Dynamin 1xA interacts with Endophilin A1 via its spliced long C-terminus for ultrafast endocytosis.

Dynamin 1 mediates fission of endocytic synaptic vesicles in the brain and has two major splice variants, Dyn1xA and Dyn1xB, which are nearly identical apart from the extended C-terminal region of Dyn1xA. Despite a similar set of binding partners, only Dyn1xA is enriched at endocytic zones and accelerates vesicle fission during ultrafast endocytosis. Here, we report that Dyn1xA achieves this localization by preferentially binding to Endophilin A1 through a newly defined binding site within its long C-terminal tail extension. Endophilin A1 binds this site at higher affinity than the previously reported site, and the affinity is determined by amino acids within the Dyn1xA tail but outside the binding site. This interaction is regulated by the phosphorylation state of two serine residues specific to the Dyn1xA variant. Dyn1xA and Endophilin A1 colocalize in patches near the active zone, and mutations disrupting Endophilin A binding to the long tail cause Dyn1xA mislocalization and stalled endocytic pits on the plasma membrane during ultrafast endocytosis. Together, these data suggest that the specificity for ultrafast endocytosis is defined by the phosphorylation-regulated interaction of Endophilin A1 with the C-terminal extension of Dyn1xA.

Synaptotagmin 7 docks synaptic vesicles to support facilitation and Doc2α-triggered asynchronous release.

Despite decades of intense study, the molecular basis of asynchronous neurotransmitter release remains enigmatic. Synaptotagmin (syt) 7 and Doc2 have both been proposed as Ca2+ sensors that trigger this mode of exocytosis, but conflicting findings have led to controversy. Here, we demonstrate that at excitatory mouse hippocampal synapses, Doc2α is the major Ca2+ sensor for asynchronous release, while syt7 supports this process through activity-dependent docking of synaptic vesicles. In synapses lacking Doc2α, asynchronous release after single action potentials is strongly reduced, while deleting syt7 has no effect. However, in the absence of syt7, docked vesicles cannot be replenished on millisecond timescales. Consequently, both synchronous and asynchronous release depress from the second pulse onward during repetitive activity. By contrast, synapses lacking Doc2α have normal activity-dependent docking, but continue to exhibit decreased asynchronous release after multiple stimuli. Moreover, disruption of both Ca2+ sensors is non-additive. These findings result in a new model whereby syt7 drives activity-dependent docking, thus providing synaptic vesicles for synchronous (syt1) and asynchronous (Doc2 and other unidentified sensors) release during ongoing transmission.

Dynamin 1xA interacts with Endophilin A1 via its spliced long C-terminus for ultrafast endocytosis.

Dynamin 1 (Dyn1) has two major splice variants, xA and xB, with unique C-terminal extensions of 20 and 7 amino acids, respectively. Of these, only Dyn1xA is enriched at endocytic zones and accelerates vesicle fission during ultrafast endocytosis. Here, we report that the long tail variant, Dyn1xA, achieves this localization by preferentially binding to Endophilin A through a newly defined Class II binding site overlapping with its extension, at a site spanning the splice boundary. Endophilin binds this site at higher affinity than the previously reported site, and this affinity is determined by amino acids outside the binding sites acting as long distance elements within the xA tail. Their interaction is regulated by the phosphorylation state of two serine residues specific to the xA variant. Dyn1xA and Endophilin colocalize in patches near the active zone of synapses. Mutations selectively disrupting Endophilin binding to the long extension cause Dyn1xA mislocalization along axons. In these mutants, endocytic pits are stalled on the plasma membrane during ultrafast endocytosis. These data suggest that the specificity for ultrafast endocytosis is defined by the phospho-regulated interaction of Endophilin A through a newly identified site of Dyn1xA's long tail.

ActuAtor, a Listeria-inspired molecular tool for physical manipulation of intracellular organizations through de novo actin polymerization.

Form and function are often interdependent throughout biology. Inside cells, mitochondria have particularly attracted attention since both their morphology and functionality are altered under pathophysiological conditions. However, directly assessing their causal relationship has been beyond reach due to the limitations of manipulating mitochondrial morphology in a physiologically relevant manner. By engineering a bacterial actin regulator, ActA, we developed tools termed "ActuAtor" that inducibly trigger actin polymerization at arbitrary subcellular locations. The ActuAtor-mediated actin polymerization drives striking deformation and/or movement of target organelles, including mitochondria, Golgi apparatus, and nucleus. Notably, ActuAtor operation also disperses non-membrane-bound entities such as stress granules. We then implemented ActuAtor in functional assays, uncovering the physically fragmented mitochondria being slightly more susceptible to degradation, while none of the organelle functions tested are morphology dependent. The modular and genetically encoded features of ActuAtor should enable its application in studies of the form-function interplay in various intracellular contexts.

