Mitochondrial morphology is governed by the balance of mitochondrial fusion, mediated by mitofusins and optic atrophy 1 (OPA1), and fission, mediated by dynamin-related protein 1 (Drp1). Disordered mitochondrial dynamics alters metabolism, proliferation, apoptosis and mitophagy, contributing to human diseases, including neurodegenerative syndromes, pulmonary arterial hypertension (PAH), cancer and ischemia/reperfusion injury. Post-translational regulation of Drp1 (by phosphorylation and SUMOylation) is an established means of modulating Drp1 activation and translocation to the outer mitochondrial membrane (OMM). This review focuses on Drp1 adaptor proteins that also regulate fission. The proteins include fission 1 (Fis1), mitochondrial fission factor (Mff) and mitochondrial dynamics proteins of 49 kDa and 51 kDa (MiD49, MiD51). Heterologous MiD overexpression sequesters inactive Drp1 on the OMM, promoting fusion; conversely, increased endogenous MiD creates focused Drp1 multimers that optimize OMM scission. The triggers that activate MiD-bound Drp1 in disease states are unknown; however, MiD51 has a unique capacity for ADP binding at its nucleotidyltransferase domain. Without ADP, MiD51 inhibits Drp1, whereas ADP promotes MiD51-mediated fission, suggesting a link between metabolism and fission. Confusion over whether MiDs mediate fusion (by sequestering inactive Drp1) or fission (by guiding Drp1 assembly) relates to a failure to consider cell types used and to distinguish endogenous compared with heterologous changes in expression. We speculate that endogenous MiDs serve as Drp1-binding partners that are dysregulated in disease states and may be important targets for inhibiting cell proliferation and ischemia/reperfusion injury. Moreover, it appears that the composition of the fission apparatus varies between disease states and amongst individuals. MiDs may be important targets for inhibiting cell proliferation and attenuating ischemia/reperfusion injury.

INTRODUCTION

Mitochondria are the primary sites of ATP production [1]. They are also key regulators of intracellular calcium homeostasis through their ability to take up calcium, by means of the mitochondrial calcium uniporter, and release it back to the cytosol, via several transporters [2,3]. However, mitochondria have important non-canonical functions. For example, mitochondria exist in dynamic networks. Individual organelles rapidly divide by a process called fission and join together by a process called fusion [4]. The balance of fusion and fission regulates mitochondrial morphology. Mitochondrial number can be increased by the process of mitochondrial biogenesis. Abnormal mitochondria (or portions of mitochondria) can be eliminated through a quality control process called mitophagy [5]. The mitochondria's non-canonical repertoire also includes intracellular transport (trafficking). In aggregate these functions are referred to as mitochondrial dynamics [6].

Fission creates a greater number of discrete non-networked mitochondria, whereas fusion increases connectivity, thereby allowing sharing of matrix proteins and mitochondrial DNA among these organelles [7]. The causes and consequences of mitochondrial fission and fusion are highly contextual. For example, fission may be either a physiological step in the process of mitosis (mitotic fission), the beginning of programmed cell death (apoptosis) or a normal part of a cell's quality control mechanism, allowing elimination of dysfunctional mitochondria (mitophagy) [5,8,9]. Mitochondrial dynamics also alters the network in order to fulfill the specific metabolic and energetic demands of the cell. Mitochondrial dynamics participate in cell cycle progression, apoptosis, production of oxygen-derived free radicals, mitochondrial DNA stability, oxygen sensing and the cell's stress response [10,11]. Disorders of mitochondrial dynamics are emerging as mechanisms of pathogenesis in diseases that had not classically been considered to be linked to mitochondria. Acquired, pathological alterations in mitochondrial dynamics contribute to many complex human diseases, including cardiovascular diseases, such as pulmonary arterial hypertension (PAH), degenerative neurological diseases, such as Parkinson's disease, ischaemia/reperfusion injury and cancer [10].

During mitosis, mitochondrial fission ensures equitable distribution of mitochondria to daughter cells [12]. Rapid cell division is a hallmark of both PAH and non-small-cell lung cancer (NSCLC). In dividing cells, a burst of dynamin-related protein 1 (Drp1)-mediated fission during mitosis coordinates mitochondrial and nuclear division [8]. This mitotic fission results in part from cyclin B1/cyclin-dependent kinase 1 (CDK1), which simultaneously triggers mitosis and fission by phosphorylation of Drp1 at Ser616 [1315]). The interaction of Drp1 with its binding partner, fission 1 (Fis1), contributes to mitotic fission [16]. Inhibiting mitotic fission [by inhibiting Drp1 or augmenting the fusion mediator mitofusin 2 (Mfn2)] prevents cell cycle progression, arresting the cell in the G2M phase and also induces apoptosis [14,15].

The opposing process, fusion, allows for the mixing of mitochondrial contents between organelles for maintenance of a homogeneous mitochondrial network [17]. Fusion also allows a healthy mitochondrion to compensate for oxidative damage in a failing mitochondrion by admixture of its healthy mitochondrial proteins and mitochondrial DNA [6]. When fusion is no longer adequate to compensate for accumulated damage, the diseased portion of the mitochondria depolarizes and undergoes fission, with a simultaneous suppression of fusion. This isolates damaged sections of mitochondria, allowing their removal in a mitophagic vacuole, thereby protecting the cell [5].

