Mitochondrial fission and fusion and motility in mitophagy

Mitochondria are dynamic organelles that continuously undergo fission and fusion, which are necessary for cell survival and adaptation to changing conditions needed for cell growth, division, and distribution of mitochondria during differentiation [4].

Mitochondrial fusion in mammals is mediated by the fusion proteins mitofusin 1 (Mfn1) and Mfn2 and optic atrophy 1 (OPA1). Mfn1 and Mfn2 are dynamin-related GTPases that are responsible for fusion of outer mitochondrial membranes. OPA1 is also a dynamin-related GTPase, which is responsible for fusion of inner mitochondrial membranes (Fig. 1A). Presenilin-associatedrhomboid-like (PARL) [13] and paraplegin (an AAA protease present in the mitochondrial matrix) [14] induce alternative splicing and alternative processing of OPA1 to generate eight OPA1 isoforms. However, OPA1 processing still occurs in PARL or paraplegin knockout MEF cells, suggesting that other factors may also be involved in OPA1 processing [15]. Yme can further cleave OPA1 under normal conditions to generate Short and Long forms of OPA1 (S-OPA1 and L-OPA1) [16], where L-OPA1 is integral in the inner membrane and S-OPA1 is located in the intermembrane space. L-OPA1 is further cleaved by the inducible protease OMA1 when mitochondria are depolarized by the mitochondrial uncoupler carbonyl cyanide m-chloro phenyl hydrazine (CCCP), resulting in mitochondrial fragmentation by preventing mitochondrial fusion [17,18]. The mitochondrial deacetylase SIRT3 is capable of deacetylating OPA1 and elevating its GTPase activity [19].

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Fig. 1

Mitochondrial fusion and fission in mammalian cells. (A) Mitochondrial fusion is mediated by large dynamin-related GTPase proteins Mfn1, Mfn2 and OPA1. Outer mitochondria membrane (OM) fusion is mediated by Mfn1 and Mfn2, whereas inner mitochondria membrane (IM) fusion is mediated by OPA1. (B) Mitochondria fission requires the recruitment of Drp1 from cytosol to mitochondria. Drp1 is also a dynamin-related GTPase protein that binds to four Drp1 receptor proteins Fis1, Mff, MID49 and MID51, which are mitochondria OM proteins.

Mitochondrial fission in mammals is mediated by dynamin-related protein 1 (Drp1), which is also a large GTPase. Drp1 is a cytosolic protein that can be recruited to the outer mitochondrial membrane to constrict mitochondria resulting in eventual division of a mitochondrion into two separate organelles. Drp1 interacts with four mitochondrial receptor proteins: fission 1 (Fis1), mitochondria fission factor (Mff), mitochondrial dynamics protein of 49 kDa (MID49) and MID51 (Fig. 1B). In mammalian cells, it seems that the interaction between Fis1 and Drp1 has a minor role in regulating mitochondrial fission whereas the interactions of Drp1 with the other three receptor proteins play prominent roles for fission [20–24]. In addition, Drp1 can also localize at the endoplasmic reticulum–mitochondria contact site, and the endoplasmic reticulum may play a role in the process of mitochondrial fission [25]. Accumulating evidence indicates that posttranslational modification of Drp1 is an important mechanism for regulating its function. During cell division, mitochondrial fission is essential for separating mitochondria into two daughter cells. At the onset of mitosis, Drp1 is phosphorylated by Cdk1/Cyclin B at Ser585, which increases Drp1 GTPase activity [26]. In contrast, reversible phosphorylation of Drp1 by cyclic AMP-dependent protein kinase (PKA) and its dephosphorylation by phosphatase calcineurin at Ser656 leads to elongated mitochondria [27]. In addition, the mitochondrial phosphatase phosphoglycerate mutase family member 5 (PGAM5), dephosphorylates Drp1 at Ser637 and recruits Drp1 to mitochondria to induce mitochondrial fragmentation. This PGAM5-Drp1-mediated mitochondrial fragmentation was initially thought to play a critical role in programmed necrosis, but this notion has been challenged by recent findings that Drp1 knockout mouse embryonic fibroblasts (MEFs) were equally as sensitive to necrosis as wild type MEFs [28,29]. In addition to phosphorylation, Drp1 can also be ubiquitinated by MARCH V, a mitochondrial E3 ligase, or sumoylated by SUMO-1. The ubiquitination and sumoylation of Drp1 can either regulate the stability of Drp1 or recruit Drp1 to the actual mitochondrial dividing site and in turn regulate mitochondria fission [30–32].

