RESUMO
The emerging role of mitochondria as signaling organelles raises the question of whether individual mitochondria can initiate heterotypic communication with neighboring organelles. Using fluorescent probes targeted to the endoplasmic-reticulum-mitochondrial interface, we demonstrate that single mitochondria generate oxidative bursts, rapid redox oscillations, confined to the nanoscale environment of the interorganellar contact sites. Using probes fused to inositol 1,4,5-trisphosphate receptors (IP3Rs), we show that Ca2+ channels directly sense oxidative bursts and respond with Ca2+ transients adjacent to active mitochondria. Application of specific mitochondrial stressors or apoptotic stimuli dramatically increases the frequency and amplitude of the oxidative bursts by enhancing transient permeability transition pore openings. Conversely, blocking interface Ca2+ transport via elimination of IP3Rs or mitochondrial calcium uniporter channels suppresses ER-mitochondrial Ca2+ feedback and cell death. Thus, single mitochondria initiate local retrograde signaling by miniature oxidative bursts and, upon metabolic or apoptotic stress, may also amplify signals to the rest of the cell.
Assuntos
Mitocôndrias/metabolismo , Transporte Proteico/fisiologia , Explosão Respiratória/fisiologia , Cálcio/metabolismo , Canais de Cálcio , Sinalização do Cálcio/fisiologia , Permeabilidade da Membrana Celular/fisiologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/fisiologia , Células HEK293 , Células Hep G2 , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Membranas Mitocondriais/metabolismo , Oxirredução , Explosão Respiratória/genética , Análise de Célula Única/métodosRESUMO
How mitochondrial shape and substrate-specific metabolism are related has been a difficult question to address. Here, new work by Ngo et al (2023) reports that mitochondrial shape-long versus fragmented-determines the activity of ß-oxidation of long-chain fatty acids, supporting a novel role for mitochondrial fission products as ß-oxidation hubs.
Assuntos
Ácidos Graxos , Mitocôndrias , Mitocôndrias/metabolismo , Oxirredução , Ácidos Graxos/metabolismo , Dinâmica MitocondrialRESUMO
Activating Ca2+-sensitive enzymes of oxidative metabolism while preventing calcium overload that leads to mitochondrial and cellular injury requires dynamic control of mitochondrial Ca2+ uptake. This is ensured by the mitochondrial calcium uptake (MICU)1/2 proteins that gate the pore of the mitochondrial calcium uniporter (mtCU). MICU1 is relatively sparse in the heart, and recent studies claimed the mammalian heart lacks MICU1 gating of mtCU. However, genetic models have not been tested. We find that MICU1 is present in a complex with MCU in nonfailing human hearts. Furthermore, using murine genetic models and pharmacology, we show that MICU1 and MICU2 control cardiac mitochondrial Ca2+ influx, and that MICU1 deletion alters cardiomyocyte mitochondrial calcium signaling and energy metabolism. MICU1 loss causes substantial compensatory changes in the mtCU composition and abundance, increased turnover of essential MCU regulator (EMRE) early on and, later, of MCU, that limit mitochondrial Ca2+ uptake and allow cell survival. Thus, both the primary consequences of MICU1 loss and the ensuing robust compensation highlight MICU1's relevance in the beating heart.
Assuntos
Sinalização do Cálcio , Proteínas de Ligação ao Cálcio , Cálcio , Proteínas de Transporte de Cátions , Proteínas de Transporte da Membrana Mitocondrial , Miócitos Cardíacos , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Humanos , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/genética , Miócitos Cardíacos/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Mitocôndrias Cardíacas/metabolismo , Canais de Cálcio/metabolismo , Canais de Cálcio/genética , Camundongos Knockout , Miocárdio/metabolismo , MasculinoRESUMO
Most cancer deaths result from progression of therapy resistant disease, yet our understanding of this phenotype is limited. Cancer therapies generate stress signals that act upon mitochondria to initiate apoptosis. Mitochondria isolated from neuroblastoma cells were exposed to tBid or Bim, death effectors activated by therapeutic stress. Multidrug-resistant tumor cells obtained from children at relapse had markedly attenuated Bak and Bax oligomerization and cytochrome c release (surrogates for apoptotic commitment) in comparison with patient-matched tumor cells obtained at diagnosis. Electron microscopy identified reduced ER-mitochondria-associated membranes (MAMs; ER-mitochondria contacts, ERMCs) in therapy-resistant cells, and genetically or biochemically reducing MAMs in therapy-sensitive tumors phenocopied resistance. MAMs serve as platforms to transfer Ca2+ and bioactive lipids to mitochondria. Reduced Ca2+ transfer was found in some but not all resistant cells, and inhibiting transfer did not attenuate apoptotic signaling. In contrast, reduced ceramide synthesis and transfer was common to resistant cells and its inhibition induced stress resistance. We identify ER-mitochondria-associated membranes as physiologic regulators of apoptosis via ceramide transfer and uncover a previously unrecognized mechanism for cancer multidrug resistance.
