Fis1, Mff,MiD49, and MiD51 mediate Drp1 recruitment in mitochondrial fission

Several mitochondrial outer membrane proteins—mitochondrial fission protein 1 (Fis1), mitochondrial fission factor (Mff), mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51, respectively)—have been proposed to promote mitochondrial fission by recruiting the GTPase dynamin-related protein 1 (Drp1), but fundamental issues remain concerning their function. A recent study supported such a role for Mff but not for Fis1. In addition, it is unclear whether MiD49 and MiD51 activate or inhibit fission, because their overexpression causes extensive mitochondrial elongation. It is also unknown whether these proteins can act in the absence of one another to mediate fission. Using Fis1-null, Mff-null, and Fis1/Mff-null cells, we show that both Fis1 and Mff have roles in mitochondrial fission. Moreover, immunofluorescence analysis of Drp1 suggests that Fis1 and Mff are important for the number and size of Drp1 puncta on mitochondria. Finally, we find that either MiD49 or MiD51 can mediate Drp1 recruitment and mitochondrial fission in the absence of Fis1 and Mff. These results demonstrate that multiple receptors can recruit Drp1 to mediate mitochondrial fission.     

Inhibiting Drp1-mediated mitochondrial fission selectively prevents the release of cytochrome c during apoptosis

Most cell death stimuli trigger the mitochondrial release of cytochrome c and other cofactors that induce caspase activation and ensuing apoptosis. Apoptosis is also associated with massive mitochondrial fragmentation and cristae remodeling. Dynamin-related protein 1 (Drp1), a protein of the mitochondrial fission machinery, has been reported to participate in apoptotic mitochondrial fragmentation. Several theories explaining the mechanisms of cytochrome c release have been proposed. One suggests that it relies on the activation of Drp1-mediated mitochondrial fission. Here, we report that downregulation of Drp1 inhibits fragmentation of the mitochondrial network and partially prevents the release of cytochrome c but fails to prevent the release of other mitochondrial factors such as second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI, Omi/HtrA2, adenylate kinase 2 and deafness dystonia peptide/TIMM8a. An explanation for the prevention of cytochrome c release is provided by our observation that inhibiting Drp1-mediated mitochondrial fission prevents the mitochondrial release of soluble OPA1 that was proposed to regulate cristae remodeling and complete cytochrome c release during apoptosis. Finally, we observed that downregulation of Drp1 delays but does not inhibit apoptosis, suggesting that mitochondrial fragmentation is not a prerequisite for apoptosis.     

Adaptor Proteins MiD49 and MiD51 Can Act Independently of Mff and Fis1 in Drp1 Recruitment and Are Specific for Mitochondrial Fission

Drp1 (dynamin-related protein 1) is recruited to both mitochondrial and peroxisomal membranes to execute fission. Fis1 and Mff are Drp1 receptor/effector proteins of mitochondria and peroxisomes. Recently, MiD49 and MiD51 were also shown to recruit Drp1 to the mitochondrial surface; however, different reports have ascribed opposing roles in fission and fusion. Here, we show that MiD49 or MiD51 overexpression blocked fission by acting in a dominant-negative manner by sequestering Drp1 specifically at mitochondria, causing unopposed fusion events at mitochondria along with elongation of peroxisomes. Mitochondrial elongation caused by MiD49/51 overexpression required the action of fusion mediators mitofusins 1 and 2. Furthermore, at low level overexpression when MiD49 and MiD51 form discrete foci at mitochondria, mitochondrial fission events still occurred. Unlike Fis1 and Mff, MiD49 and MiD51 were not targeted to the peroxisomal surface, suggesting that they specifically act to facilitate Drp1-directed fission at mitochondria. Moreover, when MiD49 or MiD51 was targeted to the surface of peroxisomes or lysosomes, Drp1 was specifically recruited to these organelles. Moreover, the Drp1 recruitment activity of MiD49/51 appeared stronger than that of Mff or Fis1. We conclude that MiD49 and MiD51 can act independently of Mff and Fis1 in Drp1 recruitment and suggest that they provide specificity to the division of mitochondria.     

