RESUMEN
Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode (MELAS) syndrome, caused by a single base substitution in mitochondrial DNA (m.3243A>G), is one of the most common maternally inherited mitochondrial diseases accompanied by neuronal damage due to defects in the oxidative phosphorylation system. There is no established treatment. Our previous study reported a superior restoration of mitochondrial function and bioenergetics in mitochondria-deficient cells using highly purified mesenchymal stem cells (RECs). However, whether such exogenous mitochondrial donation occurs in mitochondrial disease models and whether it plays a role in the recovery of pathological neuronal functions is unknown. Here, utilizing induced pluripotent stem cells (iPSC), we differentiated neurons with impaired mitochondrial function from patients with MELAS. MELAS neurons and RECs/mesenchymal stem cells (MSCs) were cultured under contact or non-contact conditions. Both RECs and MSCs can donate mitochondria to MELAS neurons, but RECs are more excellent than MSCs for mitochondrial transfer in both systems. In addition, REC-mediated mitochondrial transfer significantly restored mitochondrial function, including mitochondrial membrane potential, ATP/ROS production, intracellular calcium storage, and oxygen consumption rate. Moreover, mitochondrial function was maintained for at least three weeks. Thus, REC-donated exogenous mitochondria might offer a potential therapeutic strategy for treating neurological dysfunction in MELAS.
Asunto(s)
Acidosis Láctica , Síndrome MELAS , Células Madre Mesenquimatosas , Enfermedades Mitocondriales , Humanos , Síndrome MELAS/genética , Síndrome MELAS/terapia , Mitocondrias/genética , Acidosis Láctica/metabolismo , Acidosis Láctica/patología , ADN Mitocondrial/metabolismo , Enfermedades Mitocondriales/metabolismo , Neuronas/patología , Células Madre Mesenquimatosas/metabolismoRESUMEN
Skeletal muscle is highly developed after birth, consisting of glycolytic fast-twitch and oxidative slow-twitch fibers; however, the mechanisms of fiber-type-specific differentiation are poorly understood. Here, we found an unexpected role of mitochondrial fission in the differentiation of fast-twitch oxidative fibers. Depletion of the mitochondrial fission factor dynamin-related protein 1 (Drp1) in mouse skeletal muscle and cultured myotubes results in specific reduction of fast-twitch muscle fibers independent of respiratory function. Altered mitochondrial fission causes activation of the Akt/mammalian target of rapamycin (mTOR) pathway via mitochondrial accumulation of mTOR complex 2 (mTORC2), and rapamycin administration rescues the reduction of fast-twitch fibers in vivo and in vitro. Under Akt/mTOR activation, the mitochondria-related cytokine growth differentiation factor 15 is upregulated, which represses fast-twitch fiber differentiation. Our findings reveal a crucial role of mitochondrial dynamics in the activation of mTORC2 on mitochondria, resulting in the differentiation of muscle fibers.
Asunto(s)
Dinámicas Mitocondriales , Enfermedades Musculares , Ratones , Animales , Fibras Musculares de Contracción Lenta/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Redes y Vías Metabólicas , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Mamíferos/metabolismoRESUMEN
Imeglimin is a recently launched antidiabetic drug structurally related to metformin. To provide insight into the pharmacological properties of imeglimin, we investigated its effects on hepatocytes and compared them with those of metformin. The effects of imeglimin on mitochondrial function in HepG2 cells or mouse primary hepatocytes were examined with an extracellular flux analyzer and on gene expression in HepG2 cells by comprehensive RNA-sequencing analysis. The effects of the drug on AMPK activity in HepG2 cells, mouse primary hepatocytes, and mouse liver were also examined. Treatment of HepG2 cells or mouse primary hepatocytes with imeglimin reduced the oxygen consumption rate coupled to ATP production. Imeglimin activated AMPK in these cells whereas the potency was smaller than metformin. Bolus administration of imeglimin in mice also activated AMPK in the liver. Whereas the effects of imeglimin and metformin on gene expression in HepG2 cells were similar overall, the expression of genes encoding proteins of mitochondrial respiratory complex III and complex I was upregulated by imeglimin but not by metformin. Our results suggest that imeglimin and metformin exert similar pharmacological effects on mitochondrial respiration, AMPK activity, and gene expression in cultured hepatocytes, whereas the two drugs differ in their effects on the expression of certain genes related to mitochondrial function.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Metformina , Animales , Ratones , Proteínas Quinasas Activadas por AMP/metabolismo , Expresión Génica , Hepatocitos/metabolismo , Hipoglucemiantes/farmacología , Metformina/farmacología , Metformina/metabolismo , Mitocondrias/metabolismo , Células Hep G2 , HumanosRESUMEN
