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The changing shape of mitochondrial apoptosis
Review
The changing shape of mitochondrial apoptosis Michał Wasilewski1 and Luca Scorrano1,2 1 2
Dulbecco-Telethon Institute, Venetian Institute of Molecular Medicine, Via Orus 2, 35129 Padova, Italy Department of Cell Physiology and Metabolism, University of Geneva Medical School, 1 Rue M. Servet, 1211 Gene`ve, Switzerland
Mitochondria are key organelles in conversion of energy, regulation of cellular signaling and amplification of programmed cell death. The anatomy of the organelle matches this functional versatility in complexity and is modulated by the concerted action of proteins that impinge on its fusion–fission equilibrium. A growing body of evidence implicates changes in mitochondrial shape in the progression of apoptosis and, therefore, proteins governing such changes are likely candidates for involvement in pathogenetic mechanisms in neurodegeneration and cancer. Here, we discuss the recent advancements in our knowledge about the machinery that regulates mitochondrial shape and on the role of molecular mechanisms controlling mitochondrial morphology during cell death. Mitochondrial shape Mitochondria are essential organelles for the life and death of a cell and participate in energy conversion, regulation of signaling cascades and apoptosis [1] (Box 1). Although the conventional image of these organelles is of a static nature, mitochondria can fuse and divide. This ability has been known for years but obscured by the fine yet static details provided by electron microscopy. The advanced tools of live-cell imaging and 3D reconstruction, as well as electron tomography, have enabled us to fully appreciate their complexity and dynamic nature. The spectrum of mitochondrial shapes ranges from spherical, grain-like, individual entities to long, branched filaments that, ultimately, might form one interconnected ‘megamitochondrion’ per cell. Mitochondrial complexity begins at the level of their external appearance and continues to a similarly complex and dynamic internal structure [2]. A major leap forward in understanding the mechanisms and consequences of mitochondrial fusion–fission came with the discovery that the morphology of the organelle, like its ultrastructure, undergoes dynamic changes during apoptosis. Interfering with these changes slows apoptotic progression, indicating a functional role for mitochondrial shape in this process [3–5]. This finding instigated research on the molecular details, mechanisms and pathophysiological consequences of mitochondrial shape changes (Box 2). Here, we review the current understanding of mitochondrial fusion and fission in mammals and discuss evidence that supports a role for morphological changes of mitochondria in certain physiological and pathological processes, including apoptosis (Box 3). Corresponding author: Scorrano, L. ([email protected]).
Mitochondrial fusion and fission Mitochondrial shape results from the balance of two ongoing antagonistic processes, fusion and fission. When either process is blocked, the final morphology of the mitochondria is the consequence of unopposed progression towards the other side of the equilibrium [6]. Members of the machinery regulating mitochondrial fusion have been identified by analyzing organelle morphology in Saccharomices cerevisiae mutants [7,8]. The shape of mitochondria depends on several large ubiquitous GTPases with structural homology to dynamins [9] that participate in fusion, fission and tubulation of biological membranes. Proteins that regulate mitochondrial shape share at least the GTPase domain and a C-terminal coiled-coil domain with the prototypical dynamins, and these domains can function as GTPase effectors or to mediate protein–protein interaction [10]. The dynamin-related proteins that impinge on mitochondrial fusion are peculiar in that most of them are integral membrane proteins. We next discuss the properties of ‘mitochondria-shaping’ proteins of mammals in particular. A scheme of the players known to participate in mitochondrial fission and fusion in mammalian cells is presented in Figure 1 and 2, respectively. Fission In mammalian cells, mitochondrial fission depends on dynamin-related protein 1 (Drp1), a cytoplasmic large GTPase similar to dynamin that mediates the fragmentation of mitochondria and peroxisomes [11]. Drp1 translocates to mitochondria in response to cellular and mitochondrial cues. After mitochondrial dysfunction, cytoplasmic Ca2+ rises, leading to activation of calcineurin and dephosphorylation of the conserved Ser637 of Drp1 [12], inducing translocation of Drp1 to mitochondria. Conflicting data exists surrounding the kinase responsible for phosphorylation of this residue. Protein kinase A was reported to fulfill this function, linking mitochondrial morphology to another crucial second messenger, cyclic AMP [13,14]. Alternatively, Ser637 could be phosphorylated by calmodulin-dependent protein kinase Ia, although in this case, phosphorylation of Drp1 induced its mitochondrial localization [15]. The phosphorylation status of this site is dominant over that of Ser616 [12], which is controlled by cyclin-dependent kinase 1 to drive mitochondrial fission during mitosis [16]. Mitochondrial Drp1 can then be stabilized on the surface of the organelle by SUMOylation [17,18], a process known to protect molecules from degradation by the ubiquitin-proteasome system.
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Box 1. Mitochondria Mitochondria are double-membrane organelles, the origin of which has been traced to an ancient prokaryotic endosymbiont. The outer mitochondrial membrane (OMM) has a composition similar to that of other eukaryotic membranes and originates from a vesicle engulfing the endosymbiont. It is permeable to metabolites and small peptides up to 3000 Da, thanks to the presence of voltage-dependent anion channels. The inner mitochondrial membrane (IMM) has a clearer prokaryotic origin and differs from the OMM in its physical properties and composition. Its ion conductance and metabolite permeability are tightly controlled. IMM comprises a larger fraction of proteins and approximately 20% of cardiolipin, a lipid also found in bacterial membranes, which is crucial for proper activity of several IMM enzymes. IMM folds into a complicated network of tubules and lamellae called cristae, reflecting its unique function. IMM is the site of oxidative phosphorylation (OXPHOS), which provides most of the ATP produced during aerobic metabolism. According to the chemiosmotic theory, the basic principle of OXPHOS is to pump protons across the proton-impermeable IMM, generating an electrochemical potential, which is then used to drive phosphorylation of ADP to ATP by F1F0-ATPase synthase. Energy required for proton pumping is provided by electrons from NADH and FADH produced during the tricarboxylic acid cycle and other catabolic processes. The energy of
Fis1 is a membrane protein homogenously distributed in the outer mitochondrial membrane (OMM) via a transmembrane domain located in the C-terminal region, and only a small portion of the molecule protrudes into the intermembrane space (IMS) (Figure 1). The cytoplasmic region contains six alpha helices, four of which (a2–a5) form two tetratricopeptide-repeat (TPR)-like domains, predicted to allow protein–protein interactions [19]. Overexpression of Fis1 induces mitochondrial fragmentation, but because it does not possess enzymatic activity, its role is probably restricted to anchoring effector proteins to mitochondria. Accordingly, mitochondrial fragmentation caused by Fis1 overexpression can be blocked by expression of a dominant-negative mutant of Drp1 [20]. Moreover, Drp1 and Fis1 seem to interact, as judged by crosslinking and coimmunoprecipitation [21]. The Drp1– Fis interaction is a transient event, and efficient fission
electrons, liberated in a number of redox reactions, is translated into proton pumping by three large protein complexes in IMM (complex I, III and IV), called together with the non-proton-pumping complex II, the respiratory chain (RC) [1]. Mitochondria contain an autonomous circular small genome that is located in the internal compartment, the matrix. mtDNA encodes only 13 proteins, mitochondrial ribosomal RNA and transfer RNA, but mutations in these genes result in severe diseases, mostly because of impaired energy production. Apart from OXPHOS, mitochondria participate in other important metabolic pathways, such as fatty acid synthesis, gluconeogenesis, steroidogenesis, synthesis of haeme and urea cycle [2]. Moreover, mitochondria cooperate with the ER to control calcium homeostasis and integrate information during apoptosis. The balance between pro- and antiapoptotic proteins of the Bcl-2 family controls release of cytochrome c, apoptosis-induction factor, Smac/DIABLO and endonuclease G from mitochondria. Sequestered in mitochondria, these proteins carry out routine tasks (e.g. cytochrome c shuttles electrons in RC). When released to the cytoplasm, they function as proapoptotic factors, either by inducing apoptosome formation (cytochrome c) or by a direct enzymatic activity (endonuclease G) [74].
