OMA1-Mediated Mitochondrial Dynamics Balance Organellar Homeostasis Upstream of Cellular Stress Responses.

In response to cellular metabolic and signaling cues, the mitochondrial network employs distinct sets of membrane-shaping factors to dynamically modulate organellar structures through a balance of fission and fusion. While these organellar dynamics mediate mitochondrial structure/function homeostasis, they also directly impact critical cell-wide signaling pathways such as apoptosis, autophagy, and the integrated stress response (ISR). Mitochondrial fission is driven by the recruitment of the cytosolic dynamin-related protein-1 (DRP1), while fusion is carried out by mitofusins 1 and 2 (in the outer membrane) and optic atrophy-1 (OPA1) in the inner membrane. This dynamic balance is highly sensitive to cellular stress; when the transmembrane potential across the inner membrane (Δψm) is lost, fusion-active OPA1 is cleaved by the overlapping activity with m-AAA protease-1 (OMA1 metalloprotease, disrupting mitochondrial fusion and leaving dynamin-related protein-1 (DRP1)-mediated fission unopposed, thus causing the collapse of the mitochondrial network to a fragmented state. OMA1 is a unique regulator of stress-sensitive homeostatic mitochondrial balance, acting as a key upstream sensor capable of priming the cell for apoptosis, autophagy, or ISR signaling cascades. Recent evidence indicates that higher-order macromolecular associations within the mitochondrial inner membrane allow these specialized domains to mediate crucial organellar functionalities.

Correction to: A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone.

The original article [1] contains three erroneous mentions of usage of a restriction enzyme-BstZ17I-in the Methods section as displayed in the following sentences.

A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone.

BACKGROUND: Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone. RESULTS: We describe a step-by-step method to truncate genes of interest in mammalian cell lines using custom-made donor vectors. Our method employs 2 guide RNAs, mutant Cas9D10A nickase (Cas9 = CRISPR associated sequence 9), and a custom-made donor vector for homologous recombination to precisely truncate a gene of interest with a selectable neomycin resistance cassette (NPTII: Neomycin Phosphotransferase II). We provide a detailed protocol on how to design and construct a custom donor vector using Gibson assembly (and the commonly utilized pcDNA3 vector as the backbone) allowing researchers to obtain specific gene modifications of interest (gene truncation, gene deletion, epitope tagging or knock-in mutation). Selection of mutants in mammalian cell lines with G418 (Geneticin) combined with several screening methods: western blot analysis, polymerase chain reaction, and Sanger sequencing resulted in streamlined mutant isolation. Proof of principle experiments were done in several mammalian cell lines. CONCLUSIONS: Here we describe a detailed protocol to employ CRISPR Cas9 genome editing to truncate genes of interest using the commonly employed expression vector pcDNA3 as the backbone for the donor vector. Providing a detailed protocol for custom donor vector design and construction will enable researchers to develop unique genome editing tools. To date, detailed protocols for CRISPR Cas9 custom donor vector construction are limited (Lee et al. in Sci Rep 5:8572, 2015; Ma et al. in Sci Rep 4:4489, 2014). Custom donor vectors are commercially available, but can be expensive. Our goal is to share this protocol to aid researchers in performing genetic investigations that require custom donor vectors for specialized applications (specific gene truncations, knock-in mutations, and epitope tagging applications).

A threshold of transmembrane potential is required for mitochondrial dynamic balance mediated by DRP1 and OMA1.

