Human MIEF1 recruits Drp1 to mitochondrial outer membranes and promotes mitochondrial fusion rather than fission

Mitochondrial morphology is controlled by two opposing processes: fusion and fission. Drp1 (dynamin-related protein 1) and hFis1 are two key players of mitochondrial fission, but how Drp1 is recruited to mitochondria and how Drp1-mediated mitochondrial fission is regulated in mammals is poorly understood. Here, we identify the vertebrate-specific protein MIEF1 (mitochondrial elongation factor 1; independently identified as MiD51), which is anchored to the outer mitochondrial membrane. Elevated MIEF1 levels induce extensive mitochondrial fusion, whereas depletion of MIEF1 causes mitochondrial fragmentation. MIEF1 interacts with and recruits Drp1 to mitochondria in a manner independent of hFis1, Mff (mitochondrial fission factor) and Mfn2 (mitofusin 2), but inhibits Drp1 activity, thus executing a negative effect on mitochondrial fission. MIEF1 also interacts with hFis1 and elevated hFis1 levels partially reverse the MIEF1-induced fusion phenotype. In addition to inhibiting Drp1, MIEF1 also actively promotes fusion, but in a manner distinct from mitofusins. In conclusion, our findings uncover a novel mechanism which controls the mitochondrial fusion-fission machinery in vertebrates. As MIEF1 is vertebrate-specific, these data also reveal important differences between yeast and vertebrates in the regulation of mitochondrial dynamics.     

The mitochondrial fission protein hFis1 requires the endoplasmic reticulum gateway to induce apoptosis

Mitochondrial fission ensures organelle inheritance during cell division and participates in apoptosis. The fission protein hFis1 triggers caspase-dependent cell death, by causing the release of cytochrome c from mitochondria. Here we show that mitochondrial fission induced by hFis1 is genetically distinct from apoptosis. In cells lacking the multidomain proapoptotic Bcl-2 family members Bax and Bak (DKO), hFis1 caused mitochondrial fragmentation but not organelle dysfunction and apoptosis. Similarly, a mutant in the intermembrane region of hFis1-induced fission but not cell death, further dissociating mitochondrial fragmentation from apoptosis induction. Selective correction of the endoplasmic reticulum (ER) defect of DKO cells restored killing by hFis1, indicating that death by hFis1 relies on the ER gateway of apoptosis. Consistently, hFis1 did not directly activate BAX and BAK, but induced Ca(2+)-dependent mitochondrial dysfunction. Thus, hFis1 is a bifunctional protein that independently regulates mitochondrial fragmentation and ER-mediated apoptosis.     

Selective actions of mitochondrial fission/fusion genes on metabolism-secretion coupling in insulin-releasing cells

Mitochondria form filamentous networks that undergo continuous fission/fusion. In the pancreatic beta-cells, mitochondria are essential for the transduction of signals linking nutrient metabolism to insulin granule exocytosis. Here we have studied mitochondrial networks in the insulinoma cell line INS-1E, primary rat and human beta-cells. We have further investigated the impact of mitochondrial fission/fusion on metabolism-secretion coupling in INS-1E cells. Overexpression of hFis1 caused dramatic mitochondrial fragmentation, whereas Mfn1 evoked hyperfusion and the aggregation of mitochondria. Cells overexpressing hFis1 or Mfn1 showed reduced mitochondrial volume, lowered cellular ATP levels, and as a consequence, impaired glucose-stimulated insulin secretion. Decreased mitochondrial ATP generation was partially compensated for by enhanced glycolysis as indicated by increased lactate production in these cells. Dominant-negative Mfn1 elicited mitochondrial shortening and fragmentation of INS-1E cell mitochondria, similar to hFis1. However, the mitochondrial volume, cytosolic ATP levels, and glucose-stimulated insulin secretion were little affected. We conclude that mitochondrial fragmentation per se does not impair metabolism-secretion coupling. Through their impact on mitochondrial bioenergetics and distribution, hFis1 and Mfn1 activities influence mitochondrial signal generation thereby insulin exocytosis.     

hFis1, a novel component of the mammalian mitochondrial fission machinery

The balance between the fission and fusion mechanisms regulate the morphology of mitochondria. In this study we have identified a mammalian protein that we call hFis1, which is the orthologue of the yeast Fis1p known to participate in yeast mitochondrial division. hFis1, when overexpressed in various cell types, localized to the outer mitochondrial membrane and induced mitochondrial fission. This event was inhibited by a dominant negative mutant of Drp1 (Drp1(K38A)), a major component of the fission apparatus. Fragmentation of the mitochondrial network by hFis1 was followed by the release of cytochrome c and ultimately apoptosis. Bcl-xL was able to block cytochrome c release and apoptosis but failed to prevent mitochondrial fragmentation. Our studies show that hFis1 is part of the mammalian fission machinery and suggest that regulation of the fission processes might be involved in apoptotic mechanisms.     