Mitochondrial fission and fusion are mediated by large GTPases. Drp1 mediates fission. Mitofusin 1 (Mfn1) and Mfn2 and optic atrophy 1 (OPA1) mediate fusion of the outer and inner mitochondrial membranes respectively. Upon activation Drp1 is recruited from the cytosol to the outer mitochondrial membrane (OMM) [18]. Here, in a microenvironment shaped by the endoplasmic reticulum (ER) [1921], Drp1 interacts with binding partners and multimerizes, creating a collar-like fission apparatus. The fission apparatus relies on Drp1’s GTPase activity to cause scission and divide the organelle. Drp1 is regulated by post-translational modifications, such as phosphorylation [8,2224] and SUMOylation [25,26]. This post-translational regulation seems to be largely involved in Drp1 activation or pacification, which permits or prevents movement from cytosol to the OMM respectively. Once activated, Drp1 moves to the OMM where it interacts with binding proteins, including fission protein 1 (Fis1) [2729], mitochondrial fission factor (Mff) [30] and/or the newly discovered mitochondrial dynamics proteins of 49 kDa and 51 kDa (MiD49 and MiD51) [27]. The relative roles for each of the four binding proteins, particularly that of MiD49 and MiD51, are poorly understood. There are conflicting reports whether MiD49 and MiD51 promote fission [27] or fusion [31,32]. The role of the Drp1-binding partners in human diseases is largely unknown.

Previous studies involving inhibition of Drp1 either by the pharmacological inhibitor, mdivi-1 or by siRNA support the interpretation that the forced restoration of the mitochondrial network can decrease cell proliferation and induce apoptosis in human NSCLC and human PAH pulmonary artery smooth muscle cells (PASMCs). Furthermore, studies have also shown that forced induction of mitochondrial fusion by the overexpression of the fusion mediator Mfn2 inhibits proliferation and induces apoptosis in vascular smooth muscle, lung cancer and hepatocellular carcinoma cells [15,33,34]. Drp1 is also activated in diseases where the concentrations of intracellular calcium are pathologically increased such as in cardiac arrest. For example, in cardiac ischaemia/reperfusion injury the activation of Drp1 increases reactive oxygen species (ROS) which causes cardiac dysfunction that can be therapeutically targeted using Drp1 inhibitors [35].

MEDIATORS OF MITOCHONDRIAL FUSION

The outer and inner membranes of a mitochondrion join to the corresponding membrane on a second mitochondrion in distinct but co-ordinated fusion events during the course of mitochondrial fusion (Figure 1). These events are almost simultaneous, with content mixing occurring soon after mitochondrial contact [36]. When two mitochondria fuse the result is the admixture of their membranes and lipids, the contents of their intermembrane spaces and the contents of their matrices, which includes their mitochondrial DNA.

Mitochondrial dynamics

Figure 1
Mitochondrial dynamics

(A) Joining of mitochondria by the process of fusion which is mediated by the OMM fusion proteins, Mfn1 and Mfn2, and the inner mitochondrial (IMM) fusion protein OPA1. Fusion causes the intermixing of the mitochondrial matrix labelled in yellow and green and results in an elongated mitochondrion. (B) Division of mitochondria by the process of fission. Fission is mediated by Drp1, which is recruited to the mitochondria from the cytosol by adaptor proteins such as MFF, Fis1, MiD49 and MiD51. Drp1 assembles and surrounds the mitochondria forming a ring-like structure, which cleaves the organelle causing fission. (C) Heterogeneity of disease and patient type in which either MiD49 or MiD51 may be up-regulated. We hypothesize that up-regulation of these Drp1 adaptors can recruit active Drp1 (phosphorylated at Ser616) causing mitochondrial fission or can recruit inactive Drp1 (phosphorylated at Ser637) causing mitochondrial fusion. In addition, the binding partners may interact co-operatively or competitively, affecting fission. Drp1 multimerization is also an important, but incompletely understood, contributor to the regulation of the fission complex.

Figure 1
Mitochondrial dynamics

(A) Joining of mitochondria by the process of fusion which is mediated by the OMM fusion proteins, Mfn1 and Mfn2, and the inner mitochondrial (IMM) fusion protein OPA1. Fusion causes the intermixing of the mitochondrial matrix labelled in yellow and green and results in an elongated mitochondrion. (B) Division of mitochondria by the process of fission. Fission is mediated by Drp1, which is recruited to the mitochondria from the cytosol by adaptor proteins such as MFF, Fis1, MiD49 and MiD51. Drp1 assembles and surrounds the mitochondria forming a ring-like structure, which cleaves the organelle causing fission. (C) Heterogeneity of disease and patient type in which either MiD49 or MiD51 may be up-regulated. We hypothesize that up-regulation of these Drp1 adaptors can recruit active Drp1 (phosphorylated at Ser616) causing mitochondrial fission or can recruit inactive Drp1 (phosphorylated at Ser637) causing mitochondrial fusion. In addition, the binding partners may interact co-operatively or competitively, affecting fission. Drp1 multimerization is also an important, but incompletely understood, contributor to the regulation of the fission complex.

Different proteins are responsible for the fusion of the outer and inner mitochondrial membranes. Mfn1 and Mfn2 are large transmembrane GTPases embedded in the OMM that execute membrane fusion [37,38]. Inner mitochondrial membrane fusion is mediated by OPA1 protein, a dynamin-related GTPase associated with the inner mitochondrial membrane or intermembrane space [39,40] (Figure 1A).

When any of these three fusion mediators is depleted, mitochondrial fusion is profoundly impaired [11]. Mfn1 or Mfn2 single-knockout cells, created in mouse embryonic fibroblasts (MEFs), have fragmented mitochondria that display reduced fusion. Mitochondria in MEFs lacking both mitofusins display a complete lack of fusion [11,17]. Tethering of adjacent mitochondria by Mfn1 and Mfn2 proceeds via the formation of hetero-oligomeric or homo-oligomeric complexes. This docking of adjacent mitochondria is mediated by a heptad repeat in the mitofusins’ C-termini, which forms an antiparallel coiled-coil structure, bringing OMMs of fusing mitochondria within 100 Å proximity [41].

