Importance of mitochondrial dynamics during meiosis and sporulation

Opposing fission and fusion events maintain the yeast mitochondrial network. Six proteins regulate these membrane dynamics during mitotic growth-Dnm1p, Mdv1p, and Fis1p mediate fission; Fzo1p, Mgm1p, and Ugo1p mediate fusion. Previous studies established that mitochondria fragment and rejoin at distinct stages during meiosis and sporulation, suggesting that mitochondrial fission and fusion are required during this process. Here we report that strains defective for mitochondrial fission alone, or both fission and fusion, complete meiosis and sporulation. However, visualization of mitochondria in sporulating cultures reveals morphological defects associated with the loss of fusion and/or fission proteins. Specifically, mitochondria collapse to one side of the cell and fail to fragment during presporulation. In addition, mitochondria are not inherited equally by newly formed spores, and mitochondrial DNA nucleoid segregation defects give rise to spores lacking nucleoids. This nucleoid inheritance defect is correlated with an increase in petite spore colonies. Unexpectedly, mitochondria fragment in mature tetrads lacking fission proteins. The latter finding suggests either that novel fission machinery operates during sporulation or that mechanical forces generate the mitochondrial fragments observed in mature spores. These results provide evidence of fitness defects caused by fission mutations and reveal new phenotypes associated with fission and fusion mutations.     

Mitochondrial fission in apoptosis, neurodegeneration and aging

A decline in mitochondrial function is well recognized in neurodegenerative diseases and aging, and is thought to play a causal role in their biology. Unfortunately, the molecular basis underlying this detrimental loss in mitochondrial function remains mysterious. Interestingly, mitochondria undergo frequent fission and fusion. This process is regulated by molecular machinery that has been highly conserved during evolution, including dynamin-related GTPases that manifest opposing effects. A balance between mitochondrial fission and fusion events is required for normal mitochondrial and cellular function. Emerging evidence indicates that mitochondria undergo rapid and extensive fission at an early stage during apoptosis. A clue that these new findings are of significance for the pathogenesis of neurodegenerative disease is provided by the observation that OPA-1, a dynamin-related GTPase regulating mitochondrial fusion, is mutated in humans with dominant optic atrophy, which is characterized by degeneration of retinal ganglion cells and childhood blindness. Loss of function of OPA-1, analogous to deficiency of its yeast homologue, Mgm1p, is expected to lead to mitochondrial fission, loss of mitochondrial DNA, respiratory deficits and an increase in reactive oxygen species. Here we review the molecular mediators controlling mitochondrial fission and fusion, and how death effector molecules may hijack this ancient machinery. A shift in the rate of mitochondrial fission or fusion may provide a new mechanistic explanation for the mitochondrial dysfunction in neurodegenerative diseases and normal aging, and may offer a new target for therapeutic intervention.     

Inhibiting the mitochondrial fission machinery does not prevent Bax/Bak-dependent apoptosis

Apoptosis, induced by a number of death stimuli, is associated with a fragmentation of the mitochondrial network. These morphological changes in mitochondria have been shown to require proteins, such as Drp1 or hFis1, which are involved in regulating the fission of mitochondria. However, the precise role of mitochondrial fission during apoptosis remains elusive. Here we report that inhibiting the fission machinery in Bax/Bak-mediated apoptosis, by down-regulating of Drp1 or hFis1, prevents the fragmentation of the mitochondrial network and partially inhibits the release of cytochrome c from the mitochondria but fails to block the efflux of Smac/DIABLO. In addition, preventing mitochondrial fragmentation does not inhibit cell death induced by Bax/Bak-dependent death stimuli, in contrast to the effects of Bcl-xL or caspase inhibition. Therefore, the fission of mitochondria is a dispensable event in Bax/Bak-dependent apoptosis.     

Tying trafficking to fusion and fission at the mighty mitochondria

The mitochondrion is a unique organelle that serves as the main site of ATP generation needed for energy in the cell. However, mitochondria also play essential roles in cell death through apoptosis and necrosis, as well as a variety of crucial functions related to stress regulation, autophagy, lipid synthesis and calcium storage. There is a growing appreciation that mitochondrial function is regulated by the dynamics of its membrane fusion and fission; longer, fused mitochondria are optimal for ATP generation, whereas fission of mitochondria facilitates mitophagy and cell division. Despite the significance of mitochondrial homeostasis for such crucial cellular events, the intricate regulation of mitochondrial fusion and fission is only partially understood. Until very recently, only a single mitochondrial fission protein had been identified. Moreover, only now have researchers turned to address the upstream machinery that regulates mitochondrial fusion and fission proteins. Herein, we review the known GTPases involved in mitochondrial fusion and fission, but also highlight recent studies that address the mechanisms by which these GTPases are regulated. In particular, we draw attention to a substantial new body of literature linking endocytic regulatory proteins, such as the retromer VPS35 cargo selection complex subunit, to mitochondrial homeostasis. These recent studies suggest that relationships and cross‐regulation between endocytic and mitochondrial pathways may be more widespread than previously assumed.      

