WD40 protein Mda1 is purified with Dnm1 and forms a dividing ring for mitochondria before Dnm1 in Cyanidioschyzon merolae

Mitochondria are not produced de novo but are maintained by division. Mitochondrial division is a coordinated process of positioning and constriction of the division site and fission of double membranes, in which dynamin-related protein is believed to mediate outer membrane fission. Part of the mitochondrial division machinery was purified from M phase-arrested Cyanidioschyzon merolae cells through biochemical fractionation. The dynamin-related protein Dnm1 was one of the two major proteins in the purified fraction and was accompanied by a newly identified protein CMR185C, named Mda1. Mda1 contained a predictable coiled-coil region and WD40 repeats, similarly to Mdv1 and Caf4 in yeasts. Immunofluorescence and immunoelectron microscopy showed that Mda1 localizes as a medial belt or ring on the mitochondrial outer surface throughout the division. The ring formation of Mda1 followed the plane of the ring of FtsZ, a protein that resides in the matrix. Dnm1 consistently colocalized with Mda1 only in the late stages of division. Mda1 protein was expressed through S to M phases and was phosphorylated specifically in M phase when Mda1 transformed from belt into foci and became colocalizing with Dnm1. Dephosphorylation of Mda1 in vitro increased its sedimentation coefficient, suggesting conformational changes of the macromolecule. Disassembly of the purified mitochondrial division machinery was performed by adding GTP to independently release Dnm1, suggesting that Mda1 forms a stable homo-oligomer by itself as a core structure of the mitochondrial division machinery.     

The membrane remodeling protein Pex11p activates the GTPase Dnm1p during peroxisomal fission

The initial phase of peroxisomal fission requires the peroxisomal membrane protein Peroxin 11 (Pex11p), which remodels the membrane, resulting in organelle elongation. Here, we identify an additional function for Pex11p, demonstrating that Pex11p also plays a crucial role in the final step of peroxisomal fission: dynamin-like protein (DLP)-mediated membrane scission. First, we demonstrate that yeast Pex11p is necessary for the function of the GTPase Dynamin-related 1 (Dnm1p) in vivo. In addition, our data indicate that Pex11p physically interacts with Dnm1p and that inhibiting this interaction compromises peroxisomal fission. Finally, we demonstrate that Pex11p functions as a GTPase activating protein (GAP) for Dnm1p in vitro. Similar observations were made for mammalian Pex11β and the corresponding DLP Drp1, indicating that DLP activation by Pex11p is conserved. Our work identifies a previously unknown requirement for a GAP in DLP function.Peroxisomes are ubiquitous, single-membrane–bounded cell organelles that harbor enzymes involved in a large number of metabolic processes. Common functions are the β-oxidation of fatty acids and hydrogen peroxide metabolism. Specialized functions include the metabolism of various carbon and organic nitrogen sources in fungi and the production of plasmalogens and bile acids in mammals, to name but a few (1). Their importance is underlined by the severe, often lethal human disorders caused by defects in peroxisome biogenesis or metabolism (2). Importantly, defects in peroxisome multiplication, caused by mutations in genes that control peroxisome fission, also result in severe human disorders (3, 4).Based on data from yeast and mammals, the current model for peroxisomal fission describes a three-step process, consisting of (i) organelle elongation, (ii) constriction, and (iii) the actual scission step (5–7). So far, Peroxin 11 (Pex11p), a highly conserved and abundant peroxisomal membrane protein, is the only protein known to play a crucial role in the first step (8). Its vital role in peroxisome multiplication is illustrated by the observation that in all organisms studied so far, Pex11p overproduction results in enhanced peroxisome proliferation, whereas PEX11 deletion causes a decrease in number, together with an increase in peroxisome size (8). The function of Pex11p in organelle elongation is mediated by the extreme N-terminal region of Pex11p, which can adopt the structure of an amphipathic helix, which upon insertion into membranes induces their curvature, resulting in organelle tubulation (9).The molecular mechanisms of peroxisome constriction are poorly understood. In contrast, several proteins required for the final stage of the fission process are known. The first protein shown to be involved in this process was Saccharomyces cerevisiae Vps1p, a dynamin-like protein (DLP) (10). Later studies revealed that in this organism the DLP Dynamin-related 1 (Dnm1p) is also involved in peroxisome fission, especially under peroxisome-inducing growth conditions (11). Dnm1p forms a fission machinery together with the tail-anchored fission protein Fis1p and (in S. cerevisiae) the accessory proteins Mdv1p and Caf4p (12). Interestingly these proteins are also responsible for mitochondrial fission in yeast (13).Dnm1p (Drp1 in mammals) (11, 14) is a large GTPase that achieves membrane fission by forming oligomeric, ring-like structures around constricted sites on organelle membranes (15). Powered by GTP hydrolysis, these ring-like structures then tighten further until the membrane severs. Interestingly, Dnm1p is recruited to Pex11p-enriched elongated peroxisomal membranes, suggesting that Pex11p and Dnm1p are functionally linked (16, 17).Here, we identify a previously unknown role for Pex11p in peroxisomal fission. We show that Pex11p directly interacts with Dnm1p and that this interaction stimulates the GTPase activity of Dnm1p, establishing Pex11p as a GTPase activating protein (GAP) that plays a crucial role in the last step of the peroxisome fission process.     