Using matrix mixing assays to detect complete fusion, it is clear that MEFs with null alleles for OPA1 fail to fuse [40]. OPA1-deficient cells have a number of other cellular defects, notably increased apoptotic sensitivity, reduction in respiratory capacity and disorganization of cristae [11]. There are both ‘long’ and ‘short’ isoforms of OPA1; the long isoform is membrane-anchored, whereas the short isoform is devoid of a membrane anchor but retains the ability to interact with membranes. In normal cellular conditions, a combination of these two isoforms appears necessary for mitochondrial fusion; however, the long isoform alone may be capable of mediating fusion during times of stress [42,43]. Further investigations, which tracked outer and inner mitochondrial membrane fusion separately, revealed that OPA1-null MEFs still retain outer membrane fusion capability and only lack inner membrane fusion [40]. This supports the idea that the mitofusins and OPA1 are distinct actors in the fusion process, with mitofusins mediating OMM fusion followed by OPA1-mediated inner membrane fusion.

CLASSICAL MITOCHONDRIAL FISSION MACHINERY

The mitochondrial fission machinery has two major components: Drp1 and its adaptor proteins. Drp1 is a member of the dynamin GTPase family and is homologous with dynamin, the mitochondrial fission mediator in yeast [18]. Drp1 has an essential role in fission and mice lacking Drp1 die before birth [44]. Fluorescent microscopy provides evidence that Drp1 is recruited from the cytosol to mitochondria, where it forms puncta around the mitochondrial tubule [45]. In the cytosol, Drp1 exists as dimers or tetramers and only assembles into higher-order complexes when it binds to the OMM [18,46,47]. Oligomerized Drp1 in mitochondrial puncta are proposed to wrap around the mitochondrial tubule to mediate membrane scission, analogous to the pinching off of endocytic vesicles by dynamin [48]. In this regard, it is noteworthy that Drp1, in interaction with Fis1 and Mff, also mediates peroxisomal fission [49].

Despite the central role of Drp1 in mitochondrial fission, the role of different adaptor proteins in supporting the recruitment and oligomerization of Drp1 remains unclear. In yeast, dynamin is recruited by Fis1, Mdv1 and Caf4p [5052]. No Mdv1 or Caf4p homologues exist in mammals, suggesting that there are alter-native binding proteins involved in mammalian mitochondrial fission. The role of Fis1 in the mammalian fission machinery is unclear and its relevance in homeostatic fission is controversial [29,30,53,54], with recent proposals suggesting that it plays a role in mitophagy [55,56].

In addition to Fis1, three other proteins have been implicated as adaptor proteins for Drp1: Mff, MiD49 and MiD51 [32,57,58] (Figure 1B). Mff is capable of recruiting Drp1 independently of Fis1 and transiently interacts with Drp1 via its cytoplasmic domain [30,57]. In this review, we focus on the role of the most recently discovered components of the mitochondrial fission machinery, the OMM proteins MiD49 and MiD51. The exact role of these proteins remains to be thoroughly explained and their role in disease is unclear; however, they will probably prove important in explaining contextual differences in mitochondrial fission and variability in the role of mitochondrial dynamics in various diseases (Figure 1C).

EARLY INSIGHTS INTO MiD49 AND MiD51

Two independent groups proposed that MiD49 and MiD51 are Drp1 adaptor proteins [32,58]. Palmer et al. [58] identified MiD49 and MiD51 from a prior random cellular localization screen of uncharacterized human proteins. They found that expression of one novel protein, SMCR7L (Smith–Magenis syndrome chromosome region candidate gene 7-like), altered mitochondrial morphology [59]. A second mitochondrial protein, SMCR7 (Smith–Magenis syndrome chromosome region candidate gene 7), which shares 45% sequence identity with SMCR7L, was also discovered and proposed as a mediator of mitochondrial dynamics. SMCR7 was subsequently redesignated MIEF2 (mitochondrial elongation factor 2) and SMCR7L as MIEF1 (mitochondrial elongation factor 1) to reflect their role in mitochondrial dynamics. MiD49 is encoded by the MIEF2 gene and MiD51 is encoded by MIEF1. Both MiD49 and MiD51 are nuclear-encoded proteins. Zhao et al. [32] independently identified MiD51 from the same intracellular localization database, noting that ectopic overexpression of this 52 kDa, 463 amino acid, protein triggered mitochondrial elongation. They found that although MiD51 reduces Drp1’s GTP-binding activity, it did not affect upstream events (Drp1 oligomerization or phosphorylation). Zhao et al. [32] also noted that the phosphorylation status of Drp1-Ser637 was not critical for its interaction with MiD51.

Sequence alignment of MiD51 orthologues indicates that MiD51 is conserved in vertebrates, but is not present in yeast, invertebrates or plants [32]. MiD49 mRNA is expressed ubiquitously, with highest expression in heart and skeletal muscle [60]. Expression of MiD51 is also relatively high in adult human heart, skeletal muscle, pancreas and kidney and low in lung, brain and placenta [32]. MiD49 and MiD51 appear to be expressed differentially in development; MiD49 mRNA is more abundant in adult organs, whereas MiD51 mRNA is more abundant in fetal organs [31]. MiD49 and MiD51 are also expressed differentially in human disease cell lines such as glioma, neuroblastoma and other cancer cell lines [31].

CONFLICTING EARLY REPORTS OF MiD PROTEIN FUNCTION

Both MiD49 and MiD51 are integral mitochondrial membrane proteins anchored in the OMM by their N-termini, with C-terminal cytosolic domains [32,58]. Early studies reported different roles for the MiD proteins in mitochondrial dynamics. Palmer et al. [58] observed that MiD49 and MiD51 directly recruit Drp1 to the mitochondria surface and that Drp1-mediated fission events took place early after the induction of MiD49 or MiD51 expression. However, at later time points, the mitochondrial network becomes extensively fused, as did the peroxisomes. This led the authors to suggest that MiD49 or MiD51 overexpression leads to sequestration and inactivation of Drp1 [58]. This MiD-mediated sequestration of Drp1 leaves the fusion apparatus, notably mitofusins, unopposed, resulting in fusion. Conversely, knockdown of both MiD49 and MiD51 reduced Drp1–MiD associations and caused unopposed fusion. These findings support a role for MiD49 and MiD51 in promoting mitochondrial fission, although they suggest the amount of MiD expressed may be critically important in determining the effects on mitochondrial morphology, with substantial overexpression driving fusion. Single knockdown did not affect mitochondrial morphology [58]. The changes in mitochondrial phenotype with different expression levels suggest that MiD protein expression is tightly regulated in the cell to produce fission events. In preliminary work in human PAH PASMCs we find that knockdown of endogenously elevated levels of MiD49 or MiD51 causes fusion of the mitochondria, which are fragmented at baseline in this disease (Figure 2). It is also noteworthy that expression of both MiDs appears to be up-regulated in PAH compared with control subjects (Figure 3).

