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Mitochondrial Carrier Homolog 2 (MTCH2) acts as a receptor for the BH3 interacting-domain death agonist (BID) in the mitochondrial outer membrane. Loss of MTCH2 affects mitochondria energy metabolism and function. MTCH2 forebrain conditional KO (MTCH2 BKO) display a deficit in hippocampus-dependent cognitive functions. Here we study age-related MTCH2 BKO behavioral and electrophysiological aspects of hippocampal functions. MTCH2 BKO exhibit impaired spatial but not motor learning and an impairment in long-term potentiation (LTP) in hippocampal slices. Moreover, MTCH2 BKO express an increase in activated microglia, in addition to a reduction in neuron density in the hippocampus, but do not express amyloid-β plaques or neurofibrillary tangles. These results highlight the role of mitochondria in the normal hippocampus-dependent memory formation.

The role of mitochondria dynamics and its molecular regulators remains largely unknown during naïve-to-primed pluripotent cell interconversion. Here we report that mitochondrial MTCH2 is a regulator of mitochondrial fusion, essential for the naïve-to-primed interconversion of murine embryonic stem cells (ESCs). During this interconversion, wild-type ESCs elongate their mitochondria and slightly alter their glutamine utilization. In contrast, MTCH2−/− ESCs fail to elongate their mitochondria and to alter their metabolism, maintaining high levels of histone acetylation and expression of naïve pluripotency markers. Importantly, enforced mitochondria elongation by the pro-fusion protein Mitofusin (MFN) 2 or by a dominant negative form of the pro-fission protein dynamin-related protein (DRP) 1 is sufficient to drive the exit from naïve pluripotency of both MTCH2−/− and wild-type ESCs. Taken together, our data indicate that mitochondria elongation, governed by MTCH2, plays a critical role and constitutes an early driving force in the naïve-to-primed pluripotency interconversion of murine ESCs., Reprogramming of mitochondria metabolism occurs during naïve to primed pluripotency differentiation in mouse embryonic stem cells (ESCs). Here the authors show that mitochondrial MTCH2 regulates mitochondrial fusion and that this fusion is required for naïve to primed pluripotency conversion

Nonalcoholic fatty liver disease is one of the most prevalent metabolic disorders and it tightly associates with obesity, type 2 diabetes, and cardiovascular disease. Reduced mitochondrial lipid oxidation contributes to hepatic fatty acid accumulation. Here, we show that the Fas cell surface death receptor (Fas/CD95/Apo-1) regulates hepatic mitochondrial metabolism. Hepatic Fas overexpression in chow-fed mice compromises fatty acid oxidation, mitochondrial respiration, and the abundance of mitochondrial respiratory complexes promoting hepatic lipid accumulation and insulin resistance. In line, hepatocyte-specific ablation of Fas improves mitochondrial function and ameliorates high-fat-diet-induced hepatic steatosis, glucose tolerance, and insulin resistance. Mechanistically, Fas impairs fatty acid oxidation via the BH3 interacting-domain death agonist (BID). Mice with genetic or pharmacological inhibition of BID are protected from Fas-mediated impairment of mitochondrial oxidation and hepatic steatosis. We suggest Fas as a potential novel therapeutic target to treat obesity-associated fatty liver and insulin resistance., published version, peerReviewed

Summary Tafazzin (TAZ) is a mitochondrial transacylase that remodels the mitochondrial cardiolipin into its mature form. Through a CRISPR screen, we identified TAZ as necessary for the growth and viability of acute myeloid leukemia (AML) cells. Genetic inhibition of TAZ reduced stemness and increased differentiation of AML cells both in vitro and in vivo. In contrast, knockdown of TAZ did not impair normal hematopoiesis under basal conditions. Mechanistically, inhibition of TAZ decreased levels of cardiolipin but also altered global levels of intracellular phospholipids, including phosphatidylserine, which controlled AML stemness and differentiation by modulating toll-like receptor (TLR) signaling.

Through a clustered regularly insterspaced short palindromic repeats (CRISPR) screen to identify mitochondrial genes necessary for the growth of acute myeloid leukemia (AML) cells, we identified the mitochondrial outer membrane protein mitochondrial carrier homolog 2 (MTCH2). In AML, knockdown of MTCH2 decreased growth, reduced engraftment potential of stem cells, and induced differentiation. Inhibiting MTCH2 in AML cells increased nuclear pyruvate and pyruvate dehydrogenase (PDH), which induced histone acetylation and subsequently promoted the differentiation of AML cells. Thus, we have defined a new mechanism by which mitochondria and metabolism regulate AML stem cells and gene expression.