Assuntos
Mitocôndrias , Neuroblastoma , Apoptose , Ceramidas , Resistência a Múltiplos Medicamentos , Humanos , Membranas Mitocondriais , Neuroblastoma/tratamento farmacológicoRESUMO
Proper control of the mitochondrial Ca2+ uniporter's pore (MCU) is required to allow Ca2+-dependent activation of oxidative metabolism and to avoid mitochondrial Ca2+ overload and cell death. The MCU's gatekeeping and cooperative activation is mediated by the Ca2+-sensing MICU1 protein, which has been proposed to form dimeric complexes anchored to the EMRE scaffold of MCU. We unexpectedly find that MICU1 suppresses inhibition of MCU by ruthenium red/Ru360, which bind to MCU's DIME motif, the selectivity filter. This led us to recognize in MICU1's sequence a putative DIME interacting domain (DID), which is required for both gatekeeping and cooperative activation of MCU and for cell survival. Thus, we propose that MICU1 has to interact with the D-ring formed by the DIME domains in MCU to control the uniporter.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Compostos de Rutênio/farmacologia , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte de Cátions/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HEK293 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genéticaRESUMO
Mitochondrial Ca2+ uptake is mediated by the mitochondrial uniporter complex (mtCU) that includes a tetramer of the pore-forming subunit, MCU, a scaffold protein, EMRE, and the EF-hand regulatory subunit, MICU1 either homodimerized or heterodimerized with MICU2/3. MICU1 has been proposed to regulate Ca2+ uptake via the mtCU by physically occluding the pore and preventing Ca2+ flux at resting cytoplasmic [Ca2+] (free calcium concentration) and to increase Ca2+ flux at high [Ca2+] due to cooperative activation of MICUs EF-hands. However, mtCU and MICU1 functioning when its EF-hands are unoccupied by Ca2+ is poorly studied due to technical limitations. To overcome this barrier, we have studied the mtCU in divalent-free conditions by assessing the Ru265-sensitive Na+ influx using fluorescence-based measurement of mitochondrial matrix [Na+] (free sodium concentration) rise and the ensuing depolarization and swelling. We show an increase in all these measures of Na+ uptake in MICU1KO cells as compared to wild-type (WT) and rescued MICU1KO HEK cells. However, mitochondria in WT cells and MICU1 stable-rescued cells still allowed some Ru265-sensitive Na+ influx that was prevented by MICU1 in excess upon acute overexpression. Thus, MICU1 restricts the cation flux across the mtCU in the absence of Ca2+, but even in cells with high endogenous MICU1 expression such as HEK, some mtCU seem to lack MICU1-dependent gating. We also show rearrangement of the mtCU and altered number of functional channels in MICU1KO and different rescues, and loss of MICU1 during mitoplast preparation, that together might have obscured the pore-blocking function of MICU1 in divalent-free conditions in previous studies.
