Modulation of Dynamin-related Protein 1 (DRP1) Function by Increased O-linked-β-N-acetylglucosamine Modification (O-GlcNAc) in Cardiac Myocytes

O-linked-N-acetyl-glucosamine glycosylation (O-GlcNAcylation) of the serine and threonine residues of cellular proteins is a dynamic process and affects phosphorylation. Prolonged O-GlcNAcylation has been linked to diabetes-related complications, including mitochondrial dysfunction. Mitochondria are dynamically remodeling organelles, that constantly fuse (fusion) and divide (fission). An imbalance of this process affects mitochondrial function. In this study, we found that dynamin-related protein 1 (DRP1) is O-GlcNAcylated in cardiomyocytes at threonine 585 and 586. O-GlcNAcylation was significantly enhanced by the chemical inhibition of N-acetyl-glucosaminidase. Increased O-GlcNAcylation decreases the phosphorylation of DRP1 at serine 637, which is known to regulate DRP1 function. In fact, increased O-GlcNAcylation augments the level of the GTP-bound active form of DRP1 and induces translocation of DRP1 from the cytoplasm to mitochondria. Mitochondrial fragmentation and decreased mitochondrial membrane potential also accompany the increased O-GlcNAcylation. In conclusion, this report shows, for the first time, that O-GlcNAcylation modulates DRP1 functionality in cardiac muscle cells.     

MAPL is a new mitochondrial SUMO E3 ligase that regulates mitochondrial fission

The modification of proteins by the small ubiquitin‐like modifier (SUMO) is known to regulate an increasing array of cellular processes. SUMOylation of the mitochondrial fission GTPase dynamin‐related protein 1 (DRP1) stimulates mitochondrial fission, suggesting that SUMOylation has an important function in mitochondrial dynamics. The conjugation of SUMO to its substrates requires a regulatory SUMO E3 ligase; however, so far, none has been functionally associated with the mitochondria. By using biochemical assays, overexpression and RNA interference experiments, we characterized the mitochondrial‐anchored protein ligase (MAPL) as the first mitochondrial‐anchored SUMO E3 ligase. Furthermore, we show that DRP1 is a substrate for MAPL, providing a direct link between MAPL and the fission machinery. Importantly, the large number of unidentified mitochondrial SUMO targets suggests a global role for SUMOylation in mitochondrial function, placing MAPL as a crucial component in the regulation of multiple conjugation events.     

Mitochondrial morphology and dynamics: actors, mechanisms and functions

Mitochondria are dynamic organelles that continuously move, fuse and divide. Their overall morphology, ranging from a filamentous network to a collection of isolated dots, is determined by fusion-fission equilibrium, which depends on the cellular and physiological context. The machineries of fusion and fission, that are conserved throughout evolution, include three large GTPases of the dynamin-superfamily: Dnm1/DRP1 - involved in fission?- as well as Fzo1/MFN and Mgm1/OPA1?- required for fusion. While the activities, mecanisms and regulations of mitochondrial fusion and fission machineries continue to be unravelled, the relevance of mitochondrial dynamics is witnessed by their impact on organelle functions, cell survival and cell differenciation, their requirement for embryonic development and their involvement in neurological diseases.     

Accelerated dentinogenesis by inhibiting the mitochondrial fission factor,dynamin related protein 1

Undifferentiated odontogenic epithelium and dental papilla cells differentiate into ameloblasts and odontoblasts, respectively, both of which are essential for tooth development. These differentiation processes involve dramatic functional and morphological changes of the cells. For these changes to occur, activation of mitochondrial functions, including ATP production, is extremely important. In addition, these changes are closely related to mitochondrial fission and fusion, known as mitochondrial dynamics. However, few studies have focused on the role of mitochondrial dynamics in tooth development. The purpose of this study was to clarify this role. We used mouse tooth germ organ cultures and a mouse dental papilla cell line with the ability to differentiate into odontoblasts, in combination with knockdown of the mitochondrial fission factor, dynamin related protein (DRP)1. In organ cultures of the mouse first molar, tooth germ developed to the early bell stage. The amount of dentin formed under DRP1 inhibition was significantly larger than that of the control. In experiments using a mouse dental papilla cell line, differentiation into odontoblasts was enhanced by inhibiting DRP1. This was associated with increased mitochondrial elongation and ATP production compared to the control. These results suggest that DRP1 inhibition accelerates dentin formation through mitochondrial elongation and activation. This raises the possibility that DRP1 might be a therapeutic target for developmental disorders of teeth.     

