Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization

Mitochondrial fusion and division play important roles in the regulation of apoptosis. Mitochondrial fusion proteins attenuate apoptosis by inhibiting release of cytochrome c from mitochondria, in part by controlling cristae structures. Mitochondrial division promotes apoptosis by an unknown mechanism. We addressed how division proteins regulate apoptosis using inhibitors of mitochondrial division identified in a chemical screen. The most efficacious inhibitor, mdivi-1 (for mitochondrial division inhibitor) attenuates mitochondrial division in yeast and mammalian cells by selectively inhibiting the mitochondrial division dynamin. In cells, mdivi-1 retards apoptosis by inhibiting mitochondrial outer membrane permeabilization. In vitro, mdivi-1 potently blocks Bid-activated Bax/Bak-dependent cytochrome c release from mitochondria. These data indicate the mitochondrial division dynamin directly regulates mitochondrial outer membrane permeabilization independent of Drp1-mediated division. Our findings raise the interesting possibility that mdivi-1 represents a class of therapeutics for stroke, myocardial infarction, and neurodegenerative diseases.     

A Human Dynamin-related Protein Controls the Distribution of Mitochondria

Mitochondria exist as a dynamic tubular network with projections that move, break, and reseal in response to local environmental changes. We present evidence that a human dynamin-related protein (Drp1) is specifically required to establish this morphology. Drp1 is a GTPase with a domain structure similar to that of other dynamin family members. To identify the function of Drp1, we transiently transfected cells with mutant Drp1. A mutation in the GTPase domain caused profound alterations in mitochondrial morphology. The tubular projections normally present in wild-type cells were retracted into large perinuclear aggregates in cells expressing mutant Drp1. The morphology of other organelles was unaffected by mutant Drp1. There was also no effect of mutant Drp1 on the transport functions of the secretory and endocytic pathways. By EM, the mitochondrial aggregates found in cells that were transfected with mutant Drp1 appear as clusters of tubules rather than a large mass of coalescing membrane. We propose that Drp1 is important for distributing mitochondrial tubules throughout the cell. The function of this new dynamin-related protein in organelle morphology represents a novel role for a member of the dynamin family of proteins.     

Mitochondrial Safeguard: a stress response that offsets extreme fusion and protects respiratory function via flickering‐induced Oma1 activation

The connectivity of mitochondria is regulated by a balance between fusion and division. Many human diseases are associated with excessive mitochondrial connectivity due to impaired Drp1, a dynamin‐related GTPase that mediates division. Here, we report a mitochondrial stress response, named mitochondrial safeguard, that adjusts the balance of fusion and division in response to increased mitochondrial connectivity. In cells lacking Drp1, mitochondria undergo hyperfusion. However, hyperfusion does not completely connect mitochondria because Opa1 and mitofusin 1, two other dynamin‐related GTPases that mediate fusion, become proteolytically inactivated. Pharmacological and genetic experiments show that the activity of Oma1, a metalloprotease that cleaves Opa1, is regulated by short pulses of the membrane depolarization without affecting the overall membrane potential in Drp1‐knockout cells. Re‐activation of Opa1 and Mitofusin 1 in Drp1‐knockout cells further connects mitochondria beyond hyperfusion, termed extreme fusion, leading to bioenergetic deficits. These findings reveal an unforeseen safeguard mechanism that prevents extreme fusion of mitochondria, thereby maintaining mitochondrial function when the balance is shifted to excessive connectivity.     

A role for septin 2 in Drp1‐mediated mitochondrial fission

Mitochondria are essential eukaryotic organelles often forming intricate networks. The overall network morphology is determined by mitochondrial fusion and fission. Among the multiple mechanisms that appear to regulate mitochondrial fission, the ER and actin have recently been shown to play an important role by mediating mitochondrial constriction and promoting the action of a key fission factor, the dynamin‐like protein Drp1. Here, we report that the cytoskeletal component septin 2 is involved in Drp1‐dependent mitochondrial fission in mammalian cells. Septin 2 localizes to a subset of mitochondrial constrictions and directly binds Drp1, as shown by immunoprecipitation of the endogenous proteins and by pulldown assays with recombinant proteins. Depletion of septin 2 reduces Drp1 recruitment to mitochondria and results in hyperfused mitochondria and delayed FCCP‐induced fission. Strikingly, septin depletion also affects mitochondrial morphology in Caenorhabditis elegans, strongly suggesting that the role of septins in mitochondrial dynamics is evolutionarily conserved.     