Assuntos
Canais de Cálcio , Proteínas de Transporte da Membrana Mitocondrial , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Canais de Cálcio/metabolismo , Mitocôndrias/metabolismo , Transporte Biológico , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismoRESUMO
Inner mitochondrial membrane fusion and cristae shape depend on optic atrophy protein 1, OPA1. Mutations in OPA1 lead to autosomal dominant optic atrophy (ADOA), an important cause of inherited blindness. The Guanosin Triphosphatase (GTPase) and GTPase effector domains (GEDs) of OPA1 are essential for mitochondrial fusion; yet, their specific roles remain elusive. Intriguingly, patients carrying OPA1 GTPase mutations have a higher risk of developing more severe multisystemic symptoms in addition to optic atrophy, suggesting pathogenic contributions for the GTPase and GED domains, respectively. We studied OPA1 GTPase and GED mutations to understand their domain-specific contribution to protein function by analyzing patient-derived cells and gain-of-function paradigms. Mitochondria from OPA1 GTPase (c.870+5G>A and c.889C>T) and GED (c.2713C>T and c.2818+5G>A) mutants display distinct aberrant cristae ultrastructure. While all OPA1 mutants inhibited mitochondrial fusion, some GTPase mutants resulted in elongated mitochondria, suggesting fission inhibition. We show that the GED is dispensable for fusion and OPA1 oligomer formation but necessary for GTPase activity. Finally, splicing defect mutants displayed a posttranslational haploinsufficiency-like phenotype but retained domain-specific dysfunctions. Thus, OPA1 domain-specific mutants result in distinct impairments in mitochondrial dynamics, providing insight into OPA1 function and its contribution to ADOA pathogenesis and severity.
Assuntos
Mitocôndrias , Atrofia Óptica Autossômica Dominante , Humanos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Atrofia Óptica Autossômica Dominante/genética , Atrofia Óptica Autossômica Dominante/metabolismo , Atrofia Óptica Autossômica Dominante/patologia , MutaçãoRESUMO
Ca2+ is a ubiquitous intracellular messenger that controls diverse cellular functions but can become toxic and cause cell death. Selective control of specific targets depends on spatiotemporal patterning of the calcium signal and decoding it by multiple, tunable, and often strategically positioned Ca2+-sensing elements. Ca2+ is detected by specialized motifs on proteins that have been biochemically characterized decades ago. However, the field of Ca2+ sensing has been reenergized by recent progress in fluorescent technology, genetics, and cryo-EM. These approaches exposed local Ca2+-sensing mechanisms inside organelles and at the organellar interfaces, revealed how Ca2+ binding might work to open some channels, and identified human mutations and disorders linked to a variety of Ca2+-sensing proteins. Here we attempt to place these new developments in the context of intracellular calcium homeostasis and signaling.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Proteínas Sensoras de Cálcio Intracelular/metabolismo , Motivos de Aminoácidos , Animais , Canais de Cálcio/metabolismo , Microscopia Crioeletrônica , Predisposição Genética para Doença , Homeostase , Humanos , Proteínas Sensoras de Cálcio Intracelular/genética , Proteínas Sensoras de Cálcio Intracelular/ultraestrutura , Ativação do Canal Iônico , Mutação , Fenótipo , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
The ER-mitochondrial interface is central to calcium signaling, organellar dynamics, and lipid biosynthesis. The ER and mitochondrial membranes also host sources and targets of reactive oxygen species (ROS), but their local dynamics and relevance remained elusive since measurement and perturbation of ROS at the organellar interface has proven difficult. Employing drug-inducible synthetic ER-mitochondrial linkers, we overcame this problem and demonstrate that the ER-mitochondrial interface hosts a nanodomain of H2O2, which is induced by cytoplasmic [Ca(2+)] spikes and exerts a positive feedback on calcium oscillations. H2O2 nanodomains originate from the mitochondrial cristae, which are compressed upon calcium signal propagation to the mitochondria, likely due to Ca(2+)-induced K(+) and concomitant water influx to the matrix. Thus, ER-mitochondrial H2O2 nanodomains represent a component of inter-organelle communication, regulating calcium signaling and mitochondrial activities.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Microdomínios da Membrana/metabolismo , Mitocôndrias Hepáticas/metabolismo , Membranas Mitocondriais/metabolismo , Canais de Cálcio/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Genes Reporter , Células Hep G2 , Humanos , Peróxido de Hidrogênio/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/ultraestrutura , Microscopia de Fluorescência , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/ultraestrutura , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/ultraestrutura , Oxirredução , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