Membrane compression by synaptic vesicle exocytosis triggers ultrafast endocytosis.

Compensatory endocytosis keeps the membrane surface area of secretory cells constant following exocytosis. At chemical synapses, clathrin-independent ultrafast endocytosis maintains such homeostasis. This endocytic pathway is temporally and spatially coupled to exocytosis; it initiates within 50 ms at the region immediately next to the active zone where vesicles fuse. However, the coupling mechanism is unknown. Here, we demonstrate that filamentous actin is organized as a ring, surrounding the active zone at mouse hippocampal synapses. Assuming the membrane area conservation is due to this actin ring, our theoretical model suggests that flattening of fused vesicles exerts lateral compression in the plasma membrane, resulting in rapid formation of endocytic pits at the border between the active zone and the surrounding actin-enriched region. Consistent with model predictions, our data show that ultrafast endocytosis requires sufficient compression by exocytosis of multiple vesicles and does not initiate when actin organization is disrupted, either pharmacologically or by ablation of the actin-binding protein Epsin1. Our work suggests that membrane mechanics underlie the rapid coupling of exocytosis to endocytosis at synapses.

Mitophagy initiates retrograde mitochondrial-nuclear signaling to guide retinal pigment cell heterogeneity.

Age-related macular degeneration (AMD), the leading cause of blindness among the elderly, is without treatment for early disease. Degenerative retinal pigment epithelial (RPE) cell heterogeneity is a well-recognized but understudied pathogenic factor. Due to the daily phagocytosis of photoreceptor outer segments, unique photo-oxidative stress, and high metabolism for maintaining vision, the RPE has robust macroautophagy/autophagy, and mitochondrial and antioxidant networks. However, the autophagy subtype, mitophagy, in the RPE and AMD is understudied. Here, we found decreased PINK1 (PTEN induced kinase 1) in perifoveal RPE of early AMD eyes. PINK1-deficient RPE have impaired mitophagy and mitochondrial function that triggers death-resistant epithelial-mesenchymal transition (EMT). This reprogramming is mediated by novel retrograde mitochondrial-nuclear signaling (RMNS) through superoxide, NFE2L2 (NFE2 like bZIP transcription factor 2), TXNRD1 (thioredoxin reductase 1), and phosphoinositide 3-kinase (PI3K)-AKT (AKT serine/threonine kinase) that induced canonical transcription factors ZEB1 (zinc finger E-box binding homeobox 1) and SNAI1 (Snail family transcriptional repressor 1) and an EMT transcriptome. NFE2L2 deficiency disrupted RMNS that paradoxically normalized morphology but decreased function and viability. Thus, RPE heterogeneity is defined by the interaction of two cytoprotective pathways that is triggered by mitophagy function. By neutralizing the consequences of impaired mitophagy, an antioxidant dendrimer tropic for the RPE and mitochondria, EMT (a recognized AMD alteration) was abrogated to offer potential therapy for early AMD, a stage without treatment.Abbreviations: ACTB: actin beta; AKT: AKT serine/threonine kinase; AMD: age-related macular degeneration; CCCP: cyanide m-chlorophenyl hydrazone; CDH1: cadherin 1; DAVID: Database for Annotation, Visualization and Integrated Discovery; DHE: dihydroethidium; D-NAC: N-acetyl-l-cysteine conjugated to a poly(amido amine) dendrimer; ECAR: extracellular acidification rate; EMT: epithelial-mesenchymal transition; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSEA: Gene Set Enrichment Analysis; HSPD1: heat shock protein family D (Hsp60) member 1; IVT: intravitreal; KD: knockdown; LMNA, lamin A/C; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MMP: mitochondrial membrane potential; NAC: N-acetyl-l-cysteine; NQO1: NAD(P)H quinone dehydrogenase 1; NFE2L2: NFE2 like bZIP transcription factor 2; O2-: superoxide anion; OCR: oxygen consumption rate; PI3K: phosphoinositide 3-kinase; PINK1: PTEN induced kinase 1; RMNS: retrograde mitochondrial-nuclear signaling; ROS: reactive oxygen species; RPE: retinal pigment epithelium; SNAI1: snail family transcriptional repressor 1; TJP1: tight junction protein 1; TPP-D-NAC: triphenyl phosphinium and N-acetyl-l-cysteine conjugated to a poly(amido amine) dendrimer; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; Trig: trigonelline; TXNRD1: thioredoxin reductase 1; VIM: vimentin; WT: wild-type; ZEB1: zinc finger E-box binding homeobox 1.