Pink1 regulates the oxidative phosphorylation machinery via mitochondrial fission

Mutations in PTEN-induced kinase 1 (PINK1), a mitochondrial Ser/Thr kinase, cause an autosomal recessive form of Parkinson''s disease (PD), PARK6. To investigate the mechanism of PINK1 pathogenesis, we used the Drosophila Pink1 knockout (KO) model. In mitochondria isolated from Pink1-KO flies, mitochondrial respiration driven by the electron transport chain (ETC) is significantly reduced. This reduction is the result of a decrease in ETC complex I and IV enzymatic activity. As a consequence, Pink1-KO flies also display a reduced mitochondrial ATP synthesis. Because mitochondrial dynamics is important for mitochondrial function and Pink1-KO flies have defects in mitochondrial fission, we explored whether fission machinery deficits underlie the bioenergetic defect in Pink1-KO flies. We found that the bioenergetic defects in the Pink1-KO can be ameliorated by expression of Drp1, a key molecule in mitochondrial fission. Further investigation of the ETC complex integrity in wild type, Pink1-KO, PInk1-KO/Drp1 transgenic, or Drp1 transgenic flies indicates that the reduced ETC complex activity is likely derived from a defect in the ETC complex assembly, which can be partially rescued by increasing mitochondrial fission. Taken together, these results suggest a unique pathogenic mechanism of PINK1 PD: The loss of PINK1 impairs mitochondrial fission, which causes defective assembly of the ETC complexes, leading to abnormal bioenergetics.     

Pink1 regulates mitochondrial dynamics through interaction with the fission/fusion machinery

Mitochondria form dynamic tubular networks that undergo frequent morphological changes through fission and fusion, the imbalance of which can affect cell survival in general and impact synaptic transmission and plasticity in neurons in particular. Some core components of the mitochondrial fission/fusion machinery, including the dynamin-like GTPases Drp1, Mitofusin, Opa1, and the Drp1-interacting protein Fis1, have been identified. How the fission and fusion processes are regulated under normal conditions and the extent to which defects in mitochondrial fission/fusion are involved in various disease conditions are poorly understood. Mitochondrial malfunction tends to cause diseases with brain and skeletal muscle manifestations and has been implicated in neurodegenerative diseases such as Parkinson's disease (PD). Whether abnormal mitochondrial fission or fusion plays a role in PD pathogenesis has not been shown. Here, we show that Pink1, a mitochondria-targeted Ser/Thr kinase linked to familial PD, genetically interacts with the mitochondrial fission/fusion machinery and modulates mitochondrial dynamics. Genetic manipulations that promote mitochondrial fission suppress Drosophila Pink1 mutant phenotypes in indirect flight muscle and dopamine neurons, whereas decreased fission has opposite effects. In Drosophila and mammalian cells, overexpression of Pink1 promotes mitochondrial fission, whereas inhibition of Pink1 leads to excessive fusion. Our genetic interaction results suggest that Fis1 may act in-between Pink1 and Drp1 in controlling mitochondrial fission. These results reveal a cell biological role for Pink1 and establish mitochondrial fission/fusion as a paradigm for PD research. Compounds that modulate mitochondrial fission/fusion could have therapeutic value in PD intervention.     

Atypical mitochondrial fission upon bacterial infection

We recently showed that infection by Listeria monocytogenes causes mitochondrial network fragmentation through the secreted pore-forming toxin listeriolysin O (LLO). Here, we examine factors involved in canonical fusion and fission. Strikingly, LLO-induced mitochondrial fragmentation does not require the traditional fission machinery, as Drp1 oligomers are absent from fragmented mitochondria following Listeria infection or LLO treatment, as the dynamin-like protein 1 (Drp1) receptor Mff is rapidly degraded, and as fragmentation proceeds efficiently in cells with impaired Drp1 function. LLO does not cause processing of the fusion protein optic atrophy protein 1 (Opa1), despite inducing a decrease in the mitochondrial membrane potential, suggesting a unique Drp1- and Opa1-independent fission mechanism distinct from that triggered by uncouplers or the apoptosis inducer staurosporine. We show that the ER marks LLO-induced mitochondrial fragmentation sites even in the absence of functional Drp1, demonstrating that the ER activity in regulating mitochondrial fission can be induced by exogenous agents and that the ER appears to regulate fission by a mechanism independent of the canonical mitochondrial fission machinery.Mitochondria are essential organelles that perform a multitude of functions, ranging from the production of biosynthetic intermediates and energy to innate immune signaling and cellular calcium buffering or the storage of proapoptotic components (1). To perform these diverse functions, mitochondria respond to cellular cues and display a highly variable and dynamic morphology, constantly undergoing fusion and fission. It is becoming increasingly clear that mitochondrial dynamics and function are deeply interconnected, and mitochondrial dysfunction is associated with a range of diseases.Wild-type mitochondrial morphology and function are maintained by a balance between mitochondrial fusion and fission. Fusion allows exchange of genetic material between single mitochondria and is mediated by two large guanosine triphosphate phosphohydrolases (GTPases) embedded in the outer membrane (mitofusin 1 and 2) and an inner membrane GTPase, Opa1 (2). Deletion mutants affecting these three proteins accumulate dysfunctional mitochondria, leading to neurodegenerative phenotypes and different forms of myopathy (1, 3).Mitochondrial fusion is balanced by fission, which is essential to ensure proper distribution of mitochondria and energy supply to daughter cells in mitosis or within a single cell. This necessity is particularly evident in neurons, where fission defects prevent efficient mitochondrial transport to synapses, the crucial sites of energy consumption (4, 5). The physiological importance of mitochondrial fission is further highlighted by its essential role in embryonic development in mice and nematodes (6–8).Mitochondrial fission is thought to be accomplished by the dynamin-like protein Drp1, a mainly cytosolic protein that is recruited to future fission sites, where it oligomerizes to form spirals that constrict mitochondria. Mitochondrial fission is regulated at several levels: by initial ER- and actin-mediated mitochondrial constriction (9, 10), leading to the accumulation of the membrane-bound Drp1 receptor Mff and by several posttranslational modifications of Drp1, which modulate its activity (11).Listeria monocytogenes is a foodborne pathogen capable of invading nonphagocytic cells, where it can replicate and spread. The pathogenic potential of L. monocytogenes correlates with the expression of several virulence genes (12). One of the most important virulence factors is listeriolysin O (LLO), a highly regulated secreted pore-forming toxin (reviewed in ref. 13). LLO belongs to the family of cholesterol-dependent cytolysins (CDCs), most of which are produced by extracellular bacteria such as Streptococci or Clostridia. CDCs oligomerize on cholesterol-containing membranes to form nonselective ion-permeable pores of variable sizes (14) that act in concert with bacterial phospholipases to allow bacterial escape from the phagosome. More recently, LLO has been found to have several intracellular and extracellular roles that extend beyond phagosomal escape. For example, we have shown that infection with L. monocytogenes causes fragmentation of the host mitochondrial network by action of its pore-forming toxin LLO before bacterial entry (15).In this study, we demonstrate that LLO-induced mitochondrial fragmentation does not follow canonical pathways, because it is independent of key fusion and fission components, such as Opa1 and Drp1. We demonstrate that the ER marks mitochondrial fragmentation sites even in the absence of functional Drp1, and that the actin cytoskeleton also facilitates fragmentation. LLO-induced fragmentation is distinct from that observed upon treatment with uncouplers [such as carbonyl cyanide m-chlorophenylhydrazone (CCCP)] and apoptosis inducers (such as staurosporine), revealing a unique pathway for mitochondrial fragmentation that can be induced by an exogenous agent.     