Neutrophils simultaneously restrict Staphylococcus aureus dissemination and facilitate bactericidal activity during infection through the formation of neutrophil extracellular traps (NETs). Neutrophils that produce higher levels of mitochondrial superoxide undergo enhanced terminal NET formation (suicidal NETosis) in response to S. aureus; however, mechanisms regulating mitochondrial homeostasis upstream of neutrophil antibacterial processes are not fully resolved. Here, we demonstrate that mitochondrial calcium uptake 1 (MICU1)-deficient (MICU1-/-) neutrophils accumulate higher levels of calcium and iron within the mitochondria in a mitochondrial calcium uniporter (MCU)-dependent manner. Corresponding with increased ion flux through the MCU, mitochondrial superoxide production is elevated, thereby increasing the propensity for MICU1-/- neutrophils to undergo suicidal NETosis rather than primary degranulation in response to S. aureus. Increased NET formation augments macrophage killing of bacterial pathogens. Similarly, MICU1-/- neutrophils alone are not more antibacterial toward S. aureus, but rather, enhanced suicidal NETosis by MICU1-/- neutrophils facilitates increased bactericidal activity in the presence of macrophages. Similarly, mice with a deficiency in MICU1 restricted to cells expressing LysM exhibit lower bacterial burdens in the heart with increased survival during systemic S. aureus infection. Coinciding with the decrease in S. aureus burdens, MICU1-/- neutrophils in the heart produce higher levels of mitochondrial superoxide and undergo enhanced suicidal NETosis. These results demonstrate that ion flux by the MCU affects the antibacterial function of neutrophils during S. aureus infection.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Animais , Antibacterianos , Cálcio/metabolismo , Canais de Cálcio , Proteínas de Ligação ao Cálcio , Humanos , Camundongos , Proteínas de Transporte da Membrana Mitocondrial , Neutrófilos/metabolismo , Staphylococcus aureus/metabolismo , SuperóxidosRESUMO
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.
Assuntos
Dinâmica Mitocondrial , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Linhagem Celular , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Técnicas de Inativação de Genes , Humanos , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Transporte Proteico , Ratos , Canal de Ânion 2 Dependente de Voltagem/genéticaRESUMO
Mitochondrial Ca2+ uptake has long been considered crucial for meeting the fluctuating energy demands of cells in the heart and other tissues. Increases in mitochondrial matrix [Ca2+] drive mitochondrial ATP production via stimulation of Ca2+-sensitive dehydrogenases. Mitochondria-targeted sensors have revealed mitochondrial matrix [Ca2+] rises that closely follow the cytoplasmic [Ca2+] signals in many paradigms. Mitochondrial Ca2+ uptake is mediated by the Ca2+ uniporter (mtCU). Pharmacological manipulation of the mtCU is potentially key to understanding its physiological significance, but no specific, cell-permeable inhibitors were identified. In the past decade, as the molecular identity of the mtCU was brought to light, efforts have focused on genetic targeting. However, in the cells/animals that are able to survive impaired mtCU function, robust compensatory changes were found in the mtCU as well as other mechanisms. Thus, the discovery, through chemical library screens on normal and mtCU-deficient cells, of new small-molecule inhibitors with improved cell permeability and specificity might offer a better chance to test the relevance of mitochondrial Ca2+ uptake. Success with the development of small molecule mtCU inhibitors is also expected to have clinical impact, considering the growing evidence for the role of mitochondrial Ca2+ uptake in a variety of diseases, including heart attack, stroke and various neurodegenerative disorders. Here, we review the progress in pharmacological targeting of mtCU and illustrate the challenges in this field using data obtained with MCU-i11, a new small molecule inhibitor.
Assuntos
Canais de Cálcio/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Marcação de Genes , Humanos , Modelos Biológicos , Preparações Farmacêuticas/metabolismoRESUMO
The use of fixed fibroblasts from familial and sporadic Alzheimer's disease patients has previously indicated an upregulation of mitochondria-ER contacts (MERCs) as a hallmark of Alzheimer's disease. Despite its potential significance, the relevance of these results is limited because they were not extended to live neurons. Here we performed a dynamic in vivo analysis of MERCs in hippocampal neurons from McGill-R-Thy1-APP transgenic rats, a model of Alzheimer's disease-like amyloid pathology. Live FRET imaging of neurons from transgenic rats revealed perturbed 'lipid-MERCs' (gap width <10â nm), while 'Ca2+-MERCs' (10-20â nm gap width) were unchanged. In situ TEM showed no significant differences in the lipid-MERCs:total MERCs or lipid-MERCs:mitochondria ratios; however, the average length of lipid-MERCs was significantly decreased in neurons from transgenic rats as compared to controls. In accordance with FRET results, untargeted lipidomics showed significant decreases in levels of 12 lipids and bioenergetic analysis revealed respiratory dysfunction of mitochondria from transgenic rats. Thus, our results reveal changes in MERC structures coupled with impaired mitochondrial functions in Alzheimer's disease-related neurons.This article has an associated First Person interview with the first author of the paper.