Mitochondria have their own DNA (mtDNA), which encodes essential respiratory subunits. Under live imaging, mitochondrial nucleoids, composed of several copies of mtDNA and DNA-binding proteins, such as mitochondrial transcription factor A (TFAM), actively move inside mitochondria and change the morphology, in concert with mitochondrial membrane fission. Here we found the mitochondrial inner membrane-anchored AAA-ATPase protein ATAD3A mediates the nucleoid dynamics. Its ATPase domain exposed to the matrix binds directly to TFAM and mediates nucleoid trafficking along mitochondria by ATP hydrolysis. Nucleoid trafficking also required ATAD3A oligomerization via an interaction between the coiled-coil domains in intermembrane space. In ATAD3A deficiency, impaired nucleoid trafficking repressed the clustered and enlarged nucleoids observed in mitochondrial fission-deficient cells resulted in dispersed distribution of small nucleoids observed throughout the mitochondrial network, and this enhanced respiratory complex formation. Thus, mitochondrial fission and nucleoid trafficking cooperatively determine the size, number, and distribution of nucleoids in mitochondrial network, which should modulate respiratory complex formation.
Asunto(s)
Mitocondrias , Proteínas Mitocondriales , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Dinámicas Mitocondriales/genética , ADN Mitocondrial/genéticaRESUMEN
Regulation of mitochondrial respiration and morphology is important for maintaining steady-state hematopoiesis, yet few studies have comparatively evaluated the effects of abnormal mitochondrial respiration and dynamics on blood-cell differentiation in isolation or combination. This study sought to explore these effects in mouse models with one or both of the following deficits: a large-scale deletion of mitochondrial DNA (ΔmtDNA), accumulated to varying extents, or knockout of the mitochondrial fission factor Drp1. Each deficit was found to independently provoke anemia but with clearly different manifestations. The former showed signs of aberrant respiration, analogous to Pearson syndrome, while the latter showed signs of abnormal mitochondrial dynamics and was associated with changes in the relative proportions of leukocyte lineages. Combining these deficits acted to amplify abnormal iron metabolism in erythropoiesis, exacerbating anemia in an additive manner. Our results indicate that mitochondrial respiration and dynamics play distinct roles in different sets of processes and cell lineages in hematopoietic differentiation.
Asunto(s)
Anemia , ADN Mitocondrial , Ratones , Animales , ADN Mitocondrial/genética , Modelos Animales de Enfermedad , Anemia/genética , LeucocitosRESUMEN
AIMS/HYPOTHESIS: Mitochondria are highly dynamic organelles continuously undergoing fission and fusion, referred to as mitochondrial dynamics, to adapt to nutritional demands. Evidence suggests that impaired mitochondrial dynamics leads to metabolic abnormalities such as non-alcoholic steatohepatitis (NASH) phenotypes. However, how mitochondrial dynamics are involved in the development of NASH is poorly understood. This study aimed to elucidate the role of mitochondrial fission factor (MFF) in the development of NASH. METHODS: We created mice with hepatocyte-specific deletion of MFF (MffLiKO). MffLiKO mice fed normal chow diet (NCD) or high-fat diet (HFD) were evaluated for metabolic variables and their livers were examined by histological analysis. To elucidate the mechanism of development of NASH, we examined the expression of genes related to endoplasmic reticulum (ER) stress and lipid metabolism, and the secretion of triacylglycerol (TG) using the liver and primary hepatocytes isolated from MffLiKO and control mice. RESULTS: MffLiKO mice showed aberrant mitochondrial morphologies with no obvious NASH phenotypes during NCD, while they developed full-blown NASH phenotypes in response to HFD. Expression of genes related to ER stress was markedly upregulated in the liver from MffLiKO mice. In addition, expression of genes related to hepatic TG secretion was downregulated, with reduced hepatic TG secretion in MffLiKO mice in vivo and in primary cultures of MFF-deficient hepatocytes in vitro. Furthermore, thapsigargin-induced ER stress suppressed TG secretion in primary hepatocytes isolated from control mice. CONCLUSIONS/INTERPRETATION: We demonstrated that ablation of MFF in liver provoked ER stress and reduced hepatic TG secretion in vivo and in vitro. Moreover, MffLiKO mice were more susceptible to HFD-induced NASH phenotype than control mice, partly because of ER stress-induced apoptosis of hepatocytes and suppression of TG secretion from hepatocytes. This study provides evidence for the role of mitochondrial fission in the development of NASH.
Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , Animales , Modelos Animales de Enfermedad , Hepatocitos/metabolismo , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Dinámicas Mitocondriales/genética , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismoRESUMEN
BACKGROUND: Mitochondria, which play a critical role in energy production by oxidative respiration, are highly dynamic organelles and their double membranes undergo frequent events of fusion and fission. Mitochondria are believed to be derived from the endosymbiosis of proteobacteria, and thus mitochondria still contain their own DNA, mitochondrial DNA (mtDNA). Several copies of mtDNA form mitochondrial nucleoid with DNA-binding proteins. Recently, the morphology and distribution of the mitochondrial membrane and nucleoid were reported to be cooperatively regulated during their dynamic movement. However, the molecular mechanism is unclear, because the involved molecules are poorly understood, and suitable techniques to analyze nucleoid have not been fully developed. RESULTS: To solve these issues, we examined the molecular mechanism of nucleoid dynamics by two approaches. First, we constructed a new probe to perform live imaging of nucleoid dynamics using the DNA-binding domain of mitochondrial transcriptional factor A (TFAM) and the photo-convertible fluorescent protein Kikume Green-Red (KikGR). Nucleoids were visualized stably for a long period using the new probe. Second, we searched for nucleoid regulatory factors by small interfering RNA screening using HeLa cells and identified a subset of MARCH family ubiquitin ligases that affect nucleoid morphology. CONCLUSION: The factors and probe, reported in this study, would be useful to reveal novel mechanisms of mitochondrial regulation. GENERAL SIGNIFICANCE: The mtDNA dynamics should be concerned in the regulation of mitochondrial activity and its quality control, associated with mitochondrial membrane dynamics.
Asunto(s)
Bioensayo/métodos , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/metabolismo , Mitocondrias/metabolismo , Dinámicas Mitocondriales , Membranas Mitocondriales/metabolismo , ADN Mitocondrial/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Células HeLa , HumanosRESUMEN
Mitochondria are multifunctional organelles that produce energy and are critical for various signaling pathways. Mitochondrial antiviral signaling (MAVS) is a mitochondrial outer membrane protein essential for the anti-RNA viral immune response, which is regulated by mitochondrial dynamics and energetics; however, the molecular link between mitochondrial metabolism and immunity is unclear. Here we show in cultured mammalian cells that MAVS is activated by mitochondrial fission factor (Mff), which senses mitochondrial energy status. Mff mediates the formation of active MAVS clusters on mitochondria, independent of mitochondrial fission and dynamin-related protein 1. Under mitochondrial dysfunction, Mff is phosphorylated by the cellular energy sensor AMP-activated protein kinase (AMPK), leading to the disorganization of MAVS clusters and repression of the acute antiviral response. Mff also contributes to immune tolerance during chronic infection by disrupting the mitochondrial MAVS clusters. Taken together, Mff has a critical function in MAVS-mediated innate immunity, by sensing mitochondrial energy metabolism via AMPK signaling.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Interacciones Huésped-Patógeno/fisiología , Inmunidad Innata/fisiología , Proteínas de la Membrana/genética , Proteínas Mitocondriales/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Citocinas/metabolismo , Fibroblastos/inmunología , Células HeLa/virología , Humanos , Proteínas de la Membrana/metabolismo , Ratones Noqueados , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Fosforilación , Infecciones por Respirovirus/inmunologíaRESUMEN
Mitochondria are dynamic organelles that frequently divide and fuse with each other. The dynamin-related GTPase protein Drp1 has a key role in mitochondrial fission. To analyse the physiological roles of Drp1 in cultured human cells, we analysed Drp1-deficient HeLa cells established by genome editing using CRISPR/Cas9. Under fluorescent microscopy, not only mitochondria were elongated but their DNA (mtDNA) nucleoids were extremely enlarged in bulb-like mitochondrial structures ('mito-bulbs') in the Drp1-deficient HeLa cells. We further found that respiratory activity, as measured by oxygen consumption rates, was severely repressed in Drp1-deficient HeLa cells and that this was reversible by the co-repression of mitochondrial fusion factors. Although mtDNA copy number was not affected, several respiratory subunits were repressed in Drp1-deficient HeLa cells. These results suggest that mitochondrial fission is required for the maintenance of active respiratory activity and the morphology of mtDNA nucleoids in human cells.