Mitochondrial fission is an upstream and required event for bax foci formation in response to nitric oxide in cortical neurons

Mitochondrial dysfunction is an underpinning event in many neurodegenerative disorders. Less clear, however, is how mitochondria become injured during neuronal demise. Nitric oxide (NO) evokes rapid mitochondrial fission in cortical neurons. Interestingly, proapoptotic Bax relocates from the cytoplasm into large foci on mitochondrial scission sites in response to nitrosative stress. Antiapoptotic Bcl-xL does not prevent mitochondrial fission despite its ability to block Bax puncta formation on mitochondria and to mitigate neuronal cell death. Mitofusin 1 (Mfn1) or dominant-negative dynamin-related protein 1(K38A) (Drp1(k38A)) inhibits mitochondrial fission and Bax accumulation on mitochondria induced by exposure to an NO donor. Although NO is known to cause a bioenergetic crisis, lowering ATP by glycolytic or mitochondrial inhibitors neither induces mitochondrial fission nor Bax foci formation on mitochondria. Taken together, these data indicate that the mitochondrial fission machinery acts upstream of the Bcl-2 family of proteins in neurons challenged with nitrosative stress.     

Mitochondrial fusion is regulated by Reaper to modulate Drosophila programmed cell death

In most multicellular organisms, the decision to undergo programmed cell death in response to cellular damage or developmental cues is typically transmitted through mitochondria. It has been suggested that an exception is the apoptotic pathway of Drosophila melanogaster, in which the role of mitochondria remains unclear. Although IAP antagonists in Drosophila such as Reaper, Hid and Grim may induce cell death without mitochondrial membrane permeabilization, it is surprising that all three localize to mitochondria. Moreover, induction of Reaper and Hid appears to result in mitochondrial fragmentation during Drosophila cell death. Most importantly, disruption of mitochondrial fission can inhibit Reaper and Hid-induced cell death, suggesting that alterations in mitochondrial dynamics can modulate cell death in fly cells. We report here that Drosophila Reaper can induce mitochondrial fragmentation by binding to and inhibiting the pro-fusion protein MFN2 and its Drosophila counterpart dMFN/Marf. Our in vitro and in vivo analyses reveal that dMFN overexpression can inhibit cell death induced by Reaper or γ-irradiation. In addition, knockdown of dMFN causes a striking loss of adult wing tissue and significant apoptosis in the developing wing discs. Our findings are consistent with a growing body of work describing a role for mitochondrial fission and fusion machinery in the decision of cells to die.     

The role of mitochondria in apoptosis

Apoptosis (programmed cell death) is a cellular self-destruction mechanism that is essential for a variety of biological events, such as developmental sculpturing, tissue homeostasis, and the removal of unwanted cells. Mitochondria play a crucial role in regulating cell death. Ca2+ has long been recognized as a participant in apoptotic pathways. Mitochondria are known to modulate and synchronize Ca2+ signaling. Massive accumulation of Ca2+ in the mitochondria leads to apoptosis. The Ca2+ dynamics of ER and mitochondria appear to be modulated by the Bcl-2 family proteins, key factors involved in apoptosis. The number and morphology of mitochondria are precisely controlled through mitochondrial fusion and fission process by numerous mitochondria-shaping proteins. Mitochondrial fission accompanies apoptotic cell death and appears to be important for progression of the apoptotic pathway. Here, we highlight and discuss the role of mitochondrial calcium handling and mitochondrial fusion and fission machinery in apoptosis.     

Mitochondrial fission in apoptosis

Mitochondria fuse and divide continuously within cells to form a dynamic network. One of the steps in apoptosis is the fragmentation of mitochondria, and recent evidence indicates that the mitochondrial fission machinery actively participates in the process of programmed cell death.     