Silencing MiD49 and MiD51 in PAH PASMCs inhibits mitochondrial fission

Figure 2
Silencing MiD49 and MiD51 in PAH PASMCs inhibits mitochondrial fission

Representative images of mitochondrial networks of PAH PASMCs and PAH PASMCs transfected with the small interfering RNA (siRNA) against MiD49 or MiD51. Cells were loaded with the potentiometric dye tetramethylrhodamine methyl ester (TMRM) (red) and imaged under a confocal microscope to assess the mitochondrial network structure. Note the fusion of the mitochondrial network caused by down-regulation of the MiD proteins. This suggests that in these cells the MiDs are fissogenic. Scale bar=10 μm.

Figure 2
Silencing MiD49 and MiD51 in PAH PASMCs inhibits mitochondrial fission

Representative images of mitochondrial networks of PAH PASMCs and PAH PASMCs transfected with the small interfering RNA (siRNA) against MiD49 or MiD51. Cells were loaded with the potentiometric dye tetramethylrhodamine methyl ester (TMRM) (red) and imaged under a confocal microscope to assess the mitochondrial network structure. Note the fusion of the mitochondrial network caused by down-regulation of the MiD proteins. This suggests that in these cells the MiDs are fissogenic. Scale bar=10 μm.

Confocal and stimulated emission depletion (STED) microscopy images of MiD49, and MiD51 in normal and PAH PASMCs

Figure 3
Confocal and stimulated emission depletion (STED) microscopy images of MiD49, and MiD51 in normal and PAH PASMCs

PASMCs from normal and PAH patients were stained with Mitotracker Red (confocal) and Tom20 (super-resolution STED imaging). Note the mitochondrial localization of MiDs on the OMM. The nucleus was stained with DAPI. Mitochondria shown in ‘red’, MiD49 and MiD51 shown in ‘green’ and nucleus shown in ‘blue’. Note the presence of ‘green’ in the nucleus suggesting the novel possibility of extra-mitochondrial location of MiD adaptor proteins. Scale bar=20 μm.

Figure 3
Confocal and stimulated emission depletion (STED) microscopy images of MiD49, and MiD51 in normal and PAH PASMCs

PASMCs from normal and PAH patients were stained with Mitotracker Red (confocal) and Tom20 (super-resolution STED imaging). Note the mitochondrial localization of MiDs on the OMM. The nucleus was stained with DAPI. Mitochondria shown in ‘red’, MiD49 and MiD51 shown in ‘green’ and nucleus shown in ‘blue’. Note the presence of ‘green’ in the nucleus suggesting the novel possibility of extra-mitochondrial location of MiD adaptor proteins. Scale bar=20 μm.

Palmer et al. [58] found that the Drp1 recruitment activity of MiD49 and MiD51 was stronger than that of Mff or Fis1. Unlike other Drp1-binding partners, MiDs are not found in peroxisomes and appear to be uniquely localized to the OMM. MiD proteins were found at mitochondrial constriction sites, independently of Drp1 expression. They noted that MiD49 and MiD51 could form foci and rings around mitochondria, suggesting that they may act as Drp1 scaffolds.

Contrary to the designation of MiD49 and MiD51 as components of the mitochondrial fission machinery by Palmer et al. [58], Zhao et al. [32] reported that MiD51 is an active promoter of mitochondrial fusion. They observed that overexpression of MiD51 produced mitochondrial fusion which persisted post-knockdown of Mfn2 [32]. Also in contrast with Palmer et al. [58] (and our preliminary results–Figure 2), Zhao et al. [32] found that knockdown of MiD51 induced mitochondrial fragmentation rather than fusion. They concluded that MiD51 recruits Drp1 to mitochondria and inhibits its GTPase activity to block fission, while acting as a mitofusin independent fusion factor [32]. In contrast Palmer et al. [59] found an obligatory role for the mitofusins in fusion elicited by heterologous MiD overexpression. Although MiD49 or MiD51 overexpression causes fusion in cells lacking either Mfn1 or Mfn2 no fusion is induced in Mfn1/Mfn2 double-knockout cells [59]. Therefore, it appears that the presence of at least one mitofusin is necessary to produce the elongated mitochondrial phenotype observed post-MiD49 or -MiD51 overexpression. This probably indicates that the MiD proteins do not function as independent fusion mediators. It is noteworthy that this fusion is occurring with heterologous overexpression of the MiDs, rather than with normal MiD expression or even disease-mediated up-regulation of the MiDs. These contextual difference may ultimately prove important.

Interestingly, Drp1 overexpression did not alter mitochondrial morphology and overexpression of MiD51 induced fusion was partially reversed by co-expressing Fis1 [32,54]. This may suggest a competitive interaction between MiD51 and Fis1 as a Drp1-binding protein. Zhao et al. [32] also assessed the effects of overexpression of MiD51 on cell death and found no increase in apoptotic signals, such as second mitochondria-derived activator of caspases (SMAC-Diablo), but did note a rise in light chain 3B II (LC3B-II), consistent with increased mitophagy. Conversely, a recent study by Otera et al. [61] indicated that mitochondrial fission mediated by Drp1 through MiD49 and MiD51 is important for cristae remodelling to enable the release of cytochrome c into the cytoplasm during the initial phase of intrinsic apoptosis.