Assuntos
Doença de Alzheimer , Retículo Endoplasmático , Mitocôndrias , Neurônios , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Modelos Animais de Doenças , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neurônios/metabolismo , Neurônios/patologia , Ratos , Ratos TransgênicosRESUMO
AIMS: MICU1 encodes the gatekeeper of the mitochondrial Ca2+ uniporter, MICU1 and biallelic loss-of-function mutations cause a complex, neuromuscular disorder in children. Although the role of the protein is well understood, the precise molecular pathophysiology leading to this neuropaediatric phenotype has not been fully elucidated. Here we aimed to obtain novel insights into MICU1 pathophysiology. METHODS: Molecular genetic studies along with proteomic profiling, electron-, light- and Coherent anti-Stokes Raman scattering microscopy and immuno-based studies of protein abundances and Ca2+ transport studies were employed to examine the pathophysiology of MICU1 deficiency in humans. RESULTS: We describe two patients carrying MICU1 mutations, two nonsense (c.52C>T; p.(Arg18*) and c.553C>T; p.(Arg185*)) and an intragenic exon 2-deletion presenting with ataxia, developmental delay and early onset myopathy, clinodactyly, attention deficits, insomnia and impaired cognitive pain perception. Muscle biopsies revealed signs of dystrophy and neurogenic atrophy, severe mitochondrial perturbations, altered Golgi structure, vacuoles and altered lipid homeostasis. Comparative mitochondrial Ca2+ transport and proteomic studies on lymphoblastoid cells revealed that the [Ca2+ ] threshold and the cooperative activation of mitochondrial Ca2+ uptake were lost in MICU1-deficient cells and that 39 proteins were altered in abundance. Several of those proteins are linked to mitochondrial dysfunction and/or perturbed Ca2+ homeostasis, also impacting on regular cytoskeleton (affecting Spectrin) and Golgi architecture, as well as cellular survival mechanisms. CONCLUSIONS: Our findings (i) link dysregulation of mitochondrial Ca2+ uptake with muscle pathology (including perturbed lipid homeostasis and ER-Golgi morphology), (ii) support the concept of a functional interplay of ER-Golgi and mitochondria in lipid homeostasis and (iii) reveal the vulnerability of the cellular proteome as part of the MICU1-related pathophysiology.
Assuntos
Proteínas de Ligação ao Cálcio/deficiência , Cálcio/metabolismo , Proteínas de Transporte de Cátions/deficiência , Proteínas de Transporte da Membrana Mitocondrial/genética , Doenças Musculares/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/deficiência , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Doenças Musculares/patologia , ProteômicaRESUMO
Exchanges of matrix contents are essential to the maintenance of mitochondria. Cardiac mitochondrial exchange matrix content in two ways: by direct contact with neighboring mitochondria and over longer distances. The latter mode is supported by thin tubular protrusions, called nanotunnels, that contact other mitochondria at relatively long distances. Here, we report that cardiac myocytes of heterozygous mice carrying a catecholaminergic polymorphic ventricular tachycardia-linked RyR2 mutation (A4860G) show a unique and unusual mitochondrial response: a significantly increased frequency of nanotunnel extensions. The mutation induces Ca2+ imbalance by depressing RyR2 channel activity during excitation-contraction coupling, resulting in random bursts of Ca2+ release probably due to Ca2+ overload in the sarcoplasmic reticulum. We took advantage of the increased nanotunnel frequency in RyR2A4860G+/- cardiomyocytes to investigate and accurately define the ultrastructure of these mitochondrial extensions and to reconstruct the overall 3D distribution of nanotunnels using electron tomography. Additionally, to define the effects of communication via nanotunnels, we evaluated the intermitochondrial exchanges of matrix-targeted soluble fluorescent proteins, mtDsRed and photoactivable mtPA-GFP, in isolated cardiomyocytes by confocal microscopy. A direct comparison between exchanges occurring at short and long distances directly demonstrates that communication via nanotunnels is slower.