As an organellar network, mitochondria dynamically regulate their organization via opposing fusion and fission pathways to maintain bioenergetic homeostasis and contribute to key cellular pathways. This dynamic balance is directly linked to bioenergetic function: loss of transmembrane potential across the inner membrane (Δψ m) disrupts mitochondrial fission/fusion balance, causing fragmentation of the network. However, the level of Δψ m required for mitochondrial dynamic balance, as well as the relative contributions of fission and fusion pathways, have remained unclear. To explore this, mitochondrial morphology and Δψ m were examined via confocal imaging and tetramethyl rhodamine ester (TMRE) flow cytometry, respectively, in cultured 143B osteosarcoma cells. When normalized to the TMRE value of untreated 143B cells as 100%, both genetic (mtDNA-depleted ρ0) and pharmacological [carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-treated] cell models below 34% TMRE fluorescence were unable to maintain mitochondrial interconnection, correlating with loss of fusion-active long OPA1 isoforms (L-OPA1). Mechanistically, this threshold is maintained by mechanistic coordination of DRP1-mediated fission and OPA1-mediated fusion: cells lacking either DRP1 or the OMA1 metalloprotease were insensitive to loss of Δψ m, instead maintaining an obligately fused morphology. Collectively, these findings demonstrate a mitochondrial 'tipping point' threshold mediated by the interaction of Δψ m with both DRP1 and OMA1; moreover, DRP1 appears to be required for effective OPA1 maintenance and processing, consistent with growing evidence for direct interaction of fission and fusion pathways. These results suggest that Δψ m below threshold coordinately activates both DRP1-mediated fission and OMA1 cleavage of OPA1, collapsing mitochondrial dynamic balance, with major implications for a range of signaling pathways and cellular life/death events.

Commentary: Mitochondrial DNA damage and loss in diabetes.

This commentary discusses damage and loss of mitochondrial DNA (mtDNA) in type 2 diabetes mellitus from both the clinical and experimental perspectives. Increasingly, an array of studies in experimental models and patients suggests that the cellular stresses of insulin resistance in type 2 diabetes damage mtDNA, leading to loss of mitochondrial genetic content. As such, mtDNA is emerging as both a valuable monitoring tool and translational preventive target for metabolic disease. Copyright © 2016 John Wiley & Sons, Ltd.

Mitochondrial autophagy in cells with mtDNA mutations results from synergistic loss of transmembrane potential and mTORC1 inhibition.

Autophagy has emerged as a key cellular process for organellar quality control, yet this pathway apparently fails to eliminate mitochondria containing pathogenic mutations in mitochondrial DNA (mtDNA) in patients with a variety of human diseases. In order to explore how mtDNA-mediated mitochondrial dysfunction interacts with endogenous autophagic pathways, we examined autophagic status in a panel of human cytoplasmic hybrid (cybrid) cell lines carrying a variety of pathogenic mtDNA mutations. We found that both genetic- and chemically induced loss of mitochondrial transmembrane potential (Δψ(m)) caused recruitment of the pro-mitophagic factor Parkin to mitochondria. Strikingly, however, the loss of Δψ(m) alone was insufficient to prompt delivery of mitochondria to the autophagosome (mitophagy). We found that mitophagy could be induced following treatment with the mTORC1 inhibitor rapamycin in cybrids carrying either large-scale partial deletions of mtDNA or complete depletion of mtDNA. Further, we found that the level of endogenous Parkin is a crucial determinant of mitophagy. These results suggest a two-hit model, in which the synergistic induction of both (i) mitochondrial recruitment of Parkin following the loss of Δψ(m) and (ii) mTORC1-controlled general macroautophagy is required for mitophagy. It appears that mitophagy can be accomplished by the endogenous autophagic machinery, but requires the full engagement of both of these pathways.

Differentiation activates mitochondrial OPA1 processing in myoblast cell lines.

Mitochondrial optic atrophy-1 (OPA1) plays key roles in adapting mitochondrial structure to bioenergetic function. When transmembrane potential across the inner membrane (Δψm) is intact, long (L-OPA1) isoforms shape the inner membrane through membrane fusion and the formation of cristal junctions. When Δψm is lost, however, OPA1 is cleaved to short, inactive S-OPA1 isoforms by the OMA1 metalloprotease, disrupting mitochondrial structure and priming cellular stress responses such as apoptosis. Previously, we demonstrated that L-OPA1 of H9c2 cardiomyoblasts is insensitive to loss of Δψm via challenge with the protonophore carbonyl cyanide chlorophenyl hydrazone (CCCP), but that CCCP-induced OPA1 processing is activated upon differentiation in media with low serum supplemented with all-trans retinoic acid (ATRA). Here, we show that this developmental induction of OPA1 processing in H9c2 cells is independent of ATRA; moreover, pretreatment of undifferentiated H9c2s with chloramphenicol (CAP), an inhibitor of mitochondrial protein synthesis, recapitulates the Δψm-sensitive OPA1 processing observed in differentiated H9c2s. L6.C11 and C2C12 myoblast lines display the same developmental and CAP-sensitive induction of OPA1 processing, demonstrating a general mechanism of OPA1 regulation in mammalian myoblast cell settings. Restoration of CCCP-induced OPA1 processing correlates with increased apoptotic sensitivity. Moreover, OPA1 knockdown indicates that intact OPA1 is necessary for effective myoblast differentiation. Taken together, our results indicate that a novel developmental mechanism acts to regulate OMA1-mediated OPA1 processing in myoblast cell lines, in which differentiation engages mitochondrial stress sensing.

