mitochondrial localization of Kv1.3, we employed a genetic model and stably transfected CTLL-2 cells, which are genetically deficient for this channel, with Kv1.3. Mitochondria isolated from Kv1.3-reconstituted CTLL-2 expressed the channel protein and displayed an activity, which was identical to that observed in Jurkat mitochondria, whereas mitochondria of mock-transfected cells lacked a channel with the characteristics of Kv1.3. Our data provide the first molecular identification of a mitochondrial potassium conductance.     

Regulated Targeting of BAX to Mitochondria

The proapoptotic protein BAX contains a single predicted transmembrane domain at its COOH terminus. In unstimulated cells, BAX is located in the cytosol and in peripheral association with intracellular membranes including mitochondria, but inserts into mitochondrial membranes after a death signal. This failure to insert into mitochondrial membrane in the absence of a death signal correlates with repression of the transmembrane signal-anchor function of BAX by the NH₂-terminal domain. Targeting can be instated by deleting the domain or by replacing the BAX transmembrane segment with that of BCL-2. In stimulated cells, the contribution of the NH₂ terminus of BAX correlates with further exposure of this domain after membrane insertion of the protein. The peptidyl caspase inhibitor zVAD-fmk partly blocks the stimulated mitochondrial membrane insertion of BAX in vivo, which is consistent with the ability of apoptotic cell extracts to support mitochondrial targeting of BAX in vitro, dependent on activation of caspase(s). Taken together, our results suggest that regulated targeting of BAX to mitochondria in response to a death signal is mediated by discrete domains within the BAX polypeptide. The contribution of one or more caspases may reflect an initiation and/or amplification of this regulated targeting.     

Knockdown of interferon-induced transmembrane protein 1 inhibited proliferation,induced cell cycle arrest and apoptosis,and suppressed MAPK signaling pathway in pancreatic cancer cells

ABSTRACT

Pancreatic cancer (PC), highly malignant, is one of the most lethal cancers. Interferon-induced transmembrane protein 1 (IFITM1) has recently been regarded as a new molecular marker in human cancers. However, the role of IFITM1 in PC remains unclear. In this study, a short hairpin RNA (shRNA) was constructed to assess the effect of IFITM1 on PANC-1 and ASPC-1 cells. The level of IFITM1 was downregulated in cells transfected with shRNA targeting IFITM1 (sh-IFITM1). Silencing of IFITM1 significantly decreased cell viability, downregulated the level of Ki-67, arrested cell at G1/S phase, reduced the number of cells in S phase, and decreased cyclinD1, cyclinE, CDK2, and CDK4 levels. Moreover, Hoechst staining and Western blotting analysis showed that cell apoptosis was induced by IFITM1. IFITM1 knockdown suppressed the MAPK signaling pathway by downregulation of p-ERK, p-P38, and p-JNK levels. These findings suggested that IFITM1 could be considered a potential therapeutic target for PC.     

Mitochondrial ROS--radical detoxification, mediated by protein kinase D

The mitochondrial electron transport chain is the major source for the production of oxygen radicals. Mitochondria-generated reactive oxygen species (mROS) have been implicated in decreasing the life span and contributing to age-related diseases (known as the free radical theory of aging). Recently, the serine/threonine kinase protein kinase D1 (PKD1) was identified as a mitochondrial sensor for oxidative stress. mROS-activated PKD regulates a radical-sensing signaling pathway, which relays mROS production to the induction of nuclear genes that mediate cellular detoxification and survival. This PKD regulated signaling pathway is the first known mitochondria located and mitochondrially regulated antioxidant system that protects these organelles and cells from oxidative stress-mediated damage or cell death. The identification of this and further intracellular protective signaling pathways provides an opportunity to manipulate the effects of mROS, and might provide the key to targeting aging effects and age-related diseases that have been linked to mitochondrial dysfunctions.     

Transmembrane domain of IFITM3 is responsible for its interaction with influenza virus HA2 subunit

Interferon-inducible transmembrane protein 3 (IFITM3) inhibits influenza virus infection by blocking viral membrane fusion, but the exact mechanism remains elusive. Here, we investigated the function and key region of IFITM3 in blocking influenza virus entry mediated by hemagglutinin (HA). The restriction of IFITM3 on HA-mediated viral entry was confirmed by pseudovirus harboring HA protein from H5 and H7 influenza viruses. Subcellular co-localization and immunocoprecipitation analyses revealed that IFITM3 partially co-located with the full-length HA protein and could directly interact with HA₂ subunit but not HA₁ subunit of H5 and H7 virus. Truncated analyses showed that the transmembrane domain of the IFITM3 and HA₂ subunit might play an important role in their interaction. Finally, this interaction of IFITM3 was also verified with HA₂ subunits from other subtypes of influenza A virus and influenza B virus. Overall, our data demonstrate for the first time a direct interaction between IFITM3 and influenza HA protein via the transmembrane domain, providing a new perspective for further exploring the biological significance of IFITM3 restriction on influenza virus infection or HA-mediated antagonism or escape.     

Acute effect of fatty acids on metabolism and mitochondrial coupling in skeletal muscle

Acute effects of free fatty acids (FFA) were investigated on: (1) glucose oxidation, and UCP-2 and -3 mRNA and protein levels in 1 h incubated rat soleus and extensor digitorium longus (EDL) muscles, (2) mitochondrial membrane potential in cultured skeletal muscle cells, (3) respiratory activity and transmembrane electrical potential in mitochondria isolated from rat skeletal muscle, and (4) oxygen consumption by anesthetized rats. Long-chain FFA increased both basal and insulin-stimulated glucose oxidation in incubated rat soleus and EDL muscles and reduced mitochondrial membrane potential in C2C12 myotubes and rat skeletal muscle cells. Caprylic, palmitic, oleic, and linoleic acid increased O₂ consumption and decreased electrical membrane potential in isolated mitochondria from rat skeletal muscles. FFA did not alter UCP-2 and -3 mRNA and protein levels in rat soleus and EDL muscles. Palmitic acid increased oxygen consumption by anesthetized rats. These results suggest that long-chain FFA acutely lead to mitochondrial uncoupling in skeletal muscle.     

Apoptotic microtubule network organization and maintenance depend on high cellular ATP levels and energized mitochondria

Microtubule cytoskeleton is reformed during apoptosis, forming a cortical structure beneath plasma membrane, which plays an important role in preserving cell morphology and plasma membrane integrity. However, the maintenance of the apoptotic microtubule network (AMN) during apoptosis is not understood. In the present study, we examined apoptosis induced by camptothecin (CPT), a topoisomerase I inhibitor, in human H460 and porcine LLCPK-1α cells. We demonstrate that AMN was organized in apoptotic cells with high ATP levels and hyperpolarized mitochondria and, on the contrary, was dismantled in apoptotic cells with low ATP levels and mitochondrial depolarization. AMN disorganization after mitochondrial depolarization was associated with increased plasma membrane permeability assessed by enhancing LDH release and increased intracellular calcium levels. Living cell imaging monitoring of both, microtubule dynamics and mitochondrial membrane potential, showed that AMN persists during apoptosis coinciding with cycles of mitochondrial hyperpolarization. Eventually, AMN was disorganized when mitochondria suffered a large depolarization and cell underwent secondary necrosis. AMN stabilization by taxol prevented LDH release and calcium influx even though mitochondria were depolarized, suggesting that AMN is essential for plasma membrane integrity. Furthermore, high ATP levels and mitochondria polarization collapse after oligomycin treatment in apoptotic cells suggest that ATP synthase works in “reverse” mode during apoptosis. These data provide new explanations for the role of AMN and mitochondria during apoptosis.