BIN1 modulation in vivo rescues dynamin-related myopathy

The mechanoenzyme dynamin 2 (DNM2) is crucial for intracellular organization and trafficking. DNM2 is mutated in dominant centronuclear myopathy (DNM2-CNM), a muscle disease characterized by defects in organelle positioning in myofibers. It remains unclear how the in vivo functions of DNM2 are regulated in muscle. Moreover, there is no therapy for DNM2-CNM to date. Here, we overexpressed human amphiphysin 2 (BIN1), a membrane remodeling protein mutated in other CNM forms, in Dnm2^RW/+ and Dnm2^RW/RW mice modeling mild and severe DNM2-CNM, through transgenesis or with adeno-associated virus (AAV). Increasing BIN1 improved muscle atrophy and main histopathological features of Dnm2^RW/+ mice and rescued the perinatal lethality and survival of Dnm2^RW/RW mice. In vitro experiments showed that BIN1 binds and recruits DNM2 to membrane tubules, and that the BIN1-DNM2 complex regulates tubules fission. Overall, BIN1 is a potential therapeutic target for dominant centronuclear myopathy linked to DNM2 mutations.

Membrane remodeling is a key process for intracellular organization and intercellular communication. A number of proteins regulating this process are mutated in human diseases. We focus on centronuclear myopathy due to mutations in the large GTPase dynamin 2 (DNM2), the first protein shown to catalyze membrane fission (1, 2). The pathological implication of DNM2 is unclear and there is no therapy to date for this disease. Here we validated an approach that ameliorated the disease in a mouse model and investigated the pathological and rescue mechanisms.DNM2 is a mechanoenzyme implicated mainly in vesicle budding in endocytosis and recycling and in cytoskeleton organization (1, 3). Upon membrane binding DNM2 oligomerizes around the neck of nascent vesicles, and the increase in the GTPase activity correlates with membrane fission. Several SH3 (Src homology 3) containing proteins as endophilins or amphiphysins can bind to the proline-rich domain (PRD) of dynamins. Among these, BIN1 is a N-BAR (N-terminal amphipathic helix Bin Amphiphysin Rvs) domain protein sensing and promoting membrane curvature and tubulation (4, 5). BAR-SH3 proteins recruit dynamins to membranes and promote their functions at specific sites (6). Recent findings suggested endophilin structurally inhibits dynamin-mediated membrane fission (7). However, it remains unclear how BAR proteins modulate dynamins activity and functions, especially in vivo.Centronuclear myopathies (CNMs) are rare congenital myopathies linked to muscle weakness, hypotonia, and muscle atrophy correlated with hypotrophic muscle fibers and mislocalized or altered organelles as nuclei, mitochondria, and triads (8, 9). Apart from DNM2, loss-of-function mutations in the membrane remodeling protein amphiphysin 2 (BIN1; MIM [Mendelian Inheritance in Man]#255200) (10), in the lipid phosphatase myotubularin (MTM1; MIM#310400) (11) and in the triad calcium channel ryanodine receptor (RYR1; MIM#117000) (12–14), lead to CNMs. The incidence of CNM is about 24 per million births, leading to a calculated prevalence for DNM2-CNM of about 550 in total for the European Union, the United States, Australia, and Japan (15). Heterozygous DNM2 mutations cause dominant CNMs (MIM#160150), ranging from severe muscle involvement with neonatal onset to mild phenotype with adult onset, partly correlating with the site of mutation (16). A homozygous DNM2 mutation was reported in patients with recessive lethal congenital contracture syndrome (LCCS5; MIM#615368) while their heterozygous parents displayed a mild CNM (17). Several lines of evidence suggest that DNM2-CNM mutations are gain of function. They increase the GTPase activity and oligomer stability in vitro (18, 19). Moreover, in vivo, overexpression of wild-type (WT) DNM2 cause a CNM-like phenotype in mice (20, 21). Human and mouse genetics suggested that the BIN1-DNM2 complex is important for skeletal muscle. In particular, some BIN1-CNM mutations abrogate the binding to DNM2 while others decrease the membrane tubulation properties of BIN1 (10). Moreover, the perinatal lethality of Bin1^−/− mice was rescued by decreasing Dnm2 levels to 50% (22).BIN1 overexpression was shown to rescue MTM1-CNM in mice (23). Here, we hypothesize that modulating BIN1 can rescue the muscular phenotypes due to DNM2-CNM mutations. As a DNM2-CNM model, we selected the Dnm2^RW/+ mouse that mimics the most common CNM mutation, R465W, in the middle/stalk domain, and displays a mild muscle weakness with decreased muscle force, muscle atrophy due to myofiber hypotrophy, and central accumulation of oxidative staining from 2 mo of age (24). Dnm2^RW/+ mice do not display centralized nuclei in myofibers unlike patients; albeit myonuclei domains seem altered (25). The homozygous Dnm2^RW/RW mice developed a more severe phenotype and died at birth potentially from hypoglycemia and altered neonatal autophagy (25).In this study, we overexpressed human BIN1 in Dnm2^RW/+ and Dnm2^RW/RW mice, which model mild and severe forms of DNM2-CNM, respectively. Of note, we validate the proof of concept that increasing BIN1 rescues DNM2-CNM and decipher the regulation of the BIN1-DNM2 complex in vitro and in vivo.     