Ca(2+) homeostasis during mitochondrial fragmentation and perinuclear clustering induced by hFis1

Mitochondria modulate Ca(2+) signals by taking up, buffering, and releasing Ca(2+) at key locations near Ca(2+) release or influx channels. The role of such local interactions between channels and organelles is difficult to establish in living cells because mitochondria form an interconnected network constantly remodeled by coordinated fusion and fission reactions. To study the effect of a controlled disruption of the mitochondrial network on Ca(2+) homeostasis, we took advantage of hFis1, a protein that promotes mitochondrial fission by recruiting the dynamin-related protein, Drp1. hFis1 expression in HeLa cells induced a rapid and complete fragmentation of mitochondria, which redistributed away from the plasma membrane and clustered around the nucleus. Despite the dramatic morphological alteration, hFis1-fragmented mitochondria maintained a normal transmembrane potential and pH and took up normally the Ca(2+) released from intracellular stores upon agonist stimulation, as measured with a targeted ratiometric pericam probe. In contrast, hFis1-fragmented mitochondria took up more slowly the Ca(2+) entering across plasma membrane channels, because the Ca(2+) ions reaching mitochondria propagated faster and in a more coordinated manner in interconnected than in fragmented mitochondria. In parallel cytosolic fura-2 measurements, the capacitative Ca(2+) entry (CCE) elicited by store depletion was only marginally reduced by hFis1 expression. Regardless of mitochondria shape and location, disruption of mitochondrial potential with uncouplers or oligomycin/rotenone reduced CCE by approximately 35%. These observations indicate that close contact to Ca(2+) influx channels is not required for CCE modulation and that the formation of a mitochondrial network facilitates Ca(2+) propagation within interconnected mitochondria.     

The mitochondrial protein hFis1 regulates mitochondrial fission in mammalian cells through an interaction with the dynamin-like protein DLP1

The yeast protein Fis1p has been shown to participate in mitochondrial fission mediated by the dynamin-related protein Dnm1p. In mammalian cells, the dynamin-like protein DLP1/Drp1 functions as a mitochondrial fission protein, but the mechanisms by which DLP1/Drp1 and the mitochondrial membrane interact during the fission process are undefined. In this study, we have tested the role of a mammalian homologue of Fis1p, hFis1, and provided new and mechanistic information about the control of mitochondrial fission in mammalian cells. Through differential tagging and deletion experiments, we demonstrate that the intact C-terminal structure of hFis1 is essential for mitochondrial localization, whereas the N-terminal region of hFis1 is necessary for mitochondrial fission. Remarkably, an increased level of cellular hFis1 strongly promotes mitochondrial fission, resulting in an accumulation of fragmented mitochondria. Conversely, cell microinjection of hFis1 antibodies or treatment with hFis1 antisense oligonucleotides induces an elongated and collapsed mitochondrial morphology. Further, fluorescence resonance energy transfer and coimmunoprecipitation studies demonstrate that hFis1 interacts with DLP1. These results suggest that hFis1 participates in mitochondrial fission through an interaction that recruits DLP1 from the cytosol. We propose that hFis1 is a limiting factor in mitochondrial fission and that the number of hFis1 molecules on the mitochondrial surface determines fission frequency.     

The mitochondrial outer membrane protein hFis1 regulates mitochondrial morphology and fission through self-interaction

Mitochondrial fission in mammals is mediated by at least two proteins, DLP1/Drp1 and hFis1. DLP1 mediates the scission of mitochondrial membranes through GTP hydrolysis, and hFis1 is a putative DLP1 receptor anchored at the mitochondrial outer membrane by a C-terminal single transmembrane domain. The cytosolic domain of hFis1 contains six α-helices (α1-α6) out of which α2-α5 form two tetratricopeptide repeat (TPR) folds. In this study, by using chimeric constructs, we demonstrated that the cytosolic domain contains the necessary information for hFis1 function during mitochondrial fission. By using transient expression of different mutant forms of the hFis1 protein, we found that hFis1 self-interaction plays an important role in mitochondrial fission. Our results show that deletion of the α1 helix greatly increased the formation of dimeric and oligomeric forms of hFis1, indicating that α1 helix functions as a negative regulator of the hFis1 self-interaction. Further mutational approaches revealed that a tyrosine residue in the α5 helix and the linker between α3 and α4 helices participate in hFis1 oligomerization. Mutations causing oligomerization defect greatly reduced the ability to induce not only mitochondrial fragmentation by full-length hFis1 but also the formation of swollen ball-shaped mitochondria caused by α1-deleted hFis1. Our data suggest that oligomerization of hFis1 in the mitochondrial outer membrane plays a role in mitochondrial fission, potentially through participating in fission factor recruitment.     