Although these processes remain somewhat unclear it demonstrates that the MiDs are Drp1-binding partners and depending on temporal and dose consideration may guide Drp1 to form a fission apparatus or act as bait that binds and inactivates Drp1, allowing fusion mechanisms to predominate [58,59]. In PAH and cancer, fusion mechanisms are compromised, perhaps explaining why (in unpublished preliminary studies) increased endogenous expression of MiD49 and MiD51 are associated with fission, not fusion (Figure 3).

MiD49 AND MiD51 ARE COMPONENTS OF THE FISSION MACHINERY

MiD proteins are not independent fission mediators, rather they act through their ability to bind Drp1. Overexpression of MiD49 or MiD51 is not sufficient to induce mitochondrial fission in Drp1-null cells [62]. Drp1-null cells have a more severe elongation phenotype than cells lacking the Fis1 or Mff [27]. Fis1 and Mff single-knockout cells retain adaptor proteins capable of recruiting Drp1 from the cytosol to mitochondria and initiating fission. However, in Fis1/Mff double-knockout cells, knockdown of either MiD49 or MiD51 enhances mitochondrial elongation, whereas overexpression of either MiD protein partially restores Drp1 recruitment to the mitochondria [27]. MiD49/MiD51 double-knockout MEFs are resistant to carbonyl cyanide m-chlorophenyl hydrazine (CCCP)-induced fragmentation [53], confirming a role for MiDs in mitochondrial fission.

In contrast with Zhao et al. [32], studies that were conducted in human embryonic kidney (HEK)-293T and HeLa cells, and our preliminary studies in primary isolates of human PASMCs, suggest that silencing of MiD49 and MiD51 promotes fusion (Figure 2).

WHY IS THERE THE CONTRADICTION IN THE MiD LITERATURE?

We suspect much of the differences between studies relate to the importance of cell type and endogenous rather than heterologous expression of MiDs. One needs to consider whether adaptor proteins are being exogenously overexpressed rather than up-regulated by endogenous mechanisms. In addition, the type of cells studied and their state of metabolism and proliferation are important considerations. Our unpublished preliminary findings are consistent with those of Loson et al. [27], who reported that Fis1, Mff, MiD49 and MiD51 can each recruit Drp1 and can each serve as binding partners that promote fission. Loson et al. [27] noted that the MiDs are particularly able to promote fission. Perhaps the physiological state of the cell and its complement of binding partners and post-translationally modified forms of Drp1 determines the consequences of MiD49 and MiD51 expression, as we illustrate schematically in Figure 1(C). Additional support for the MiDs as fission mediators comes from Koirala et al. [62]. Using a yeast strain lacking all fission proteins, they identified the minimal combinations of GTPases and adaptors necessary to achieve mitochondrial fission [62]. They found that Fis1 was dispensable for fission, whereas Mff and the MiDs bound Drp1 and mediated fission. Not only did the MiDs facilitate Drp1 recruitment they also co-assembled with Drp1 in vitro [62]. Drp1–MiD heteropolymers had narrower diameters than Drp1 homopolymers, suggesting that the MiDs might assist in narrowing or focusing the fission apparatus, thereby facilitating membrane scission [62].

Clinton et al. [63] conducted an experiment that reminds us of the importance of ensuring physiological conditions when assessing binding partners. They showed that Mff is capable of recruiting dimeric Drp1, but noted the importance of anchoring Mff to a lipid scaffold to better detect Mff–Drp1 interactions [63]. Thus cell physiology, and its alterations by disease, may limit direct extrapolations from reductionist models of fission. Certainly the fission mediators are heavily regulated by mitochondrial phospholipids, such as cardiolipin and phosphatidic acid. Thus, it remains unclear how the different adaptor proteins interact with each other and with Drp1 in vivo. One possible avenue for interaction might be that the MiD proteins act as initial recruiters of inactive Drp1, which undergoes oligomerization and subsequent Ser637 dephosphorylation prior to inducing fission.

MiD49 AND MiD51 RECRUIT INACTIVE Drp1

The MiD-mediated rescue of Drp1 recruitment to mitochondria in Fis1/Mff-knockout cells provides insight into the role of MiDs in fission. This recruitment activity is independent of Mff and Fis1, as the cytosolic domain of MiD51 is sufficient to recruit Drp1 to lysosomes [59]. In contrast with Mff, whose overexpression induces mitochondrial fragmentation, overexpression of the MiD proteins induces mitochondrial fusion, as mentioned previously [30,32,58]. As an explanation for this discrepancy in behaviour between the MiD proteins and Mff, Palmer et al. [58] proposed that MiD49 and MiD51 recruit inactive forms of Drp1. As a result, MiD protein overexpression causes an accumulation of this fission-incompetent Drp1 at the OMM.

ROLE OF Drp1 PHOSPHORYLATION IN INTERACTION WITH MiD PROTEINS

Drp1 is inhibited by phosphorylation at Ser637 and activated by phosphorylation at Ser616 [8,23,24,46,64]. The interaction between Mff and Drp1 is dependent on Drp1’s phosphorylation status. Dephosphorylation of Drp1 at Ser637 is necessary for Mff interaction with Drp1 [65], whereas the MiDs can bind the phosphorylated form of Drp1 at Ser637. Mitochondrial Drp1 Ser637 levels are enhanced in cells overexpressing MiD49 or MiD51, suggesting increased recruitment of phosphorylated Drp1 Ser637 [27]. In this study, Drp1 phosphorylation at Ser637 was a prerequisite for complex formation with the MiD proteins. Supporting this, wild-type Drp1 (presumably phosphorylated) co-immunoprecipitated more efficiently with MiD49 and MiD51 than did a phospho-null Drp1S637A mutant [27]. Likewise, dephosphorylation of Drp1-Ser637, induced by UV irradiation, decreased MiD51 binding to Drp1 [65]. Dephosphorylation of Drp1 at Ser637 can also be induced by treatment with CCCP. CCCP treatment of cells overexpressing either MiD49 or MiD51 causes rapid mitochondrial fission, providing further evidence that the mitochondrial elongation that is associated with MiD49 and MiD51 overexpression is linked to excessive recruitment of inactive Drp1 [27]. Perhaps Mff and the MiD proteins act as differential regulators of Drp1, with Mff recruiting the active form of Drp1 whereas MiD49 and MiD51 recruit the inactive form. The recruitment of inactive Drp1 by MiD49 and MiD51 suggests that additional triggers may be required to activate fission events to take place at MiD–Drp1 mitochondrial foci.