Caspase cleavage product of BAP31 induces mitochondrial fission through endoplasmic reticulum calcium signals,enhancing cytochrome c release to the cytosol

Stimulation of cell surface death receptors activates caspase-8, which targets a limited number of substrates including BAP31, an integral membrane protein of the endoplasmic reticulum (ER). Recently, we reported that a caspase-resistant BAP31 mutant inhibited several features of Fas-induced apoptosis, including the release of cytochrome c (cyt.c) from mitochondria (Nguyen, M., D.G. Breckenridge, A. Ducret, and G.C. Shore. 2000. Mol. Cell. Biol. 20:6731-6740), implicating ER-mitochondria crosstalk in this pathway. Here, we report that the p20 caspase cleavage fragment of BAP31 can direct pro-apoptotic signals between the ER and mitochondria. Adenoviral expression of p20 caused an early release of Ca2+ from the ER, concomitant uptake of Ca2+ into mitochondria, and mitochondrial recruitment of Drp1, a dynamin-related protein that mediates scission of the outer mitochondrial membrane, resulting in dramatic fragmentation and fission of the mitochondrial network. Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8-induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway. Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.     

Apoptosis in <Emphasis Type="Italic">Drosophila</Emphasis>: which role for mitochondria?

It is now well established that the mitochondrion is a central regulator of mammalian cell apoptosis. However, the importance of this organelle in non-mammalian apoptosis has long been regarded as minor, mainly because of the absence of a crucial role for cytochrome c in caspase activation. Recent results indicate that the control of caspase activation and cell death in Drosophila occurs at the mitochondrial level. Numerous proteins, including RHG proteins and proteins of the Bcl-2 family that are key regulators of Drosophila apoptosis, constitutively or transiently localize in mitochondria. These proteins participate in the cell death process at different levels such as degradation of Diap1, a Drosophila IAP, production of mitochondrial reactive oxygen species or stimulation of the mitochondrial fission machinery. Here, we review these mitochondrial events that might have their counterpart in human.     

Mitochondrial dynamics in the regulation of neuronal cell death

Mitochondria undergo continuous fission and fusion events in physiological situations. Fragmentation of mitochondria during cell death has been shown to play a key role in cell death progression, including release of the mitochondrial apoptotic proteins. Ultrastructural changes in mitochondria, such as cristae remodeling, is also involved in cell death initiation. Here, we emphasize the important role of mitochondrial fission/fusion machinery in neuronal cell death. Unlike many other cell types such as immortalized cell lines, neurons are distinct morphologically and functionally. We will discuss how this uniqueness presents special challenges in the cellular response to neurotoxic stresses, and how this affects the mitochondrial dynamics in the regulation of cell death in neurons.     

Mitochondrial fusion and fission in the control of apoptosis

Mitochondrial outer membrane permeabilization (MOMP) determines the point-of-no-return of most if not all signal-transduction cascades leading to cell death. It has been postulated that the molecular mechanism leading to MOMP could depend on the activation of the mitochondrial fission machinery mediated by proteins from the dynamin superfamily. However, recent work suggests that, depending on the specific apoptosis induction pathway, mitochondrial fission can occur independently or downstream from MOMP. Moreover, fragmentation of the mitochondrial network can inhibit MOMP and apoptosis in response to a particular range of lethal stimuli, namely those relying on Ca(2+) waves. Failure to transmit the Ca(2+) wave through disconnected mitochondria then interrupts the propagation of the pro-apoptotic signal. Thus, mitochondrial fission can either enhance or reduce the probability of MOMP and consequent cell death, depending on the initial lethal stimulus.     

Death-associated Protein 3 Regulates Mitochondrial-encoded Protein Synthesis and Mitochondrial Dynamics

Mitochondrial morphologies change over time and are tightly regulated by dynamic machinery proteins such as dynamin-related protein 1 (Drp1), mitofusion 1/2, and optic atrophy 1 (OPA1). However, the detailed mechanisms of how these molecules cooperate to mediate fission and fusion remain elusive. DAP3 is a mitochondrial ribosomal protein that involves in apoptosis, but its biological function has not been well characterized. Here, we demonstrate that DAP3 specifically localizes in the mitochondrial matrix. Knockdown of DAP3 in mitochondria leads to defects in mitochondrial-encoded protein synthesis and abnormal mitochondrial dynamics. Moreover, depletion of DAP3 dramatically decreases the phosphorylation of Drp1 at Ser-637 on mitochondria, enhancing the retention time of Drp1 puncta on mitochondria during the fission process. Furthermore, autophagy is inhibited in the DAP3-depleted cells, which sensitizes cells to different types of death stimuli. Together, our results suggest that DAP3 plays important roles in mitochondrial function and dynamics, providing new insights into the mechanism of a mitochondrial ribosomal protein function in cell death.     