Thapsigargin induces biphasic fragmentation of mitochondria through calcium-mediated mitochondrial fission and apoptosis

Mitochondrial fission and fusion are the main components mediating the dynamic change of mitochondrial morphology observed in living cells. While many protein factors directly participating in mitochondrial dynamics have been identified, upstream signals that regulate mitochondrial morphology are not well understood. In this study, we tested the role of intracellular Ca(2+) in regulating mitochondrial morphology. We found that treating cells with the ER Ca(2+)-ATPase inhibitor thapsigargin (TG) induced two phases of mitochondrial fragmentation. The initial fragmentation of mitochondria occurs rapidly within minutes dependent on an increase in intracellular Ca(2+) levels, and Ca(2+) influx into mitochondria is necessary for inducing mitochondrial fragmentation. The initial mitochondrial fragmentation is a transient event, as tubular mitochondrial morphology was restored as the Ca(2+) level decreased. We were able to block the TG-induced mitochondrial fragmentation by inhibiting mitochondrial fission proteins DLP1/Drp1 or hFis1, suggesting that increased mitochondrial Ca(2+) acts upstream to activate the cellular mitochondrial fission machinery. We also found that prolonged incubation with TG induced the second phase of mitochondrial fragmentation, which was non-reversible and led to cell death as reported previously. These results suggest that Ca(2+) is involved in controlling mitochondrial morphology via intra-mitochondrial Ca(2+) signaling as well as the apoptotic process.     

A novel mitochondrial ubiquitin ligase plays a critical role in mitochondrial dynamics

In this study, we have identified a novel mitochondrial ubiquitin ligase, designated MITOL, which is localized in the mitochondrial outer membrane. MITOL possesses a Plant Homeo-Domain (PHD) motif responsible for E3 ubiquitin ligase activity and predicted four-transmembrane domains. MITOL displayed a rapid degradation by autoubiquitination activity in a PHD-dependent manner. HeLa cells stably expressing a MITOL mutant lacking ubiquitin ligase activity or MITOL-deficient cells by small interfering RNA showed an aberrant mitochondrial morphology such as fragmentation, suggesting the enhancement of mitochondrial fission by MITOL dysfunction. Indeed, a dominant-negative expression of Drp1 mutant blocked mitochondrial fragmentation induced by MITOL depletion. We found that MITOL associated with and ubiquitinated mitochondrial fission protein hFis1 and Drp1. Pulse-chase experiment showed that MITOL overexpression increased turnover of these fission proteins. In addition, overexpression phenotype of hFis1 could be reverted by MITOL co-overexpression. Our finding indicates that MITOL plays a critical role in mitochondrial dynamics through the control of mitochondrial fission proteins.     

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.     

Roles of the mammalian mitochondrial fission and fusion mediators Fis1, Drp1, and Opa1 in apoptosis

During apoptosis, the mitochondrial network fragments. Using short hairpin RNAs for RNA interference, we manipulated the expression levels of the proteins hFis1, Drp1, and Opa1 that are involved in mitochondrial fission and fusion in mammalian cells, and we characterized their functions in mitochondrial morphology and apoptosis. Down-regulation of hFis1 powerfully inhibits cell death to an extent significantly greater than down-regulation of Drp1 and at a stage of apoptosis distinct from that induced by Drp1 inhibition. Cells depleted of Opa1 are extremely sensitive to exogenous apoptosis induction, and some die spontaneously by a process that requires hFis1 expression. Wild-type Opa1 may function normally as an antiapoptotic protein, keeping spontaneous apoptosis in check. However, if hFis1 is down-regulated, cells do not require Opa1 to prevent apoptosis, suggesting that Opa1 may be normally counteracting the proapoptotic action of hFis1. We also demonstrate in this study that mitochondrial fragmentation per se does not result in apoptosis. However, we provide further evidence that multiple components of the mitochondrial morphogenesis machinery can positively and negatively regulate apoptosis.     