Structural mitochondrial abnormalities as well as genetic aberrations in mitochondrial proteins have been known in Myelodysplastic syndrome (MDS) , yet there is currently little data regarding the metabolic properties and energy production of MDS cells. In the current study we used state-of-the-art methods to assess OXPHOS in peripheral blood cells obtained from MDS patients and healthy controls We then assessed the effect of food supplements- Coenzyme Q10 and carnitine on mitochondrial function and hematological response .We show here for the first time that in low risk MDS there is a significant impairment of mitochondrial respiration in peripheral blood cells and this can be improved with food supplements. We also show that such myelodysplastic syndrome, mitochondria, oxidative phosphorylation, coenzyme Q10, seahorse XF analyzer. supplements lead to improvement in cytopenia's and quality of life.

Living organisms require a constant supply of safe and efficient energy to maintain homeostasis and to allow locomotion of single cells, tissues and the entire organism. The source of energy can be glycolysis, a simple series of enzymatic reactions in the cytosol, or a much more complex process in the mitochondria, oxidative phosphorylation (OXPHOS). In this review we will examine how does the organism balance its source of energy in two seemingly distinct and unrelated processes: hematopoiesis and exercise. In both processes we will show the importance of the metabolic program and its regulation. We will also discuss the importance of oxygen availability not as a sole determinant, but in the context of the nutrient and cellular state, and address the emerging role of lactate as an energy source and signaling molecule in health and disease.

Haematopoietic stem and progenitor cells (HSPCs), which sustain lifelong production of all haematopoietic cell types, are known to be exquisitely sensitive to DNA-damaging events, like γ-irradiatio...

Mitochondrial Carrier Homolog 2 (MTCH2) is a novel regulator of mitochondria metabolism, which was recently associated with Alzheimer’s disease. Here we demonstrate that deletion of forebrain MTCH2 increases mitochondria and whole-body energy metabolism, increases locomotor activity, but impairs motor coordination and balance. Importantly, mice deficient in forebrain MTCH2 display a deficit in hippocampus-dependent cognitive functions, including spatial memory, long term potentiation (LTP) and rates of spontaneous excitatory synaptic currents. Moreover, MTCH2-deficient hippocampal neurons display a deficit in mitochondria motility and calcium handling. Thus, MTCH2 is a critical player in neuronal cell biology, controlling mitochondria metabolism, motility and calcium buffering to regulate hippocampal-dependent cognitive functions.

Bid is a Bcl-2 family protein that promotes apoptosis by activating Bax and eliciting mitochondrial outer membrane permeabilization (MOMP). Full-length Bid is cleaved in response to apoptotic stimuli into two fragments, p7 and tBid (p15), that are held together by strong hydrophobic interactions until the complex binds to membranes. The detailed mechanism(s) of fragment separation including tBid binding to membranes and release of the p7 fragment to the cytoplasm remain unclear. Using liposomes or isolated mitochondria with fluorescently labeled proteins at physiological concentrations as in vitro models, we report that the two components of the complex quickly separate upon interaction with a membrane. Once tBid binds to the membrane, it undergoes slow structural rearrangements that result in an equilibrium between two major tBid conformations on the membrane. The conformational change of tBid is a prerequisite for interaction with Bax and is, therefore, a novel step that can be modulated to promote or inhibit MOMP. Using automated high-throughput image analysis in cells, we show that down-regulation of Mtch2 causes a significant delay between tBid and Bax relocalization in cells. We propose that by promoting insertion of tBid via a conformational change at the mitochondrial outer membrane, Mtch2 accelerates tBid-mediated Bax activation and MOMP. Thus the interaction of Mtch2 and tBid is a potential target for therapeutic control of Bid initiated cell death.