U50，488H抑制心肌细胞缺氧/复氧诱导Drp1线粒体转位的作用及机制

目的 研究κ-阿片受体（κ-OR）激动剂U50,488H对缺氧/复氧（H/R）原代心肌细胞Drp1线粒体转位的作用与机制。 方法 将心肌细胞共分为6组,分别为常氧（Control）组、H/R组、H/R+U50,488H组、Control+Scramble RNAi组、H/R+Scramble RNAi组、H/R+Mid51 RNAi组。利用CCK-8试剂盒检测细胞活力;AnnexinV、PI试剂盒检测细胞凋亡;采用激光共聚焦显微镜观察线粒体的形态变化和Drp1与线粒体共定位情况,Western blotting检测细胞内线粒体外膜Drp1相关结合蛋白（Fis1、Mff、Mid49和Mid51）的表达水平。 结果 与常氧组相比,H/R组细胞凋亡与死亡率明显增加（P<0.01）,线粒体分裂明显增加（P<0.01）,Drp1的线粒体转位明显增多,Mid51表达显著上调（P<0.01）,但Fis1、Mff与Mid49的表达无明显差异;而κ-OR激动剂U50,488H可降低H/R后细胞凋亡与死亡率（P<0.01）,抑制线粒体分裂（P<0.01）,减少Drp1与线粒体的结合同时下调Mid51的表达水平（P<0.01）,但Fis1、Mff与Mid49的表达无明显变化;进一步研究发现,敲低Mid51可降低H/R后细胞凋亡与死亡率（P<0.01,P<0.05）,抑制线粒体的分裂（P<0.01）,减少Drp1的线粒体的转位。 结论 H/R可引起线粒体分裂与心肌细胞损伤,激活κ-OR可通过下调Mid51抑制Drp1的线粒体转位,进而抑制线粒体分裂,从而减轻H/R损伤。     

Integrity of the yeast mitochondrial genome,but not its distribution and inheritance,relies on mitochondrial fission and fusion

Mitochondrial DNA (mtDNA) is essential for mitochondrial and cellular function. In Saccharomyces cerevisiae, mtDNA is organized in nucleoprotein structures termed nucleoids, which are distributed throughout the mitochondrial network and are faithfully inherited during the cell cycle. How the cell distributes and inherits mtDNA is incompletely understood although an involvement of mitochondrial fission and fusion has been suggested. We developed a LacO-LacI system to noninvasively image mtDNA dynamics in living cells. Using this system, we found that nucleoids are nonrandomly spaced within the mitochondrial network and observed the spatiotemporal events involved in mtDNA inheritance. Surprisingly, cells deficient in mitochondrial fusion and fission distributed and inherited mtDNA normally, pointing to alternative pathways involved in these processes. We identified such a mechanism, where we observed fission-independent, but F-actin–dependent, tip generation that was linked to the positioning of mtDNA to the newly generated tip. Although mitochondrial fusion and fission were dispensable for mtDNA distribution and inheritance, we show through a combination of genetics and next-generation sequencing that their absence leads to an accumulation of mitochondrial genomes harboring deleterious structural variations that cluster at the origins of mtDNA replication, thus revealing crucial roles for mitochondrial fusion and fission in maintaining the integrity of the mitochondrial genome.Mitochondrial DNA (mtDNA) is essential for respiratory growth of all eukaryotic cells, and all multicellular organisms depend on mtDNA for their development. Not surprisingly, given the fundamental importance of mtDNA, mutations within mtDNA have been identified as the cause for a plethora of human diseases (1). mtDNA in Saccharomyces cerevisiae encodes for seven essential subunits of the respiratory chain, one protein and two RNA subunits of the mitochondrial ribosome, 24 tRNAs, and the RNA subunit of RNase P (2). Every cell contains 50–100 copies of mtDNA that are organized into nucleoprotein complexes termed nucleoids, each containing 1–10 copies of mtDNA (3, 4). Nucleoids are distributed throughout the mitochondrial network, which is likely important for equivalently supplying spatially separated mitochondrial segments with mitochondrially encoded proteins.How the distribution of mtDNA throughout the mitochondrial network is established and maintained is not fully understood. Previous work from our laboratory and others has shown that the movement of nucleoids within the mitochondrial network is limited, suggesting that the mechanisms of nucleoid distribution are tightly interlinked with the dynamics of mitochondria themselves (5, 6). Mitochondria undergo constant fusion and fission events that are mediated by dedicated machineries, with the central components Fzo1 and Dnm1 required for fusion and fission, respectively (7). Recently, we have provided support for a role of mitochondrial fission in mtDNA distribution. We have shown that mtDNA localizes to sites of Dnm1-dependent mitochondrial fission and that it is segregated after scission to both of the newly generated mitochondrial tips (8). Localizing mtDNA to the newly formed tips would then allow transport of mitochondrial tips and mtDNA to distal parts in the cell, where fusion with the mitochondrial network may drive mtDNA distribution. Such a mechanism would be particularly important during inheritance of mtDNA to daughter buds during cell division, where mtDNA needs to be transported over a relatively large distance. In S. cerevisiae, mitochondria are inherited in a myosin- and F-actin–dependent process, in which a mitochondrial tubule invades the budding daughter cell and is subsequently anchored at the distal membrane (9). An active mtDNA partition and inheritance apparatus has been postulated (6); however, the spatiotemporal relationship between the inheritance of mitochondria and the inheritance of mtDNA has not been examined.If mitochondrial fusion and fission were essential for the distribution and inheritance of mtDNA, their loss would impair the process. Indeed, fusion-defective cells lose mtDNA (10, 11), most likely due to excessive fragmentation. By contrast, however, fission-defective cells, as well as cells defective in fusion and fission, remain capable of respiratory growth, indicating that a functional mitochondrial genome must be maintained (10, 12). These observations suggest that fission-independent mechanisms must exist that facilitate mtDNA inheritance.In this work, we investigated the role of mitochondrial fusion and fission in mtDNA distribution and inheritance. Through the development of a noninvasive method to quantify the spatial organization of mtDNA within mitochondrial tubules, we found that cells deficient in fusion and fission maintain a WT distribution of mtDNA. Live-cell imaging showed that this distribution is facilitated by the de novo generation of tubules from the sides of existing tubules, a process coupled to the spatial positioning of mtDNA to the newly formed tip. Unexpectedly, although dispensable for maintaining mtDNA distribution and inheritance, fusion and fission were required to maintain the integrity of the mitochondrial genome.     