Development of pullulan/chitosan/salvianolic acid ternary fibrous membranes and their potential for chemotherapeutic applications.

This study investigates the feasibility of centrifugal spinning for producing fibrous membranes containing pullulan, chitosan, and danshen extract. The danshen extract is composed of 20 wt% salvianolic acid B (SA). Citric acid was added to the mixture as a crosslinking agent to promote its use in the aqueous medium. The influence of the danshen concentration (25 wt% and 33 wt%) on fiber morphology, thermal behavior, and the biochemical effect was analyzed. Developed fiber-based membranes consist of long, continuous, and uniform fibers with a sparse scattering of beads. Fiber diameter analysis shows values ranging from 384 ± 123 nm to 644 ± 141 nm depending on the concentration of danshen. The nanofibers show adequate aqueous stability after crosslinking. Thermal analysis results prove that SA is loaded into nanofibers without compromising their structural integrity. Cell-based results indicate that the developed nanofiber membranes promote cell growth and are not detrimental to fibroblast cells. Anticancer studies reveal a promising inhibition to the proliferation of HCT116 colon cancer cells. The developed systems show potential as innovative systems to be used as a bioactive chemotherapeutic drug that could be placed on the removed tumor site to prevent development of colon cancer microdeposits.

FOXO1 promotes the expression of canonical WNT target genes in examined basal-like breast and glioblastoma multiforme cancer cells.

Basal-like breast cancer (BBC) and glioblastoma multiforme (GBM) are aggressive cancers associated with poor prognosis. BBC and GBM have stem cell-like gene expression signatures, which are in part driven by forkhead box O (FOXO) transcription factors. To gain further insight into the impact of FOXO1 in BBC, we treated BT549 cells with AS1842856 and performed RNA sequencing. AS1842856 binds to unphosphorylated FOXO1 and inhibits its ability to directly bind to DNA. Gene Set Enrichment Analysis indicated that a set of WNT pathway target genes, including lymphoid enhancer-binding factor 1 (LEF1) and transcription factor 7 (TCF7), were robustly induced after AS1842856 treatment. These same genes were also induced in GBM cell lines U87MG, LN18, LN229, A172, and DBTRG upon AS1842856 treatment. By contrast, follow-up RNA interference (RNAi) targeting of FOXO1 led to reduced LEF1 and TCF7 gene expression in BT549 and U87MG cells. In agreement with RNAi experiments, CRISPR Cas9-mediated FOXO1 disruption reduced the expression of canonical WNT genes LEF1 and TCF7 in U87MG cells. The loss of TCF7 gene expression in FOXO1 disruption mutants was restored by exogenous expression of the DNA-binding-deficient FOXO1-H215R. Therefore, FOXO1 induces TCF7 in a DNA-binding-independent manner, similar to other published FOXO1-activated genes such as TCF4 and hes family bHLH transcription factor 1. Our work demonstrates that FOXO1 promotes canonical WNT gene expression in examined BBC and GBM cells, similar to results found in Drosophila melanogaster, T-cell development, and murine acute myeloid leukemia models.

Oxymatrine Loaded Cross-Linked PVA Nanofibrous Scaffold: Design and Characterization and Anticancer Properties.