Cellular reactive oxygen species (ROS) are tightly regulated to prevent tissue damage. ROS also help to monitor different cell fates, suggesting that a 'ROS rheostat' exists in cells. It is well established that ROS are crucial for stem cell biology; in this review, we discuss how mitochondrial ROS influence hematopoietic cell fates. We also examine the importance in this process of BID and other BCL-2 family members, many of which have been implicated in regulating cell fates by modulating mitochondrial integrity/activity and cell cycle progression in the hematopoietic lineage. Based on this knowledge, we propose that selected BCL-2 proteins coordinate mitochondria and nuclear activities via ROS to enable 'synchronized' cell fate decisions.

The BH3-only Bid protein is a critical sentinel of cellular stress in the liver and the hematopoietic system. Bid's initial `claim to fame' came from its ability—as a caspase-truncated product—to trigger the mitochondrial apoptotic program following death receptor activation. Today we know that Bid can response to multiple types of proteases, which are activated under different conditions such as T-cell activation, ischemical reperfusion injury and lysosomal injury. Activation of the mitochondrial apoptotic program by Bid—via its recently identified receptor mitochondrial carrier homolog 2—involves multiple mechanisms, including release of cytochrome c and second mitochondria-derived activator of caspase (Smac), alteration of mitochondrial cristae organization, generation of reactive oxygen species and engagement of the permeability transition pore. Bid is also emerging—in its full-length form—as a pivotal sentinel of DNA damage in the bone marrow regulated by the ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and Rad3-related (ATR) kinases. The ATM/ATR-Bid pathway is critically involved in preserving the quiescence and survival of hematopoietic stem cells both in the absence and presence of external stress, and a large part of this review will be dedicated to recent advances in this area of research.

The molecular basis of the interaction between mitochondrial carrier homologue 2 (MTCH2) and truncated BID (tBID) was characterized. These proteins participate in the apoptotic pathway, and the interaction between them may serve as a target for anticancer lead compounds. In response to apoptotic signals, MTCH2 recruits tBID to the mitochondria, where it activates apoptosis. A combination of peptide arrays screening with biochemical and biophysical techniques was used to characterize the mechanism of the interaction between tBID and MTCH2 at the structural and molecular levels. The regions that mediate the interaction between the proteins were identified. The two specific binding sites between the proteins were determined to be tBID residues 59–73 that bind MTCH2 residues 140–161, and tBID residues 111–125 that bind MTCH2 residues 240–290. Peptides derived from tBID residues 111–125 and 59–73 induced cell death in osteosarcoma cells. These peptides may serve as lead compounds for anticancer drugs that act by targeting the tBID-MTCH2 interaction.

BID, a BH3-only BCL2 family member, functions in apoptosis as well as the DNA-damage response. Our previous data demonstrated that BID is an ATM effector acting to induce cell-cycle arrest and inhibition of apoptosis following DNA damage. Here we show that ATM-mediated BID phosphorylation plays an unexpected role in maintaining the quiescence of haematopoietic stem cells (HSCs). Loss of BID phosphorylation leads to escape from quiescence of HSCs, resulting in exhaustion of the HSC pool and a marked reduction of HSC repopulating potential in vivo. We also demonstrate that BID phosphorylation plays a role in protecting HSCs from irradiation, and that regulating both quiescence and survival of HSCs depends on BID's ability to regulate oxidative stress. Moreover, loss of BID phosphorylation, ATM knockout or exposing mice to irradiation leads to an increase in mitochondrial BID, which correlates with an increase in mitochondrial oxidative stress. These results show that the ATM-BID pathway serves as a critical checkpoint for coupling HSC homeostasis and the DNA-damage stress response to enable long-term regenerative capacity.

SummaryMitochondrial carrier homolog 2 (MTCH2) is a repressor of mitochondrial oxidative phosphorylation (OXPHOS), and its locus is associated with increased BMI in humans. Here, we demonstrate that mice deficient in muscle MTCH2 are protected from diet-induced obesity and hyperinsulinemia and that they demonstrate increased energy expenditure. Deletion of muscle MTCH2 also increases mitochondrial OXPHOS and mass, triggers conversion from glycolytic to oxidative fibers, increases capacity for endurance exercise, and increases heart function. Moreover, metabolic profiling of mice deficient in muscle MTCH2 reveals a preference for carbohydrate utilization and an increase in mitochondria and glycolytic flux in muscles. Thus, MTCH2 is a critical player in muscle biology, modulating metabolism and mitochondria mass as well as impacting whole-body energy homeostasis.