A conserved MutS homolog connector domain interface interacts with MutL homologs

Escherichia coli MutS forms a mispair-dependent ternary complex with MutL that is essential for initiating mismatch repair (MMR) but is structurally uncharacterized, in part owing to its dynamic nature. Here, we used hydrogen/deuterium exchange mass spectrometry and other methods to identify a region in the connector domain (domain II) of MutS that binds MutL and is required for mispair-dependent ternary complex formation and MMR. A structurally conserved region in Msh2, the eukaryotic homolog, was required for formation of a mispair-dependent Msh2–Msh6–Mlh1–Pms1 ternary complex. These data indicate that the connector domain of MutS and Msh2 contains the interface for binding MutL and Mlh1–Pms1, respectively, and support a mechanism whereby mispair and ATP binding induces a conformational change that allows the MutS and Msh2 interfaces to interact with their partners.     

Melatonin prevents Drp1‐mediated mitochondrial fission in diabetic hearts through SIRT1‐PGC1α pathway

Myocardial contractile dysfunction is associated with an increase in mitochondrial fission in patients with diabetes. However, whether mitochondrial fission directly promotes diabetes‐induced cardiac dysfunction is still unknown. Melatonin exerts a substantial influence on the regulation of mitochondrial fission/fusion. This study investigated whether melatonin protects against diabetes‐induced cardiac dysfunction via regulation of mitochondrial fission/fusion and explored its underlying mechanisms. Here, we show that melatonin prevented diabetes‐induced cardiac dysfunction by inhibiting dynamin‐related protein 1 (Drp1)‐mediated mitochondrial fission. Melatonin treatment decreased Drp1 expression, inhibited mitochondrial fragmentation, suppressed oxidative stress, reduced cardiomyocyte apoptosis, improved mitochondrial function and cardiac function in streptozotocin (STZ )‐induced diabetic mice, but not in SIRT 1^?/? diabetic mice. In high glucose‐exposed H9c2 cells, melatonin treatment increased the expression of SIRT 1 and PGC ‐1α and inhibited Drp1‐mediated mitochondrial fission and mitochondria‐derived superoxide production. In contrast, SIRT 1 or PGC ‐1α siRNA knockdown blunted the inhibitory effects of melatonin on Drp1 expression and mitochondrial fission. These data indicated that melatonin exerted its cardioprotective effects by reducing Drp1‐mediated mitochondrial fission in a SIRT 1/PGC ‐1α‐dependent manner. Moreover, chromatin immunoprecipitation analysis revealed that PGC ‐1α directly regulated the expression of Drp1 by binding to its promoter. Inhibition of mitochondrial fission with Drp1 inhibitor mdivi‐1 suppressed oxidative stress, alleviated mitochondrial dysfunction and cardiac dysfunction in diabetic mice. These findings show that melatonin attenuates the development of diabetes‐induced cardiac dysfunction by preventing mitochondrial fission through SIRT 1‐PGC 1α pathway, which negatively regulates the expression of Drp1 directly. Inhibition of mitochondrial fission may be a potential target for delaying cardiac complications in patients with diabetes.     

Impaired complex IV activity in response to loss of LRPPRC function can be compensated by mitochondrial hyperfusion