To mdivi‐1 or not to mdivi‐1: Is that the question?

The fission/division and fusion of mitochondria are fundamental aspects of mitochondrial biology. The balance of fission and fusion sets the length of mitochondria in cells to serve their physiological requirements. The fission of mitochondria is markedly induced in many disease states and in response to cellular injury, resulting in the fragmentation of mitochondria into dysfunctional units. The mechanism that drives fission is dependent on the dynamin related protein 1 (Drp1) GTPase. mdivi‐1 is a quinazolinone originally described as a selective inhibitor of Drp1, over other dynamin family members, and reported to inhibit mitochondrial fission. A recent study has challenged the activity of mdivi‐1 as an inhibitor of Drp1. This study raises serious issues regarding the interpretation of data addressing the effects of mdivi‐1 as reflective of the inhibition of Drp1 and thus fission. This commentary considers the evidence for and against mdivi‐1 as an inhibitor of Drp1 and presents the following considerations; (1) the activity of mdivi‐1 toward Drp1 GTPase activity requires further biochemical investigation, (2) as there is a large body of literature using mdivi‐1 in vitro with effects as predicted for inhibition of Drp1 and mitochondrial fission, reviewed herein, the evidence is in favor of mdivi‐1's originally described bioactivity, and (3) until the issue is resolved, experimental interpretations for the effects of mdivi‐1 on inhibition of fission in cell and tissue experiments warrants stringent positive controls directly addressing the effects of mdivi‐1 on fission. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1260–1268, 2017     

Mff oligomerization is required for Drp1 activation and synergy with actin filaments during mitochondrial division

Mitochondrial division is an important cellular process in both normal and pathological conditions. The dynamin GTPase Drp1 is a central mitochondrial division protein, driving constriction of the outer mitochondrial membrane (OMM). In mammals, the OMM protein mitochondrial fission factor (Mff) is a key receptor for recruiting Drp1 from the cytosol to the mitochondrion. Actin filaments are also important in Drp1 recruitment and activation. The manner in which Mff and actin work together in Drp1 activation is unknown. Here we show that Mff is an oligomer (most likely a trimer) that dynamically associates and disassociates through its C-terminal coiled coil, with a K_d in the range of 10 µM. Dynamic Mff oligomerization is required for Drp1 activation. While not binding Mff directly, actin filaments enhance Mff-mediated Drp1 activation by lowering the effective Mff concentration 10-fold. Total internal reflection microscopy assays using purified proteins show that Mff interacts with Drp1 on actin filaments in a manner dependent on Mff oligomerization. In U2OS cells, oligomerization-defective Mff does not effectively rescue three defects in Mff knockout cells: mitochondrial division, mitochondrial Drp1 recruitment, and peroxisome division. The ability of Mff to assemble into puncta on mitochondria depends on its oligomerization, as well as on actin filaments and Drp1.     

A Highlights from MBoC Selection: A dimeric equilibrium intermediate nucleates Drp1 reassembly on mitochondrial membranes for fission

The GTPase dynamin-related protein 1 (Drp1) catalyzes mitochondrial division, but the mechanisms remain poorly understood. Much of what is attributed to Drp1’s mechanism of action in mitochondrial membrane fission parallels that of prototypical dynamin in endocytic vesicle scission. Unlike the case for dynamin, however, no lipid target for Drp1 activation at the mitochondria has been identified. In addition, the oligomerization properties of Drp1 have not been well established. We show that the mitochondria-specific lipid cardiolipin is a potent stimulator of Drp1 GTPase activity, as well as of membrane tubulation. We establish further that under physiological conditions, Drp1 coexists as two morphologically distinct polymeric species, one nucleotide bound in solution and the other membrane associated, which equilibrate via a dimeric assembly intermediate. With two mutations, C300A and C505A, that shift Drp1 polymerization equilibria in opposite directions, we demonstrate that dimers, and not multimers, potentiate the reassembly and reorganization of Drp1 for mitochondrial membrane remodeling both in vitro and in vivo.     