Mitochondrial outer-membrane permeabilization and remodelling in apoptosis

Many human pathologies are associated with defects in mitochondria such as diabetes, neurodegenerative diseases or cancer. This tiny organelle is involved in a plethora of processes in mammalian cells, including energy production, lipid metabolism and cell death. In the so-called intrinsic apoptotic pathway, the outer mitochondrial membrane (MOM) is premeabilized by the pro-apoptotic Bcl-2 members Bax and Bak, allowing the release of apoptogenic factors such as cytochrome c from the inter-membrane space into the cytosol. At the same time, mitochondria fragment in response to Drp-1 activation suggesting that mitochondrial fission could play a role in mitochondrial outer-membrane permeabilization (MOMP). In this review, we will discuss the link that could exist between mitochondrial fission and fusion machinery, Bcl-2 family members and MOMP.     

Fzo1, a protein involved in mitochondrial fusion, inhibits apoptosis

Mitochondrial morphology and physiology are regulated by the processes of fusion and fission. Some forms of apoptosis are reported to be associated with mitochondrial fragmentation. We showed that overexpression of Fzo1A/B (rat) proteins involved in mitochondrial fusion, or silencing of Dnm1 (rat)/Drp1 (human) (a mitochondrial fission protein), increased elongated mitochondria in healthy cells. After apoptotic stimulation, these interventions inhibited mitochondrial fragmentation and cell death, suggesting that a process involved in mitochondrial fusion/fission might play a role in the regulation of apoptosis. Consistently, silencing of Fzo1A/B or Mfn1/2 (a human homolog of Fzo1A/B) led to an increase of shorter mitochondria and enhanced apoptotic death. Overexpression of Fzo1 inhibited cytochrome c release and activation of Bax/Bak, as assessed from conformational changes and oligomerization. Silencing of Mfn or Drp1 caused an increase or decrease of mitochondrial sensitivity to apoptotic stimulation, respectively. These results indicate that some of the proteins involved in mitochondrial fusion/fission modulate apoptotic cell death at the mitochondrial level.     

The mitochondrial fission regulator DRP3B does not regulate cell death in plants

BACKGROUND AND AIMS: Recent reports have described dramatic alterations in mitochondrial morphology during metazoan apoptosis. A dynamin-related protein (DRP) associated with mitochondrial outer membrane fission is known to be involved in the regulation of apoptosis. This study analysed the relationship between mitochondrial fission and regulation of plant cell death. METHODS: Transgenic plants were generated possessing Arabidopsis DRP3B (K56A), the dominant-negative form of Arabidopsis DRP, mitochondrial-targeted green fluorescent protein and mouse Bax. KEY RESULTS: Arabidopsis plants over-expressing DRP3B (K56A) exhibited long tubular mitochondria. In these plants, mitochondria appeared as a string-of-beads during cell death. This indicates that DRP3B (K56A) prevented mitochondrial fission during plant cell death. However, in contrast to results for mammalian cells and yeast, Bax-induced cell death was not inhibited in DRP3B (K56A)-expressing plant cells. Similarly, hydrogen peroxide-, menadione-, darkness- and salicylic acid-induced cell death was not inhibited by DRP3B (K56A) expression. CONCLUSIONS: These results indicate that the systems controlling cell death in animals and plants are not common in terms of mitochondrial fission.     

Dynamics of mitochondrial morphology in healthy cells and during apoptosis

Mitochondria exist as dynamic networks that often change shape and subcellular distribution. The number and morphology of mitochondria within a cell are controlled by precisely regulated rates of organelle fusion and fission. Recent reports have described dramatic alterations in mitochondrial morphology during the early stages of apoptotic cell death, a fragmentation of the network and the remodeling of the cristae. Surprisingly, proteins discovered to control mitochondrial morphology appear to also participate in apoptosis and proteins associated with the regulation of apoptosis have been shown to affect mitochondrial ultrastructure. In this review the recent progress in understanding the mechanisms governing mitochondrial morphology and the latest advances connecting the regulation of mitochondrial morphology with programmed cell death are discussed.     