Mitochondrial morphology changes in response to various stimuli but the significance of this is unclear. In a screen for mutants with abnormal mitochondrial morphology, we identified MMA-1, the Caenorhabditis elegans homolog of the French Canadian Leigh Syndrome protein LRPPRC (leucine-rich pentatricopeptide repeat containing). We demonstrate that reducing mma-1 or LRPPRC function causes mitochondrial hyperfusion. Reducing mma-1/LRPPRC function also decreases the activity of complex IV of the electron transport chain, however without affecting cellular ATP levels. Preventing mitochondrial hyperfusion in mma-1 animals causes larval arrest and embryonic lethality. Furthermore, prolonged LRPPRC knock-down in mammalian cells leads to mitochondrial fragmentation and decreased levels of ATP. These findings indicate that in a mma-1/LRPPRC–deficient background, hyperfusion allows mitochondria to maintain their functions despite a reduction in complex IV activity. Our data reveal an evolutionary conserved mechanism that is triggered by reduced complex IV function and that induces mitochondrial hyperfusion to transiently compensate for a drop in the activity of the electron transport chain.Mitochondria are dynamic organelles that frequently fuse and divide, and their steady-state morphology is determined by the relative rates of fusion and fission (1–5). Mitochondrial fusion and fission are under the control of a conserved family of GTPases, the dynamin-related proteins (DRPs). For example, mammalian Drp1 or Caenorhabditis elegans DRP-1 are required for mitochondrial fission (6, 7). Conversely, mammalian Mfn1/2 and Opa1 or C. elegans FZO-1 and EAT-3 are required for the fusion of the outer and inner mitochondrial membrane, respectively (8–13).Mitochondrial morphology can change in response to various cellular stimuli, including metabolic signals. Electron microscopic studies revealed that the ultrastructure of mitochondria changes depending on their rate of oxidative phosphorylation. For example, inner mitochondrial membrane cristae change from an “orthodox” to a “condensed” conformation in response to increased oxidative phosphorylation [i.e., increased activity of the electron transport chain (ETC)] (14). Furthermore, there is evidence for a bidirectional, functional link between the activity of the ETC and mitochondrial dynamics; that is, the ability of mitochondria to fuse and divide and, hence, change their steady-state morphology. On the one hand, mutations that reduce the activities of specific complexes of the ETC affect mitochondrial morphology and cause mitochondrial fragmentation. For example, fibroblasts from patients with defects in complexes I, III, or IV have fragmented mitochondria (15–17). Similarly, pharmacological inhibition of any of the ETC complexes in primary human fibroblasts and rat cortical neurons leads to mitochondrial fragmentation (18, 19). On the other hand, mutations that impair mitochondrial dynamics lead to defects in oxidative phosphorylation. For example, inactivation in HeLa cells of Drp1, which is required for mitochondrial fission, causes a decrease in mitochondrial membrane potential, respiration, and cellular ATP (20). Similarly, primary skin cells derived from patients with Optic Dominant Atrophy, who carry mutations in the mitochondrial fusion protein Opa1, exhibit reduced levels of complex IV subunits, resulting in decreased complex IV activity (21). Furthermore, the inactivation of the mitochondrial fusion protein Mfn2 in muscle cells grown in culture causes a reduction in the levels of subunits of several ETC complexes, as well as a decrease in mitochondrial membrane potential and respiration (22).However, rather than fragmenting, mitochondria have recently also been shown to undergo a process referred to as “hyperfusion.” Specifically, in response to different forms of stress, such as the inhibition of cytosolic protein synthesis or starvation, mitochondria in cultured mammalian cells form large, hyperfused mitochondria (23–26). This hyperfusion appears to be mediated through the activation of the mitochondrial fusion proteins Mfn and Opa1, or the inactivation of the mitochondrial fission protein Drp1. In the case of mitochondrial hyperfusion in response to the inhibition of cytosolic protein synthesis, cells with hyperfused mitochondria have higher levels of cellular ATP than control cells (23). In the case of mitochondrial hyperfusion in response to starvation, cells with hyperfused mitochondria maintain normal levels of cellular ATP despite starvation conditions (24). Therefore, hyperfused mitochondria are likely to produce ATP more efficiently. It has been proposed that mitochondrial hyperfusion is a prosurvival response to various forms of stress because cells defective in mitochondrial hyperfusion are more sensitive to stress and undergo apoptosis (23, 24).The mammalian LRPPRC gene is mutated in patients that suffer from French Canadian Leigh Syndrome, a neurodegenerative disease associated with complex IV deficiency (27). LRPPRC encodes a leucine-rich pentatricopeptide repeat containing protein that is targeted to the mitochondrial matrix (28). In the mitochondrial matrix, LRPPRC forms a ribonucleoprotein complex with the stem-loop RNA binding protein SLIRP and mitochondrial mRNAs (17, 29). The LRPPRC/SLIRP complex activates the mitochondrial poly(A) polymerase MTPAP, and thereby promotes the polyadenylation of mitochondrial mRNAs (29, 30). LRPPRC/SLIRP/MTPAP-dependent polyadenylation stabilizes mitochondrial mRNAs, such as COXI and COXII, which encode two subunits of complex IV (29, 30). In addition, LRPPRC plays an important role in the control of mitochondrial translation because its inactivation leads to abnormal patterns of mitochondrial translation, specifically resulting in complex IV deficiency (29).Whether a homolog of mammalian LRPPRC exists in C. elegans has been unclear (28). We now report that C. elegans mma-1 encodes a structural and functional homolog of LRPPRC. Furthermore, we show that reducing the function of mma-1 in C. elegans or LRPPRC in mammalian cells causes a reduction in the level of complex IV of the ETC but not in the level of cellular ATP. In addition, we provide evidence that essential mitochondrial functions are maintained through mitochondrial hyperfusion, which is triggered by reducing complex IV activity. Our results reveal an evolutionary conserved compensatory mechanism that is induced by a deficiency in a specific complex of the ETC, complex IV, and whose role is to trigger mitochondrial hyperfusion to maintain mitochondrial function. Finally, we show that this compensatory mechanism is effective only transiently and that prolonged reduction of LRPPRC/mma-1 function causes a collapse of cellular functions.     

Amyloid-β overproduction causes abnormal mitochondrial dynamics via differential modulation of mitochondrial fission/fusion proteins