The mitochondrial fission receptor Mff selectively recruits oligomerized Drp1

Dynamin-related protein 1 (Drp1) is the GTP-hydrolyzing mechanoenzyme that catalyzes mitochondrial fission in the cell. Residing in the cytosol as dimers and tetramers, Drp1 is recruited by receptors on the mitochondrial outer membrane, where it further assembles into a helical ring that drives division via GTP-dependent constriction. The Drp1 receptor Mff is a major regulator of mitochondrial fission, and its overexpression results in increased fission. In contrast, the alternative Drp1 receptors MiD51 and MiD49 appear to recruit inactive forms of Drp1, because their overexpression inhibits fission. Using genetic and biochemical assays, we studied the interaction of Drp1 with Mff. We show that the insert B region of Drp1 inhibits Mff–Drp1 interactions, such that recombinant Drp1 mutants lacking insert B form a stable complex with Mff. Mff cannot bind to assembly-deficient mutants of Drp1, suggesting that Mff selectively interacts with higher-order complexes of Drp1. In contrast, the alternative Drp1 receptors MiD51 and MiD49 can recruit Drp1 dimers. Therefore Drp1 recruitment by Mff versus MiD51 and MiD49 may result in different outcomes because they recruit different subpopulations of Drp1 from the cytosol.     

Crystal structure and functional analysis of MiD49, a receptor for the mitochondrial fission protein Drp1

Mitochondrial fission requires recruitment of dynamin‐related protein 1 (Drp1) to the mitochondrial surface, where assembly leads to activation of its GTP‐dependent scission function. MiD49 and MiD51 are two receptors on the mitochondrial outer membrane that can recruit Drp1 to facilitate mitochondrial fission. Structural studies indicated that MiD51 has a variant nucleotidyl transferase fold that binds an ADP co‐factor essential for activation of Drp1 function. MiD49 shares sequence homology with MiD51 and regulates Drp1 function. However, it is unknown if MiD49 binds an analogous co‐factor. Because MiD49 does not readily crystallize, we used structural predictions and biochemical screening to identify a surface entropy reduction mutant that facilitated crystallization. Using molecular replacement, we determined the atomic structure of MiD49 to 2.4 Å. Like MiD51, MiD49 contains a nucleotidyl transferase domain; however, the electron density provides no evidence for a small‐molecule ligand. Structural changes in the putative nucleotide‐binding pocket make MiD49 incompatible with an extended ligand like ADP, and critical nucleotide‐binding residues found in MiD51 are not conserved. MiD49 contains a surface loop that physically interacts with Drp1 and is necessary for Drp1 recruitment to the mitochondrial surface. Our results suggest a structural basis for the differential regulation of MiD51‐ versus MiD49‐mediated fission.     

Structural and functional analysis of MiD51, a dynamin receptor required for mitochondrial fission

Mitochondrial fission is important for organelle transport, inheritance, and turnover, and alterations in fission are seen in neurological disease. In mammals, mitochondrial fission is executed by dynamin-related protein 1 (Drp1), a cytosolic guanosine triphosphatase that polymerizes and constricts the organelle. Recruitment of Drp1 to mitochondria involves receptors including Mff, MiD49, and MiD51. MiD49/51 form foci at mitochondrial constriction sites and coassemble with Drp1 to drive fission. Here, we solved the crystal structure of the cytosolic domain of human MiD51, which adopts a nucleotidyltransferase fold. Although MiD51 lacks catalytic residues for transferase activity, it specifically binds guanosine diphosphate and adenosine diphosphate. MiD51 mutants unable to bind nucleotides were still able to recruit Drp1. Disruption of an additional region in MiD51 that is not part of the nucleotidyltransferase fold blocked Drp1 recruitment and assembly of MiD51 into foci. MiD51 foci are also dependent on the presence of Drp1, and after scission they are distributed to daughter organelles, supporting the involvement of MiD51 in the fission apparatus.     