Mst1 deletion reduces hyperglycemia-mediated vascular dysfunction via attenuating mitochondrial fission and modulating the JNK signaling pathway

Diabetes is a leading cause of microvascular complications, such as nephropathy and retinopathy. Recent studies have proposed that hyperglycemia-induced endothelial cell dysfunction is modulated by mitochondrial stress. Therefore, our experiment was to detect the upstream mediator of mitochondrial stress in hyperglycemia-treated endothelial cells with a focus on macrophage-stimulating 1 (Mst1) and mitochondrial fission. Our data illuminated that hyperglycemia incubation reduced cell viability, as well as increased apoptosis ratio in endothelial cell, and this alteration seemed to be associated with Mst1 upregulation. Inhibition of Mst1 via transfection of Mst1 siRNA into an endothelial cell could sustain cell viability and maintain mitochondrial function. At the molecular levels, endothelial cell death was accompanied with the activation of mitochondrial oxidative stress, mitochondrial apoptosis, and mitochondrial fission. Genetic ablation of Mst1 could reduce mitochondrial oxidative injury, block mitochondrial apoptosis, and repress mitochondrial fission. Besides, we also found Mst1 triggered mitochondrial dysfunction as well as endothelial cell damage through augmenting JNK pathway. Suppression of JNK largely ameliorated the protective actions of Mst1 silencing on hyperglycemia-treated endothelial cells and sustain mitochondrial function. The present study identifies Mst1 as a primary key mediator for hyperglycemia-induced mitochondrial damage and endothelial cell dysfunction. Increased Mst1 impairs mitochondrial function and activates endothelial cell death via opening mitochondrial death pathway through JNK.     

Mitofusin 2 protects cerebellar granule neurons against injury-induced cell death

Of the GTPases involved in the regulation of the fusion machinery, mitofusin 2 (Mfn2) plays an important role in the nervous system as point mutations of this isoform are associated with Charcot Marie Tooth neuropathy. Here, we investigate whether Mfn2 plays a role in the regulation of neuronal injury. We first examine mitochondrial dynamics following different modes of injury in cerebellar granule neurons. We demonstrate that neurons exposed to DNA damage or oxidative stress exhibit extensive mitochondrial fission, an early event preceding neuronal loss. The extent of mitochondrial fragmentation and remodeling is variable and depends on the mode and the severity of the death stimuli. Interestingly, whereas mitofusin 2 loss of function significantly induces cell death in the absence of any cell death stimuli, expression of mitofusin 2 prevents cell death following DNA damage, oxidative stress, and K+ deprivation induced apoptosis. More importantly, whereas wild-type Mfn2 and the hydrolysis-deficient mutant of Mfn2 (Mfn2(RasG12V)) function equally to promote fusion and lengthening of mitochondria, the activated Mfn2(RasG12V) mutant shows a significant increase in the protection of neurons against cell death and release of proapoptotic factor cytochrome c. These findings highlight a signaling role for Mfn2 in the regulation of apoptosis that extends beyond its role in mitochondrial fusion.     

Oxidative stress-mediated mitochondrial fission promotes hepatic stellate cell activation via stimulating oxidative phosphorylation

Previous studies have demonstrated dysregulated mitochondrial dynamics in fibrotic livers and hepatocytes. Little is currently known about how mitochondrial dynamics are involved, nor is it clear how mitochondrial dynamics participate in hepatic stellate cell (HSC) activation. In the present study, we investigated the role of mitochondrial dynamics in HSC activation and the underlying mechanisms. We verified that mitochondrial fission was enhanced in human and mouse fibrotic livers and active HSCs. Moreover, increased mitochondrial fission driven by fis1 overexpression could promote HSC activation. Inhibiting mitochondrial fission using mitochondrial fission inhibitor-1 (Mdivi-1) could inhibit activation and induce apoptosis of active HSCs, indicating that increased mitochondrial fission is essential for HSC activation. Mdivi-1 treatment also induced apoptosis in active HSCs in vivo and thus ameliorated CCl₄-induced liver fibrosis. We also found that oxidative phosphorylation (OxPhos) was increased in active HSCs, and OxPhos inhibitors inhibited activation and induced apoptosis in active HSCs. Moreover, increasing mitochondrial fission upregulated OxPhos, while inhibiting mitochondrial fission downregulated OxPhos, suggesting that mitochondrial fission stimulates OxPhos during HSC activation. Next, we found that inhibition of oxidative stress using mitoquinone mesylate (mitoQ) and Tempol inhibited mitochondrial fission and OxPhos and induced apoptosis in active HSCs, suggesting that oxidative stress contributes to excessive mitochondrial fission during HSC activation. In conclusion, our study revealed that oxidative stress contributes to enhanced mitochondrial fission, which triggers OxPhos during HSC activation. Importantly, inhibiting mitochondrial fission has huge prospects for alleviating liver fibrosis by eliminating active HSCs.Subject terms: Endocrine system and metabolic diseases, Cell biology