In addition to fission and fusion, the movement of mitochondria through the cytoskeleton is also important for the cellular distribution and turnover of mitochondria [33]. Mitochondria in mammalian cells are mostly transported on microtubules using a kinesin motor towards the plus end and a dynein motor towards the minus end of microtubules [34] (Fig. 2). The attachment of mitochondria to the kinesin motor is regulated by a series of molecular adapters. The adapter protein Milton directly interacts with the outer mitochondrial membrane protein Mitochondrial Rho GTPase (Miro) and in turn links mitochondria to kinesin [34]. Interestingly, both Mfn1 and Mfn2 interact with Miro and Milton, and loss of Mfn2 alone is sufficient to cause a defect in mitochondrial transport in neurons [35]. In addition, the availability of extracellular glucose can also regulate mitochondrial motility in neurons. Most mitochondria in mouse axons are immobile, which is regulated by syntaphilin (SNPH). SNPH anchors axonal mitochondria to microtubules resulting in the local stalling of mitochondria. SNPH serves as a docking protein, and deletion of SNPH in mice increases axonal mitochondrial motility. In response to elevated Ca₂+, Miro releases Kinesin-1, which then binds with SNPH to inhibit the mobilization of axonal mitochondria [36]. In addition, Milton is GlcNAcylated by O-GlcNac Transferase (OGT) in response to increased extracellular glucose levels, which diminishes mitochondrial mobility [37].

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Fig. 2

Regulation of mitochondrial motility. Mitochondria are transported on cytoskeleton microtubules by molecular motor proteins kinesin and dynein. Milton acts as an adapter molecule to link the motor proteins to the outer mitochondrial membrane protein miro, and kinesin transports mitochondria towards the plus end of microtubules whereas dynein transports mitochondria to the minus end of microtubules. When intracellular calcium levels rise, kinesin is dissociated from the Milton–miro complex and then binds to SNPH. SNPH inhibits the ATPase activity of kinesin to block the movement of mitochondria.

It has been suggested that mitochondria fission and fusion may serve as important quality control mechanisms for preserving mitochondria. For example, dysfunctional mitochondria may lose their fusion capacity by inactivating fusion or activating fission machineries to prevent the damaged mitochondria from incorporating back into the healthy mitochondrial network. After photo-labeling, mitochondria undergo continuous cycles of fission and fusion and show a striking heterogeneity with two sets of daughter mitochondria that either have increased or decreased membrane potential. Daughter mitochondria with higher membrane potential (presumably good quality mitochondria) proceed to fusion while depolarized daughter mitochondria (presumably bad quality mitochondria) are degraded by mitophagy [5]. Therefore, mitochondrial fission and fusion may serve as a quality control mechanism to preserve the better mitochondria via fusion and rid of the bad quality mitochondria via fission and subsequent mitophagy. When mitochondrial damage is considered mild, it will trigger the proteolytic mediated processing and inactivation of Opa1 resulting in the impairment of inner membrane fusion without affecting the outer membrane fusion (which is mediated by Mfn1 and Mfn2). Severe damage may activate Pink1–Parkin-mediated events resulting in the ubiquitination and proteasomal degradation of Mfn1 and Mfn2 and subsequent mitophagy (discussed in detail below). However, under nutrient replete conditions, Mfn1 and Mfn2 knockout MEFs do not show increased mitophagy, suggesting that mitochondrial fission alone is not enough to induce mitophagy. It seems that in addition to mitochondrial fragmentation, mitochondria must also be dysfunctional/depolarized and may also possibly need to recruit other autophagy receptor proteins for mitophagy induction to occur [10]. However, it is also likely that small mitochondria are relatively easier to be enwrapped by autophagosomes compared to larger elongated mitochondria. In addition to mitochondrial fission, the motility of mitochondria also seems to be important for mitophagy. Overexpression of Pink1 and Parkin can lead to Miro ubiquitination and degradation, suggesting a link between Pink1–Parkin and Miro in mitophagy [38]. Indeed, loss of Miro results in perinuclear clustering of mitochondria, which promotes mitophagy in HeLa cells. However, it remains unclear whether Miro would act as a mitophagy receptor or Miro–Milton–Kinesin-mediated mitochondria transport may normally prevent the mitophagy process. It will be interesting to determine whether SNPH-mediated mitochondrial motility would also be involved in mitophagy.