MiD49 and MiD51, new components of the mitochondrial fission machinery

Mitochondria form intricate networks through fission and fusion events. Here, we identify mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51, respectively) anchored in the mitochondrial outer membrane. MiD49/51 form foci and rings around mitochondria similar to the fission mediator dynamin-related protein 1 (Drp1). MiD49/51 directly recruit Drp1 to the mitochondrial surface, whereas their knockdown reduces Drp1 association, leading to unopposed fusion. Overexpression of MiD49/51 seems to sequester Drp1 from functioning at mitochondria and cause fused tubules to associate with actin. Thus, MiD49/51 are new mediators of mitochondrial division affecting Drp1 action at mitochondria.     

Mitochondrial E3 ubiquitin ligase MARCH5 controls mitochondrial fission and cell sensitivity to stress-induced apoptosis through regulation of MiD49 protein

Ubiquitin- and proteasome-dependent outer mitochondrial membrane (OMM)-associated degradation (OMMAD) is critical for mitochondrial and cellular homeostasis. However, the scope and molecular mechanisms of the OMMAD pathways are still not well understood. We report that the OMM-associated E3 ubiquitin ligase MARCH5 controls dynamin-related protein 1 (Drp1)-dependent mitochondrial fission and cell sensitivity to stress-induced apoptosis. MARCH5 knockout selectively inhibited ubiquitination and proteasomal degradation of MiD49, a mitochondrial receptor of Drp1, and consequently led to mitochondrial fragmentation. Mitochondrial fragmentation in MARCH5^−/− cells was not associated with inhibition of mitochondrial fusion or bioenergetic defects, supporting the possibility that MARCH5 is a negative regulator of mitochondrial fission. Both MARCH5 re-expression and MiD49 knockout in MARCH5^−/− cells reversed mitochondrial fragmentation and reduced sensitivity to stress-induced apoptosis. These findings and data showing MARCH5-dependent degradation of MiD49 upon stress support the possibility that MARCH5 regulation of MiD49 is a novel mechanism controlling mitochondrial fission and, consequently, the cellular response to stress.     

Proteins That Fuse and Fragment Mitochondria in Apoptosis: Con-Fissing a Deadly Con-Fusion?

During apoptosis, mitochondria undergo multiple changes that culminate in the release of cytochrome c and other proapoptotic cofactors. Recently, a role for previously overlooked morphological changes, fission of the mitochondrial reticulum and remodeling of mitochondrial cristae, has been suggested in mammalian cells and in developmental apoptosis of C. elegans. Mitochondrial morphology is determined by fusion and fission processes, controlled by a growing set of “mitochondria-shaping” proteins, whose levels and function appear to regulate the mitochondrial pathways of cell death. Expression of pro-fusion proteins, as well as of inhibition of pro-fission molecules reduces apoptosis, suggesting a linear relationship between fragmentation and death. Mechanisms by which mitochondrial fragmentation promotes apoptosis and interactions between fragmentation and remodeling of the inner membrane are largely unclear. A tempting, unifying hypothesis suggests that fission is coupled to cristae remodeling to maximize cytochrome c release.     

Novel roles of RTN4 and CLIMP-63 in regulating mitochondrial structure,bioenergetics and apoptosis

The recruitment of DRP1 to mitochondrial membranes prior to fission is facilitated by the wrapping of endoplasmic reticulum (ER) membranes around the mitochondria. To investigate the complex interplay between the ER membranes and DRP1 in the context of mitochondrial structure and function, we downregulate two key ER shaping proteins, RTN4 and CLIMP-63, and demonstrate pronounced mitochondrial hyperfusion and reduced ER-mitochondria contacts, despite their differential regulation of ER architecture. Although mitochondrial recruitment of DRP1 is unaltered in cells lacking RTN4 or CLIMP-63, several aspects of mitochondrial function, such as mtDNA-encoded translation, respiratory capacity and apoptosis are significantly hampered. Further mechanistic studies reveal that CLIMP-63 is required for cristae remodeling (OPA1 proteolysis) and DRP1-mediated mitochondrial fission, whereas both RTN4 and CLIMP-63 regulate the recruitment of BAX to ER and mitochondrial membranes to enable cytochrome c release and apoptosis, thereby performing novel and distinct roles in the regulation of mitochondrial structure and function.Subject terms: Cell biology, Cancer     

OPA1 controls apoptotic cristae remodeling independently from mitochondrial fusion