This study focuses on the fabrication, characterization and anticancer properties of biocompatible and biodegradable composite nanofibers consisting of poly(vinyl alcohol) (PVA), oxymatrine (OM), and citric acid (CA) using a facile and high-yield centrifugal spinning process known as Forcespinning. The effects of varying concentrations of OM and CA on fiber diameter and molecular cross-linking are investigated. The morphological and thermo-physical properties, as well as water absorption of the developed nanofiber-based mats are characterized using microscopical analysis, energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, and thermogravimetric analysis. In vitro anticancer studies are conducted with HCT116 colorectal cancer cells. Results show a high yield of long fibers embedded with beads. Fiber average diameters range between 462 and 528 nm depending on OM concentration. The thermal analysis results show that the fibers are stable at room temperature. The anticancer study reveals that PVA nanofiber membrane with high concentrations of OM can suppress the proliferation of HCT116 colorectal cancer cells. The study provides a comprehensive investigation of OM embedded into nanosized PVA fibers and the prospective application of these membranes as a drug delivery system.

Development of zinc oxide/hydroxyapatite/poly(D,L-lactic acid) fibrous scaffold for tissue engineering applications.

Scaffolds based on polymeric fibers represent an engaging biomedical device due to their particular morphology and similarity with extracellular matrices. The biggest challenge to use fibrous materials in the biomedical field is related to their favorable platform for the adhesion of pathogenic microorganisms. Therefore, their optimum performance not only depends on their bioactive potential but also on their antimicrobial properties. The aim of this work was the design of antimicrobial (zinc oxide, ZnO) and bioactive (hydroxyapatite, Hap) fibrous materials using poly(D, L-lactic acid) (PDLLA) as the polymer fiber substrate. Fiber based composite scaffolds were developed using the Forcespinning® technique. For analysis purposes, the morphological, thermal, antimicrobial and biological properties of the fibrous hybrid system obtained at a concentration of 5 wt% of ZnO and 5 wt% of Hap were studied. The incorporation of the aforementioned nanoparticles (NPs) mixture in PDLLA led to an increase in viscosity and a pseudo-plastic tendency of the precursor solution, which caused an increase in fiber diameters and their dispersion of values. Small cavities and certain roughness were the main surface morphology observed on the fibers before and after NPs incorporation. The fiber thermal stability decreased due to the presence of the NPs. The antimicrobial properties of the hybrid fibrous scaffold presented a growth inhibition (GI) of 70 and 85% for E. coli and S. aureus strains, respectively. Concerning the osteoblast-cell compatibility, PDLLA and hybrid PDLLA scaffold showed low toxicity (cell viabilities above 80%), allowing cell growth inside its three-dimension structure and favorable cell morphology extended along the fibers. This behavior suggests a promising potential of this hybrid PDLLA scaffold for bone application.

Functional complementation of mitochondrial DNAs: mobilizing mitochondrial genetics against dysfunction.

Human mitochondrial DNA (mtDNA) is a 16.6-kb circular genome that is typically found in approximately 1000 copies per cell. Frequently, one or more forms of mtDNA (i.e. wildtype (WT) and one or more mutant variants) will co-exist within an individual cell, a situation termed heteroplasmy; however, it has been unclear how different mitochondria and mtDNA populations interact functionally in a heteroplasmic cell system. Using sequence-specific microscopic methods to examine mtDNA at suborganellar resolution, we examined the submitochondrial organization of mtDNA heteroplasmy in nucleoids, the DNA-protein complexes that organize and package mtDNA. Our recent results reveal that, while heterologous mtDNAs are generally maintained stably in separate nucleoid populations, the two mtDNAs transcomplement each other to restore WT-like levels of mitochondrial function and morphology. These findings reveal that the diffusion of mtDNA-derived transcripts through the mitochondrial matrix allows for transcomplementation, despite the apparent genetic autonomy of nucleoids. The fundamental ability of mtDNAs to complement each other within the matrix of the mitochondrial network provides a mechanistic basis for therapeutic strategies designed to restore mitochondrial function in heteroplasmic cells by increasing WT mtDNA content, particularly in light of the emerging connection between the processes of mitochondrial fission/fusion and mtDNA nucleoid organization.

Reactive oxygen species, oxidative stress, and cell death correlate with level of CoQ10 deficiency.