requires dissociation of the complex. Accordingly, genetic modifications of the N-terminal domain of Fis1, which stabilize its binding to Drp1, abolish mitochondrial fragmentation [22]. However, the absolute requirement of Fis1 for binding Drp1 to mitochondria is somewhat weakened by the observation that downregulation of Fis1 only partially diminishes Drp1 recruitment to mitochondria [6]. Another question on the mechanism of interaction between Fis1 and Drp1 arises from the yeast model, in which this interaction requires the adaptor Mdv1 [23]. A mammalian orthologue for Mdv1 has not been yet identified, but considering the conservation of fission mechanisms, additional elements could be involved. Mechanism of fission By comparing Drp1 with the prototypical dynamins and considering data obtained in yeast, it was postulated that
Box 2. Mitochondrial dynamics and bioenergetics One of the most prominent functions of mitochondria is ATP production by oxidative phosphorylation. Research on the relationship between mitochondrial shape and function dates back to 1966, when Hackenbrock [75] observed that mitochondria isolated from rat livers shrunk and displayed a denser matrix during respiration. This conformation was called ‘condensed’, as opposed to the ‘orthodox’ conformation of inactive mitochondria. The matrix conformation transition was ascribed to osmotic changes caused by K+ movements across the inner mitochondrial membrane and thought to be only an artefact of isolated mitochondria. Indeed, mitochondria observed in most mammalian tissues manifest orthodox conformation [76,77]. Nevertheless, mitochondria from muscles with high energy requirements seem to have condensed matrix [78]. Moreover, matrix condensation is observed during apoptosis [79]. Overall, mitochondrial morphology depends on the energetic state of the cell. In general, treatments that compromise mitochondrial membrane potential induce mitochondrial fragmentation [12], an observation that correlates well with data from other organisms. Fusion of the outer membrane in yeast, for example, requires a chemical component of electrochemical potential, and fusion of the inner membrane depends on an electrical component [80]. Interestingly, recovery of mitochondrial morphology after wash-out of uncouplers requires de novo protein synthesis, suggesting a defect
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in mitochondrial fusion [81] probably caused by Opa1 degradation [42]. It is tempting to speculate that decreased potential blocks fusion by Opa1 degradation and ultimately leads to mitochondrial fragmentation by activating the fission machinery [12]. In turn, the bioenergetic state of a cell can be modified by changing the levels of mitochondria-shaping proteins. Loss of fusion and resulting mitochondrial fragmentation can cause mitochondrial dysfunction, perhaps through the impaired exchange of matrix material in fusion-incompetent mitochondria [82] and consequent accumulation of stochastic functional errors. It is more difficult to explain why failure to divide results in mitochondrial dysfunction [83]. Blockage of fission could impair elimination of defective elements of the network by autophagy, leading to accumulation of dysfunctional organelles and to dysfunction via secondary mechanisms, such as those induced by reactive oxygen species [83,84]. Another emerging link between bioenergetics and mitochondrial structure comes from studies of the F1F0-ATP synthase [85]. Dimer formation of this enzyme does not influence its activity but does affect cristae structure [85]. Accordingly, ribbons of enzyme dimers are located in domains of high membrane curvature (e.g. cristae tubules and on the sides of flat lamellar cristae) [86], and overexpression of IF1, the natural inhibitor of F1F0-ATP synthase, stimulates both enzyme dimerization and cristae biogenesis [87].
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Box 3. Diseases of mitochondria-shaping proteins Charcot-Marie-Tooth Charcot-Marie-Tooth (CMT) is an inherited peripheral neuropathy that causes progressive deterioration of nerve conduction velocity in peripheral neurons, clinically apparent as muscle atrophy and sensory defects. CMT is a heterogeneous disease caused by demyelination of nerves (CMTI, CMTIII and CMTIV) or axonal degeneration without demyelination (CMTII). Mutations in Mfn2 are associated with most cases of CMT type IIa (CMTIIa) [36]. Molecular mechanisms underlying CMTIIa are not clear. Axonal degeneration could be a consequence of insufficient energy supply caused by improper mitochondria localization. In fact, Mfn2 dysfunction in neurons leads to perinuclear aggregation of mitochondria and a decrease in their axonal transport [82,88]. Alternatively, given the low expression of Mfn1 in these cells, mutated Mfn2 might not be functionally complemented by its Mfn1 homologue [82]. Accordingly, aggregation of mitochondria is not observed in fibroblasts from CMTIIa patients, which express higher levels of Mfn1 [89]. Mutations in GDAP1 are also associated with another type of CMT, type IVa. GDAP1 is an integral protein of the outer mitochondrial membrane expressed in neurons of the peripheral and central nervous system. GDAP1 mutants have heterogeneous influence on mitochondria morphology, ranging from fragmentation to perinuclear aggregation [90]. The mechanism or mechanisms by which mutations in GDAP1 cause CMT remain(s) elusive. Autosomal dominant optic atrophy Autosomal dominant optic atrophy (ADOA) is the most common inherited optic neuropathy and is characterized by mild-to-moderate progressive loss of visual acuity, often accompanied by impaired color perception, central visual field defects and temporal optic disc pallor. ADOA is caused by loss of retinal ganglion (RG) neurons and is associated with Opa1 mutations [91,92]. ADOA is probably caused by
haploinsufficiency [93]; accordingly, decreased Opa1 levels are found in samples collected from patients [94]. However, the relationship between Opa1 levels and RG cell death remains unsolved. Some evidence points to impaired oxidative phosphorylation as a possible underlying mechanism, whereas other data suggest involvement of mitochondrial dynamics and increased susceptibility to apoptosis [94]. Parkinson’s disease Parkinson’s disease (PD) is a neurodegenerative disorder caused by loss of dopaminergic neurons in the midbrain. The phosphatase and tensin homolog-induced kinase 1 (Pink1) and Parkin, two genes involved in the inherited form of PD, have been recently connected to mitochondrial dynamics. Pink1 is a mitochondrial serine/threonine kinase, and Parkin is a cytoplasmic E3 ubiquitin ligase. Null mutants of Pink1 and Parkin in Drosophila melanogaster display mitochondrial dysfunction and PD-like symptoms [95]. Of note, Parkin complements loss of Pink1 [96]. Considerable interest was stirred by the finding that induction of mitochondrial fission or attenuation of fusion ameliorates the inherited PD in these fly models [97], suggesting that Pink1 and Parkin might regulate mitochondrial shape. However, the main question is whether the effects of Pink1 and Parkin on mitochondrial morphology are direct or epiphenomena of a primary function; for example, in the regulation of mitochondrial (dys)function and degradation by autophagy. So far, the available evidence supports the latter hypothesis. Loss of Pink1 is associated with defects in the mitochondrial respiratory chain [98,99] and Parkin targets dysfunctional mitochondria to autophagosomes, where they are degraded [100]. Thus, fragmentation observed in Pink1-deficient cells might well be a consequence of organelle dysfunction, and the finding that induction of fusion worsens the phenotype could be a consequence of impaired mitophagy that obviously requires organelles of discrete size to be targeted to the autophagosomes.
ing and lateral tension, which in vivo might be provided by pulling the organelle along microtubules, its intracellular tracks [25,26]. It should be noted that Drp1 differs from conventional dynamins because it does not possess a lipidbinding domain, perhaps explaining its requirement for a membrane receptor such as Fis1 (and perhaps others that are yet unknown). Indeed, fission sites are often characterized by high Fis1 concentrations [27].