Mitochondria amplify activation of caspases during apoptosis by releasing cytochrome c and other cofactors. This is accompanied by fragmentation of the organelle and remodeling of the cristae. Here we provide evidence that Optic Atrophy 1 (OPA1), a profusion dynamin-related protein of the inner mitochondrial membrane mutated in dominant optic atrophy, protects from apoptosis by preventing cytochrome c release independently from mitochondrial fusion. OPA1 does not interfere with activation of the mitochondrial "gatekeepers" BAX and BAK, but it controls the shape of mitochondrial cristae, keeping their junctions tight during apoptosis. Tightness of cristae junctions correlates with oligomerization of two forms of OPA1, a soluble, intermembrane space and an integral inner membrane one. The proapoptotic BCL-2 family member BID, which widens cristae junctions, also disrupts OPA1 oligomers. Thus, OPA1 has genetically and molecularly distinct functions in mitochondrial fusion and in cristae remodeling during apoptosis.     

Loss of Drp1 function alters OPA1 processing and changes mitochondrial membrane organization

RNAi mediated loss of Drp1 function changes mitochondrial morphology in cultured HeLa and HUVEC cells by shifting the balance of mitochondrial fission and fusion towards unopposed fusion. Over time, inhibition of Drp1 expression results in the formation of a highly branched mitochondrial network along with “bulge”-like structures. These changes in mitochondrial morphology are accompanied by a reduction in levels of Mitofusin 1 (Mfn1) and 2 (Mfn2) and a modified proteolytic processing of OPA1 isoforms, resulting in the inhibition of cell proliferation. In addition, our data imply that bulge formation is driven by Mfn1 action along with particular proteolytic short-OPA1 (s-OPA1) variants: Loss of Mfn2 in the absence of Drp1 results in an increase of Mfn1 levels along with processed s-OPA1-isoforms, thereby enhancing continuous “fusion” and bulge formation. Moreover, bulge formation might reflect s-OPA1 mitochondrial membrane remodeling activity, resulting in the compartmentalization of cytochrome c deposits. The proteins Yme1L and PHB2 appeared not associated with the observed enhanced OPA1 proteolysis upon RNAi of Drp1, suggesting the existence of other OPA1 processing controlling proteins. Taken together, Drp1 appears to affect the activity of the mitochondrial fusion machinery by unbalancing the protein levels of mitofusins and OPA1.     

Mitochondrial remodeling following fission inhibition by 15d-PGJ2 involves molecular changes in mitochondrial fusion protein OPA1

We showed earlier that 15 deoxy Δ^12,14 prostaglandin J2 (15d-PGJ2) inactivates Drp1 and induces mitochondrial fusion [1]. However, prolonged incubation of cells with 15d-PGJ2 resulted in remodeling of fused mitochondria into large swollen mitochondria with irregular cristae structure. While initial fusion of mitochondria by 15d-PGJ2 required the presence of both outer (Mfn1 and Mfn2) and inner (OPA1) mitochondrial membrane fusion proteins, later mitochondrial changes involved increased degradation of the fusion protein OPA1 and ubiquitination of newly synthesized OPA1 along with decreased expression of Mfn1 and Mfn2, which likely contributed to the loss of tubular rigidity, disorganization of cristae, and formation of large swollen degenerated dysfunctional mitochondria. Similar to inhibition of Drp1 by 15d-PGJ2, decreased expression of fission protein Drp1 by siRNA also resulted in the loss of fusion proteins. Prevention of 15d-PGJ2 induced mitochondrial elongation by thiol antioxidants prevented not only loss of OPA1 isoforms but also its ubiquitination. These findings provide novel insights into unforeseen complexity of molecular events that modulate mitochondrial plasticity.     