Defunctionalizing intracellular organelles such as mitochondria and peroxisomes with engineered phospholipase A/acyltransferases.

Organelles vitally achieve multifaceted functions to maintain cellular homeostasis. Genetic and pharmacological approaches to manipulate individual organelles are powerful in probing their physiological roles. However, many of them are either slow in action, limited to certain organelles, or rely on toxic agents. Here, we design a generalizable molecular tool utilizing phospholipase A/acyltransferases (PLAATs) for rapid defunctionalization of organelles via remodeling of the membrane phospholipids. In particular, we identify catalytically active PLAAT truncates with minimal unfavorable characteristics. Chemically-induced translocation of the optimized PLAAT to the mitochondria surface results in their rapid deformation in a phospholipase activity dependent manner, followed by loss of luminal proteins as well as dissipated membrane potential, thus invalidating the functionality. To demonstrate wide applicability, we then adapt the molecular tool in peroxisomes, and observe leakage of matrix-resident functional proteins. The technique is compatible with optogenetic control, viral delivery and operation in primary neuronal cultures. Due to such versatility, the PLAAT strategy should prove useful in studying organelle biology of diverse contexts.

Dynamin is primed at endocytic sites for ultrafast endocytosis.

Dynamin mediates fission of vesicles from the plasma membrane during endocytosis. Typically, dynamin is recruited from the cytosol to endocytic sites, requiring seconds to tens of seconds. However, ultrafast endocytosis in neurons internalizes vesicles as quickly as 50 ms during synaptic vesicle recycling. Here, we demonstrate that Dynamin 1 is pre-recruited to endocytic sites for ultrafast endocytosis. Specifically, Dynamin 1xA, a splice variant of Dynamin 1, interacts with Syndapin 1 to form molecular condensates on the plasma membrane. Single-particle tracking of Dynamin 1xA molecules confirms the liquid-like property of condensates in vivo. When Dynamin 1xA is mutated to disrupt its interaction with Syndapin 1, the condensates do not form, and consequently, ultrafast endocytosis slows down by 100-fold. Mechanistically, Syndapin 1 acts as an adaptor by binding the plasma membrane and stores Dynamin 1xA at endocytic sites. This cache bypasses the recruitment step and accelerates endocytosis at synapses.

C9orf72 regulates energy homeostasis by stabilizing mitochondrial complex I assembly.

The haploinsufficiency of C9orf72 is implicated in the most common forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the full spectrum of C9orf72 functions remains to be established. Here, we report that C9orf72 is a mitochondrial inner-membrane-associated protein regulating cellular energy homeostasis via its critical role in the control of oxidative phosphorylation (OXPHOS). The translocation of C9orf72 from the cytosol to the inter-membrane space is mediated by the redox-sensitive AIFM1/CHCHD4 pathway. In mitochondria, C9orf72 specifically stabilizes translocase of inner mitochondrial membrane domain containing 1 (TIMMDC1), a crucial factor for the assembly of OXPHOS complex I. C9orf72 directly recruits the prohibitin complex to inhibit the m-AAA protease-dependent degradation of TIMMDC1. The mitochondrial complex I function is impaired in C9orf72-linked ALS/FTD patient-derived neurons. These results reveal a previously unknown function of C9orf72 in mitochondria and suggest that defective energy metabolism may underlie the pathogenesis of relevant diseases.

Molecular Basis of Mitochondrial and Peroxisomal Division Machineries.