Assuntos
Sinalização do Cálcio/fisiologia , Mitocôndrias Cardíacas/fisiologia , Animais , Acoplamento Excitação-Contração/fisiologia , Camundongos , Microscopia Confocal , Microscopia Eletrônica , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/ultraestrutura , Dinâmica Mitocondrial/fisiologia , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Canal de Liberação de Cálcio do Receptor de Rianodina/deficiência , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Taquicardia Ventricular/genéticaRESUMO
Mitochondrial fusion is thought to be important for supporting cardiac contractility, but is hardly detectable in cultured cardiomyocytes and is difficult to directly evaluate in the heart. We overcame this obstacle through in vivo adenoviral transduction with matrix-targeted photoactivatable GFP and confocal microscopy. Imaging in whole rat hearts indicated mitochondrial network formation and fusion activity in ventricular cardiomyocytes. Promptly after isolation, cardiomyocytes showed extensive mitochondrial connectivity and fusion, which decayed in culture (at 24-48 h). Fusion manifested both as rapid content mixing events between adjacent organelles and slower events between both neighboring and distant mitochondria. Loss of fusion in culture likely results from the decline in calcium oscillations/contractile activity and mitofusin 1 (Mfn1), because (i) verapamil suppressed both contraction and mitochondrial fusion, (ii) after spontaneous contraction or short-term field stimulation fusion activity increased in cardiomyocytes, and (iii) ryanodine receptor-2-mediated calcium oscillations increased fusion activity in HEK293 cells and complementing changes occurred in Mfn1. Weakened cardiac contractility in vivo in alcoholic animals is also associated with depressed mitochondrial fusion. Thus, attenuated mitochondrial fusion might contribute to the pathogenesis of cardiomyopathy.
Assuntos
Sinalização do Cálcio/fisiologia , Mitocôndrias Cardíacas/fisiologia , Dinâmica Mitocondrial/fisiologia , Contração Miocárdica/fisiologia , Animais , Linhagem Celular , Genes Reporter , Vetores Genéticos , Humanos , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Masculino , Microscopia Confocal , Mitocôndrias Cardíacas/ultraestrutura , Ratos , Ratos Sprague-Dawley , Transdução GenéticaRESUMO
A sensitization of inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release is associated with oxidative stress in multiple cell types. These effects are thought to be mediated by alterations in the redox state of critical thiols in the IP3R, but this has not been directly demonstrated in intact cells. Here, we utilized a combination of gel-shift assays with MPEG-maleimides and LC-MS/MS to monitor the redox state of recombinant IP3R1 expressed in HEK293 cells. We found that under basal conditions, â¼5 of the 60 cysteines are oxidized in IP3R1. Cell treatment with 50 µm thimerosal altered gel shifts, indicating oxidation of â¼20 cysteines. By contrast, the shifts induced by 0.5 mm H2O2 or other oxidants were much smaller. Monitoring of biotin-maleimide attachment to IP3R1 by LC-MS/MS with 71% coverage of the receptor sequence revealed modification of two cytosolic (Cys-292 and Cys-1415) and two intraluminal cysteines (Cys-2496 and Cys-2533) under basal conditions. The thimerosal treatment modified an additional eleven cysteines, but only three (Cys-206, Cys-767, and Cys-1459) were consistently oxidized in multiple experiments. H2O2 also oxidized Cys-206 and additionally oxidized two residues not modified by thimerosal (Cys-214 and Cys-1397). Potentiation of IP3R channel function by oxidants was measured with cysteine variants transfected into a HEK293 IP3R triple-knockout cell line, indicating that the functionally relevant redox-sensitive cysteines are predominantly clustered within the N-terminal suppressor domain of IP3R. To our knowledge, this study is the first that has used proteomic methods to assess the redox state of individual thiols in IP3R in intact cells.