Figure 1. A cartoon depicting some of the players involved in fission of mammalian mitochondria. The site of fission, represented by the ring around a constricted mitochondrion, is boxed and enlarged. Drp1, dynamin-related protein 1; IMM, inner mitochondrial membrane; Mff, mitochondrial fission factor; Mtp18, mitochondrial protein of 18 kDa; OMM, outer mitochondrial membrane.
Drp1 assembles in spirals around the fission sites, constricting them in a GTP-dependent manner [24]. However, the earlier model of action of prototypical dynamins has been challenged recently by the discovery that fission depends on the concerted action of dynamin and of mechanical forces acting on the membrane. This novel model postulates that fission is executed not only by the constricting force generated by dynamin but also by lipid remodel-
Fusion Fusion of the OMM is governed by two dynamin-related GTPases: mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2). They display a high degree of homology and, accordingly, a similar structure, composed of a terminal GTPase domain, two hydrophobic heptad repeats (HR) and two transmembrane domains connected by a very short intramembrane space region (only five residues) [28]. Nevertheless, they are not functionally equal. The GTPase activity of Mfn1 is much higher, although its affinity for GTP is lower, than that of Mfn2 [29]. Thus, they seem to have slightly different roles in mitochondrial fusion. Mfn1 is responsible for mitochondria tethering by antiparallel interaction of HR2 of proteins from adjacent mitochondria [30]. The role of Mfn2 is somewhat elusive, but this protein can be retrieved in hetero-oligomers with Mfn1 and perhaps participates in later steps of mitochondrial fusion. Overexpression of an inactive mutant of Mfn2 leads to aggregation of mitochondria, as if their fusion was stalled at the step of initial tethering [31,32]. In addition, Mfn2 levels correlate with oxidative metabolism of skeletal muscle [33] and the proliferative ability of vascular smooth muscle cells by 289
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Figure 2. A cartoon depicting some of the players involved in fusion of mammalian mitochondria. The inner (IMM) and outer (OMM) membranes of a mitochondrion are depicted with the main players in mitochondrial fusion and regulation of the shape of cristae. The OMM of an adjacent fusing mitochondrion is also represented. Mfn, mitofusin; Mib, mitofusins-binding protein; Opa1, optic atrophy 1.
sequestering the proto-oncogene Ras [34]. Mfn2 also controls the shape of the endoplasmic reticulum ER and tethers it to mitochondria [35]. Finally, Mfn2 mutations are associated with Charcot-Marie-Tooth type IIa peripheral neuropathy [36] (Box 3). Thus, it seems that Mfn2 has a broader spectrum of functions than Mfn1. However, at least in fibroblasts, Mfn1 (but not Mfn2) is required for fusion triggered by the inner membrane dynamin-related protein Opa1 [37], further complicating the picture. Mfns communicate with Opa1, probably through the small region exposed to the IMS; a single mutation in this region compromises their pro-fusion ability [38]. Opa1 belongs to the family of dynamin-related proteins. It is anchored to the inner mitochondrial membrane (IMM) by a transmembrane domain located close to the N terminus, and most of the protein is exposed to the IMS. In humans, there are eight splice variants of Opa1, whereas in mice, there are only four [39]. All alternatively spliced exons are located between the GTPase domain and the N terminus, close to the region inserted in the IMM. Alternatively spliced Opa1 isoforms are subject to a complex post-translational cleavage, represented experimentally by five bands on a Western blot. Two higher molecular weight MW bands represent proteins integrated into the IMM, whereas three lower MW bands reflect forms that can be released into the IMS [40]. Controversies remain on how the cleavage is exerted, but a unifying model has been proposed in which the m-AAA protease paraplegin or AFG3L2 [40,41] or the i-AAA protease Yme1L [42,43] produce the lower MW forms of Opa1, on which the rhomboid protease Parl acts to release soluble Opa1 to the IMS space [44]. 290
Two functions of Opa1 have been defined thus far. Opa1 drives Mfn1-dependent fusion of mitochondria [37], perhaps contacting Mfn1 directly through its short IMS domain [38]. Fusion requires both long and short forms of Opa1 [43], suggesting that different forms of Opa1 govern mitochondrial fusion. Opa1 is also crucial for IMM structure. In fact, dynamics of IMM seem to depend exclusively on Opa1. Its downregulation causes the appearance of vacuolar cristae and widening of cristae junctions [45,46], owing to disruption of a complex comprising IMM and IMS forms of the protein [44,46] (Figure 2). Other proteins regulating mitochondrial dynamics Besides the canonical mitochondria-shaping proteins, other factors have been suggested to regulate the morphology of the organelle. Overexpression of mitochondrial protein 18kDa (Mtp18) causes Drp1-dependent fragmentation, and its knockdown leads to highly interconnected mitochondria [47]. It has been proposed, therefore, that Mtp18 acts downstream of Drp1-Fis1, transmitting a division signal to the IMM [47,48]. Mtp18 has an antiapoptotic function; its silencing leads to cytochrome c release and cell death and sensitizes cells to apoptotic stimuli. Knockdown of Mtp18 is a rare example of increased susceptibility to apoptosis associated with highly interconnected mitochondria. Endophilin B1 is a cytoplasmic protein involved in regulating membrane curvature that partially colocalizes with mitochondria. Its downregulation causes dissociation of the OMM from the IMM, resulting in Drp1-dependent formation of OMM tubules and vesicles [49]. It was also
Review implicated in apoptotic signaling because it translocates to mitochondria, where it mediates Bcl-2-associated X protein (Bax) activation, during cell death [50]. However, it is unclear whether membrane shaping by endophilin B1 is necessary for Bax activation [51]. Mitofusin-binding protein (Mib) is a cytoplasmic protein identified through its interaction with Mfn1. It is postulated that Mib inhibits mitochondrial fusion by directly interfering with Mfn1 and possibly with Mfn2 [52]. Screens for mitochondrial morphology defects in Drosophila melanogaster brought to light another candidate component of the fission machinery. Mitochondrial fission factor, the human homolog of the identified protein, is an integral protein of the OMM, and its knockdown induces mitochondrial elongation and partly protects from fragmentation caused by uncoupler or dominant-negative Mfn1, even though it does not interact with Drp1 or hFis1 [53]. In addition to protein factors, the lipid milieu is also involved in the control of mitochondrial dynamics. Phosphatidic acid, a fusogenic lipid produced in OMM from cardiolipin by phospholipase D, is required for proper fusion of mitochondria mediated by Mfns [54]. Moreover, recruitment of inositol 50 -phosphatase synaptojanin 2A, another enzyme responsible for lipid remodeling, to mitochondria causes mitochondria aggregation [55]. Apoptosis and changes in mitochondrial shape As we mentioned earlier, several reports indicate that mitochondrial shape varies during apoptosis. However, whether these changes represent an epiphenomenon of the cell death cascade or play a key part in the amplification of this process remains the subject of much debate. We next review evidence implicating mitochondria-shaping proteins in cell death and the proposed mechanisms by which they potentially influence apoptosis. The outer side of death: mitochondrial fragmentation The original study associating mitochondrial fragmentation with neuronal apoptosis [3] was further substantiated by the discovery that when fragmentation is blocked by a dominant-negative mutant of Drp1, progression towards cell death slows [4]. It has to be stressed that although mechanisms of apoptosis and mitochondrial fission overlap to some extent, they are not inevitably connected. For example, in Caenorhabditis elegans, mitochondria fission is executed by full length DRP-1, whereas apoptosis is amplified by processing of DRP-1 by the caspase CED-3 [56]. Drp1 forms foci in the mitochondrial membrane, ultimately colocalizing with scission sites. Interestingly, upon apoptotic stimulation, at least two other proteins colocalize at these foci: Mfn2 and Bax [27]. Our understanding of this remains elusive, but several lines of evidence suggest that foci serve as platforms for crosstalk between pro- and anti-apoptotic, as well as fusion and fission, molecules. For example, formation of fission sites could be a requisite for Bax translocation to mitochondria [57]. In line with this, overexpression of Bcl-xL prevents apoptosis but not fragmentation [58]. Drp1 has been reported to act downstream of Bax because its silencing has no effect on Bax translocation [6,27]. This is corrobo-