Parkin-dependent mitophagy

One of the best studied mechanisms for mitophagy in mammalian cells is the Pink1–Parkin-mediated mitophagy pathway [10,11]. Parkin is a cytosolic E3 ubiquitin ligase that translocates to depolarized mitochondria after treatment with the mitochondrial uncoupler CCCP and initiates their degradation via mitophagy [39]. The Parkin-dependent mitophagy pathway has been shown to require both Parkin and Pink1. Pink1 is a mitochondria serine/threonine kinase. Genetic studies from drosophila indicate that Pink1 and Parkin work on the same pathway, and Pink1 acts upstream of Parkin because overexpression of Parkin in Pink1-deficient drosophila partially rescued the Pink1 mutant phenotype while overexpression of Pink1 failed to do so in Parkin-deficient drosophila [40–42]. In addition, overexpression of Pink1 alone can initiate translocation of Parkin to mitochondria without mitochondrial damage [43]. In humans, mutations of both Pink1 and Parkin have been linked to familiar autosomal recessive Parkinsonism [44]. Taken together, all evidence supports the notion that Pink1 and Parkin work on the same pathway.

The level of Pink1 is normally undetectable in most cells because Pink1 is cleaved in the mitochondria by PARL and then degraded by mitochondrial peptidases [45]. However, Pink1 is no longer cleaved and becomes stabilized on the outer mitochondrial membrane when mitochondria are depolarized [45]. Pink1 then promotes Parkin-mediated mitophagy by recruiting Parkin to mitochondria and promoting Parkin E3 ligase activity. Pink1 directly phosphorylates Thr175 and Thr217 within Parkin's linker region, which promotes Parkin mitochondrial translocation [46]. In addition, phosphorylation of Parkin by Pink1 also activates Parkin's E3 ubiquitin ligase activity, enabling it to ubiquitinate mitochondrial proteins [47]. Pink1 also phosphorylates ubiquitin at Ser65, and the phospho-ubiquitin activates Parkin E3 ubiquitin ligase activity through a feed-forward mechanism [48,49]. Using a genome-wide small interfering RNA (siRNA) screening for genes that regulate Parkin mitochondrial translocation, the translocase of outer mitochondrial membrane 7 (TOMM7) was found to stabilize Pink1 on the outer mitochondrial membrane whereas SIAH3, a mitochondrial resident protein, de-stabilized Pink1 on mitochondria. Furthermore, two other proteins, HSPA1L (an HSP70 family protein) and Bcl2-associated athanogene 4 (BAG4, a nucleotide exchange factor for HSP70), positively and negatively regulate Parkin mitochondrial translocation, respectively [50]. In addition to Pink1, four of Parkin's different cognate E2 co-enzymes (UBE2D, UBE2L3, UBE2N and UBE2R1) also positively or negatively regulate Parkin’s activation, translocation and enzymatic functions during mitophagy [51,52]. Neuregulin receptor degradation protein 1 (Nrdp1), an E3 ubiquitin ligase, regulates the ubiquitination and proteasomal degradation of Parkin. Nrdp1 interacts with C-type lectin domain family 16, member A (Clec16a), a protein that is associated with type 1 diabetes mellitus, and deletion of Clec16a increases Parkin expression. Intriguingly, despite an increased expression of Parkin, pancreas-specific Clec16a knockout mice had abnormal mitochondria with reduced oxygen consumption and ATP concentration in pancreatic β cells due to defects of mitophagy. Subsequent studies revealed that Clec16a deficient fibroblasts had defective fusion of autophagosomes with lysosomes [53], suggesting that Clec16a also regulates autophagy in addition to Parkin-mediated mitophagy.

Once recruited to the mitochondria, Parkin ubiquitinates several mitochondrial outer membrane proteins including the mitochondrial fusion proteins Mfn1 and Mfn2, Miro, Translocase of outer mitochondrial membrane 20 (TOMM20), and vbltage-dependent anion channel (VDAC) to initiate mitophagy. Ubiquitination and proteasomal degradation of Mfn1 and Mfn2 results in mitochondrial fission and fragmentation [54–57]. Mitochondrial fission has been shown to be important for mitophagy induction in liver hepatocytes among other cell types [5,58]. Fragmented mitochondria can fuse together if they have normal membrane potential, but loss of membrane potential prevents fusion and leads to mitochondria segregation and subsequent degradation by mitophagy [5]. In contrast, excessive fusion of mitochondria has been shown to inhibit the mitophagy process [59]. These observations support the notion that mitochondrial fragmentation promotes mitophagy induction. In addition to regulating mitochondria fusion and fission, Parkin-induced ubiquitination of Miro induces mitochondrial arrest, which can segregate damaged mitochondria from healthy mitochondria prior to mitophagy [60]. Parkin-mediated mitophagy can be antagonized by Ubiquitin-specific peptidase 30 (USP30), a deubiquitinase localized to mitochondria, which removes ubiquitin from damaged mitochondria previously attached by Parkin. Defective mitophagy caused by pathogenic mutations in Parkin is rescued by knockdown of USP30. Moreover, knockdown of USP30 improves mitochondrial integrity in Parkin- or Pink1-deficient flies and protects flies against paraquat toxicity in vivo by ameliorating defects in dopamine levels, motor function and organismal survival [61].