Depletion of mitochondrial fission factor DRP1 causes increased apoptosis in human colon cancer cells

Mitochondria play a critical role in regulation of apoptosis, a form of programmed cell death, by releasing apoptogenic factors including cytochrome c. Growing evidence suggests that dynamic changes in mitochondrial morphology are involved in cellular apoptotic response. However, whether DRP1-mediated mitochondrial fission is required for induction of apoptosis remains speculative. Here, we show that siRNA-mediated DRP1 knockdown promoted accumulation of elongated mitochondria in HCT116 and SW480 human colon cancer cells. Surprisingly, DRP1 down-regulation led to decreased proliferation and increased apoptosis of these cells. A higher rate of cytochrome c release and reductions in mitochondrial membrane potential were also revealed in DRP1-depleted cells. Taken together, our present findings suggest that mitochondrial fission factor DRP1 inhibits colon cancer cell apoptosis through the regulation of cytochrome c release and mitochondrial membrane integrity.     

PINK1 disables the anti-fission machinery to segregate damaged mitochondria for mitophagy

Mitochondrial fission is essential for the degradation of damaged mitochondria. It is currently unknown how the dynamin-related protein 1 (DRP1)–associated fission machinery is selectively targeted to segregate damaged mitochondria. We show that PTEN-induced putative kinase (PINK1) serves as a pro-fission signal, independently of Parkin. Normally, the scaffold protein AKAP1 recruits protein kinase A (PKA) to the outer mitochondrial membrane to phospho-inhibit DRP1. We reveal that after damage, PINK1 triggers PKA displacement from A-kinase anchoring protein 1. By ejecting PKA, PINK1 ensures the requisite fission of damaged mitochondria for organelle degradation. We propose that PINK1 functions as a master mitophagy regulator by activating Parkin and DRP1 in response to damage. We confirm that PINK1 mutations causing Parkinson disease interfere with the orchestration of selective fission and mitophagy by PINK1.     

The Arabidopsis tail-anchored protein PEROXISOMAL AND MITOCHONDRIAL DIVISION FACTOR1 is involved in the morphogenesis and proliferation of peroxisomes and mitochondria

Peroxisomes and mitochondria are multifunctional eukaryotic organelles that are not only interconnected metabolically but also share proteins in division. Two evolutionarily conserved division factors, dynamin-related protein (DRP) and its organelle anchor FISSION1 (FIS1), mediate the fission of both peroxisomes and mitochondria. Here, we identified and characterized a plant-specific protein shared by these two types of organelles. The Arabidopsis thaliana PEROXISOMAL and MITOCHONDRIAL DIVISION FACTOR1 (PMD1) is a coiled-coil protein tethered to the membranes of peroxisomes and mitochondria by its C terminus. Null mutants of PMD1 contain enlarged peroxisomes and elongated mitochondria, and plants overexpressing PMD1 have an increased number of these organelles that are smaller in size and often aggregated. PMD1 lacks physical interaction with the known division proteins DRP3 and FIS1; it is also not required for DRP3's organelle targeting. Affinity purifications pulled down PMD1's homolog, PMD2, which exclusively targets to mitochondria and plays a specific role in mitochondrial morphogenesis. PMD1 and PMD2 can form homo- and heterocomplexes. Organelle targeting signals reside in the C termini of these proteins. Our results suggest that PMD1 facilitates peroxisomal and mitochondrial proliferation in a FIS1/DRP3-independent manner and that the homologous proteins PMD1 and PMD2 perform nonredundant functions in organelle morphogenesis.     