MiD49 AND MiD51 PROMOTE Drp1 OLIGOMERIZATION

Oligomerization of Drp1 may also regulate the MiD-mediated execution of fission events [48,66]. Palmer et al. [58] found that MiD49 and MiD51 were incapable of sequestering Drp1 defective in higher-order assembly. However, assembly-deficient Drp1 mutants are, in fact, still capable of forming dimers and trimers [67].

MiD49–Drp1 COMPLEXES

MiD49 may serve as part of the Drp1 assembly, rather than as a nucleating site for Drp1 homopolymer assembly on the OMM. MiD49 and Drp1 associate stoichiometrically, suggesting that the proteins co-polymerize. These MiD49–Drp1 complexes may form in order to produce a Drp1 assembly that is narrow enough to drive fission, as the external diameter of an assembly composed only of Drp1 is too great for initiation of inner leaflet hemifusion and membrane scission [62]. Although MiD51 also promotes the oligomerization of Drp1 [68], no evidence currently exists to support its co-polymerization with Drp1. The exact mechanism by which the MiD proteins promote Drp1 assembly and alter the structure of Drp1 oligomers in the fission complex remains a key subject for study.

POSSIBLE TRIGGERS FOR FISSION INDUCTION AT MiD–Drp1 FOCI

Mitochondrial fission occurs at sites where the ER is wrapped around the mitochondria tubule [69]. MiD49 and MiD51 foci are present at both constricted and unconstricted sites where the ER contacts the OMM [70]. These MiD foci, where Drp1 is usually present, are capable of undergoing several constriction–relaxation cycles prior to the execution of a fission event [70]. Although the precise signal required for initiation of fission at these foci remains unclear, a recent study has implicated Fis1 binding to MiD51 as being important. Zhang et al. [65] observed that the interaction between MiD51 and Fis1 increased markedly after UV irradiation, which decreased the interaction between Drp1 and MiD51. These observations provide some evidence that binding of Fis1 to MiD51 is important for activation of mitochondrial fission, at least in this cell type.

MiD49 AND MiD51 RECRUITMENT OF Drp1 IS SPECIFIC FOR MITOCHONDRIAL FISSION

Drp1 is capable of mediating both mitochondrial and peroxisomal fission, and Drp1 puncta are observed at the fission sites of both types of organelle [18,71,72]. Fis1 and Mff have also been observed at peroxisomes and function in concert with PEX11 and Drp1 to mediate peroxisomal fission [49]. In contrast, the MiD proteins appear to localize uniquely to mitochondria [59]. Deletion of Mff results in peroxisomal elongation, whereas additional deletion of MiD49 and MiD51 does not further increase peroxisome length [53]. The lack of membrane promiscuity observed for the MiD proteins may be a result of their N-terminal anchor. Fis1 and Mff are C-terminally anchored in the OMM and C-terminal anchors are associated with greater alternative membrane targeting [73]. Peroxisomal elongation is observed in cells overexpressing MiD51, although this effect is likely to be produced by a reduction in the amount of Drp1 available to mediate peroxisomal fission [59]. Indeed, Drp1 levels are reduced at peroxisomes and increased in mitochondria after induction of MiD51 expression [59].

STRUCTURAL FEATURES OF MiD49 AND MiD51

In 2014, the first crystal structures of the cytosolic domains of the MiD proteins were reported [68,74,75]. The cytosolic domains of MiD49 and MiD51 are responsible for Drp1 binding [58] (Figure 4). Crystal structures of the cytosolic domains of MiD49 and MiD51 indicate that they both have nucleotidyltransferase folds with a central β-strand region and two α-helical side regions [68,74,75]. However, both proteins lack the catalytic residues required for transferase activity [68,74,75]. MiD51 does have a nucleotidyltransferase fold that binds an ADP cofactor, which is essential for activation of Drp1 [74]. MiD49 lacks the capacity for nucleotide cofactor binding. Thus, there is a plausible structural basis for differential regulation of MiD51- compared with MiD49-mediated fission [74].

Crystal structure of a MiD51 cytosolic domain that is sufficient for Drp1 recruitment

Figure 4
Crystal structure of a MiD51 cytosolic domain that is sufficient for Drp1 recruitment

(A) Structure of MiD51ΔN118 displaying the nucleotidyltransferase domain in blue and DRR domain in green. (B) Surface representation of MiD51ΔN118; view and colouring as in (A). Reproduced from [75]: Richter, V., Palmer, C.S., Osellame, L.D., Singh, A.P., Elgass, K., Stroud, D.A., Sesaki, H., Kvansakul, M. and Ryan, M.T. (2014) Structural and functional analysis of MiD51, a dynamin receptor required for mitochondrial fission. J. Cell Biol. 204, 477–486, with permission.

Figure 4
Crystal structure of a MiD51 cytosolic domain that is sufficient for Drp1 recruitment

(A) Structure of MiD51ΔN118 displaying the nucleotidyltransferase domain in blue and DRR domain in green. (B) Surface representation of MiD51ΔN118; view and colouring as in (A). Reproduced from [75]: Richter, V., Palmer, C.S., Osellame, L.D., Singh, A.P., Elgass, K., Stroud, D.A., Sesaki, H., Kvansakul, M. and Ryan, M.T. (2014) Structural and functional analysis of MiD51, a dynamin receptor required for mitochondrial fission. J. Cell Biol. 204, 477–486, with permission.