Asunto(s)
ADN Mitocondrial/metabolismo , Dinaminas/metabolismo , Mitocondrias/metabolismo , Sistemas CRISPR-Cas , Respiración de la Célula/genética , ADN Mitocondrial/genética , Dinaminas/genética , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Microscopía Fluorescente , Mitocondrias/genética , Mitocondrias/ultraestructura , Dinámicas Mitocondriales/genética , Dinámicas Mitocondriales/fisiología , ARN Interferente PequeñoRESUMEN
Mitochondria are highly dynamic organelles that undergo frequent fusion and fission. The large GTPase optic atrophy 1 (OPA1) is identified as a core component of inner membrane (IM) fusion. OPA1 exists as the membrane-anchored L-OPA1 and the proteolytically cleavage soluble S-OPA1. Recently, we showed that OPA1 and mitochondria-localized lipid cardiolipin (CL) cooperate in heterotypic IM fusion [Ban et al., Nat. Cell Biol. 19 (2017) 856-863]. We reconstituted an in vitro membrane fusion reaction using purified human L-OPA1 and S-OPA1 expressed in silkworm and found that L-OPA1 on one side of the membrane and CL on the other side were sufficient for mitochondrial fusion. L-OPA1 is the major fusion-prone factor in heterotypic fusion. However, the role of S-OPA1 remains unknown as S-OPA1 promoted L-OPA1-dependent heterotypic membrane fusion and homotypic CL-containing membrane fusion, but S-OPA1 alone was not sufficient for heterotypic membrane fusion. L-OPA1- and CL-mediated heterotypic mitochondrial fusion was confirmed in living cells, but tafazzin (Taz1), the causal gene product of Barth syndrome, was not essential for mitochondrial fusion. Taz1-dependent CL maturation might have other roles in the remodeling of mitochondrial DNA nucleoids.
Asunto(s)
Cardiolipinas/metabolismo , GTP Fosfohidrolasas/metabolismo , Fusión de Membrana , Mitocondrias/metabolismo , Dinámicas Mitocondriales , Células HeLa , HumanosRESUMEN
Aclarubicin (Acla), an effective anthracycline chemotherapeutic agent for hematologic cancers and solid tumors, is documented to perturb chromatin function via histone eviction and DNA topoisomerase inhibition in the nucleus, but much less attention has been paid to cytotoxic function in the cytoplasm. Here, we showed that Acla emitted fluorescence and that human cervical cancer HeLa cells exposed to Acla exhibited bright fluorescence signals in the cytoplasm when fluorescence microscopy was performed using the red filter (excitation 530-550nm/emission 575nm). Intriguingly, most of the signals appeared to be partitioned and enriched in entangled tubule-like structures; moreover, these signals merged with the mitochondria-specific MitoTracker signals. Notably, analysis of mitochondrial respiratory activity revealed that the oxygen consumption rate was decreased in Acla-treated cells. These findings suggest that Acla accumulates efficiently in the mitochondria of living human cells and leads to mitochondrial dysfunction, implying a previously overlooked cytotoxicity of Acla in the cytoplasm and adding mechanistic insight of the anti-cancer activity, as well as the side effects, of Acla/anthracycline-based chemotherapy.
Asunto(s)
Aclarubicina/farmacología , Antibióticos Antineoplásicos/farmacología , Microscopía Confocal , Microscopía Fluorescente , Mitocondrias/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Neoplasias del Cuello Uterino/tratamiento farmacológico , Aclarubicina/metabolismo , Aclarubicina/toxicidad , Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/toxicidad , Muerte Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Colorantes Fluorescentes/metabolismo , Células HeLa , Humanos , Mitocondrias/metabolismo , Mitocondrias/patología , Factores de Tiempo , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patologíaRESUMEN
Mitochondria are highly dynamic organelles that undergo frequent fusion and fission. Optic atrophy 1 (OPA1) is an essential GTPase protein for both mitochondrial inner membrane (IM) fusion and cristae morphology. Under mitochondria-stress conditions, membrane-anchored L-OPA1 is proteolytically cleaved to form peripheral S-OPA1, leading to the selection of damaged mitochondria for mitophagy. However, molecular details of the selective mitochondrial fusion are less well understood. Here, we showed that L-OPA1 and cardiolipin (CL) cooperate in heterotypic mitochondrial IM fusion. We reconstituted an in vitro membrane fusion reaction using purified human L-OPA1 protein expressed in silkworm, and found that L-OPA1 on one side of the membrane and CL on the other side are sufficient for fusion. GTP-independent membrane tethering through L-OPA1 and CL primes the subsequent GTP-hydrolysis-dependent fusion, which can be modulated by the presence of S-OPA1. These results unveil the most minimal intracellular membrane fusion machinery. In contrast, independent of CL, a homotypic trans-OPA1 interaction mediates membrane tethering, thereby supporting the cristae structure. Thus, multiple OPA1 functions are modulated by local CL conditions for regulation of mitochondrial morphology and quality control.