MARCH-V is a novel mitofusin 2- and Drp1-binding protein able to change mitochondrial morphology

Mitofusins and Drp1 are key components in mitochondrial membrane fusion and division, but the molecular mechanism underlying the regulation of their activities remains to be clarified. Here, we identified human membrane-associated RING-CH (MARCH)-V as a novel transmembrane protein of the mitochondrial outer membrane. Immunoprecipitation studies demonstrated that MARCH-V interacts with mitofusin 2 (MFN2) and ubiquitinated forms of Drp1. Overexpression of MARCH-V promoted the formation of long tubular mitochondria in a manner that depends on MFN2 activity. By contrast, mutations in the RING finger caused fragmentation of mitochondria. We also show that MARCH-V promotes ubiquitination of Drp1. These results indicate that MARCH-V has a crucial role in the control of mitochondrial morphology by regulating MFN2 and Drp1 activities.     

Regulation of mitochondrial morphology by APC/CCdh1-mediated control of Drp1 stability

Homeostatic maintenance of cellular mitochondria requires a dynamic balance between fission and fusion, and controlled changes in morphology are important for processes such as apoptosis and cellular division. Interphase mitochondria have been described as an interconnected network that fragments as cells enter mitosis, and this mitotic mitochondrial fragmentation is known to be regulated by the dynamin-related GTPase Drp1 (dynamin-related protein 1), a key component of the mitochondrial division machinery. Loss of Drp1 function and the subsequent failure of mitochondrial division during mitosis lead to incomplete cytokinesis and the unequal distribution of mitochondria into daughter cells. During mitotic exit and interphase, the mitochondrial network reforms. Here we demonstrate that changes in mitochondrial dynamics as cells exit mitosis are driven in part through ubiquitylation of Drp1, catalyzed by the APC/C(Cdh1) (anaphase-promoting complex/cyclosome and its coactivator Cdh1) E3 ubiquitin ligase complex. Importantly, inhibition of Cdh1-mediated Drp1 ubiquitylation and proteasomal degradation during interphase prevents the normal G1 phase regrowth of mitochondrial networks following cell division.     

Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage

Mitochondria divide and fuse continuously, and the balance between these two processes regulates mitochondrial shape. Alterations in mitochondrial dynamics are associated with neurodegenerative diseases. Here we investigate the physiological and cellular functions of mitochondrial division in postmitotic neurons using in vivo and in vitro gene knockout for the mitochondrial division protein Drp1. When mouse Drp1 was deleted in postmitotic Purkinje cells in the cerebellum, mitochondrial tubules elongated due to excess fusion, became large spheres due to oxidative damage, accumulated ubiquitin and mitophagy markers, and lost respiratory function, leading to neurodegeneration. Ubiquitination of mitochondria was independent of the E3 ubiquitin ligase parkin in Purkinje cells lacking Drp1. Treatment with antioxidants rescued mitochondrial swelling and cell death in Drp1KO Purkinje cells. Moreover, hydrogen peroxide converted elongated tubules into large spheres in Drp1KO fibroblasts. Our findings suggest that mitochondrial division serves as a quality control mechanism to suppress oxidative damage and thus promote neuronal survival.     

Cyclic AMP-dependent protein kinase phosphorylation of Drp1 regulates its GTPase activity and mitochondrial morphology