The metabolic state of stem cells is emerging as an important determinant of their fate. In the bone marrow, haematopoietic stem cell (HSC) entry into cycle, triggered by an increase in intracellular reactive oxygen species (ROS), corresponds to a critical metabolic switch from glycolysis to mitochondrial oxidative phosphorylation (OXPHOS). Here we show that loss of mitochondrial carrier homologue 2 (MTCH2) increases mitochondrial OXPHOS, triggering HSC and progenitor entry into cycle. Elevated OXPHOS is accompanied by an increase in mitochondrial size, increase in ATP and ROS levels, and protection from irradiation-induced apoptosis. In contrast, a phosphorylation-deficient mutant of BID, MTCH2's ligand, induces a similar increase in OXPHOS, but with higher ROS and reduced ATP levels, and is associated with hypersensitivity to irradiation. Thus, our results demonstrate that MTCH2 is a negative regulator of mitochondrial OXPHOS downstream of BID, indispensible in maintaining HSC homeostasis.

BCL-2 family members are pivotal regulators of the apoptotic process. Mitochondria are a major site-of-action for these proteins. Several prominent alterations occur to mitochondria during apoptosis that seem to be part of the "mitochondrial apoptotic program." The BCL-2 family members are believed to be the major regulators of this program, however their exact mechanism of action still remains a mystery. BID, a pro-apoptotic BCL-2 family member plays an essential role in initiating this program. Recently, we have revealed that in apoptotic cells the activated/truncated form of BID, tBID, interacts with a novel, uncharacterized protein named mitochondrial carrier homolog 2 (Mtch2). Mtch2 is a conserved protein that is similar to members of the mitochondrial carrier protein (MCP) family. This review summarizes the current knowledge regarding BCL-2 family members and the mitochondrial apoptotic program and examines the possible involvement of Mtch2 in this program.

Cells deficient in mitochondrial fusion have been shown to have defects linked to the exchange of inner membrane and matrix components. Because outer-mitochondrial membrane (OMM) constituents insert directly from the cytoplasm, a role for fusion in their intermitochondrial transfer was unanticipated. Here, we show that fibroblasts lacking the GTPases responsible for OMM fusion, mitofusins 1 and 2 (MFN1 and MFN2), display more heterogeneous distribution of OMM proteins. Proteins with different modes of OMM association display varying degrees of heterogeneity in Mfn1/2(-/-) cells and different kinetics of transfer during fusion in fusion-competent cells. Proapoptotic Bak exhibits marked heterogeneity, which is normalized upon expression of MFN2. Bak is critical for Bid-induced OMM permeabilization and cytochrome c release, and Mfn1/2(-/-) cells show dysregulation of Bid-dependent apoptotic signaling. Bid sensitivity of Bak-deficient mitochondria is regained upon fusion with Bak-containing mitochondria. Thus, OMM protein distribution depends on mitochondrial fusion and is a locus of apoptotic dysfunction in conditions of fusion deficiency.

BCL-2 family members are pivotal regulators of the apoptotic process. Mitochondria seem to be a major site-of-action for these proteins. Several prominent alterations occur to mitochondria during apoptosis that include the release of intermembrane space molecules, changes in the membrane potential, ionic changes, and more. All these changes seem to be part of the mitochondrial apoptotic process. The BCL-2 family members are believed to be the major regulators of this program; however, their exact mechanism of action still remains a mystery. In addition, the exact contribution of mitochondria to the apoptotic process is still unclear. This review summarizes and examines the current knowledge regarding these two issues.