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rated by Drp1 ‘selectivity’ (i.e. Drp1 controls the release of cytochrome c from mitochondria but does not control the release of Smac/DIABLO) [59]. Under non-apoptotic conditions, the majority of Drp1 cycles between the cytoplasm and mitochondria, and the remaining 20% is stably bound to mitochondria; Bax translocation increases the percentage of Drp1 that is stably bound to mitochondria. Interestingly, accumulation of Drp1 on the mitochondrial surface occurs between completion of fission and initiation of cytochrome c release [18]. This biphasic behavior suggests that although fission and cytochrome c release are dependent on the same protein and occur very close in time, they might be distinct events. Dissociation between pro-fission and pro-apoptotic functions of Drp1 was also observed in experiments with a novel inhibitor of Drp1, which attenuated permeabilization of the OMM in isolated mitochondria, even though fission did not take place in this system [60]. Several post-translational modifications of Drp1 (including phosphorylation, ubiquitination and SUMOylation) influence its cellular localization. Phosphorylation of Ser637 by protein kinase A prevents translocation of Drp1 to mitochondria and Drp1dependent fission [14], ultimately slowing apoptosis [13]. In response to a sustained increase in cytosolic Ca2+, Ser637 is dephosphorylated by calcineurin and Drp1 translocates to mitochondria [12]. SUMOylation of Drp1 controls Drp1 levels and mitochondrial localization, as well as mitochondrial fragmentation [17,18]. Drp1 can also be ubiquitinated by membrane-associated RING-CH 5, which probably promotes translocation of Drp1 to mitochondria [61–63]. The balance between these post-translational modifications can ultimately determine the morphology of the organelle. Mfn2 is another proposed partner of Bax in the OMM. Like Drp1, it colocalizes with Bax in foci [27]. Interestingly, point mutants that favor the GTP-bound form display a homogenous distribution in the OMM [38], wheras inactivation of the GTPase domain blocks Mfn2 from localizing to foci [64]. In addition, Bax translocation is blocked when Mfn2 is restricted outside foci and, similarly, Mfn2 spreads homogenously in the OMM in Bax/Bcl-2 homologous antagonist killer (Bak) deficient cells [38,64]. Overexpression of wild-type Mfn2, localized in foci, triggers apoptosis [65]. In contrast, constitutively active mutants of Mfn2 do not differ from wild-type Mfn2 in their influence on mitochondrial shape [66]. Thus, localization of Mfn2 in foci is important for Bax-mediated permeabilization of the OMM. An intriguing possibility is that Mfn2-enriched foci represent Mfn2-mediated tethering between ER and mitochondria [35], where lipids required for Bax-mediated permeabilization of the OMM [67] can be readily transferred to mitochondria [68]. Mfn2 not only participates in Bax-mediated permeabilization of the OMM but also interferes with complex survival signaling cascades, especially in cells of muscular lineage [34]. However, involvement of Mfn2 in ER–mitochondria tethering and in the regulation of survival signaling cascades seems to be independent from its mitochondrial localization and its effect on dynamics of the organelle [34,69], further stressing the multifaceted character of this protein. One would expect, given the interaction between Fis1 and Drp1 in mitochondrial fission, that this same interaction occurs in apoptosis. However, it seems that cell 291
Review death evokes Fis1 overexpression independent of Drp1 [20]. This concept is strengthened by the fact that apoptosis induced by Fis1 does not involve Bax or Bak activation but is instead a Ca2+-dependent process involving events in the IMM. This is substantiated by the ability of scavengers of reactive oxygen species and of mutations in the short domain of Fis1 in the IMS to block Fis1-induced apoptosis [70]. Accordingly, Fis1 promotes rearrangements in IMM structure [21], suggesting crosstalk between pathways of mitochondrial fission and changes in the ultrastructure of mitochondria during cell death. The inner side of death: cristae remodeling Under basal conditions, the majority of cytochrome c is located in the cristae where the complexes of the respiratory chain reside. To enable its complete release during apoptosis, cytochrome c is redistributed to the peripheral IMS in a process called cristae remodeling, characterized by fusion of individual cristae and widening of the narrow cristae junctions [5]. Remodeling of the cristae occurs in response to a variety of apoptotic stimuli, including members of the Bcl-2 family such as Bid [5], Bim [71] and Bik [72]. In the case of Bik, however, the effect seems indirect and mediated by Drp1dependent mitochondrial fission, thereby linking the outer to the inner side of mitochondrial shape changes during cell death. It has been questioned whether changes in the shape of the IMM are a late event following caspase activation or a required step for complete cytochrome c release [73]. In accordance with its proposed function in cristae biogenesis, Opa1 is the master regulator of cristae shape and, therefore, of the rate and extent of cytochrome c release during apoptosis [44,46,71]. This function depends on its GTPase activity and on the formation of an oligomer that comprises both the membrane-bound and soluble forms of the protein [46]. The rhomboid protease Parl has a crucial role in the proper assembly of the oligomer, which is destabilized early in the course of apoptosis, before the release of cytochrome c. In cells lacking Parl, oligomerization of Opa1 is impaired, resulting in a higher sensitivity to apoptosis [44]. Thus, evidence supports the latter hypothesis that cristae remodeling is associated with disassembly of the Opa1-oligomer and required for cytochrome c release and the progression of the apoptotic cascade. However, a number of questions remain: how is cristae remodeling triggered? What are the functional consequences of this remodeling on mitochondrial function? Can the Opa1-dependent arm be exploited to devise novel therapeutics to increase apoptosis in cancer cells? Future perspectives Extensive research on the role of mitochondrial dynamics in cell death has led to the consensus that mitochondrial shape changes occur during apoptosis. However, given the multiple roles of mitochondria-shaping proteins in regulating neuronal differentiation and dendrogenesis, lymphocyte migration, and aging, genetic tools to conditionally ablate or overexpress mitochondrial fusion/fission players remain an outstanding requirement to address the functional significance of these shaping proteins. 292
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Trends in Endocrinology and Metabolism Vol.20 No.6 91 Alexander, C. et al. (2000) OPA1, encoding a dynamin-related GTPase, is mutated in autosomal dominant optic atrophy linked to chromosome 3q28. Nat. Genet. 26, 211–215 92 Delettre, C. et al. (2000) Nuclear gene OPA1, encoding a mitochondrial dynamin-related protein, is mutated in dominant optic atrophy. Nat. Genet. 26, 207–210 93 Davies, V.J. et al. (2007) Opa1 deficiency in a mouse model of autosomal dominant optic atrophy impairs mitochondrial morphology, optic nerve structure and visual function. Hum. Mol. Genet. 16, 1307–1318 94 Spinazzi, M. et al. (2008) A novel deletion in the GTPase domain of OPA1 causes defects in mitochondrial morphology and distribution, but not in function. Hum. Mol. Genet. 17, 3291–3302 95 Clark, I.E. et al. (2006) Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. Nature 441, 1162– 1166 96 Park, J. et al. (2006) Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin. Nature 441, 1157– 1161 97 Poole, A.C. et al. (2008) The PINK1/Parkin pathway regulates mitochondrial morphology. Proc. Natl. Acad. Sci. U. S. A. 105, 1638–1643 98 Gautier, C.A. et al. (2008) Loss of PINK1 causes mitochondrial functional defects and increased sensitivity to oxidative stress. Proc. Natl. Acad. Sci. U. S. A. 105, 11364–11369 99 Morais, V.A. et al. (2009) Parkinson’s disease mutations in PINK1 result in decreased Complex I activity and deficient synaptic function. EMBO Mol Med 1, 1–14 100 Narendra, D. et al. (2008) Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J. Cell Biol. 183, 795–803
The changing shape of mitochondrial apoptosis Michał Wasilewski1 and Luca Scorrano1,2 1 2
Dulbecco-Telethon Institute, Venetian Institute of Molecular Medicine, Via Orus 2, 35129 Padova, Italy Department of Cell Physiology and Metabolism, University of Geneva Medical School, 1 Rue M. Servet, 1211 Gene`ve, Switzerland