Mitochondria and peroxisomes are ubiquitous subcellular organelles that are highly dynamic and possess a high degree of plasticity. These organelles proliferate through division of pre-existing organelles. Studies on yeast, mammalian cells, and unicellular algae have led to a surprising finding that mitochondria and peroxisomes share the components of their division machineries. At the heart of the mitochondrial and peroxisomal division machineries is a GTPase dynamin-like protein, Dnm1/Drp1, which forms a contractile ring around the neck of the dividing organelles. During division, Dnm1/Drp1 functions as a motor protein and constricts the membrane. This mechanochemical work is achieved by utilizing energy from GTP hydrolysis. Over the last two decades, studies have focused on the structure and assembly of Dnm1/Drp1 molecules around the neck. However, the regulation of GTP during the division of mitochondrion and peroxisome is not well understood. Here, we review the current understanding of Dnm1/Drp1-mediated divisions of mitochondria and peroxisomes, exploring the mechanisms of GTP regulation during the Dnm1/Drp1 function, and provide new perspectives on their potential contribution to mitochondrial and peroxisomal biogenesis.

Brain-specific Drp1 regulates postsynaptic endocytosis and dendrite formation independently of mitochondrial division.

Dynamin-related protein 1 (Drp1) divides mitochondria as a mechano-chemical GTPase. However, the function of Drp1 beyond mitochondrial division is largely unknown. Multiple Drp1 isoforms are produced through mRNA splicing. One such isoform, Drp1ABCD, contains all four alternative exons and is specifically expressed in the brain. Here, we studied the function of Drp1ABCD in mouse neurons in both culture and animal systems using isoform-specific knockdown by shRNA and isoform-specific knockout by CRISPR/Cas9. We found that the expression of Drp1ABCD is induced during postnatal brain development. Drp1ABCD is enriched in dendritic spines and regulates postsynaptic clathrin-mediated endocytosis by positioning the endocytic zone at the postsynaptic density, independently of mitochondrial division. Drp1ABCD loss promotes the formation of ectopic dendrites in neurons and enhanced sensorimotor gating behavior in mice. These data reveal that Drp1ABCD controls postsynaptic endocytosis, neuronal morphology and brain function.

Maintenance of Cardiolipin and Crista Structure Requires Cooperative Functions of Mitochondrial Dynamics and Phospholipid Transport.

Mitochondria are dynamic organelles that constantly fuse and divide to maintain their proper morphology, which is essential for their normal functions. Energy production, a central role of mitochondria, demands highly folded structures of the mitochondrial inner membrane (MIM) called cristae and a dimeric phospholipid (PL) cardiolipin (CL). Previous studies identified a number of factors involved in mitochondrial dynamics, crista formation, and CL biosynthesis, yet it is still enigmatic how these events are interconnected and cooperated. Here, we first report that mitochondrial fusion-division dynamics are important to maintain CL abundance. Second, our genetic and biochemical analyses revealed that intra-mitochondrial PL transport plays an important role in crista formation. Finally, we show that simultaneous defects in MIM fusion and intra-mitochondrial PL transport cause a drastic decrease in crista structure, resulting in CL depletion. These results expand our understanding of the integrated functional network among the PL transport, crista formation, and CL biogenesis.

Loss of MICOS complex integrity and mitochondrial damage, but not TDP-43 mitochondrial localisation, are likely associated with severity of CHCHD10-related diseases.

Following the involvement of CHCHD10 in FrontoTemporal-Dementia-Amyotrophic Lateral Sclerosis (FTD-ALS) clinical spectrum, a founder mutation (p.Gly66Val) in the same gene was identified in Finnish families with late-onset spinal motor neuronopathy (SMAJ). SMAJ is a slowly progressive form of spinal muscular atrophy with a life expectancy within normal range. In order to understand why the p.Ser59Leu mutation, responsible for severe FTD-ALS, and the p.Gly66Val mutation could lead to different levels of severity, we compared their effects in patient cells. Unlike affected individuals bearing the p.Ser59Leu mutation, patients presenting with SMAJ phenotype have neither mitochondrial myopathy nor mtDNA instability. The expression of CHCHD10S59L mutant allele leads to disassembly of mitochondrial contact site and cristae organizing system (MICOS) with mitochondrial dysfunction and loss of cristae in patient fibroblasts. We also show that G66V fibroblasts do not display the loss of MICOS complex integrity and mitochondrial damage found in S59L cells. However, S59L and G66V fibroblasts show comparable accumulation of phosphorylated mitochondrial TDP-43 suggesting that the severity of phenotype and mitochondrial damage do not depend on mitochondrial TDP-43 localization. The expression of the CHCHD10G66V allele is responsible for mitochondrial network fragmentation and decreased sensitivity towards apoptotic stimuli, but with a less severe effect than that found in cells expressing the CHCHD10S59L allele. Taken together, our data show that cellular phenotypes associated with p.Ser59Leu and p.Gly66Val mutations in CHCHD10 are different; loss of MICOS complex integrity and mitochondrial dysfunction, but not TDP-43 mitochondrial localization, being likely essential to develop a severe motor neuron disease.