Mitochondria in cells comprise a tubulovesicular reticulum shaped by dynamic fission and fusion events. The multimeric dynamin-like GTPase Drp1 is a critical protein mediating mitochondrial division. It harbors multiple motifs including GTP-binding, middle, and GTPase effector (GED) domains that are important for both intramolecular and intermolecular interactions. As for other members of the dynamin superfamily, such interactions are critical for assembly of higher-order structures and cooperative increases in GTPase activity. Although the functions of Drp1 in cells have been extensively studied, mechanisms underlying its regulation remain less clear. Here, we have identified cAMP-dependent protein kinase-dependent phosphorylation of Drp1 within the GED domain at Ser(637) that inhibits Drp1 GTPase activity. Mechanistically, this change in GTPase activity likely derives from decreased interaction of GTP-binding/middle domains with the GED domain since the phosphomimetic S637D mutation impairs this intramolecular interaction but not Drp1-Drp1 intermolecular interactions. Using the phosphomimetic S637D substitution, we also demonstrate that mitochondrial fission is prominently inhibited in cells. Thus, protein phosphorylation at Ser(637) results in clear alterations in Drp1 function and mitochondrial morphology that are likely involved in dynamic regulation of mitochondrial division in cells.     

Mitochondrial dynamic alterations regulate melanoma cell progression

Research on mitochondrial fusion and fission (mitochondrial dynamics) has gained much attention in recent years, as it is important for understanding many biological processes, including the maintenance of mitochondrial functions, apoptosis, and cancer. The rate of mitochondrial biosynthesis and degradation can affect various aspects of tumor progression. However, the role of mitochondrial dynamics in melanoma progression remains controversial and requires a mechanistic understanding to target the altered metabolism of cancer cells. Therefore, in our study, we disrupted mitochondrial fission with mdivi-1, the reported inhibitor of dynamin related protein 1 (Drp1), and knocked down Drp1 and Mfn2 to evaluate the effects of mitochondrial dynamic alterations on melanoma cell progression. Our confocal study results showed that mitochondrial fission was inhibited both in mdivi-1 and in Drp1 knockdown cells and, in parallel, mitochondrial fusion was induced. We also found that mitochondrial fission inhibition by mdivi-1 induced cell death in melanoma cells. However, silencing Drp1 and Mfn2 did not affect cell viability, but enhanced melanoma cell migration. We further show that dysregulated mitochondrial fusion by Mfn2 knockdowns suppressed the oxygen consumption rate of melanoma cells. Together, our findings suggest that mitochondrial dynamic alterations regulate melanoma cell migration and progression.     

Parkin‐independent mitophagy requires Drp1 and maintains the integrity of mammalian heart and brain

Mitochondrial dynamics and mitophagy have been linked to cardiovascular and neurodegenerative diseases. Here, we demonstrate that the mitochondrial division dynamin Drp1 and the Parkinson's disease‐associated E3 ubiquitin ligase parkin synergistically maintain the integrity of mitochondrial structure and function in mouse heart and brain. Mice lacking cardiac Drp1 exhibited lethal heart defects. In Drp1KO cardiomyocytes, mitochondria increased their connectivity, accumulated ubiquitinated proteins, and decreased their respiration. In contrast to the current views of the role of parkin in ubiquitination of mitochondrial proteins, mitochondrial ubiquitination was independent of parkin in Drp1KO hearts, and simultaneous loss of Drp1 and parkin worsened cardiac defects. Drp1 and parkin also play synergistic roles in neuronal mitochondrial homeostasis and survival. Mitochondrial degradation was further decreased by combination of Drp1 and parkin deficiency, compared with their single loss. Thus, the physiological importance of parkin in mitochondrial homeostasis is revealed in the absence of mitochondrial division in mammals.     

Cardiolipin's propensity for phase transition and its reorganization by dynamin-related protein 1 form a basis for mitochondrial membrane fission

Cardiolipin (CL) is an atypical, dimeric phospholipid essential for mitochondrial dynamics in eukaryotic cells. Dynamin-related protein 1 (Drp1), a cytosolic member of the dynamin superfamily of large GTPases, interacts with CL and functions to sustain the balance of mitochondrial division and fusion by catalyzing mitochondrial fission. Although recent studies have indicated a role for CL in stimulating Drp1 self-assembly and GTPase activity at the membrane surface, the mechanism by which CL functions in membrane fission, if at all, remains unclear. Here, using a variety of fluorescence spectroscopic and imaging approaches together with model membranes, we demonstrate that Drp1 and CL function cooperatively in effecting membrane constriction toward fission in three distinct steps. These involve 1) the preferential association of Drp1 with CL localized at a high spatial density in the membrane bilayer, 2) the reorganization of unconstrained, fluid-phase CL molecules in concert with Drp1 self-assembly, and 3) the increased propensity of CL to transition from a lamellar, bilayer arrangement to an inverted hexagonal, nonbilayer configuration in the presence of Drp1 and GTP, resulting in the creation of localized membrane constrictions that are primed for fission. Thus we propose that Drp1 and CL function in concert to catalyze mitochondrial division.     