The mitochondrial elongation factors MIEF1 and MIEF2 exert partially distinct functions in mitochondrial dynamics

Mitochondria are dynamic organelles whose morphology is regulated by a complex balance of fission and fusion processes, and we still know relatively little about how mitochondrial dynamics is regulated. MIEF1 (also called MiD51) has recently been characterized as a key regulator of mitochondrial dynamics and in this report we explore the functions of its paralog MIEF2 (also called MiD49), to learn to what extent MIEF2 is functionally distinct from MIEF1. We show that MIEF1 and MIEF2 have many functions in common. Both are anchored in the mitochondrial outer membrane, recruit Drp1 from the cytoplasm to the mitochondrial surface and cause mitochondrial fusion, and MIEF2, like MIEF1, can interact with Drp1 and hFis1. MIEF1 and MIEF2, however, also differ in certain aspects. MIEF1 and MIEF2 are differentially expressed in human tissues during development. When overexpressed, MIEF2 exerts a stronger fusion-promoting effect than MIEF1, and in line with this, hFis1 and Mff can only partially revert the MIEF2-induced fusion phenotype, whereas MIEF1-induced fusion is reverted to a larger extent by hFis1 and Mff. MIEF2 forms high molecular weight oligomers, while MIEF1 is largely present as a dimer. Furthermore, MIEF1 and MIEF2 use distinct domains for oligomerization: in MIEF1, the region from amino acid residues 109–154 is required, whereas oligomerization of MIEF2 depends on amino acid residues 1 to 49, i.e. the N-terminal end. We also show that oligomerization of MIEF1 is not required for its mitochondrial localization and interaction with Drp1. In conclusion, our data suggest that the mitochondrial regulators MIEF1 and MIEF2 exert partially distinct functions in mitochondrial dynamics.     

Bax is essential for Drp1‐mediated mitochondrial fission but not for mitochondrial outer membrane permeabilization caused by photodynamic therapy

Bcl‐2 family proteins are critical for the regulation of apoptosis, with the pro‐apoptotic members Bax essential for the release of cytochrome c from mitochondria in many instances. However, we found that Bax was activated after mitochondrial depolarization and the completion of cytochrome c release induced by photodynamic therapy (PDT) with the photosensitizer Photofrin in human lung adenocarcinoma cells (ASTC‐a‐1). Besides, knockdown of Bax expression by gene silencing had no effect on mitochondrial depolarization and cytochrome c release, indicating that Bax makes no contribution to mitochondrial outer membrane permeabilization (MOMP) following PDT. Further study revealed that Bax knockdown only slowed down the speed of cell death induced by PDT, indicating that Bax is not essential for PDT‐induced apoptosis. The fact that Bax knockdown totally inhibited the mitochondrial accumulation of dynamin‐related protein (Drp1) and Drp1 knockdown attenuated cell apoptosis suggest that Bax can promote PDT‐induced apoptosis through promoting Drp1 activation. Besides, Drp1 knockdown also failed to inhibit PDT‐induced cell death finally, indicating that Bax‐mediated Drp1's mitochondrial translocation is not essential for PDT‐induced cell apoptosis. On the other hand, we found that protein kinase Cδ (PKCδ), Bim L and glycogen synthase kinase 3β (GSK3β) were activated upon PDT treatment and might contribute to the activation of Bax under the condition. Taken together, Bax activation is not essential for MOMP but essential for Drp1‐mediated mitochondrial fission during the apoptosis caused by Photofrin‐PDT. J. Cell. Physiol. 226: 530–541, 2011. © 2010 Wiley‐Liss, Inc.     

Mitochondrial rhomboid PARL regulates cytochrome c release during apoptosis via OPA1-dependent cristae remodeling

Rhomboids, evolutionarily conserved integral membrane proteases, participate in crucial signaling pathways. Presenilin-associated rhomboid-like (PARL) is an inner mitochondrial membrane rhomboid of unknown function, whose yeast ortholog is involved in mitochondrial fusion. Parl-/- mice display normal intrauterine development but from the fourth postnatal week undergo progressive multisystemic atrophy leading to cachectic death. Atrophy is sustained by increased apoptosis, both in and ex vivo. Parl-/- cells display normal mitochondrial morphology and function but are no longer protected against intrinsic apoptotic death stimuli by the dynamin-related mitochondrial protein OPA1. Parl-/- mitochondria display reduced levels of a soluble, intermembrane space (IMS) form of OPA1, and OPA1 specifically targeted to IMS complements Parl-/- cells, substantiating the importance of PARL in OPA1 processing. Parl-/- mitochondria undergo faster apoptotic cristae remodeling and cytochrome c release. These findings implicate regulated intramembrane proteolysis in controlling apoptosis.     