Mitochondria are key organelles in conversion of energy, regulation of cellular signaling and amplification of programmed cell death. The anatomy of the organelle matches this functional versatility in complexity and is modulated by the concerted action of proteins that impinge on its fusion–fission equilibrium. A growing body of evidence implicates changes in mitochondrial shape in the progression of apoptosis and, therefore, proteins governing such changes are likely candidates for involvement in pathogenetic mechanisms in neurodegeneration and cancer. Here, we discuss the recent advancements in our knowledge about the machinery that regulates mitochondrial shape and on the role of molecular mechanisms controlling mitochondrial morphology during cell death. Mitochondrial shape Mitochondria are essential organelles for the life and death of a cell and participate in energy conversion, regulation of signaling cascades and apoptosis [1] (Box 1). Although the conventional image of these organelles is of a static nature, mitochondria can fuse and divide. This ability has been known for years but obscured by the fine yet static details provided by electron microscopy. The advanced tools of live-cell imaging and 3D reconstruction, as well as electron tomography, have enabled us to fully appreciate their complexity and dynamic nature. The spectrum of mitochondrial shapes ranges from spherical, grain-like, individual entities to long, branched filaments that, ultimately, might form one interconnected ‘megamitochondrion’ per cell. Mitochondrial complexity begins at the level of their external appearance and continues to a similarly complex and dynamic internal structure [2]. A major leap forward in understanding the mechanisms and consequences of mitochondrial fusion–fission came with the discovery that the morphology of the organelle, like its ultrastructure, undergoes dynamic changes during apoptosis. Interfering with these changes slows apoptotic progression, indicating a functional role for mitochondrial shape in this process [3–5]. This finding instigated research on the molecular details, mechanisms and pathophysiological consequences of mitochondrial shape changes (Box 2). Here, we review the current understanding of mitochondrial fusion and fission in mammals and discuss evidence that supports a role for morphological changes of mitochondria in certain physiological and pathological processes, including apoptosis (Box 3). Corresponding author: Scorrano, L. ([email protected]).
Mitochondrial fusion and fission Mitochondrial shape results from the balance of two ongoing antagonistic processes, fusion and fission. When either process is blocked, the final morphology of the mitochondria is the consequence of unopposed progression towards the other side of the equilibrium [6]. Members of the machinery regulating mitochondrial fusion have been identified by analyzing organelle morphology in Saccharomices cerevisiae mutants [7,8]. The shape of mitochondria depends on several large ubiquitous GTPases with structural homology to dynamins [9] that participate in fusion, fission and tubulation of biological membranes. Proteins that regulate mitochondrial shape share at least the GTPase domain and a C-terminal coiled-coil domain with the prototypical dynamins, and these domains can function as GTPase effectors or to mediate protein–protein interaction [10]. The dynamin-related proteins that impinge on mitochondrial fusion are peculiar in that most of them are integral membrane proteins. We next discuss the properties of ‘mitochondria-shaping’ proteins of mammals in particular. A scheme of the players known to participate in mitochondrial fission and fusion in mammalian cells is presented in Figure 1 and 2, respectively. Fission In mammalian cells, mitochondrial fission depends on dynamin-related protein 1 (Drp1), a cytoplasmic large GTPase similar to dynamin that mediates the fragmentation of mitochondria and peroxisomes [11]. Drp1 translocates to mitochondria in response to cellular and mitochondrial cues. After mitochondrial dysfunction, cytoplasmic Ca2+ rises, leading to activation of calcineurin and dephosphorylation of the conserved Ser637 of Drp1 [12], inducing translocation of Drp1 to mitochondria. Conflicting data exists surrounding the kinase responsible for phosphorylation of this residue. Protein kinase A was reported to fulfill this function, linking mitochondrial morphology to another crucial second messenger, cyclic AMP [13,14]. Alternatively, Ser637 could be phosphorylated by calmodulin-dependent protein kinase Ia, although in this case, phosphorylation of Drp1 induced its mitochondrial localization [15]. The phosphorylation status of this site is dominant over that of Ser616 [12], which is controlled by cyclin-dependent kinase 1 to drive mitochondrial fission during mitosis [16]. Mitochondrial Drp1 can then be stabilized on the surface of the organelle by SUMOylation [17,18], a process known to protect molecules from degradation by the ubiquitin-proteasome system.
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Box 1. Mitochondria Mitochondria are double-membrane organelles, the origin of which has been traced to an ancient prokaryotic endosymbiont. The outer mitochondrial membrane (OMM) has a composition similar to that of other eukaryotic membranes and originates from a vesicle engulfing the endosymbiont. It is permeable to metabolites and small peptides up to 3000 Da, thanks to the presence of voltage-dependent anion channels. The inner mitochondrial membrane (IMM) has a clearer prokaryotic origin and differs from the OMM in its physical properties and composition. Its ion conductance and metabolite permeability are tightly controlled. IMM comprises a larger fraction of proteins and approximately 20% of cardiolipin, a lipid also found in bacterial membranes, which is crucial for proper activity of several IMM enzymes. IMM folds into a complicated network of tubules and lamellae called cristae, reflecting its unique function. IMM is the site of oxidative phosphorylation (OXPHOS), which provides most of the ATP produced during aerobic metabolism. According to the chemiosmotic theory, the basic principle of OXPHOS is to pump protons across the proton-impermeable IMM, generating an electrochemical potential, which is then used to drive phosphorylation of ADP to ATP by F1F0-ATPase synthase. Energy required for proton pumping is provided by electrons from NADH and FADH produced during the tricarboxylic acid cycle and other catabolic processes. The energy of
Fis1 is a membrane protein homogenously distributed in the outer mitochondrial membrane (OMM) via a transmembrane domain located in the C-terminal region, and only a small portion of the molecule protrudes into the intermembrane space (IMS) (Figure 1). The cytoplasmic region contains six alpha helices, four of which (a2–a5) form two tetratricopeptide-repeat (TPR)-like domains, predicted to allow protein–protein interactions [19]. Overexpression of Fis1 induces mitochondrial fragmentation, but because it does not possess enzymatic activity, its role is probably restricted to anchoring effector proteins to mitochondria. Accordingly, mitochondrial fragmentation caused by Fis1 overexpression can be blocked by expression of a dominant-negative mutant of Drp1 [20]. Moreover, Drp1 and Fis1 seem to interact, as judged by crosslinking and coimmunoprecipitation [21]. The Drp1– Fis interaction is a transient event, and efficient fission
electrons, liberated in a number of redox reactions, is translated into proton pumping by three large protein complexes in IMM (complex I, III and IV), called together with the non-proton-pumping complex II, the respiratory chain (RC) [1]. Mitochondria contain an autonomous circular small genome that is located in the internal compartment, the matrix. mtDNA encodes only 13 proteins, mitochondrial ribosomal RNA and transfer RNA, but mutations in these genes result in severe diseases, mostly because of impaired energy production. Apart from OXPHOS, mitochondria participate in other important metabolic pathways, such as fatty acid synthesis, gluconeogenesis, steroidogenesis, synthesis of haeme and urea cycle [2]. Moreover, mitochondria cooperate with the ER to control calcium homeostasis and integrate information during apoptosis. The balance between pro- and antiapoptotic proteins of the Bcl-2 family controls release of cytochrome c, apoptosis-induction factor, Smac/DIABLO and endonuclease G from mitochondria. Sequestered in mitochondria, these proteins carry out routine tasks (e.g. cytochrome c shuttles electrons in RC). When released to the cytoplasm, they function as proapoptotic factors, either by inducing apoptosome formation (cytochrome c) or by a direct enzymatic activity (endonuclease G) [74].