Sumo1 conjugates mitochondrial substrates and participates in mitochondrial fission

Mitochondrial fission requires the evolutionarily conserved dynamin related protein (DRP1), which is recruited from the cytosol to the mitochondrial outer membrane to coordinate membrane scission. Currently, the mechanism of recruitment and assembly of DRP1 on the mitochondria is unclear. Here, we identify Ubc9 and Sumo1 as specific DRP1-interacting proteins and demonstrate that DRP1 is a Sumo1 substrate. In addition, a surprising number of Sumo1 conjugates were observed in the mitochondrial fractions, suggesting that sumoylation is a common mitochondrial modification. Video microscopy demonstrates that YFP:Sumo1 is often found at the site of mitochondrial fission and remains tightly associated to the tips of fragmented mitochondria. Consistent with this, fluorescence microscopy revealed that a portion of total cytosolic YFP:Sumo1 colocalizes with endogenous mitochondrial DRP1. Finally, transient transfection of Sumo1 dramatically increases the level of mitochondrial fragmentation. Analysis of endogenous DRP1 levels indicates that overexpression of Sumo1 specifically protects DRP1 from degradation, resulting in a more stable, active pool of DRP1, which at least partially accounts for the excess fragmentation. Together, these data are the first to identify a function for Sumo1 on the mitochondria and suggest a novel role for the participation of Sumo1 in mitochondrial fission.     

Endoplasmic reticulum BIK initiates DRP1-regulated remodelling of mitochondrial cristae during apoptosis

The endoplasmic reticulum (ER) can elicit proapoptotic signalling that results in transmission of Ca(2+) to the mitochondria, which in turn stimulates recruitment of the fission enzyme DRP1 to the surface of the organelle. Here, we show that BH3-only BIK activates this pathway at the ER in intact cells, resulting in mitochondrial fragmentation but little release of cytochrome c to the cytosol. The BIK-induced transformations in mitochondria are dynamic in nature and involve DRP1-dependent remodelling and opening of cristae, where the major stores of cytochrome c reside. This novel function for DRP1 is distinct from its recognized role in regulating mitochondrial fission. Selective permeabilization of the outer membrane with digitonin confirmed that BIK stimulation results in mobilization of intramitochondrial cytochrome c. Of note, BIK can cooperate with a weak BH3-only protein that targets mitochondria, such as NOXA, to activate BAX by a mechanism that is independent of DRP1 enzyme activity. When expressed together, BIK and NOXA cause rapid release of mobilized cytochrome c and activation of caspases.     

Differential Roles of Arabidopsis Dynamin-Related Proteins DRP3A,DRP3B,and DRP5B in Organelle Division

Dynamin-related proteins (DRPs) are key components of the organelle division machineries, functioning as molecular scissors during the fission process. In Arabidopsis, DRP3A and DRP3B are shared by peroxisomal and mitochondrial division, whereas the structurally-distinct DRP5B (ARC5) protein is involved in the division of chloroplasts and peroxisomes. Here, we further investigated the roles of DRP3A, DRP3B, and DRP5B in organelle division and plant development. Despite DRP5B's lack of stable association with mitochondria, drp5B mutants show defects in mitochondrial division. The drp3A-2 drp3B-2 drp5B-2 triple mutant exhibits enhanced mitochondrial division phenotypes over drp3A-2 drp3B-2, but its peroxisomal morphology and plant growth phenotypes resemble those of the double mutant. We further demonstrated that DRP3A and DRP3B form a supercomplex in vivo, in which DRP3A is the major component, yet DRP5B is not a constituent of this complex. We thus conclude that DRP5B participates in the division of three types of organelles in Arabidopsis, acting independently of the DRP3 complex. Our findings will help elucidate the precise composition of the DRP3 complex at organelle division sites, and will be instrumental to studies aimed at understanding how the same protein mediates the morphogenesis of distinct organelles that are linked by metabolism.     