Mitochondrial carrier homolog 2 (MTCH2) is a mitochondrial outer membrane protein that functions as a receptor-like protein for pro-apoptotic BID. In addition to its role in apoptosis, recent findings show that MTCH2 also regulates cellular metabolism. In murine hematopoietic cells, loss of MTCH2 increases oxidative phosphorylation and reduces the number of hematopoietic stem cells. Here, we sought to understand the role of MTCH2 in leukemogenesis and knocked down MTCH2 in leukemia cell lines using multiple independent shRNAs. Knockdown of MTCH2 reduced growth and viability of AML cells: OCI-AML2 (>90%), TEX (>80%), U937 (>65%), and HL60 (>75%). MTCH2 knockdown also decreased the clonogenic growth of OCI-AML2 (>60%), TEX (>70%), and U937 (>40%) cells compared to controls. However, MTCH2 knockdown did not induce cell death as indicated by annexin V/PI staining. In addition, knockdown of MTCH2 in TEX cells reduced engraftment into the marrow of non-obese diabetic/severe combined immunodeficiency-growth factor (NOD/SCID-GF) mice (control 17±4%, n=10) vs. sh-MTCH2 (4±0.86%, n=10). In mouse models, knockout of MTCH2 decreased the leukomogenic potential of murine hematopoietic stem cells transformed with the MLL-AF9 oncogene and increased the survival of these mice. To understand the mechanism by which MTCH2 knockdown decreased cell growth, we used genome wide transcriptome analysis with RNA-seq and observed an up regulation of genes involved in cellular differentiation. Consistent with increased MTCH2 knockdown promoting differentiation, OCI-AML2 cells with MTCH2 knockdown displayed increased non-specific esterase staining. Increased differentiation (Lin+ve cells) was also observed in MLL-AF9 with MTCH2 knockout. Knockdown of MTCH2 in TEX and OCI-AML2 cells increased levels of H3 and H4 histone acetylation as demonstrated by immunoblotting. Of note, differentiation and increased H3 and H4 acetylation was not observed after inhibiting other mitochondrial processes, such as mitochondrial protein synthesis or mitochondrial DNA replication. Although MTCH2 is a receptor for BID, the increased H3 and H4 acetylation appeared independent of BID as small molecule BID inhibitors did not alter H3 and H4 acetylation. MTCH2 regulates cell metabolism. Therefore, we measured changes in intracellular metabolites in AML cells after MTCH2 knockdown. In AML cells, MTCH2 knockdown increased levels of lactate (2 fold), but did not change the basal rate of oxygen consumption or the activity of mitochondrial respiratory chain complexes. Loss of mitochondrial pyruvate dehydrogenase complex increases lactate levels and a recent study reported that the translocation of pyruvate dehydrogenase complex from the mitochondria to the nucleus under conditions of mitochondrial stress, increases H3 and H4 histone acetylation (Cell. 2014; 158(1):84-97). Therefore, we measured changes in the localization of pyruvate dehydrogenase complex after MTCH2 knockdown. Knockdown of MTCH2 decreased mitochondrial and increased nuclear dehydrogenase complex in OCI-AML2 and TEX cells. Thus, in summary, MTCH2 regulates the differentiation of AML cells and controls the localization of pyruvate dehydrogenase complex and histone acetylation. These results also suggest a mechanism by which loss of MTCH2 leads to reductions of normal hematopoietic stem cells. Disclosures Schimmer: Novartis: Honoraria.

TNFR1/Fas engagement results in the cleavage of cytosolic BID to truncated tBID, which translocates to mitochondria. Immunodepletion and gene disruption indicate BID is required for cytochrome c release. Surprisingly, the three-dimensional structure of this BH3 domain-only molecule revealed two hydrophobic α-helices suggesting tBID itself might be a pore-forming protein. Instead, we demonstrate that tBID functions as a membrane-targeted death ligand in which an intact BH3 domain is required for cytochrome c release, but not for targeting.Bak-deficient mitochondria and blocking antibodies reveal tBID binds to its mitochondrial partner BAK to release cytochrome c, a process independent of permeability transition. Activated tBID results in an allosteric activation of BAK, inducing its intramembranous oligomerization into a proposed pore for cytochrome c efflux, integrating the pathway from death receptors to cell demise.