Following Parkin-mediated ubiquitination of outer mitochondrial membrane proteins, the selective autophagy adapter protein p62/SQSTM1 (Sequestosome 1) is recruited to mitochondria and thought to play a role in mitophagy due to its capacity to directly interact with LC3 via its LC3 interacting region (LIR) [56,62–64]. However, it seems that the role of p62 in mitophagy is not essential [65,66], likely due to the presence of other compensatory or redundant similar autophagy receptor proteins. Interestingly, it was recently found that optineurin, another autophagy receptor protein, was also recruited to ubiquitinated mitochondria via its ubiquitin binding domain after Parkin activation on mitochondria. Optineurin induces autophagosome formation around the damaged mitochondria by recruiting double FYVE-containing protein 1 (DFCP1) and LC3 to damaged mitochondria [67]. In addition, Parkin also recruits Ambra1 (a Beclin-1 interacting protein) to depolarized mitochondria to initiate engulfment of damaged mitochondria by autophagosomes [68]. Taken together, it appears that Pink1 and Parkin regulate mitophagy at multiple levels including mitochondrial fragmentation (via Mfn1 and Mfn2), mitochondrial motility (via Miro), autophagy receptor protein (via p62 or optineurin), and autophagy machinery (via Ambra1). The molecular events of Pink1–Parkin-mediated mitophagy are summarized in Fig. 3.

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Fig. 3

Molecular events for Parkin-dependent mitophagy. When mitochondria are depolarized in response to various insults, Parkin is translocated to the outer membrane of mitochondria. This process is regulated by PINK1, which is stabilized on depolarized mitochondria due to the inactivation of the mitochondrial protease PARL. Pink1 is also stabilized by the outer mitochondrial membrane protein TOMM7 but de-stabilized by SIAH3, a mitochondrial resident protein. PINK1 either directly phosphorylates Parkin or ubiquitin to promote Parkin translocation or its ligase activity. Cytosolic HSPA1L also promotes, whereas BAG4 inhibits, Parkin mitochondrial translocation. Once Parkin translocates to mitochondria, it promotes selective mitophagy through mitochondrial ubiquitination and recruitment of autophagy receptor proteins such as p62 and optineurin, which further recruit LC3 positive autophagosomes.

While there is a tremendous amount of evidence supporting the important role of Pink1–Parkin in regulating mitophagy, most of the studies so far used a cell culture model in which cells were ectopically overexpressed with Parkin and exposed to the mitochondrial uncoupler CCCP [10]. Studies on the role of Parkin in regulating mitophagy in vivo are scarce. Parkin knockout mice had decreased mitophagy and were more sensitive to myocardial infarction compared with wild type mice [69]. In aged mice or mice treated with doxorubicin, it was recently reported that cytosolic p53 bound to Parkin and inhibited Parkin mitochondrial translocation and mitophagy. Overexpression of Parkin attenuated functional decline of aged hearts [70]. However, a recent study showed that loss of Drp1 in mouse cardiomyocytes led to increased mitochondrial ubiquitination and p62 mitochondrial targeting, which was independent of Parkin [71]. We also found that Parkin is expressed in mouse livers, and Parkin translocated to hepatic mitochondria after mice were administrated with acetaminophen, which damages mitochondria in hepatocytes. Intriguingly, the levels of ubiquitination and p62 on mitochondria were not different between wild type and Parkin knockout mice after APAP treatment (Williams J. et al., manuscript under revision). These results suggest that there must be other E3 ligases compensating for the loss of Parkin to trigger Parkin-independent mitophagy. It is well known that Pink1 and Parkin knockout mice only bear subtle phenotypes related to dopaminergic neuronal degeneration. A recent elegant study discovered that mitochondrial ubiquitin ligase 1 (MUL1) compensates for the loss of Pink1 or Parkin, and MUL1 acts by increasing Mfn1 degradation. Overexpression of MUL1 in Drosophila almost completely rescued the phenotypes observed in Pink1/Parkin null Drosophila [72]. These results suggest that while Parkin plays an important role in mitophagy, Parkin-independent mechanisms may also be involved in regulating mitophagy (see below).

The research work in Wen-Xing Ding's laboratory was supported in part by the NIAAA funds R01 AA020518, R01 DK102142, National Center for Research Resources (5P20RR021940), the National Institute of General Medical Sciences (8P20 GM103549 and T32 ES007079), and an Institutional Development award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health (P20 GM103418). The authors want to thank Barbara Fegley from the electron microscopy core facility at the University of Kansas Medical Center for her excellent assistance for EM studies.