Asunto(s)
Cardiolipinas/metabolismo , GTP Fosfohidrolasas/metabolismo , Mitocondrias/enzimología , Dinámicas Mitocondriales , Membranas Mitocondriales/enzimología , Animales , Animales Modificados Genéticamente , Bombyx/enzimología , Bombyx/genética , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Marcación de Gen , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Hidrólisis , Liposomas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Interferencia de ARN , Proteínas Recombinantes/metabolismo , Transducción de Señal , Relación Estructura-Actividad , Factores de Tiempo , TransfecciónRESUMEN
Mitochondrial morphology is dynamically regulated by fusion and fission. Several GTPase proteins control fusion and fission, and posttranslational modifications of these proteins are important for the regulation. However, it has not been clarified how the fusion and fission is balanced. Here, we report the molecular mechanism to regulate mitochondrial morphology in mammalian cells. Ablation of the mitochondrial fission, by repression of Drp1 or Mff, or by over-expression of MiD49 or MiD51, results in a reduction in the fusion GTPase mitofusins (Mfn1 and Mfn2) in outer membrane and long form of OPA1 (L-OPA1) in inner membrane. RNAi- or CRISPR-induced ablation of Drp1 in HeLa cells enhanced the degradation of Mfns via the ubiquitin-proteasome system (UPS). We further found that UPS-related protein BAT3/BAG6, here we identified as Mfn2-interacting protein, was implicated in the turnover of Mfns in the absence of mitochondrial fission. Ablation of the mitochondrial fission also enhanced the proteolytic cleavage of L-OPA1 to soluble S-OPA1, and the OPA1 processing was reversed by inhibition of the inner membrane protease OMA1 independent on the mitochondrial membrane potential. Our findings showed that the distinct degradation systems of the mitochondrial fusion proteins in different locations are enhanced in response to the mitochondrial morphology.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Mitocondrias/metabolismo , Dinaminas , Técnicas de Inactivación de Genes , Células HeLa , Homeostasis , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Mitocondriales/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitinas/metabolismoRESUMEN
Mitochondria, which are double-membrane organelles thought to have originated through endosymbiosis of bacteria, play important roles in not only energy production, but also cellular signaling, differentiation, and development. The morphology of mitochondria is highly diverse among different tissues, and dynamic changes in mitochondrial morphology have been observed in response to various intracellular signals and stresses. These changes in mitochondrial morphology occur by repeated membrane fusion and fission events, which are regulated by three types of GTPase proteins: OPA1, Mfn1/2, Drp1 in mammalian cells. In recent years, the function and molecular mechanisms of mitochondrial dynamics have been demonstrated in cultured cells; however, the role of mitochondrial fission in maintaining tissue homeostasis remains poorly understood. Here, we review recent advances in research on the physiological role of mitochondrial fission in various differentiated tissue types in mammals.