The BCL-2 family includes both proapoptotic (e.g., BAX and BAK) and antiapoptotic (e.g., BCL-2 and BCL-X(L)) molecules. The cell death-regulating activity of BCL-2 members appears to depend on their ability to modulate mitochondrial function, which may include regulation of the mitochondrial permeability transition pore (PTP). We examined the function of BAX and BCL-X(L) using genetic and biochemical approaches in budding yeast because studies with yeast suggest that BCL-2 family members act upon highly conserved mitochondrial components. In this study we found that in wild-type yeast, BAX induced hyperpolarization of mitochondria, production of reactive oxygen species, growth arrest, and cell death; however, cytochrome c was not released detectably despite the induction of mitochondrial dysfunction. Coexpression of BCL-X(L) prevented all BAX-mediated responses. We also assessed the function of BCL-X(L) and BAX in the same strain of Saccharomyces cerevisiae with deletions of selected mitochondrial proteins that have been implicated in the function of BCL-2 family members. BAX-induced growth arrest was independent of the tested mitochondrial components, including voltage-dependent anion channel (VDAC), the catalytic beta subunit or the delta subunit of the F(0)F(1)-ATP synthase, mitochondrial cyclophilin, cytochrome c, and proteins encoded by the mitochondrial genome as revealed by [rho(0)] cells. In contrast, actual cell killing was dependent upon select mitochondrial components including the beta subunit of ATP synthase and mitochondrial genome-encoded proteins but not VDAC. The BCL-X(L) protection from either BAX-induced growth arrest or cell killing proved to be independent of mitochondrial components. Thus, BAX induces two cellular processes in yeast which can each be abrogated by BCL-X(L): cell arrest, which does not require aspects of mitochondrial biochemistry, and cell killing, which does.

The protein Bid is a participant in the pathway that leads to cell death (apoptosis), mediating the release of cytochrome c from mitochondria in response to signals from 'death' receptors known as TNFR1/Fas on the cell surface. It is a member of the proapoptotic Bcd-2 family and is activated as a result of its cleavage by caspase 8, one of a family of proteolytic cell-death proteins. To investigate the role of Bid in vivo, we have generated mice deficient for Bid. We find that when these mice are injected with an antibody directed against Fas, they nearly all survive, whereas wild-type mice die from hepatocellular apoptosis and haemorrhagic necrosis. About half of the Bid-deficient animals had no apparent liver injury and showed no evidence of activation of the effector caspases 3 and 7, although the initiator caspase 8 had been activated. Other Bid-deficient mice survived with only moderate damage: all three caspases (8 and 37) were activated but their cell nuclei were intact and no mitochondrial cytochrome c was released. We also investigated the effects of Bid deficiency in cultured cells treated with anti-Fas antibody (hepatocytes and thymocytes) or with TNFalpha. (fibroblasts). In these Bid-/- cells, mitochondrial dysfunction was delayed, cytochrome c was not released, effector caspase activity was reduced and the cleavage of apoptosis substrates was altered. This loss-of-function model indicates that Bid is a critical substrate in vivo for signalling by death-receptor agonists, which mediates a mitochondrial amplification loop that is essential for the apoptosis of selected cells.

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.

The ATM–BID pathway plays a critical role in the DNA damage response by regulating mitochondria metabolism

Cells undergoing apoptosis show a plethora of time-dependent changes. The available tools for imaging apoptosis in live cells rely either on the detection of the activity of caspases, or on the visualization of exposure of phosphatidyl serine in the outer leaflet of the cell membrane. We report here a novel method for the detection of mitochondrial events during apoptosis, namely translocation of Bax to mitochondria and release of cytochrome c (Cyt c) using bimolecular fluorescence complementation. Expression of split yellow fluorescent protein (YFP) fragments fused to Bax and Cyt c, resulted in robust induction of YFP fluorescence at the mitochondria of apoptotic cells with very low background. In vivo expression of split YFP protein fragments in liver hepatocytes and intra-vital imaging of subcutaneous tumor showed elevated YFP fluorescence upon apoptosis induction. Thus, YFP complementation could be applied for high-throughput screening and in vivo molecular imaging of mitochondrial events during apoptosis.

BackgroundBcl-2 homology domain (BH) 3-only proteins are pro-apoptotic proteins of the Bcl-2 family that couple stress signals to the mitochondrial cell death pathways. The BH3-only protein Bid can be activated in response to death receptor activation via caspase 8-mediated cleavage into a truncated protein (tBid), which subsequently translocates to mitochondria and induces the release of cytochrome-C. Using a single-cell imaging approach of Bid cleavage and translocation during apoptosis, we have recently demonstrated that, in contrast to death receptor-induced apoptosis, caspase-independent excitotoxic apoptosis involves a translocation of full length Bid (FL-Bid) from the cytosol to mitochondria. We induced a delayed excitotoxic cell death in cultured rat hippocampal neurons by a 5-min exposure to the glutamate receptor agonist N-methyl-D-aspartate (NMDA; 300 μM).ResultsWestern blot experiments confirmed a translocation of FL-Bid to the mitochondria during excitotoxic apoptosis that was associated with the release of cytochrome-C from mitochondria. These results were confirmed by immunofluorescence analysis of Bid translocation during excitotoxic cell death using an antibody raised against the amino acids 1–58 of mouse Bid that is not able to detect tBid. Finally, inducible overexpression of FL-Bid or a Bid mutant that can not be cleaved by caspase-8 was sufficient to induce apoptosis in the hippocampal neuron cultures.ConclusionOur data suggest that translocation of FL-Bid is sufficient for the activation of mitochondrial cell death pathways in response to glutamate receptor overactivation.