The i-AAA protease YME1L and OMA1 cleave OPA1 to balance mitochondrial fusion and fission

Mitochondrial fusion and structure depend on the dynamin-like GTPase OPA1, whose activity is regulated by proteolytic processing. Constitutive OPA1 cleavage by YME1L and OMA1 at two distinct sites leads to the accumulation of both long and short forms of OPA1 and maintains mitochondrial fusion. Stress-induced OPA1 processing by OMA1 converts OPA1 completely into short isoforms, inhibits fusion, and triggers mitochondrial fragmentation. Here, we have analyzed the function of different OPA1 forms in cells lacking YME1L, OMA1, or both. Unexpectedly, deletion of Oma1 restored mitochondrial tubulation, cristae morphogenesis, and apoptotic resistance in cells lacking YME1L. Long OPA1 forms were sufficient to mediate mitochondrial fusion in these cells. Expression of short OPA1 forms promoted mitochondrial fragmentation, which indicates that they are associated with fission. Consistently, GTPase-inactive, short OPA1 forms partially colocalize with ER–mitochondria contact sites and the mitochondrial fission machinery. Thus, OPA1 processing is dispensable for fusion but coordinates the dynamic behavior of mitochondria and is crucial for mitochondrial integrity and quality control.     

A role for Fis1 in both mitochondrial and peroxisomal fission in mammalian cells

The mammalian dynamin-like protein DLP1/Drp1 has been shown to mediate both mitochondrial and peroxisomal fission. In this study, we have examined whether hFis1, a mammalian homologue of yeast Fis1, which has been shown to participate in mitochondrial fission by an interaction with DLP1/Drp1, is also involved in peroxisomal growth and division. We show that hFis1 localizes to peroxisomes in addition to mitochondria. Through differential tagging and deletion experiments, we demonstrate that the transmembrane domain and the short C-terminal tail of hFis1 is both necessary and sufficient for its targeting to peroxisomes and mitochondria, whereas the N-terminal region is required for organelle fission. hFis1 promotes peroxisome division upon ectopic expression, whereas silencing of Fis1 by small interfering RNA inhibited fission and caused tubulation of peroxisomes. These findings provide the first evidence for a role of Fis1 in peroxisomal fission and suggest that the fission machinery of mitochondria and peroxisomes shares common components.     

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.     

Bax/Bak-dependent release of DDP/TIMM8a promotes Drp1-mediated mitochondrial fission and mitoptosis during programmed cell death

Mitochondrial morphology within cells is controlled by precisely regulated rates of fusion and fission . During programmed cell death (PCD), mitochondria undergo extensive fragmentation and ultimately caspase-independent elimination through a process known as mitoptosis . Though this increased fragmentation is due to increased fission through the recruitment of the dynamin-like GTPase Drp1 to mitochondria , as well as to a block in mitochondrial fusion , cellular mechanisms underlying these processes remain unclear. Here, we describe a mechanism for the increased mitochondrial Drp1 levels and subsequent stimulation of mitochondrial fission seen during PCD. We observed Bax/Bak-mediated release of DDP/TIMM8a, a mitochondrial intermembrane space (IMS) protein , into the cytoplasm, where it binds to and promotes the mitochondrial redistribution of Drp1, a mediator of mitochondrial fission. Using both loss- and gain-of-function assays, we also demonstrate that the Drp1- and DDP/TIMM8a-dependent mitochondrial fragmentation observed during PCD is an important step in mitoptosis, which in turn is involved in caspase-independent cell death. Thus, following Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP), IMS proteins released comprise not only apoptogenic factors such as cytochrome c involved in caspase activation but also DDP/TIMM8a, which activates Drp1-mediated fission to promote mitochondrial fragmentation and subsequently elimination during PCD.     

Inhibition of mitochondrial fusion by α‐synuclein is rescued by PINK1, Parkin and DJ‐1

Aggregation of α‐synuclein (αS) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of αS is largely unknown. We demonstrate with in vitro vesicle fusion experiments that αS has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, αS binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age‐dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous αS. In contrast, siRNA‐mediated downregulation of αS results in elongated mitochondria in cell culture. αS can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, αS prevents fusion of differently labelled mitochondrial populations. Thus, αS inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of αS is rescued by coexpression of PINK1, parkin or DJ‐1 but not the PD‐associated mutations PINK1 G309D and parkin Δ1–79 or by DJ‐1 C106A.     