Mitochondrial dysfunction is a prominent feature of Alzheimer disease but the underlying mechanism is unclear. In this study, we investigated the effect of amyloid precursor protein (APP) and amyloid β on mitochondrial dynamics in neurons. Confocal and electron microscopic analysis demonstrated that ≈40% M17 cells overexpressing WT APP (APPwt M17 cells) and more than 80% M17 cells overexpressing APPswe mutant (APPswe M17 cells) displayed alterations in mitochondrial morphology and distribution. Specifically, mitochondria exhibited a fragmented structure and an abnormal distribution accumulating around the perinuclear area. These mitochondrial changes were abolished by treatment with β-site APP-cleaving enzyme inhibitor IV. From a functional perspective, APP overexpression affected mitochondria at multiple levels, including elevating reactive oxygen species levels, decreasing mitochondrial membrane potential, and reducing ATP production, and also caused neuronal dysfunction such as differentiation deficiency upon retinoic acid treatment. At the molecular level, levels of dynamin-like protein 1 and OPA1 were significantly decreased whereas levels of Fis1 were significantly increased in APPwt and APPswe M17 cells. Notably, overexpression of dynamin-like protein 1 in these cells rescued the abnormal mitochondrial distribution and differentiation deficiency, but failed to rescue mitochondrial fragmentation and functional parameters, whereas overexpression of OPA1 rescued mitochondrial fragmentation and functional parameters, but failed to restore normal mitochondrial distribution. Overexpression of APP or Aβ-derived diffusible ligand treatment also led to mitochondrial fragmentation and reduced mitochondrial coverage in neuronal processes in differentiated primary hippocampal neurons. Based on these data, we concluded that APP, through amyloid β production, causes an imbalance of mitochondrial fission/fusion that results in mitochondrial fragmentation and abnormal distribution, which contributes to mitochondrial and neuronal dysfunction.     

Dynamin‐related protein 1‐mediated mitochondrial fission contributes to post‐traumatic cardiac dysfunction in rats and the protective effect of melatonin

Mechanical trauma (MT ) causes myocardial injury and cardiac dysfunction. However, the underlying mechanism remains largely unclear. This study investigated the role of mitochondrial dynamics in post‐traumatic cardiac dysfunction and the protective effects of melatonin. Adult male Sprague Dawley rats were subjected to 5‐minute rotations (200 revolutions at a rate of 40 rpm) to induce MT model. Melatonin was administrated intraperitoneally 5 minute after MT . Mitochondrial morphology, myocardial injury, and cardiac function were determined in vivo. There was smaller size of mitochondria and increased number of mitochondria per μm² in the hearts after MT when the secondary myocardial injury was induced. Melatonin treatment at the dose of 30 mg/kg reduced serine 616 phosphorylation of Drp1 and inhibited mitochondrial Drp1 translocation and mitochondrial fission in the hearts of rats subjected to MT , which contributed to the reduction of myocardial injury and the improvement of cardiac function. In vitro, H9c2 cells cultured in 20% traumatic plasma (TP ) for 12 hour showed enhanced mitochondrial fission, mitochondrial membrane potential (?Ψm) loss, mitochondrial cytochrome c release, and decreased mitochondrial complex I‐IV activities. Pretreatment with melatonin (100 μmol/L) efficiently inhibited TP ‐induced mitochondrial fission, ?Ψm loss, cytochrome c release, and improved mitochondrial function. Melatonin's protective effects were attributed to its role in suppressing plasma TNF ‐α overproduction, which was responsible for Drp1‐mediated mitochondrial fission. Taken together, our results demonstrate for the first time that abnormal mitochondrial dynamics is involved in post‐traumatic cardiac dysfunction. Melatonin has significant pharmacological potential in protecting against MT ‐induced cardiac dysfunction by preventing excessive mitochondrial fission.     

Structural mechanism of host Rab1 activation by the bifunctional Legionella type IV effector SidM/DrrA

Bacterial pathogens deliver effector proteins with diverse biochemical activities into host cells, thereby modulating various host functions. Legionella pneumophila hijacks host vesicle trafficking to avoid phagosome–lysosome fusion, a mechanism that is dependent on the Legionella Dot/Icm type IV secretion system. SidM/DrrA, a Legionella type IV effector, is important for the interactions of Legionella-containing vacuoles with host endoplasmic reticulum–derived vesicles. SidM is the only known protein that catalyzes both the exchange of GDP for GTP and GDI displacement from small GTPase Rab1. We determined the crystal structures of SidM alone (residues 317–647) and SidM (residues 193–550) in complex with nucleotide-free WT Rab1. The SidM structure contains an N-terminal helical domain with a potential new function, a Rab1-activation domain, and a C-terminal phosphatidylinositol 4-phosphate–binding P4M domain. The Rab1-activation domain has extensive strong interactions mainly with Rab1 switch I and II regions that undergo substantial conformational changes on SidM binding. Mutations of switch-contacting residues in SidM attenuate both the nucleotide exchange and GDI displacement activities. Structural comparisons of Rab1 in the SidM complex with Rab1-GDP and Ypt1-GDP in the GDI complex identify key conformational changes that disrupt the nucleotide and GDI binding of Rab1. Further biochemical and structural analyses reveal a unique mechanism of coupled GDP release and GDI displacement likely triggered by the SidM-induced drastic displacement of switch I of Rab1.     

A dynamin-like protein (ADL2b), rather than FtsZ, is involved in Arabidopsis mitochondrial division

Recently, the FtsZ protein, which is known as a key component in bacterial cell division, was reported to be involved in mitochondrial division in algae. In yeast and animals, however, mitochondrial fission depends on the dynamin-like proteins Dnm1p and Drp1, respectively, whereas in green plants, no potential mitochondrial division genes have been identified. BLAST searches of the nuclear and mitochondrial genome sequences of Arabidopsis thaliana did not find any obvious homologue of the alpha-proteobacterial-type ftsZ genes. To determine whether mitochondrial division of higher plants depends on a dynamin-like protein, we cloned a cDNA for ADL2b, an Arabidopsis homologue of Dnm1p, and tested its subcellular localization and its dominant-negative effect on mitochondrial division. The fusion protein of green fluorescent protein and ADL2b was observed as punctate structures localized at the tips and at the constriction sites of mitochondria in live plant cells. Cells expressing dominant-negative mutant ADL2b proteins (K56A and T77F) showed a significant fusion, aggregation, and/or tubulation of mitochondria. We propose that mitochondrial division in higher plants is conducted by dynamin-like proteins similar to ADL2b in Arabidopsis. The evolutional points of loss of mitochondrial FtsZ and the functional acquisition of dynamin-like proteins in mitochondrial division are discussed.     