BID, a proapoptotic BCL-2 family member, plays an essential role in the tumor necrosis factor alpha (TNF-alpha)/Fas death receptor pathway in vivo. Activation of the TNF-R1 receptor results in the cleavage of BID into truncated BID (tBID), which translocates to the mitochondria and induces the activation of BAX or BAK. In TNF-alpha-activated FL5.12 cells, tBID becomes part of a 45-kDa cross-linkable mitochondrial complex. Here we describe the biochemical purification of this complex and the identification of mitochondrial carrier homolog 2 (Mtch2) as part of this complex. Mtch2 is a conserved protein that is similar to members of the mitochondrial carrier protein family. Our studies with mouse liver mitochondria indicate that Mtch2 is an integral membrane protein exposed on the surface of mitochondria. Using blue-native gel electrophoresis we revealed that in viable FL5.12 cells Mtch2 resides in a protein complex of ca. 185 kDa and that the addition of TNF-alpha to these cells leads to the recruitment of tBID and BAX to this complex. Importantly, this recruitment was partially inhibited in FL5.12 cells stably expressing BCL-X(L). These results implicate Mtch2 as a mitochondrial target of tBID and raise the possibility that the Mtch2-resident complex participates in the mitochondrial apoptotic program.

"BH3 domain only" members of the BCL-2 family including the pro-apoptotic molecule BID represent candidates to connect with proximal signal transduction. Tumor necrosis factor alpha (TNFalpha) treatment induced a caspase-mediated cleavage of cytosolic, inactive p22 BID at internal Asp sites to yield a major p15 and minor p13 and p11 fragments. p15 BID translocates to mitochondria as an integral membrane protein. p15 BID within cytosol targeted normal mitochondria and released cytochrome c. Immunodepletion of p15 BID prevents cytochrome c release. In vivo, anti-Fas Ab results in the appearance of p15 BID in the cytosol of hepatocytes which translocates to mitochondria where it releases cytochrome c. Addition of activated caspase-8 to normal cytosol generates p15 BID which is also required in this system for release of cytochrome c. In the presence of BCL-XL/BCL-2, TNFalpha still induced BID cleavage and p15 BID became an integral mitochondrial membrane protein. However, BCL-XL/BCL-2 prevented the release of cytochrome c, yet other aspects of mitochondrial dysfunction still transpired and cells died nonetheless. Thus, while BID appears to be required for the release of cytochrome c in the TNF death pathway, the release of cytochrome c may not be required for cell death.

Expression of the pro-apoptotic molecule BAX has been shown to induce cell death. While BAX forms both homo- and heterodimers, questions remain concerning its native conformation in vivo and which moiety is functionally active. Here we demonstrate that a physiologic death stimulus, the withdrawal of interleukin-3 (IL-3), resulted in the translocation of monomeric BAX from the cytosol to the mitochondria where it could be cross-linked as a BAX homodimer. In contrast, cells protected by BCL-2 demonstrated a block in this process in that BAX did not redistribute or homodimerize in response to a death signal. To test the functional consequence of BAX dimerization, we expressed a chimeric FKBP-BAX molecule. Enforced dimerization of FKBP-BAX by the bivalent ligand FK1012 resulted in its translocation to mitochondria and induced apoptosis. Caspases were activated yet caspase inhibitors did not block death; cytochrome c was not released detectably despite the induction of mitochondrial dysfunction. Moreover, enforced dimerization of BAX overrode the protection by BCL-XL and IL-3 to kill cells. These data support a model in which a death signal results in the activation of BAX. This conformational change in BAX manifests in its translocation, mitochondrial membrane insertion and homodimerization, and a program of mitochondrial dysfunction that results in cell death.