Assuntos
Peróxido de Hidrogênio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Substituição de Aminoácidos , Sinalização do Cálcio , Cisteína/química , Cisteína/genética , Células HEK293 , Humanos , Receptores de Inositol 1,4,5-Trifosfato/química , Receptores de Inositol 1,4,5-Trifosfato/genética , OxirreduçãoRESUMO
The ER-mitochondrial junction provides a local calcium signaling domain that is critical for both matching energy production with demand and the control of apoptosis. Here, we visualize ER-mitochondrial contact sites and monitor the localized [Ca(2+)] changes ([Ca(2+)](ER-mt)) using drug-inducible fluorescent interorganelle linkers. We show that all mitochondria have contacts with the ER, but plasma membrane (PM)-mitochondrial contacts are less frequent because of interleaving ER stacks in both RBL-2H3 and H9c2 cells. Single mitochondria display discrete patches of ER contacts and show heterogeneity in the ER-mitochondrial Ca(2+) transfer. Pericam-tagged linkers revealed IP(3)-induced [Ca(2+)](ER-mt) signals that exceeded 9 microM and endured buffering bulk cytoplasmic [Ca(2+)] increases. Altering linker length to modify the space available for the Ca(2+) transfer machinery had a biphasic effect on [Ca(2+)](ER-mt) signals. These studies provide direct evidence for the existence of high-Ca(2+) microdomains between the ER and mitochondria and suggest an optimal gap width for efficient Ca(2+) transfer.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Imageamento Tridimensional/métodos , Mitocôndrias/metabolismo , Animais , Sinalização do Cálcio , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Permeabilidade da Membrana Celular , Sobrevivência Celular , Retículo Endoplasmático/ultraestrutura , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Ratos , Fatores de TempoRESUMO
Voltage-dependent anion channel (VDAC) proteins are major components of the outer mitochondrial membrane. VDAC has three isoforms with >70% sequence similarity and redundant roles in metabolite and ion transport. However, only Vdac2(-/-) (V2(-/-)) mice are embryonic lethal, indicating a unique and fundamental function of VDAC2 (V2). Recently, a specific V2 requirement was demonstrated for mitochondrial Bak import and truncated Bid (tBid)-induced apoptosis. To determine the relevant domain(s) of V2 involved, VDAC1 (V1) and V2 chimeric constructs were created and used to rescue V2(-/-) fibroblasts. Surprisingly, the commonly cited V2-specific N-terminal extension and cysteines were found to be dispensable for Bak import and high tBid sensitivity. In gain-of-function studies, V2 (123-179) was the minimal sequence sufficient to render V1 competent to support Bak insertion. Furthermore, in loss-of-function experiments, T168 and D170 were identified as critical residues. These motifs are conserved in zebrafish V2 (zfV2) that also rescued V2-deficient fibroblasts. Because high-resolution structures of zfV2 and mammalian V1 have become available, we could superimpose these structures and recognized that the critical V2-specific residues help to create a distinctive open "pocket" on the cytoplasmic surface that could facilitate Bak recruitment.
Assuntos
Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Mitocôndrias/metabolismo , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Motivos de Aminoácidos , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Canal de Ânion 2 Dependente de Voltagem/genética , Proteína Killer-Antagonista Homóloga a bcl-2/genéticaRESUMO
We report here the first families carrying recessive variants in the MSTO1 gene: compound heterozygous mutations were identified in two sisters and in an unrelated singleton case, who presented a multisystem complex phenotype mainly characterized by myopathy and cerebellar ataxia. Human MSTO1 is a poorly studied protein, suggested to have mitochondrial localization and to regulate morphology and distribution of mitochondria. As for other mutations affecting genes involved in mitochondrial dynamics, no biochemical defects typical of mitochondrial disorders were reported. Studies in patients' fibroblasts revealed that MSTO1 protein levels were strongly reduced, the mitochondrial network was fragmented, and the fusion events among mitochondria were decreased, confirming the deleterious effect of the identified variants and the role of MSTO1 in modulating mitochondrial dynamics. We also found that MSTO1 is mainly a cytosolic protein. These findings indicate recessive mutations in MSTO1 as a new cause for inherited neuromuscular disorders with multisystem features.