Asunto(s)
Dinaminas/metabolismo , Desarrollo Embrionario , GTP Fosfohidrolasas/metabolismo , Dinámicas Mitocondriales , Modelos Biológicos , Animales , Células Cultivadas , Dinaminas/genética , GTP Fosfohidrolasas/genética , Regulación del Desarrollo de la Expresión Génica , Ratones , Mitocondrias/enzimología , Mitocondrias/metabolismo , Mutación , Especificidad de ÓrganosRESUMEN
AIM/HYPOTHESIS: Mitochondria and the endoplasmic reticulum (ER) physically interact by close structural juxtaposition, via the mitochondria-associated ER membrane. Inter-organelle communication between the ER and mitochondria has been shown to regulate energy metabolism and to be central to the modulation of various key processes such as ER stress. We aimed to clarify the role of mitochondrial fission in this communication. METHODS: We generated mice lacking the mitochondrial fission protein dynamin-related protein 1 (DRP1) in the liver (Drp1LiKO mice). RESULTS: Drp1LiKO mice showed decreased fat mass and were protected from high-fat diet (HFD)-induced obesity. Analysis of liver gene expression profiles demonstrated marked elevation of ER stress markers. In addition, we observed increased expression of the fibroblast growth factor 21 (FGF21) gene through induction of activating transcription factor 4, master regulator of the integrated stress response. CONCLUSIONS/INTERPRETATION: Disruption of mitochondrial fission in the liver provoked ER stress, while inducing the expression of FGF21 to increase energy expenditure and protect against HFD-induced obesity.
Asunto(s)
Dinaminas/metabolismo , Hígado/metabolismo , Dinámicas Mitocondriales/genética , Obesidad/metabolismo , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Adiposidad/genética , Animales , Dieta Alta en Grasa , Dinaminas/genética , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/genética , Metabolismo Energético/genética , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Ratones Noqueados , Mitocondrias/metabolismo , Obesidad/genéticaRESUMEN
Mitochondria are dynamic organelles, and their fusion and fission regulate cellular signaling, development, and mitochondrial homeostasis, including mitochondrial DNA (mtDNA) distribution. Cardiac myocytes have a specialized cytoplasmic structure where large mitochondria are aligned into tightly packed myofibril bundles; however, recent studies have revealed that mitochondrial dynamics also plays an important role in the formation and maintenance of cardiomyocytes. Here, we precisely analyzed the role of mitochondrial fission in vivo. The mitochondrial fission GTPase, Drp1, is highly expressed in the developing neonatal heart, and muscle-specific Drp1 knockout (Drp1-KO) mice showed neonatal lethality due to dilated cardiomyopathy. The Drp1 ablation in heart and primary cultured cardiomyocytes resulted in severe mtDNA nucleoid clustering and led to mosaic deficiency of mitochondrial respiration. The functional and structural alteration of mitochondria also led to immature myofibril assembly and defective cardiomyocyte hypertrophy. Thus, the dynamics of mtDNA nucleoids regulated by mitochondrial fission is required for neonatal cardiomyocyte development by promoting homogeneous distribution of active mitochondria throughout the cardiomyocytes.
Asunto(s)
ADN Mitocondrial/genética , Dinaminas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Mitocondrias Cardíacas/metabolismo , Dinámicas Mitocondriales , Animales , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados , Microscopía Electrónica , Proteínas Mitocondriales/metabolismo , Células Musculares/citología , Miocitos Cardíacos/citología , Consumo de Oxígeno , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Mitochondria are dynamic organelles that change their morphology by active fusion and fission in response to cellular signaling and differentiation. The in vivo role of mitochondrial fission in mammals has been examined by using tissue-specific knockout (KO) mice of the mitochondria fission-regulating GTPase Drp1, as well as analyzing a human patient harboring a point mutation in Drp1, showing that Drp1 is essential for embryonic and neonatal development and neuronal function. During oocyte maturation and aging, structures of various membrane organelles including mitochondria and the endoplasmic reticulum (ER) are changed dynamically, and their organelle aggregation is related to germ cell formation and epigenetic regulation. However, the underlying molecular mechanisms of organelle dynamics during the development and aging of oocytes have not been well understood. Here, we analyzed oocyte-specific mitochondrial fission factor Drp1-deficient mice and found that mitochondrial fission is essential for follicular maturation and ovulation in an age-dependent manner. Mitochondria were highly aggregated with other organelles, such as the ER and secretory vesicles, in KO oocyte, which resulted in impaired Ca(2+) signaling, intercellular communication via secretion, and meiotic resumption. We further found that oocytes from aged mice displayed reduced Drp1-dependent mitochondrial fission and defective organelle morphogenesis, similar to Drp1 KO oocytes. On the basis of these findings, it appears that mitochondrial fission maintains the competency of oocytes via multiorganelle rearrangement.