Rotaviral nonstructural protein 4 triggers dynamin‐related protein 1‐dependent mitochondrial fragmentation during infection

Dynamic equilibrium between mitochondrial fission and mitochondrial fusion serves as an important quality control system within cells ensuring cellular vitality and homeostasis. Viruses often target mitochondrial dynamics as a part of their obligatory cellular reprogramming. The present study was undertaken to assess the status and regulation of mitochondrial dynamics during rotavirus infection. Distinct fragmentation of mitochondrial syncytia was observed during late hours of RV (SA11, Wa, A5‐13) infection. RV nonstructural protein 4 (NSP4) was identified as the viral trigger for disrupted mitochondrial morphology. Severance of mitochondrial interconnections was found to be a dynamin‐related protein 1 (Drp1)‐dependent process resulting synergistically from augmented mitochondrial fission and attenuated mitochondrial fusion. Cyclin‐dependent kinase 1 was subsequently identified as the cellular kinase responsible for fission‐active Ser616 phosphorylation of Drp1. In addition to its positive role in mitochondrial fission, Drp1 also resulted in mitochondrial translocation of E3‐ubiquitin ligase Parkin leading to degradation of mitochondrial fusion protein Mitofusin 1. Interestingly, RV‐NSP4 was found to interact with and be involved in recruiting fission‐active pool of Serine 616 phosphoDrp1 (Ser616 pDrp1) to mitochondria independent of accessory adaptors Mitochondrial fission factor and Fission protein 1 (Fis1). Inhibition of either Drp1 or Ser616 pDrp1 resulted in significant decrease in RV‐NSP4‐induced intrinsic apoptotic pathway. Overall, this study underscores an efficient strategy utilised by RV to couple apoptosis to mitochondrial fission facilitating dissemination of viral progeny.     

Dynamin-related Protein 1 (Drp1) Promotes Structural Intermediates of Membrane Division

Drp1 is a dynamin-like GTPase that mediates mitochondrial and peroxisomal division in a process dependent on self-assembly and coupled to GTP hydrolysis. Despite the link between Drp1 malfunction and human disease, the molecular details of its membrane activity remain poorly understood. Here we reconstituted and directly visualized Drp1 activity in giant unilamellar vesicles. We quantified the effect of lipid composition and GTP on membrane binding and remodeling activity by fluorescence confocal microscopy and flow cytometry. In contrast to other dynamin relatives, Drp1 bound to both curved and flat membranes even in the absence of nucleotides. We also found that Drp1 induced membrane tubulation that was stimulated by cardiolipin. Moreover, Drp1 promoted membrane tethering dependent on the intrinsic curvature of the membrane lipids and on GTP. Interestingly, Drp1 concentrated at membrane contact surfaces and, in the presence of GTP, formed discrete clusters on the vesicles. Our findings support a role of Drp1 not only in the formation of lipid tubes but also on the stabilization of tightly apposed membranes, which are intermediate states in the process of mitochondrial fission.     

The role of dynamin-related protein 1, a mediator of mitochondrial fission, in apoptosis.

In healthy cells, fusion and fission events participate in regulating mitochondrial morphology. Disintegration of the mitochondrial reticulum into multiple punctiform organelles during apoptosis led us to examine the role of Drp1, a dynamin-related protein that mediates outer mitochondrial membrane fission. Upon induction of apoptosis, Drp1 translocates from the cytosol to mitochondria, where it preferentially localizes to potential sites of organelle division. Inhibition of Drp1 by overexpression of a dominant-negative mutant counteracts the conversion to a punctiform mitochondrial phenotype, prevents the loss of the mitochondrial membrane potential and the release of cytochrome c, and reveals a reproducible swelling of the organelles. Remarkably, inhibition of Drp1 blocks cell death, implicating mitochondrial fission as an important step in apoptosis.     