requires dissociation of the complex. Accordingly, genetic modifications of the N-terminal domain of Fis1, which stabilize its binding to Drp1, abolish mitochondrial fragmentation [22]. However, the absolute requirement of Fis1 for binding Drp1 to mitochondria is somewhat weakened by the observation that downregulation of Fis1 only partially diminishes Drp1 recruitment to mitochondria [6]. Another question on the mechanism of interaction between Fis1 and Drp1 arises from the yeast model, in which this interaction requires the adaptor Mdv1 [23]. A mammalian orthologue for Mdv1 has not been yet identified, but considering the conservation of fission mechanisms, additional elements could be involved. Mechanism of fission By comparing Drp1 with the prototypical dynamins and considering data obtained in yeast, it was postulated that
Box 2. Mitochondrial dynamics and bioenergetics One of the most prominent functions of mitochondria is ATP production by oxidative phosphorylation. Research on the relationship between mitochondrial shape and function dates back to 1966, when Hackenbrock [75] observed that mitochondria isolated from rat livers shrunk and displayed a denser matrix during respiration. This conformation was called ‘condensed’, as opposed to the ‘orthodox’ conformation of inactive mitochondria. The matrix conformation transition was ascribed to osmotic changes caused by K+ movements across the inner mitochondrial membrane and thought to be only an artefact of isolated mitochondria. Indeed, mitochondria observed in most mammalian tissues manifest orthodox conformation [76,77]. Nevertheless, mitochondria from muscles with high energy requirements seem to have condensed matrix [78]. Moreover, matrix condensation is observed during apoptosis [79]. Overall, mitochondrial morphology depends on the energetic state of the cell. In general, treatments that compromise mitochondrial membrane potential induce mitochondrial fragmentation [12], an observation that correlates well with data from other organisms. Fusion of the outer membrane in yeast, for example, requires a chemical component of electrochemical potential, and fusion of the inner membrane depends on an electrical component [80]. Interestingly, recovery of mitochondrial morphology after wash-out of uncouplers requires de novo protein synthesis, suggesting a defect
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in mitochondrial fusion [81] probably caused by Opa1 degradation [42]. It is tempting to speculate that decreased potential blocks fusion by Opa1 degradation and ultimately leads to mitochondrial fragmentation by activating the fission machinery [12]. In turn, the bioenergetic state of a cell can be modified by changing the levels of mitochondria-shaping proteins. Loss of fusion and resulting mitochondrial fragmentation can cause mitochondrial dysfunction, perhaps through the impaired exchange of matrix material in fusion-incompetent mitochondria [82] and consequent accumulation of stochastic functional errors. It is more difficult to explain why failure to divide results in mitochondrial dysfunction [83]. Blockage of fission could impair elimination of defective elements of the network by autophagy, leading to accumulation of dysfunctional organelles and to dysfunction via secondary mechanisms, such as those induced by reactive oxygen species [83,84]. Another emerging link between bioenergetics and mitochondrial structure comes from studies of the F1F0-ATP synthase [85]. Dimer formation of this enzyme does not influence its activity but does affect cristae structure [85]. Accordingly, ribbons of enzyme dimers are located in domains of high membrane curvature (e.g. cristae tubules and on the sides of flat lamellar cristae) [86], and overexpression of IF1, the natural inhibitor of F1F0-ATP synthase, stimulates both enzyme dimerization and cristae biogenesis [87].
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Box 3. Diseases of mitochondria-shaping proteins Charcot-Marie-Tooth Charcot-Marie-Tooth (CMT) is an inherited peripheral neuropathy that causes progressive deterioration of nerve conduction velocity in peripheral neurons, clinically apparent as muscle atrophy and sensory defects. CMT is a heterogeneous disease caused by demyelination of nerves (CMTI, CMTIII and CMTIV) or axonal degeneration without demyelination (CMTII). Mutations in Mfn2 are associated with most cases of CMT type IIa (CMTIIa) [36]. Molecular mechanisms underlying CMTIIa are not clear. Axonal degeneration could be a consequence of insufficient energy supply caused by improper mitochondria localization. In fact, Mfn2 dysfunction in neurons leads to perinuclear aggregation of mitochondria and a decrease in their axonal transport [82,88]. Alternatively, given the low expression of Mfn1 in these cells, mutated Mfn2 might not be functionally complemented by its Mfn1 homologue [82]. Accordingly, aggregation of mitochondria is not observed in fibroblasts from CMTIIa patients, which express higher levels of Mfn1 [89]. Mutations in GDAP1 are also associated with another type of CMT, type IVa. GDAP1 is an integral protein of the outer mitochondrial membrane expressed in neurons of the peripheral and central nervous system. GDAP1 mutants have heterogeneous influence on mitochondria morphology, ranging from fragmentation to perinuclear aggregation [90]. The mechanism or mechanisms by which mutations in GDAP1 cause CMT remain(s) elusive. Autosomal dominant optic atrophy Autosomal dominant optic atrophy (ADOA) is the most common inherited optic neuropathy and is characterized by mild-to-moderate progressive loss of visual acuity, often accompanied by impaired color perception, central visual field defects and temporal optic disc pallor. ADOA is caused by loss of retinal ganglion (RG) neurons and is associated with Opa1 mutations [91,92]. ADOA is probably caused by
haploinsufficiency [93]; accordingly, decreased Opa1 levels are found in samples collected from patients [94]. However, the relationship between Opa1 levels and RG cell death remains unsolved. Some evidence points to impaired oxidative phosphorylation as a possible underlying mechanism, whereas other data suggest involvement of mitochondrial dynamics and increased susceptibility to apoptosis [94]. Parkinson’s disease Parkinson’s disease (PD) is a neurodegenerative disorder caused by loss of dopaminergic neurons in the midbrain. The phosphatase and tensin homolog-induced kinase 1 (Pink1) and Parkin, two genes involved in the inherited form of PD, have been recently connected to mitochondrial dynamics. Pink1 is a mitochondrial serine/threonine kinase, and Parkin is a cytoplasmic E3 ubiquitin ligase. Null mutants of Pink1 and Parkin in Drosophila melanogaster display mitochondrial dysfunction and PD-like symptoms [95]. Of note, Parkin complements loss of Pink1 [96]. Considerable interest was stirred by the finding that induction of mitochondrial fission or attenuation of fusion ameliorates the inherited PD in these fly models [97], suggesting that Pink1 and Parkin might regulate mitochondrial shape. However, the main question is whether the effects of Pink1 and Parkin on mitochondrial morphology are direct or epiphenomena of a primary function; for example, in the regulation of mitochondrial (dys)function and degradation by autophagy. So far, the available evidence supports the latter hypothesis. Loss of Pink1 is associated with defects in the mitochondrial respiratory chain [98,99] and Parkin targets dysfunctional mitochondria to autophagosomes, where they are degraded [100]. Thus, fragmentation observed in Pink1-deficient cells might well be a consequence of organelle dysfunction, and the finding that induction of fusion worsens the phenotype could be a consequence of impaired mitophagy that obviously requires organelles of discrete size to be targeted to the autophagosomes.
ing and lateral tension, which in vivo might be provided by pulling the organelle along microtubules, its intracellular tracks [25,26]. It should be noted that Drp1 differs from conventional dynamins because it does not possess a lipidbinding domain, perhaps explaining its requirement for a membrane receptor such as Fis1 (and perhaps others that are yet unknown). Indeed, fission sites are often characterized by high Fis1 concentrations [27].