The mitochondrial permeability transition regulates cytochrome c release for apoptosis during endoplasmic reticulum stress by remodeling the cristae junction

The role of the mitochondrial permeability transition (MPT) in apoptosis and necrosis is controversial. Here we show that the MPT regulates the release of cytochrome c for apoptosis during endoplasmic reticulum (ER) stress by remodeling the cristae junction (CJ). CEM cells, HCT116 colon cancer cells, and murine embryo fibroblast cells were treated with the ER stressor thapsigargin (THG), which led to cyclophilin D-dependent mitochondrial release of the profusion GTPase optic atrophy 1 (OPA1), which controls CJ integrity, and cytochrome c, leading to apoptosis. Interference RNA knockdown of Bax blocked OPA1 and cytochrome c release after THG treatment but did not prevent the MPT, showing that Bax was essential for the release of cytochrome c by MPT. In isolated mitochondria, MPT led to OPA1 and cytochrome c release independently of voltage-dependent anion channel and the outer membrane, indicating that the MPT is an inner membrane phenomenon. Last, the MPT was regulated by the electron transport chain but not mitochondrial reactive oxygen species, since THG-induced cell death was not blocked by antioxidants and did not occur in cells lacking mitochondrial DNA. Our results show that the MPT regulates CJ remodeling for cytochrome c-dependent apoptosis induced by ER stress and that mitochondrial electron transport is indispensable for this process.     

Release of OPA1 during apoptosis participates in the rapid and complete release of cytochrome c and subsequent mitochondrial fragmentation

Mitochondria are important participants in apoptosis, releasing cytochrome c into the cytoplasm and undergoing extensive fragmentation. However, mechanisms underlying these processes remain unclear. Here, we demonstrate that cytochrome c release during apoptosis precedes mitochondrial fragmentation. Unexpectedly, OPA1, a dynamin-like GTPase of the mitochondrial intermembrane space important for maintaining cristae structure, is co-released with cytochrome c. To mimic the loss of OPA1 occurring after its release, we knocked down OPA1 expression using RNA interference. This triggered structural changes in the mitochondrial cristae and caused increased fragmentation by blocking mitochondrial fusion. Because cytochrome c is mostly sequestered within cristae folds but released rapidly and completely during apoptosis, we examined the effect of OPA1 loss on cytochrome c release, demonstrating that it is accelerated. Thus, our results suggest that an initial mitochondrial leak of OPA1 leads to cristae structural alterations and exposure of previously sequestered protein pools, permitting continued release in a feed-forward manner to completion. Moreover, our findings indicate that the resulting OPA1 depletion causes a block in mitochondrial fusion, providing a compelling mechanism for the prominent increase in mitochondrial fragmentation seen during apoptosis.     

OPA1, a molecular regulator of dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a disease with no specific treatment, poor prognosis and high mortality. During DCM development, there is apoptosis, mitochondrial dynamics imbalance and changes in cristae structure. Optic atrophy 1 (OPA1) appears at high frequency in these three aspects. DCM LMNA (LaminA/C) gene mutation can activate TP53, and the study of P53 shows that P53 affects OPA1 through Bak/Bax and OMA1 (a metalloprotease). OPA1 can be considered the missing link between DCMp53 and DCM apoptosis, mitochondrial dynamics imbalance and changes in cristae structure. OPA1 regulates apoptosis by regulating the release of cytochrome c from the mitochondrial matrix through CJs (crisp linkages, located in the inner mitochondrial membrane) and unbalances mitochondrial fusion and fission by affecting mitochondrial inner membrane (IM) fusion. OPA1 is also associated with the formation and maintenance of mitochondrial cristae. OPA1 is not the root cause of DCM, but it is an essential mediator in P53 mediating the occurrence and development of DCM, so OPA1 also becomes a molecular regulator of DCM. This review discusses the implication of OPA1 for DCM from three aspects: apoptosis, mitochondrial dynamics and ridge structure.