Priming for destruction: septins at the crossroads of mitochondrial fission and bacterial autophagy

Mitochondria are essential organelles for cell survival, programmed cell death, and autophagy. They undergo cycles of fission and fusion, which are subverted by infectious pathogens and altered in many human diseases. Mitochondrial fission is mediated by the dynamin‐related protein Drp1, but the precise mechanism of its action is not well understood. In the last and current issues of EMBO Reports, two new studies 1 2 reveal that the filamentous septin GTPases interact directly with Drp1, promoting mitochondrial fission. Moreover, mitochondria were found to promote the assembly of septin filaments into cages around cytosolic Shigella flexneri bacteria 2 , which are targeted for autophagy. Thus, septins emerge as integral components of the machinery of mitochondrial fission and may pose a novel link between mitochondria and autophagy.     

The mitochondrial E3 ubiquitin ligase MARCH5 is required for Drp1 dependent mitochondrial division

We identify a mitochondrial E3 ubiquitin ligase, MARCH5, as a critical regulator of mitochondrial fission. MARCH5 RING mutants and MARCH5 RNA interference induce an abnormal elongation and interconnection of mitochondria indicative of an inhibition of mitochondrial division. The aberrant mitochondrial phenotypes in MARCH5 RING mutant-expressing cells are reversed by ectopic expression of Drp1, but not another mitochondrial fission protein Fis1. Moreover, as indicated by abnormal clustering and mitochondrial accumulation of Drp1, as well as decreased cellular mobility of YFP-Drp1 in cells expressing MARCH5 RING mutants, MARCH5 activity regulates the subcellular trafficking of Drp1, likely by impacting the correct assembly at scission sites or the disassembly step of fission complexes. Loss of this activity may account for the observed mitochondrial division defects. Finally, MARCH5 RING mutants and endogenous Drp1, but not wild-type MARCH5 or Fis1, co-assemble into abnormally enlarged clusters in a Drp1 GTPase-dependent manner, suggesting molecular interactions among these proteins. Collectively, our data suggest a model in which mitochondrial division is regulated by a MARCH5 ubiquitin-dependent switch.     

A Lethal de Novo Mutation in the Middle Domain of the Dynamin-related GTPase Drp1 Impairs Higher Order Assembly and Mitochondrial Division

Mitochondria dynamically fuse and divide within cells, and the proper balance of fusion and fission is necessary for normal mitochondrial function, morphology, and distribution. Drp1 is a dynamin-related GTPase required for mitochondrial fission in mammalian cells. It harbors four distinct domains: GTP-binding, middle, insert B, and GTPase effector. A lethal mutation (A395D) within the Drp1 middle domain was reported in a neonate with microcephaly, abnormal brain development, optic atrophy, and lactic acidemia (Waterham, H. R., Koster, J., van Roermund, C. W., Mooyer, P. A., Wanders, R. J., and Leonard, J. V. (2007) N. Engl. J. Med. 356, 1736–1741). Mitochondria within patient-derived fibroblasts were markedly elongated, but the molecular mechanisms underlying these findings were not demonstrated. Because the middle domain is particularly important for the self-assembly of some dynamin superfamily proteins, we tested the hypothesis that this A395D mutation, and two other middle domain mutations (G350D, G363D) were important for Drp1 tetramerization, higher order assembly, and function. Although tetramerization appeared largely intact, each of these mutations compromised higher order assembly and assembly-dependent stimulation of Drp1 GTPase activity. Moreover, mutant Drp1 proteins exhibited impaired localization to mitochondria, indicating that this higher order assembly is important for mitochondrial recruitment, retention, or both. Overexpression of these middle domain mutants markedly inhibited mitochondrial division in cells. Thus, the Drp1 A395D lethal defect likely resulted in impaired higher order assembly of Drp1 at mitochondria, leading to decreased fission, elongated mitochondria, and altered cellular distribution of mitochondria.