A single exposed loop corresponding to residues 238–242 on the surface of MiD51 was identified as the Drp1-binding loop (Figure 4) [68,74,75]. Disruption of a salt bridge below this loop abrogates Drp1 binding, as does disruption of the analogous salt bridge in MiD49 [74,75]. Alteration of residues in this loop leads to only small changes in the charge distribution but reduces Drp1 binding, providing evidence that the topology of the loop is important for Drp1 binding [75]. This β4-α4 loop is unique to the MiDs and is not present in other members of the nucleotidyltransferase family [75]. Interestingly, the residues contained in this loop are highly conserved between MiD51 and MiD49 and the topology of the loop is similar in both proteins [68,74]. When these residues are mutated, MiD51 is incapable of recruiting Drp1 to mitochondria [68].

The nucleotidyltransferase domain of MiD51 is responsible for binding ADP with high affinity, an interaction that stabilizes the protein [68]. MiD51 is also capable of binding GDP [75]. These nucleotide-binding residues are not conserved in MiD49 and no nucleotides have been identified as candidate MiD49 cofactors [74]. A dimerization interface was identified for MiD51, formed via electrostatic interactions between N-terminal α-helical segments of each monomer [68]. The residues of this dimerization interface are not conserved in MiD49 and it does not dimerize [74].

Neither ADP binding nor dimerization significantly affects the structure of MiD51 [68]. Mutant MiD51 defective for ADP binding and for dimerization are capable of recruiting Drp1, indicating that these functions are independent of Drp1 binding [68]. However, in the absence of ADP, incubation of Drp1 with MiD51 suppresses Drp1’s GTP hydrolysis activity [68]. Addition of ADP causes profound relief of this inhibition and results in a 20-fold stimulation of Drp1 GTPase activity [68]. Thus, ADP binding to MiD51 probably provides an activating signal for mitochondrial fission. Addition of ADP to purified MiD51 and Drp1 enhances Drp1 sedimentation, suggesting that ADP binding also promotes Drp1 oligomerization [68]. ADP may stabilize MiD51 folding, allowing Drp1 to assemble properly for membrane scission [68]. Cellular metabolism may act as a regulator of MiD51 and mitochondrial fission via control of ADP levels [68].

HUMAN DISEASE (TABLE 1)

Mitochondrial dynamics play an important role in cell metabolism, proliferation and apoptosis. Therefore, dysregulation of mitochondrial dynamics is linked to a number of human diseases. Mutations in the fusion protein Mfn2 is the cause for the neurodegenerative disease Charcot–Marie–Tooth disease type 2A [76]. Mutations in OPA1 cause autosomal dominant optic atrophy (ADOA) which presents as progressive blindness [77,78]. In addition, spastic paraplegia, a multiple sclerosis-like syndrome is also associated with mutations in OPA1 [79]. Other diseases caused by the perturbation of mitochondrial dynamics include Type 2 diabetes [80], Parkinson's disease [81], Huntington's disease [82] and Alzheimer's disease [83]. Dysregulation of the balance between fusion and fission, in which fission exceeds fusion, has been well documented in hyperproliferative diseases such as PAH and cancer. This increase in mitochondrial fission often results from Drp1 activation. Several strategies have been used to therapeutically target Drp1 as a preclinical treatment for PAH and cancer. Pharmacological inhibition of Drp1 activity, using the small-molecule GTPase inhibitor mdivi-1, produced encouraging results, regressing experimentally induced PAH and NSCLC in a xenotransplantation murine model [14,15].

Table 1
Mediators of mitochondrial fusion and fission and their binding partners
MediatorFunctionDisease related to mutation in mitochondrial dynamics related geneDisease related to acquired disorder of mitochondrial dynamics
Fusion mediators 
 Mitofusin-1 (Mfn1) OMM-anchored GTPase that tethers together adjacent mitochondria via homodimerization or heterodimerization with Mfn2 [37,38,41,88 Breast cancer [89
 Mitofusin-2 (Mfn2) OMM-anchored GTPase that tethers together adjacent mitochondria via homodimerization or heterodimerization with Mfn1 [37,38,41,88Charcot–Marie–Tooth type 2A disease [76Diabetes [80,90], pulmonary arterial hypertension [91], lung cancer [15
 Optic atrophy 1 (OPA1) IMM-anchored GTPase that mediates IMM fusion Autosomal dominant optic atrophy [77,78], spastic paraplegia, multiple sclerosis-like syndrome, Behr-like syndrome [79Hypertension [92
Fission mediators 
 Dynamin-related protein 1 (Drp1) Cytosolic GTPase that is recruited to OMM and executes mitochondrial membrane scission [18,45Microcephaly, abnormal brain development, optic atrophy, sudden death [93] developmental delay, pain insensitivity [94Alzheimer's disease [83] Parkinson's disease [81], Huntington's disease [82], pulmonary arterial hypertension [14], patent ductus arteriosus [95], lung cancer [15], breast cancer [89
Mitochondrial receptors of Drp1 
 Mitochondrial fission factor (Mff) OMM-anchored protein that recruits Drp1 from the cytosol to mitochondria during mitochondrial fission [27,57Leigh-like basal ganglia disease [96 
 Mitochondrial dynamics protein of 49 kDa (MiD49) OMM-anchored protein that recruits inactive Drp1 from the cytosol [58,65,68Unknown Unknown 
 Mitochondrial dynamics protein of 51 kDa (MiD51) OMM-anchored protein that recruits inactive Drp1 from the cytosol [58,65,68Unknown Unknown 
MediatorFunctionDisease related to mutation in mitochondrial dynamics related geneDisease related to acquired disorder of mitochondrial dynamics
Fusion mediators 
 Mitofusin-1 (Mfn1) OMM-anchored GTPase that tethers together adjacent mitochondria via homodimerization or heterodimerization with Mfn2 [37,38,41,88 Breast cancer [89
 Mitofusin-2 (Mfn2) OMM-anchored GTPase that tethers together adjacent mitochondria via homodimerization or heterodimerization with Mfn1 [37,38,41,88Charcot–Marie–Tooth type 2A disease [76Diabetes [80,90], pulmonary arterial hypertension [91], lung cancer [15
 Optic atrophy 1 (OPA1) IMM-anchored GTPase that mediates IMM fusion Autosomal dominant optic atrophy [77,78], spastic paraplegia, multiple sclerosis-like syndrome, Behr-like syndrome [79Hypertension [92
Fission mediators 
 Dynamin-related protein 1 (Drp1) Cytosolic GTPase that is recruited to OMM and executes mitochondrial membrane scission [18,45Microcephaly, abnormal brain development, optic atrophy, sudden death [93] developmental delay, pain insensitivity [94Alzheimer's disease [83] Parkinson's disease [81], Huntington's disease [82], pulmonary arterial hypertension [14], patent ductus arteriosus [95], lung cancer [15], breast cancer [89
Mitochondrial receptors of Drp1 
 Mitochondrial fission factor (Mff) OMM-anchored protein that recruits Drp1 from the cytosol to mitochondria during mitochondrial fission [27,57Leigh-like basal ganglia disease [96 
 Mitochondrial dynamics protein of 49 kDa (MiD49) OMM-anchored protein that recruits inactive Drp1 from the cytosol [58,65,68Unknown Unknown 
 Mitochondrial dynamics protein of 51 kDa (MiD51) OMM-anchored protein that recruits inactive Drp1 from the cytosol [58,65,68Unknown Unknown 