Mitochondrial Stasis Reveals p62-Mediated Ubiquitination in Parkin-Independent Mitophagy and Mitigates Nonalcoholic Fatty Liver Disease.

It is unknown what occurs if both mitochondrial division and fusion are completely blocked. Here, we introduced mitochondrial stasis by deleting two dynamin-related GTPases for division (Drp1) and fusion (Opa1) in livers. Mitochondrial stasis rescues liver damage and hypotrophy caused by the single knockout (KO). At the cellular level, mitochondrial stasis re-establishes mitochondrial size and rescues mitophagy defects caused by division deficiency. Using Drp1KO livers, we found that the autophagy adaptor protein p62/sequestosome-1-which is thought to function downstream of ubiquitination-promotes mitochondrial ubiquitination. p62 recruits two subunits of a cullin-RING ubiquitin E3 ligase complex, Keap1 and Rbx1, to mitochondria. Resembling Drp1KO, diet-induced nonalcoholic fatty livers enlarge mitochondria and accumulate mitophagy intermediates. Resembling Drp1Opa1KO, Opa1KO rescues liver damage in this disease model. Our data provide a new concept that mitochondrial stasis leads the spatial dimension of mitochondria to a stationary equilibrium and a new mechanism for mitochondrial ubiquitination in mitophagy.

A brain-enriched Drp1 isoform associates with lysosomes, late endosomes, and the plasma membrane.

Dynamin-related protein 1 (Drp1) constricts mitochondria as a mechanochemical GTPase during mitochondrial division. The Drp1 gene contains several alternative exons and produces multiple isoforms through RNA splicing. Here we performed a systematic analysis of Drp1 transcripts in different mouse tissues and identified a previously uncharacterized isoform that is highly enriched in the brain. This Drp1 isoform is termed Drp1ABCD because it contains four alterative exons: A, B, C, and D. Remarkably, Drp1ABCD is located at lysosomes, late endosomes, and the plasma membrane in addition to mitochondria. Furthermore, Drp1ABCD is concentrated at the interorganelle interface between mitochondria and lysosomes/late endosomes. The localizations of Drp1ABCD at lysosomes, late endosomes, and the plasma membrane require two exons, A and B, that are present in the GTPase domain. Drp1ABCD assembles onto these membranes in a manner that is regulated by its oligomerization and GTP hydrolysis. Experiments using lysosomal inhibitors show that the association of Drp1ABCD with lysosomes/late endosomes depends on lysosomal pH but not their protease activities. Thus, Drp1 may connect mitochondria to endosomal-lysosomal pathways in addition to mitochondrial division.

Nuclear PTEN deficiency causes microcephaly with decreased neuronal soma size and increased seizure susceptibility.

Defects in phosphatase and tensin homolog (PTEN) are associated with neurological disorders and tumors. PTEN functions at two primary intracellular locations: the plasma membrane and the nucleus. At the membrane, PTEN functions as a phosphatidylinositol (3,4,5)-trisphosphate phosphatase and suppresses PI 3-kinase signaling that drives cell growth and tumorigenesis. However, the in vivo function of nuclear PTEN is unclear. Here, using CRISPR/Cas9, we generated a mouse model in which PTEN levels in the nucleus are decreased. Nuclear PTEN-deficient mice were born with microcephaly and maintained a small brain during adulthood. The size of neuronal soma was significantly smaller in the cerebellum, cerebral cortex, and hippocampus. Also, these mice were prone to seizure. No changes in PI 3-kinase signaling were observed. By contrast, the size of other organs was unaffected. Therefore, nuclear PTEN is essential for the health of the brain by promoting the growth of neuronal soma size during development.

MIRO-1 Determines Mitochondrial Shape Transition upon GPCR Activation and Ca2+ Stress.