Melatonin prevents the dynamin‐related protein 1‐dependent mitochondrial fission and oxidative insult in the cortical neurons after 1‐methyl‐4‐phenylpyridinium treatment

Mitochondrial dysfunction and oxidative stress are involved in the pathogenesis of Parkinson's disease (PD). Mitochondrial morphology is dynamic and precisely regulated by the mitochondrial fission and fusion machinery. Aberrant mitochondrial fragmentation controlled by the mitochondrial fission protein, dynamin‐related protein 1 (Drp1), may result in cell death. Our previous results showed that melatonin protected neurons by inhibiting oxidative stress in a 1‐methyl‐4‐phenylpyridinium (MPP⁺)‐induced PD model. However, the effect of melatonin on mitochondrial dynamics remains uncharacterized. Herein, we investigated the effect of melatonin and the role of Drp1 on MPP⁺‐induced mitochondrial fission in rat primary cortical neurons. We found that MPP⁺ induced a rapid increase in the ratio of GSSG:total glutathione (a marker of oxidative stress) and mitochondrial fragmentation, Drp1 upregulation within 4 hours, and finally resulted in neuron loss 48 hours after the treatment. Neurons overexpressing wild‐type Drp1 promoted mitochondrial and nuclear fragmentation; however, neurons overexpressing dominant‐negative Drp1^K38A or cotreated with melatonin exhibited significantly reduced MPP⁺‐induced mitochondrial fragmentation and neuron death. Moreover, melatonin cotreatment prevented an MPP⁺‐induced high ratio of GSSG and mitochondrial Drp1 upregulation. The prevention of mitochondrial fission by melatonin was not found in neurons transfected with wild‐type Drp1. These results provide a new insight that the neuroprotective effect of melatonin against MPP⁺ toxicity is mediated by inhibiting the oxidative stress and Drp1‐mediated mitochondrial fragmentation.     

Characterization of Bovine Foamy Virus Gag Late Assembly Domain Motifs and Their Role in Recruiting ESCRT for Budding

A large number of retroviruses, such as human immunodeficiency virus (HIV) and prototype foamy virus (PFV), recruit the endosomal sorting complex required for transport (ESCRT) through the late domain (L domain) on the Gag structural protein for virus budding. However, little is known about the molecular mechanism of bovine foamy virus (BFV) budding. In the present study, we report that BFV recruits ESCRT for budding through the L domain of Gag. Specifically, knockdown of VPS4 (encoding vacuolar protein sorting 4), ALIX (encoding ALG-2-interacting protein X), and TSG101 (encoding tumor susceptibility 101) indicated that BFV uses ESCRT for budding. Mutational analysis of BFV Gag (BGag) showed that, in contrast to the classical L domain motifs, BGag contains two motifs, P₅₆LPI and Y₁₀₃GPL, with L domain functions. In addition, the two L domains are necessary for the cytoplasmic localization of BGag, which is important for effective budding. Furthermore, we demonstrated that the functional site of Alix is V498 in the V domain and the functional site of Tsg101 is N69 in the UBC-like domain for BFV budding. Taken together, these results demonstrate that BFV recruits ESCRT for budding through the PLPI and YGPL L domain motifs in BGag.     

All the little pieces. -Regulation of mitochondrial fusion and fission by ubiquitin and small ubiquitin-like modifer and their potential relevance in the heart.-

Mitochondria are dynamic organelles that undergo a constant cycle of division and fusion to maintain their function. The process of mitochondrial fusion has the effect of mixing their content, allowing complementation of protein components, mtDNA repair, and distribution of metabolic intermediates. Fission, on the other hand, enables mitochondria to increase in number and capacity, and to segregate mitochondria for autophagy by the lysosome ("mitophagy"). Disruption of these protein quality control mechanisms has recently been identified in multiple cardiac diseases, including cardiac hypertrophy, heart failure, dilated cardiomyopathy, and ischemic heart disease, and is intimately tied to mitochondrial control of apoptosis. Proteins that regulate mitochondrial fusion and fission have been discovered, including Mfn1, Mfn2, and Opa1 (fusion) and Drp1 and Fis1 (fission). In this review, we discuss how these proteins are regulated by post-translational modification with ubiquitin and SUMO (small ubiquitin-like modifier). We then present what is known about the ubiquitin and SUMO ligases that regulate these post-translational modifications and regulation of mitochondrial fusion and fission, exploring their potential as therapeutic targets of cardiac disease.     

The Salmonella virulence protein SifA is a G protein antagonist

Salmonella's success at proliferating intracellularly and causing disease depends on the translocation of a major virulence protein, SifA, into the host cell. SifA recruits membranes enriched in lysosome associated membrane protein 1 (LAMP1) and is needed for growth of Salmonella induced filaments (Sifs) and the Salmonella containing vacuole (SCV). It directly binds a host protein called SKIP (SifA and kinesin interacting protein) which is critical for membrane stability and motor dynamics at the SCV. SifA also contains a WxxxE motif, predictive of G protein mimicry in bacterial effectors, but whether and how it mimics the action of a host G protein is not known. We show that SKIP's pleckstrin homology domain, which directly binds SifA, also binds to the late endosomal GTPase Rab9. Knockdown studies suggest that both SKIP and Rab9 function to maintain peripheral LAMP1 distribution in cells. The Rab9:SKIP interaction is GTP-dependent and is inhibited by SifA binding to the SKIP pleckstrin homology domain, suggesting that SifA may be a Rab9 antagonist. SifA:SKIP binding is significantly tighter than Rab9:SKIP binding and may thus allow SifA to bring SKIP to the SCV via SKIP's Rab9-binding site. Rab9 can measurably reverse SifA-dependent LAMP1 recruitment and the perinuclear location of the SCV in cells. Importantly, binding to SKIP requires SifA residues W197 and E201 of the conserved WxxxE signature sequence, leading to the speculation that bacterial G protein mimicry may result in G protein antagonism.