Coenzyme Q(10) (CoQ(10)) is essential for electron transport in the mitochondrial respiratory chain and antioxidant defense. The relative importance of respiratory chain defects, ROS production, and apoptosis in the pathogenesis of CoQ(10) deficiency is unknown. We determined previously that severe CoQ(10) deficiency in cultured skin fibroblasts harboring COQ2 and PDSS2 mutations produces divergent alterations of bioenergetics and oxidative stress. Here, to better understand the pathogenesis of CoQ(10) deficiency, we have characterized the effects of varying severities of CoQ(10) deficiency on ROS production and mitochondrial bioenergetics in cells harboring genetic defects of CoQ(10) biosynthesis. Levels of CoQ(10) seem to correlate with ROS production; 10-15% and >60% residual CoQ(10) are not associated with significant ROS production, whereas 30-50% residual CoQ(10) is accompanied by increased ROS production and cell death. Our results confirm that varying degrees of CoQ(10) deficiency cause variable defects of ATP synthesis and oxidative stress. These findings may lead to more rational therapeutic strategies for CoQ(10) deficiency.

Mitochondrial OMA1 and OPA1 as Gatekeepers of Organellar Structure/Function and Cellular Stress Response.

Mammalian mitochondria are emerging as a critical stress-responsive contributor to cellular life/death and developmental outcomes. Maintained as an organellar network distributed throughout the cell, mitochondria respond to cellular stimuli and stresses through highly sensitive structural dynamics, particularly in energetically demanding cell settings such as cardiac and muscle tissues. Fusion allows individual mitochondria to form an interconnected reticular network, while fission divides the network into a collection of vesicular organelles. Crucially, optic atrophy-1 (OPA1) directly links mitochondrial structure and bioenergetic function: when the transmembrane potential across the inner membrane (ΔΨm) is intact, long L-OPA1 isoforms carry out fusion of the mitochondrial inner membrane. When ΔΨm is lost, L-OPA1 is cleaved to short, fusion-inactive S-OPA1 isoforms by the stress-sensitive OMA1 metalloprotease, causing the mitochondrial network to collapse to a fragmented population of organelles. This proteolytic mechanism provides sensitive regulation of organellar structure/function but also engages directly with apoptotic factors as a major mechanism of mitochondrial participation in cellular stress response. Furthermore, emerging evidence suggests that this proteolytic mechanism may have critical importance for cell developmental programs, particularly in cardiac, neuronal, and stem cell settings. OMA1's role as a key mitochondrial stress-sensitive protease motivates exciting new questions regarding its mechanistic regulation and interactions, as well as its broader importance through involvement in apoptotic, stress response, and developmental pathways.

Aloe Vera extract-based composite nanofibers for wound dressing applications.

Natural, biocompatible, and biodegradable composite nanofibers made of Aloe vera extract, pullulan, chitosan, and citric acid were successfully produced via Forcespinning® technology. The addition of Aloe vera extract at different weight percent loadings was investigated. The morphology, thermal properties, physical properties, and water absorption of the nanofibers were characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and thermogravimetric analysis (TGA). The developed nanofiber membranes exhibited good water absorption capabilities, synergistic antibacterial activity against Escherichia coli, and promoted cell attachment and growth. Its porous and high surface area structure make it a potential candidate for wound dressing applications due to its ability to absorb excessive blood and exudates, as well as provide protection from infection while maintaining good thermal stability.

Mitochondrial OPA1 cleavage is reversibly activated by differentiation of H9c2 cardiomyoblasts.

Optic atrophy-1 (OPA1) is a dynamin-like GTPase localized to the mitochondrial inner membrane, playing key roles in inner membrane fusion and cristae maintenance. OPA1 is regulated by the mitochondrial transmembrane potential (Δψm): when Δψm is intact, long OPA1 isoforms (L-OPA1) carry out inner membrane fusion. Upon loss of Δψm, L-OPA1 isoforms are proteolytically cleaved to short (S-OPA1) isoforms by the stress-inducible OMA1 metalloprotease, causing collapse of the mitochondrial network and promoting apoptosis. Here, we show that L-OPA1 isoforms of H9c2 cardiomyoblasts are retained under loss of Δψm, despite the presence of OMA1. However, when H9c2s are differentiated to a more cardiac-like phenotype via treatment with retinoic acid (RA) in low serum media, loss of Δ ψm induces robust, and reversible, cleavage of L-OPA1 and subsequent OMA1 degradation. These findings indicate that a potent developmental switch regulates Δ ψm-sensitive OPA1 cleavage, suggesting novel developmental and regulatory mechanisms for OPA1 homeostasis.