Asunto(s)
Dinaminas/metabolismo , Regulación de la Expresión Génica/fisiología , Células de la Granulosa/fisiología , Oocitos/fisiología , Folículo Ovárico/fisiología , Ovulación/fisiología , Envejecimiento , Animales , Calcio , Dinaminas/genética , Femenino , Fertilidad , Células de la Granulosa/citología , Humanos , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Mitocondrias/fisiología , Oocitos/citologíaRESUMEN
Mammalian cells typically contain thousands of copies of mitochondrial DNA assembled into hundreds of nucleoids. Here we analyzed the dynamic features of nucleoids in terms of mitochondrial membrane dynamics involving balanced fusion and fission. In mitochondrial fission GTPase dynamin-related protein (Drp1)-deficient cells, nucleoids were enlarged by their clustering within hyperfused mitochondria. In normal cells, mitochondrial fission often occurred adjacent to nucleoids, since localization of Mff and Drp1 is dependent on the nucleoids. Thus, mitochondrial fission adjacent to nucleoids should prevent their clustering by maintaining small and fragmented nucleoids. The enhanced clustering of nucleoids resulted in the formation of highly stacked cristae structures in enlarged bulb-like mitochondria (mito-bulbs). Enclosure of proapoptotic factor cytochrome c, but not of Smac/DIABLO, into the highly stacked cristae suppressed its release from mitochondria under apoptotic stimuli. In the absence of nucleoids, Drp1 deficiency failed to form mito-bulbs and to protect against apoptosis. Thus, mitochondrial dynamics by fission and fusion play a critical role in controlling mitochondrial nucleoid structures, contributing to cristae reformation and the proapoptotic status of mitochondria.
Asunto(s)
Citocromos c/metabolismo , Mitocondrias/ultraestructura , Dinámicas Mitocondriales/fisiología , Membranas Mitocondriales/fisiología , Apoptosis/efectos de los fármacos , Benzotiazoles , Diaminas , Dinaminas/deficiencia , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Immunoblotting , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Compuestos Orgánicos , Consumo de Oxígeno , Quinolinas , Interferencia de ARN , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Imagen de Lapso de TiempoRESUMEN
In yeast, C-tail-anchored mitochondrial outer membrane protein Fis1 recruits the mitochondrial-fission-regulating GTPase Dnm1 to mitochondrial fission sites. However, the function of its mammalian homologue remains enigmatic because it has been reported to be dispensable for the mitochondrial recruitment of Drp1, a mammalian homologue of Dnm1. We identified TBC1D15 as a Fis1-binding protein in HeLa cell extracts. Immunoprecipitation revealed that Fis1 efficiently interacts with TBC1D15 but not with Drp1. Bacterially expressed Fis1 and TBC1D15 formed a direct and stable complex. Exogenously expressed TBC1D15 localized mainly in cytoplasm in HeLa cells, but when coexpressed with Fis1 it localized to mitochondria. Knockdown of TBC1D15 induced highly developed mitochondrial network structures similar to the effect of Fis1 knockdown, suggesting that the TBC1D15 and Fis1 are associated with the regulation of mitochondrial morphology independently of Drp1. These data suggest that Fis1 acts as a mitochondrial receptor in the recruitment of mitochondrial morphology protein in mammalian cells.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Activadoras de GTPasa/genética , Células HeLa , Humanos , Inmunoprecipitación , Proteínas de la Membrana/genética , Microscopía Fluorescente , Proteínas Mitocondriales/genética , Unión Proteica/genética , Unión Proteica/fisiología , Interferencia de ARNRESUMEN
LAPTM5 (lysosomal-associated protein multispanning transmembrane 5) is a membrane protein on the intracellular vesicles. We have previously demonstrated that the accumulation of LAPTM5-positive vesicles was closely associated with the programmed cell death occurring during the spontaneous regression of neuroblastomas. Although the accumulation of LAPTM5 protein might occur at the post-translational level, the molecular mechanism has been unclear. Here, we found that the level of LAPTM5 protein is regulated negatively by the degradation through ubiquitination by ITCH, an E3 ubiquitin ligase. ITCH directly binds to the PPxY motif of LAPTM5 via its WW domains and promotes ubiquitination through a HECT-type ligase domain. Overexpression of ITCH led to the degradation of LAPTM5 protein, and conversely, knockdown of ITCH by siRNA resulted in the stabilization of LAPTM5 protein. In addition, the inhibition of ITCH enhanced the cell death occurred by accumulation of LAPTM5 in neuroblastoma cells. These findings suggest that LAPTM5 is a novel substrate in terms of degradation by the ubiquitin ligase ITCH, and this system might act as a negative regulator in the spontaneous regression of neuroblastomas by preventing LAPTM5-mediated cell death.