Figure 1. A cartoon depicting some of the players involved in fission of mammalian mitochondria. The site of fission, represented by the ring around a constricted mitochondrion, is boxed and enlarged. Drp1, dynamin-related protein 1; IMM, inner mitochondrial membrane; Mff, mitochondrial fission factor; Mtp18, mitochondrial protein of 18 kDa; OMM, outer mitochondrial membrane.
Drp1 assembles in spirals around the fission sites, constricting them in a GTP-dependent manner [24]. However, the earlier model of action of prototypical dynamins has been challenged recently by the discovery that fission depends on the concerted action of dynamin and of mechanical forces acting on the membrane. This novel model postulates that fission is executed not only by the constricting force generated by dynamin but also by lipid remodel-
Fusion Fusion of the OMM is governed by two dynamin-related GTPases: mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2). They display a high degree of homology and, accordingly, a similar structure, composed of a terminal GTPase domain, two hydrophobic heptad repeats (HR) and two transmembrane domains connected by a very short intramembrane space region (only five residues) [28]. Nevertheless, they are not functionally equal. The GTPase activity of Mfn1 is much higher, although its affinity for GTP is lower, than that of Mfn2 [29]. Thus, they seem to have slightly different roles in mitochondrial fusion. Mfn1 is responsible for mitochondria tethering by antiparallel interaction of HR2 of proteins from adjacent mitochondria [30]. The role of Mfn2 is somewhat elusive, but this protein can be retrieved in hetero-oligomers with Mfn1 and perhaps participates in later steps of mitochondrial fusion. Overexpression of an inactive mutant of Mfn2 leads to aggregation of mitochondria, as if their fusion was stalled at the step of initial tethering [31,32]. In addition, Mfn2 levels correlate with oxidative metabolism of skeletal muscle [33] and the proliferative ability of vascular smooth muscle cells by 289
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Figure 2. A cartoon depicting some of the players involved in fusion of mammalian mitochondria. The inner (IMM) and outer (OMM) membranes of a mitochondrion are depicted with the main players in mitochondrial fusion and regulation of the shape of cristae. The OMM of an adjacent fusing mitochondrion is also represented. Mfn, mitofusin; Mib, mitofusins-binding protein; Opa1, optic atrophy 1.
sequestering the proto-oncogene Ras [34]. Mfn2 also controls the shape of the endoplasmic reticulum ER and tethers it to mitochondria [35]. Finally, Mfn2 mutations are associated with Charcot-Marie-Tooth type IIa peripheral neuropathy [36] (Box 3). Thus, it seems that Mfn2 has a broader spectrum of functions than Mfn1. However, at least in fibroblasts, Mfn1 (but not Mfn2) is required for fusion triggered by the inner membrane dynamin-related protein Opa1 [37], further complicating the picture. Mfns communicate with Opa1, probably through the small region exposed to the IMS; a single mutation in this region compromises their pro-fusion ability [38]. Opa1 belongs to the family of dynamin-related proteins. It is anchored to the inner mitochondrial membrane (IMM) by a transmembrane domain located close to the N terminus, and most of the protein is exposed to the IMS. In humans, there are eight splice variants of Opa1, whereas in mice, there are only four [39]. All alternatively spliced exons are located between the GTPase domain and the N terminus, close to the region inserted in the IMM. Alternatively spliced Opa1 isoforms are subject to a complex post-translational cleavage, represented experimentally by five bands on a Western blot. Two higher molecular weight MW bands represent proteins integrated into the IMM, whereas three lower MW bands reflect forms that can be released into the IMS [40]. Controversies remain on how the cleavage is exerted, but a unifying model has been proposed in which the m-AAA protease paraplegin or AFG3L2 [40,41] or the i-AAA protease Yme1L [42,43] produce the lower MW forms of Opa1, on which the rhomboid protease Parl acts to release soluble Opa1 to the IMS space [44]. 290
Two functions of Opa1 have been defined thus far. Opa1 drives Mfn1-dependent fusion of mitochondria [37], perhaps contacting Mfn1 directly through its short IMS domain [38]. Fusion requires both long and short forms of Opa1 [43], suggesting that different forms of Opa1 govern mitochondrial fusion. Opa1 is also crucial for IMM structure. In fact, dynamics of IMM seem to depend exclusively on Opa1. Its downregulation causes the appearance of vacuolar cristae and widening of cristae junctions [45,46], owing to disruption of a complex comprising IMM and IMS forms of the protein [44,46] (Figure 2). Other proteins regulating mitochondrial dynamics Besides the canonical mitochondria-shaping proteins, other factors have been suggested to regulate the morphology of the organelle. Overexpression of mitochondrial protein 18kDa (Mtp18) causes Drp1-dependent fragmentation, and its knockdown leads to highly interconnected mitochondria [47]. It has been proposed, therefore, that Mtp18 acts downstream of Drp1-Fis1, transmitting a division signal to the IMM [47,48]. Mtp18 has an antiapoptotic function; its silencing leads to cytochrome c release and cell death and sensitizes cells to apoptotic stimuli. Knockdown of Mtp18 is a rare example of increased susceptibility to apoptosis associated with highly interconnected mitochondria. Endophilin B1 is a cytoplasmic protein involved in regulating membrane curvature that partially colocalizes with mitochondria. Its downregulation causes dissociation of the OMM from the IMM, resulting in Drp1-dependent formation of OMM tubules and vesicles [49]. It was also
Review implicated in apoptotic signaling because it translocates to mitochondria, where it mediates Bcl-2-associated X protein (Bax) activation, during cell death [50]. However, it is unclear whether membrane shaping by endophilin B1 is necessary for Bax activation [51]. Mitofusin-binding protein (Mib) is a cytoplasmic protein identified through its interaction with Mfn1. It is postulated that Mib inhibits mitochondrial fusion by directly interfering with Mfn1 and possibly with Mfn2 [52]. Screens for mitochondrial morphology defects in Drosophila melanogaster brought to light another candidate component of the fission machinery. Mitochondrial fission factor, the human homolog of the identified protein, is an integral protein of the OMM, and its knockdown induces mitochondrial elongation and partly protects from fragmentation caused by uncoupler or dominant-negative Mfn1, even though it does not interact with Drp1 or hFis1 [53]. In addition to protein factors, the lipid milieu is also involved in the control of mitochondrial dynamics. Phosphatidic acid, a fusogenic lipid produced in OMM from cardiolipin by phospholipase D, is required for proper fusion of mitochondria mediated by Mfns [54]. Moreover, recruitment of inositol 50 -phosphatase synaptojanin 2A, another enzyme responsible for lipid remodeling, to mitochondria causes mitochondria aggregation [55]. Apoptosis and changes in mitochondrial shape As we mentioned earlier, several reports indicate that mitochondrial shape varies during apoptosis. However, whether these changes represent an epiphenomenon of the cell death cascade or play a key part in the amplification of this process remains the subject of much debate. We next review evidence implicating mitochondria-shaping proteins in cell death and the proposed mechanisms by which they potentially influence apoptosis. The outer side of death: mitochondrial fragmentation The original study associating mitochondrial fragmentation with neuronal apoptosis [3] was further substantiated by the discovery that when fragmentation is blocked by a dominant-negative mutant of Drp1, progression towards cell death slows [4]. It has to be stressed that although mechanisms of apoptosis and mitochondrial fission overlap to some extent, they are not inevitably connected. For example, in Caenorhabditis elegans, mitochondria fission is executed by full length DRP-1, whereas apoptosis is amplified by processing of DRP-1 by the caspase CED-3 [56]. Drp1 forms foci in the mitochondrial membrane, ultimately colocalizing with scission sites. Interestingly, upon apoptotic stimulation, at least two other proteins colocalize at these foci: Mfn2 and Bax [27]. Our understanding of this remains elusive, but several lines of evidence suggest that foci serve as platforms for crosstalk between pro- and anti-apoptotic, as well as fusion and fission, molecules. For example, formation of fission sites could be a requisite for Bax translocation to mitochondria [57]. In line with this, overexpression of Bcl-xL prevents apoptosis but not fragmentation [58]. Drp1 has been reported to act downstream of Bax because its silencing has no effect on Bax translocation [6,27]. This is corrobo-