OPA1 (dys)functions

Mitochondrial morphology varies according to cell type and cellular context from an interconnected filamentous network to isolated dots. This morphological plasticity depends on mitochondrial dynamics, a balance between antagonistic forces of fission and fusion. DRP1 and FIS1 control mitochondrial outer membrane fission and Mitofusins its fusion. This review focuses on OPA1, one of the few known actors of inner membrane dynamics, whose mutations provoke an optic neuropathy. Since its first identification in 2000 the characterization of the functions of OPA1 has made rapid progress thus providing numerous clues to unravel the pathogenetic mechanisms of ADOA-1.     

Mitochondrial function and actin regulate dynamin-related protein 1-dependent mitochondrial fission

Mitochondria display a variety of shapes, ranging from small and spherical or the classical tubular shape to extended networks. Shape transitions occur frequently and include fusion, fission, and branching. It was reported that some mitochondrial shape transitions are developmentally regulated, whereas others were linked to disease or apoptosis. However, if and how mitochondrial function controls mitochondrial shape through regulation of mitochondrial fission and fusion is unclear. Here, we show that inhibitors of electron transport, ATP synthase, or the permeability transition pore (mtPTP) induced reversible mitochondrial fission. Mitochondrial fission depended on dynamin-related protein 1 (DRP1) and F-actin: Disruption of F-actin attenuated fission and recruitment of DRP1 to mitochondria. In contrast, uncoupling of electron transport and oxidative phosphorylation caused mitochondria to adopt a distinct disk shape. This shape change was independent of the cytoskeleton and DRP1 and was most likely caused by swelling. Thus, disruption of mitochondrial function rapidly and reversibly altered mitochondrial shape either by activation of DRP1-dependent fission or by swelling, indicating a close relationship between mitochondrial fission, shape, and function. Furthermore, our results suggest that the actin cytoskeleton is involved in mitochondrial fission by facilitating mitochondrial recruitment of DRP1.     

DRP1-dependent mitochondrial fission initiates follicle cell differentiation during Drosophila oogenesis

Exit from the cell cycle is essential for cells to initiate a terminal differentiation program during development, but what controls this transition is incompletely understood. In this paper, we demonstrate a regulatory link between mitochondrial fission activity and cell cycle exit in follicle cell layer development during Drosophila melanogaster oogenesis. Posterior-localized clonal cells in the follicle cell layer of developing ovarioles with down-regulated expression of the major mitochondrial fission protein DRP1 had mitochondrial elements extensively fused instead of being dispersed. These cells did not exit the cell cycle. Instead, they excessively proliferated, failed to activate Notch for differentiation, and exhibited downstream developmental defects. Reintroduction of mitochondrial fission activity or inhibition of the mitochondrial fusion protein Marf-1 in posterior-localized DRP1-null clones reversed the block in Notch-dependent differentiation. When DRP1-driven mitochondrial fission activity was unopposed by fusion activity in Marf-1-depleted clones, premature cell differentiation of follicle cells occurred in mitotic stages. Thus, DRP1-dependent mitochondrial fission activity is a novel regulator of the onset of follicle cell differentiation during Drosophila oogenesis.     

Dichotomy in hypoxia-induced mitochondrial fission in placental mesenchymal cells during development and preeclampsia: consequences for trophoblast mitochondrial homeostasis