Mitochondria shape cytosolic calcium ([Ca2+]c) transients and utilize the mitochondrial Ca2+ ([Ca2+]m) in exchange for bioenergetics output. Conversely, dysregulated [Ca2+]c causes [Ca2+]m overload and induces permeability transition pore and cell death. Ablation of MCU-mediated Ca2+ uptake exhibited elevated [Ca2+]c and failed to prevent stress-induced cell death. The mechanisms for these effects remain elusive. Here, we report that mitochondria undergo a cytosolic Ca2+-induced shape change that is distinct from mitochondrial fission and swelling. [Ca2+]c elevation, but not MCU-mediated Ca2+ uptake, appears to be essential for the process we term mitochondrial shape transition (MiST). MiST is mediated by the mitochondrial protein Miro1 through its EF-hand domain 1 in multiple cell types. Moreover, Ca2+-dependent disruption of Miro1/KIF5B/tubulin complex is determined by Miro1 EF1 domain. Functionally, Miro1-dependent MiST is essential for autophagy/mitophagy that is attenuated in Miro1 EF1 mutants. Thus, Miro1 is a cytosolic Ca2+ sensor that decodes metazoan Ca2+ signals as MiST.

Elevated mitochondrial activity distinguishes fibrogenic hepatic stellate cells and sensitizes for selective inhibition by mitotropic doxorubicin.

Activation of hepatic stellate cells (HSCs) is an integral component of the wound-healing process in liver injury/inflammation. However, uncontrolled activation of HSCs leads to constant secretion of collagen-rich extracellular matrix (ECM) proteins, resulting in liver fibrosis. The enhanced ECM synthesis/secretion demands an uninterrupted supply of intracellular energy; however, there is a paucity of data on the bioenergetics, particularly the mitochondrial (mito) metabolism of fibrogenic HSCs. Here, using human and rat HSCs in vitro, we show that the mito-respiration, mito-membrane potential (Δψm) and cellular 'bioenergetic signature' distinguish fibrogenic HSCs from normal, less-active HSCs. Ex vivo, HSCs from mouse and rat models of liver fibrosis further confirmed the altered 'bioenergetic signature' of fibrogenic HSCs. Importantly, the distinctive elevation in mito-Δψm sensitized fibrogenic HSCs for selective inhibition by mitotropic doxorubicin while normal, less-active HSCs and healthy human primary hepatocytes remained minimally affected if not, unaffected. Thus, the increased mito-Δψm may provide an opportunity to selectively target fibrogenic HSCs in liver fibrosis.

Assay to Measure Interactions between Purified Drp1 and Synthetic Liposomes.

A mitochondrion is a dynamic intracellular organelle that actively divides and fuses to control its size, number and shape in cells. A regulated balance between mitochondrial division and fusion is fundamental to the function, distribution and turnover of mitochondria (Roy et al., 2015). Mitochondrial division is mediated by dynamin-related protein 1 (Drp1), a mechano-chemical GTPase that constricts mitochondrial membranes (Tamura et al., 2011). Mitochondrial membrane lipids such as phosphatidic acid and cardiolipin bind Drp1, and Drp1-phospholipid interactions provide key regulatory mechanisms for mitochondrial division (Montessuit et al., 2010; Bustillo-Zabalbeitia et al., 2014; Macdonald et al., 2014; Stepanyants et al., 2015; Adachi et al., 2016). Here, we describe biochemical experiments that quantitatively measure interactions of Drp1 with lipids using purified recombinant Drp1 and synthetic liposomes with a defined set of phospholipids. This assay makes it possible to define the specificity of protein-lipid interaction and the role of the head group and acyl chains.

The Putative Drp1 Inhibitor mdivi-1 Is a Reversible Mitochondrial Complex I Inhibitor that Modulates Reactive Oxygen Species.

Mitochondrial fission mediated by the GTPase dynamin-related protein 1 (Drp1) is an attractive drug target in numerous maladies that range from heart disease to neurodegenerative disorders. The compound mdivi-1 is widely reported to inhibit Drp1-dependent fission, elongate mitochondria, and mitigate brain injury. Here, we show that mdivi-1 reversibly inhibits mitochondrial complex I-dependent O2 consumption and reverse electron transfer-mediated reactive oxygen species (ROS) production at concentrations (e.g., 50 µM) used to target mitochondrial fission. Respiratory inhibition is rescued by bypassing complex I using yeast NADH dehydrogenase Ndi1. Unexpectedly, respiratory impairment by mdivi-1 occurs without mitochondrial elongation, is not mimicked by Drp1 deletion, and is observed in Drp1-deficient fibroblasts. In addition, mdivi-1 poorly inhibits recombinant Drp1 GTPase activity (Ki > 1.2 mM). Overall, these results suggest that mdivi-1 is not a specific Drp1 inhibitor. The ability of mdivi-1 to reversibly inhibit complex I and modify mitochondrial ROS production may contribute to effects observed in disease models.