Disruption of fusion results in mitochondrial heterogeneity and dysfunction

Mitochondria undergo continual cycles of fusion and fission, and the balance of these opposing processes regulates mitochondrial morphology. Paradoxically, cells invest many resources to maintain tubular mitochondrial morphology, when reducing both fusion and fission simultaneously achieves the same end. This observation suggests a requirement for mitochondrial fusion, beyond maintenance of organelle morphology. Here, we show that cells with targeted null mutations in Mfn1 or Mfn2 retained low levels of mitochondrial fusion and escaped major cellular dysfunction. Analysis of these mutant cells showed that both homotypic and heterotypic interactions of Mfns are capable of fusion. In contrast, cells lacking both Mfn1 and Mfn2 completely lacked mitochondrial fusion and showed severe cellular defects, including poor cell growth, widespread heterogeneity of mitochondrial membrane potential, and decreased cellular respiration. Disruption of OPA1 by RNAi also blocked all mitochondrial fusion and resulted in similar cellular defects. These defects in Mfn-null or OPA1-RNAi mammalian cells were corrected upon restoration of mitochondrial fusion, unlike the irreversible defects found in fzodelta yeast. In contrast, fragmentation of mitochondria, without severe loss of fusion, did not result in such cellular defects. Our results showed that key cellular functions decline as mitochondrial fusion is progressively abrogated.     

Regulation of Ca2+-induced permeability transition by Bcl-2 is antagonized by Drp1 and hFis1

The regulation of mitochondrial permeability transition (MPT) is essential for cell survival. Un-controlled opening of the MPT pore is often associated with cell death. Anti-death protein Bcl-2 can block MPT as assessed by the enhanced capacity of mitochondria to accumulate and retain Ca²⁺. We report here that two proteins of the mitochondrial fission machinery, dynamin-related protein (Drp1) and human mitochondrial fission protein (hFis1), have an antagonistic effect on Bcl-2. Drp1, with the assistance of hFis1, sensitizes cells to MPT by reducing the mitochondrial Ca²⁺ retention capacity (CRC). While the reduction of CRC by Drp1/hFis1 is linked to mitochondrial fission, the antagonism between Bcl-2 and Drp1 appears to be mediated by mutually exclusive interactions of the two proteins with hFis1. The complexity of protein–protein interactions demonstrated in the present study suggests that in addition to the previously described role of Bcl-2 in the control of apoptosis, Bcl-2 may also participate directly or indirectly in the regulation of mitochondrial fission.     

Regulation of mitochondrial morphology by membrane potential,and DRP1-dependent division and FZO1-dependent fusion reaction in mammalian cells

Mitochondria are dynamic organelles that undergo frequent fission and fusion or branching. To analyze the mitochondrial fusion reaction, mitochondria were separately labeled with green or red fluorescent protein (GFP and RFP, respectively) in HeLa cells, and the cells were fused using hemagglutinating virus of Japan (HVJ). The resulting mixing of the fluorescent reporters was then followed using fluorescence microscopy. This system revealed that mitochondria fuse frequently in mammalian cells, and the fusion depends on the membrane potential across the inner membrane. The protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), led to fragmentation of the mitochondria and inhibited the fusion reaction. Removal of CCCP recovered the fusion activity to reform filamentous mitochondrial networks. Analysis of the effects of GTP-binding proteins, DRP1 and two FZO1 isoforms, and the GTPase-domain mutants on the CCCP-induced mitochondrial morphologic changes revealed that DRP1 and FZO1 are involved in membrane budding and fusion, respectively. Furthermore, a HVJ-dependent cell fusion assay combined with RNA interference (RNAi) demonstrated that both FZO1 isoforms are essential and must be acting in cis for the mitochondrial fusion reaction to occur.     

Regulation of mitochondrial morphology by APC/CCdh1-mediated control of Drp1 stability

Homeostatic maintenance of cellular mitochondria requires a dynamic balance between fission and fusion, and controlled changes in morphology are important for processes such as apoptosis and cellular division. Interphase mitochondria have been described as an interconnected network that fragments as cells enter mitosis, and this mitotic mitochondrial fragmentation is known to be regulated by the dynamin-related GTPase Drp1 (dynamin-related protein 1), a key component of the mitochondrial division machinery. Loss of Drp1 function and the subsequent failure of mitochondrial division during mitosis lead to incomplete cytokinesis and the unequal distribution of mitochondria into daughter cells. During mitotic exit and interphase, the mitochondrial network reforms. Here we demonstrate that changes in mitochondrial dynamics as cells exit mitosis are driven in part through ubiquitylation of Drp1, catalyzed by the APC/C(Cdh1) (anaphase-promoting complex/cyclosome and its coactivator Cdh1) E3 ubiquitin ligase complex. Importantly, inhibition of Cdh1-mediated Drp1 ubiquitylation and proteasomal degradation during interphase prevents the normal G1 phase regrowth of mitochondrial networks following cell division.