Perturbation of mitochondrial dynamics can also be caused by excessive translocation of Drp1 to the OMM due to high expression and activity of the Drp1 adaptor proteins (Figure 3). Although the involvement of the Drp1 adaptor proteins in human diseases is not yet well documented, the disruptor peptide P110, which prevents translocation of Drp1 to the mitochondria by blocking the interaction between Drp1 and Fis1, improves mitochondrial dynamics and cell function in the in vitro models of neurodegenerative diseases [84] and cardiac ischaemia/reperfusion injury [85].

Combined silencing of MiD49 and MiD51 in a murine atrial derived cell line protects the cells from acute ischaemia/reperfusion injury, indicating that these adaptor proteins can be therapeutically targeted for cardioprotection [86]. Another preliminary observation suggests that significant up-regulation of these two MiDs in human PAH PASMCs, contributes to the excess proliferation that characterizes this obstructive vasculopathy [87]. However, these findings are based on studies in cells and have yet to be validated in vivo. Lack of MiD49- and MD51-knockout mice has limited progress in the investigation of the physiological role of these adaptor proteins in disease models. Further studies are required to investigate the expression and activity of these adaptor proteins in human diseases.

CONCLUSIONS

MiD49 and MiD51 are Drp1-binding proteins involved in mitochondrial fission. They can recruit inactive Drp1 to the OMM. Heterologous overexpression of MiDs, sequesters inactive Drp1 and promotes fusion. In contrast, endogenous MiDs can also interact with Drp1 creating focused multimers that optimize membrane scission. Structural studies of MiD51 reveal important differences from MiD49, including MiD51’s unique capacity for GDP/ADP binding at its nucleotidyltransferase domain. The absence of ADP results in MiD51-mediated inhibition of Drp1, whereas, with ADP, MiD51 promotes fission, suggesting a link between metabolism and mitochondrial dynamics. Although substantial structural and functional characterization has been performed since the identification of MiD49 and MiD51 in 2011, the triggers that activate MiD-bound Drp1 in disease states are unknown. Further study is necessary to elucidate the complex interplay and co-ordination between Drp1 and the four binding partners that mediate mitochondrial fission. Moreover, there is confusion as to whether the MiDs primarily mediate fusion (by sequestering inactive Drp1 on the OMM) or fission (by guiding the structural assembly of Drp1 into a more focused constriction apparatus). This confusion may relate to a failure to distinguish the role of endogenous expression of the MiDs compared with heterologous overexpression of MiDs. We speculate that endogenous MiDs serve as Drp1-binding partners that are dysregulated in disease states. MiDs may themselves be important targets for chemical or molecular modulation in the treatment of proliferative disorders or in efforts to prevent cell injury. The diversity of Drp1-binding partners suggests that the composition of the fission apparatus may vary between disease states and among individuals. Were this true, the Drp1-binding partners could be appealing targets for precision medicine. Our laboratory is actively pursuing the possibility that molecular and pharmacological modulation of Drp1 adaptor proteins could offer novel therapeutic strategies for diseases caused by the dysregulation of mitochondrial dynamics.

FUNDING

This work was supported by the CIHR Foundation Grant [143261]; the Canada Foundation for Innovation (CFI) [229252]; National Institutes of Health [R01HL071115 and R01HL113003]; Tier 1 Canada Research Chair in Mitochondrial Dynamics [950-22952]; and the William J. Henderson Foundation (to S.L.A).

Abbreviations

     
  • CCCP

    carbonyl cyanide m-chlorophenyl hydrazine

  •  
  • Drp1

    dynamin-related protein 1

  •  
  • ER

    endoplasmic reticulum

  •  
  • Fis1

    fission 1

  •  
  • MEF

    mouse embryonic fibroblast

  •  
  • Mff

    mitochondrial fission factor

  •  
  • Mfn1

    mitofusin 1

  •  
  • Mfn2

    mitofusin 2

  •  
  • MiD49 and MiD51

    mitochondrial dynamics proteins of 49 kDa and 51 kDa

  •  
  • MIEF1

    mitochondrial elongation factor 1

  •  
  • MIEF2

    mitochondrial elongation factor 2

  •  
  • NSCLC

    non-small-cell lung cancer

  •  
  • OMM

    outer mitochondrial membrane

  •  
  • OPA1

    optic atrophy 1

  •  
  • PAH

    pulmonary arterial hypertension

  •  
  • PASMC

    pulmonary artery smooth muscle cell

  •  
  • SMCR7

    Smith–Magenis syndrome chromosome region candidate gene 7

  •  
  • SMCR7L

    Smith–Magenis syndrome chromosome region candidate gene 7-like

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Author notes

1

Co-first authors.