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rated by Drp1 ‘selectivity’ (i.e. Drp1 controls the release of cytochrome c from mitochondria but does not control the release of Smac/DIABLO) [59]. Under non-apoptotic conditions, the majority of Drp1 cycles between the cytoplasm and mitochondria, and the remaining 20% is stably bound to mitochondria; Bax translocation increases the percentage of Drp1 that is stably bound to mitochondria. Interestingly, accumulation of Drp1 on the mitochondrial surface occurs between completion of fission and initiation of cytochrome c release [18]. This biphasic behavior suggests that although fission and cytochrome c release are dependent on the same protein and occur very close in time, they might be distinct events. Dissociation between pro-fission and pro-apoptotic functions of Drp1 was also observed in experiments with a novel inhibitor of Drp1, which attenuated permeabilization of the OMM in isolated mitochondria, even though fission did not take place in this system [60]. Several post-translational modifications of Drp1 (including phosphorylation, ubiquitination and SUMOylation) influence its cellular localization. Phosphorylation of Ser637 by protein kinase A prevents translocation of Drp1 to mitochondria and Drp1dependent fission [14], ultimately slowing apoptosis [13]. In response to a sustained increase in cytosolic Ca2+, Ser637 is dephosphorylated by calcineurin and Drp1 translocates to mitochondria [12]. SUMOylation of Drp1 controls Drp1 levels and mitochondrial localization, as well as mitochondrial fragmentation [17,18]. Drp1 can also be ubiquitinated by membrane-associated RING-CH 5, which probably promotes translocation of Drp1 to mitochondria [61–63]. The balance between these post-translational modifications can ultimately determine the morphology of the organelle. Mfn2 is another proposed partner of Bax in the OMM. Like Drp1, it colocalizes with Bax in foci [27]. Interestingly, point mutants that favor the GTP-bound form display a homogenous distribution in the OMM [38], wheras inactivation of the GTPase domain blocks Mfn2 from localizing to foci [64]. In addition, Bax translocation is blocked when Mfn2 is restricted outside foci and, similarly, Mfn2 spreads homogenously in the OMM in Bax/Bcl-2 homologous antagonist killer (Bak) deficient cells [38,64]. Overexpression of wild-type Mfn2, localized in foci, triggers apoptosis [65]. In contrast, constitutively active mutants of Mfn2 do not differ from wild-type Mfn2 in their influence on mitochondrial shape [66]. Thus, localization of Mfn2 in foci is important for Bax-mediated permeabilization of the OMM. An intriguing possibility is that Mfn2-enriched foci represent Mfn2-mediated tethering between ER and mitochondria [35], where lipids required for Bax-mediated permeabilization of the OMM [67] can be readily transferred to mitochondria [68]. Mfn2 not only participates in Bax-mediated permeabilization of the OMM but also interferes with complex survival signaling cascades, especially in cells of muscular lineage [34]. However, involvement of Mfn2 in ER–mitochondria tethering and in the regulation of survival signaling cascades seems to be independent from its mitochondrial localization and its effect on dynamics of the organelle [34,69], further stressing the multifaceted character of this protein. One would expect, given the interaction between Fis1 and Drp1 in mitochondrial fission, that this same interaction occurs in apoptosis. However, it seems that cell 291
Review death evokes Fis1 overexpression independent of Drp1 [20]. This concept is strengthened by the fact that apoptosis induced by Fis1 does not involve Bax or Bak activation but is instead a Ca2+-dependent process involving events in the IMM. This is substantiated by the ability of scavengers of reactive oxygen species and of mutations in the short domain of Fis1 in the IMS to block Fis1-induced apoptosis [70]. Accordingly, Fis1 promotes rearrangements in IMM structure [21], suggesting crosstalk between pathways of mitochondrial fission and changes in the ultrastructure of mitochondria during cell death. The inner side of death: cristae remodeling Under basal conditions, the majority of cytochrome c is located in the cristae where the complexes of the respiratory chain reside. To enable its complete release during apoptosis, cytochrome c is redistributed to the peripheral IMS in a process called cristae remodeling, characterized by fusion of individual cristae and widening of the narrow cristae junctions [5]. Remodeling of the cristae occurs in response to a variety of apoptotic stimuli, including members of the Bcl-2 family such as Bid [5], Bim [71] and Bik [72]. In the case of Bik, however, the effect seems indirect and mediated by Drp1dependent mitochondrial fission, thereby linking the outer to the inner side of mitochondrial shape changes during cell death. It has been questioned whether changes in the shape of the IMM are a late event following caspase activation or a required step for complete cytochrome c release [73]. In accordance with its proposed function in cristae biogenesis, Opa1 is the master regulator of cristae shape and, therefore, of the rate and extent of cytochrome c release during apoptosis [44,46,71]. This function depends on its GTPase activity and on the formation of an oligomer that comprises both the membrane-bound and soluble forms of the protein [46]. The rhomboid protease Parl has a crucial role in the proper assembly of the oligomer, which is destabilized early in the course of apoptosis, before the release of cytochrome c. In cells lacking Parl, oligomerization of Opa1 is impaired, resulting in a higher sensitivity to apoptosis [44]. Thus, evidence supports the latter hypothesis that cristae remodeling is associated with disassembly of the Opa1-oligomer and required for cytochrome c release and the progression of the apoptotic cascade. However, a number of questions remain: how is cristae remodeling triggered? What are the functional consequences of this remodeling on mitochondrial function? Can the Opa1-dependent arm be exploited to devise novel therapeutics to increase apoptosis in cancer cells? Future perspectives Extensive research on the role of mitochondrial dynamics in cell death has led to the consensus that mitochondrial shape changes occur during apoptosis. However, given the multiple roles of mitochondria-shaping proteins in regulating neuronal differentiation and dendrogenesis, lymphocyte migration, and aging, genetic tools to conditionally ablate or overexpress mitochondrial fusion/fission players remain an outstanding requirement to address the functional significance of these shaping proteins. 292
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