Detection of mitochondrial DNA (mtDNA) mutations.

The maternally inherited mitochondrial DNA (mtDNA) is a circular 16,569bp double stranded DNA that encodes 37 genes, 24 of which (2 rRNAs and 22 tRNAs) are necessary for transcription and translation of 13 polypeptides that are all subunits of respiratory chain. Pathogenic mutations in mtDNA cause respiratory chain dysfunction, and are the underlying defect in an ever-increasing number of mtDNA-related encephalomyopathies with distinct phenotypes. In this chapter, we present an overview of mtDNA mutations and describe the molecular techniques currently employed in our laboratory to detect two types of mtDNA mutations: single-large-scale rearrangements and point mutations.

A Disturbance in the Force: Cellular Stress Sensing by the Mitochondrial Network.

As a highly dynamic organellar network, mitochondria are maintained as an organellar network by delicately balancing fission and fusion pathways. This homeostatic balance of organellar dynamics is increasingly revealed to play an integral role in sensing cellular stress stimuli. Mitochondrial fission/fusion balance is highly sensitive to perturbations such as loss of bioenergetic function, oxidative stress, and other stimuli, with mechanistic contribution to subsequent cell-wide cascades including inflammation, autophagy, and apoptosis. The overlapping activity with m-AAA protease 1 (OMA1) metallopeptidase, a stress-sensitive modulator of mitochondrial fusion, and dynamin-related protein 1 (DRP1), a regulator of mitochondrial fission, are key factors that shape mitochondrial dynamics in response to various stimuli. As such, OMA1 and DRP1 are critical factors that mediate mitochondrial roles in cellular stress-response signaling. Here, we explore the current understanding and emerging questions in the role of mitochondrial dynamics in sensing cellular stress as a dynamic, responsive organellar network.

Texas Sour Orange Juice Used in Scaffolds for Tissue Engineering.

Fine fibers of polyhydroxybutyrate (PHB), a biopolymer, were developed via a centrifugal spinning technique. The developed fibers have an average diameter of 1.8 µm. Texas sour orange juice (SOJ) was applied as a natural antibacterial agent and infiltrated within the fibrous membranes. The antibacterial activity against common Gram-positive and Gram-negative bacteria (Staphylococcus aureus and Escherichia coli, respectively) was evaluated as well as cell adhesion and viability. The PHB/SOJ scaffolds showed antibacterial activity of up to 152% and 71% against S. aureus and E. coli, respectively. The cell studies revealed a suitable environment for cell growth and cell attachment. The outcome of this study opens up new opportunities for fabrication of fibrous materials for biomedical applications having multifunctional properties while using natural agents.

Oxidative insults disrupt OPA1-mediated mitochondrial dynamics in cultured mammalian cells.

OBJECTIVE: To explore the impact of oxidative insults on mitochondrial dynamics. In mammalian cells, oxidative insults activate stress response pathways including inflammation, cytokine secretion, and apoptosis. Intriguingly, mitochondria are emerging as a sensitive network that may function as an early indicator of subsequent cellular stress responses. Mitochondria form a dynamic network, balancing fusion, mediated by optic atrophy-1 (OPA1), and fission events, mediated by dynamin-related protein-1 (DRP1), to maintain homeostasis. METHODS: Here, we examine the impact of oxidative insults on mitochondrial dynamics in 143B osteosarcoma and H9c2 cardiomyoblast cell lines via confocal microscopy, flow cytometry, and protein-based analyses. RESULTS: When challenged with hydrogen peroxide (H2O2), a ROS donor, both cell lines display fragmentation of the mitochondrial network and loss of fusion-active OPA1 isoforms, indicating that OPA1-mediated mitochondrial fusion is disrupted by oxidative damage in mammalian cells. Consistent with this, cells lacking OMA1, a key protease responsible for cleavage of OPA1, are protected against OPA1 cleavage and mitochondrial fragmentation in response to H2O2 challenge. DISCUSSION: Taken together, these findings indicate that oxidative insults damage OPA1-mediated mitochondrial dynamics in mammalian cells via activation of OMA1, consistent with an emerging role for mitochondrial dynamics as an early indicator of cellular stress signaling.