Dynamic changes in physiologic oxygen are required for proper placenta development; yet, when low-oxygen levels persist, placental development is halted, culminating in preeclampsia (PE), a serious complication of pregnancy. Considering mitochondria’s function is intimately linked to oxygen changes, we investigated the impact of oxygen on mitochondrial dynamics in placental mesenchymal stromal cells (pMSCs) that are vital for proper placental development. Transmission electron microscopy, proximity ligation assays for mitochondrial VDAC1 and endoplasmic reticulum IP3R, and immunoanalyses of p-DRP1 and OPA1, demonstrate that low-oxygen conditions in early 1st trimester and PE promote mitochondrial fission in pMSCs. Increased mitochondrial fission of mesenchymal cells was confirmed in whole PE placental tissue sections. Inhibition of DRP1 oligomerization with MDiVi-1 shows that low oxygen-induced mitochondrial fission is a direct consequence of DRP1 activation, likely via HIF1. Mitophagy, a downstream event prompted by mitochondrial fission, is a prominent outcome in PE, but not 1st trimester pMSCs. We also investigated whether mesenchymal–epithelial interactions affect mitochondrial dynamics of trophoblasts in PE placentae. Exposure of trophoblastic JEG3 cells to exosomes of preeclamptic pMSCs caused heightened mitochondrial fission in the cells via a sphingomyelin-dependent mechanism that was restored by MDiVi-1. Our data uncovered dichotomous regulation of mitochondrial fission and health in human placental mesenchymal cells under physiologic and pathologic hypoxic conditions and its impact on neighboring trophoblast cells.Subject terms: Mechanisms of disease, Endocrine reproductive disorders     

FUNDC1 regulates mitochondrial dynamics at the ER–mitochondrial contact site under hypoxic conditions

In hypoxic cells, dysfunctional mitochondria are selectively removed by a specialized autophagic process called mitophagy. The ER–mitochondrial contact site (MAM) is essential for fission of mitochondria prior to engulfment, and the outer mitochondrial membrane protein FUNDC1 interacts with LC3 to recruit autophagosomes, but the mechanisms integrating these processes are poorly understood. Here, we describe a new pathway mediating mitochondrial fission and subsequent mitophagy under hypoxic conditions. FUNDC1 accumulates at the MAM by associating with the ER membrane protein calnexin. As mitophagy proceeds, FUNDC1/calnexin association attenuates and the exposed cytosolic loop of FUNDC1 interacts with DRP1 instead. DRP1 is thereby recruited to the MAM, and mitochondrial fission then occurs. Knockdown of FUNDC1, DRP1, or calnexin prevents fission and mitophagy under hypoxic conditions. Thus, FUNDC1 integrates mitochondrial fission and mitophagy at the interface of the MAM by working in concert with DRP1 and calnexin under hypoxic conditions in mammalian cells.     

Fission and selective fusion govern mitochondrial segregation and elimination by autophagy

Accumulation of depolarized mitochondria within beta-cells has been associated with oxidative damage and development of diabetes. To determine the source and fate of depolarized mitochondria, individual mitochondria were photolabeled and tracked through fusion and fission. Mitochondria were found to go through frequent cycles of fusion and fission in a 'kiss and run' pattern. Fission events often generated uneven daughter units: one daughter exhibited increased membrane potential (delta psi(m)) and a high probability of subsequent fusion, while the other had decreased membrane potential and a reduced probability for a fusion event. Together, this pattern generated a subpopulation of non-fusing mitochondria that were found to have reduced delta psi(m) and decreased levels of the fusion protein OPA1. Inhibition of the fission machinery through DRP1(K38A) or FIS1 RNAi decreased mitochondrial autophagy and resulted in the accumulation of oxidized mitochondrial proteins, reduced respiration and impaired insulin secretion. Pulse chase and arrest of autophagy at the pre-proteolysis stage reveal that before autophagy mitochondria lose delta psi(m) and OPA1, and that overexpression of OPA1 decreases mitochondrial autophagy. Together, these findings suggest that fission followed by selective fusion segregates dysfunctional mitochondria and permits their removal by autophagy.     

Preventing mitochondrial fission impairs mitochondrial function and leads to loss of mitochondrial DNA

Mitochondria form a highly dynamic tubular network, the morphology of which is regulated by frequent fission and fusion events. However, the role of mitochondrial fission in homeostasis of the organelle is still unknown. Here we report that preventing mitochondrial fission, by down-regulating expression of Drp1 in mammalian cells leads to a loss of mitochondrial DNA and a decrease of mitochondrial respiration coupled to an increase in the levels of cellular reactive oxygen species (ROS). At the cellular level, mitochondrial dysfunction resulting from the lack of fission leads to a drop in the levels of cellular ATP, an inhibition of cell proliferation and an increase in autophagy. In conclusion, we propose that mitochondrial fission is required for preservation of mitochondrial function and thereby for maintenance of cellular homeostasis.     

Mitochondrial fusion and fission in cell life and death

Mitochondria are dynamic organelles that constantly fuse and divide. These processes (collectively termed mitochondrial dynamics) are important for mitochondrial inheritance and for the maintenance of mitochondrial functions. The core components of the evolutionarily conserved fusion and fission machineries have now been identified, and mechanistic studies have revealed the first secrets of the complex processes that govern fusion and fission of a double membrane-bound organelle. Mitochondrial dynamics was recently recognized as an important constituent of cellular quality control. Defects have detrimental consequences on bioenergetic supply and contribute to the pathogenesis of neurodegenerative diseases. These findings open exciting new directions to explore mitochondrial biology.     

The Arabidopsis Chloroplast Division Protein DYNAMIN-RELATED PROTEIN5B Also Mediates Peroxisome Division

Peroxisomes are highly dynamic organelles involved in various metabolic pathways. The division of peroxisomes is regulated by factors such as the PEROXIN11 (PEX11) proteins that promote peroxisome elongation and the dynamin-related proteins (DRPs) and FISSION1 (FIS1) proteins that function together to mediate organelle fission. In Arabidopsis thaliana, DRP3A/DRP3B and FIS1A (BIGYIN)/FIS1B are two pairs of homologous proteins known to function in both peroxisomal and mitochondrial division. Here, we report that DRP5B, a DRP distantly related to the DRP3s and originally identified as a chloroplast division protein, also contributes to peroxisome division. DRP5B localizes to both peroxisomes and chloroplasts. Mutations in the DRP5B gene lead to peroxisome division defects and compromised peroxisome functions. Using coimmunoprecipitation and bimolecular fluorescence complementation assays, we further demonstrate that DRP5B can interact or form a complex with itself and with DRP3A, DRP3B, FIS1A, and most of the Arabidopsis PEX11 isoforms. Our data suggest that, in contrast with DRP3A and DRP3B, whose orthologs exist across plant, fungal, and animal kingdoms, DRP5B is a plant/algal invention to facilitate the division of their organelles (i.e., chloroplasts and peroxisomes). In addition, our results support the notion that proteins involved in the early (elongation) and late (fission) stages of peroxisome division may act cooperatively.     

Inner-membrane proteins PMI/TMEM11 regulate mitochondrial morphogenesis independently of the DRP1/MFN fission/fusion pathways

Mitochondria are highly dynamic organelles that can change in number and morphology during cell cycle, development or in response to extracellular stimuli. These morphological dynamics are controlled by a tight balance between two antagonistic pathways that promote fusion and fission. Genetic approaches have identified a cohort of conserved proteins that form the core of mitochondrial remodelling machineries. Mitofusins (MFNs) and OPA1 proteins are dynamin-related GTPases that are required for outer- and inner-mitochondrial membrane fusion respectively whereas dynamin-related protein 1 (DRP1) is the master regulator of mitochondrial fission. We demonstrate here that the Drosophila PMI gene and its human orthologue TMEM11 encode mitochondrial inner-membrane proteins that regulate mitochondrial morphogenesis. PMI-mutant cells contain a highly condensed mitochondrial network, suggesting that PMI has either a pro-fission or an anti-fusion function. Surprisingly, however, epistatic experiments indicate that PMI shapes the mitochondria through a mechanism that is independent of drp1 and mfn. This shows that mitochondrial networks can be shaped in higher eukaryotes by at least two separate pathways: one PMI-dependent and one DRP1/MFN-dependent.