InteractiveFly: GeneBrief

Mitochondrial assembly regulatory factor: Biological Overview | References


Gene name - Mitochondrial assembly regulatory factor

Synonyms - Drosophila Mitofusin

Cytological map position - 5F4-5F4

Function - signaling

Keywords - target of the Yorkie pathway, regulation of mitochondrial fusion, organ size, steroid synthesis in the ring gland and ovarian follicle cell differentiation, modulates endoplasmic reticulum function, Reaper binds Marf to induce mitochondrial fragmentation

Symbol - Marf

FlyBase ID: FBgn0029870

Genetic map position - chrX:6,365,949-6,370,924

Classification - Fzo_mitofusin - DLP_2: Dynamin-like protein including dynamins, mitofusins, and guanylate-binding proteins - P-loop_NTPase: P-loop containing Nucleoside Triphosphate Hydrolases

Cellular location - cytoplasmic



NCBI links: EntrezGene

Recent literature
Wang, Z. H., Clark, C. and Geisbrecht, E. R. (2016). Drosophila Clueless is involved in Parkin-dependent mitophagy by promoting VCP-mediated Marf degradation. Hum Mol Genet [Epub ahead of print]. PubMed ID: 26931463
Summary:
PINK1/Parkin-mediated mitochondrial quality control (MQC) requires valosin-containing protein (VCP)-dependent Mitofusin/Marf degradation to prevent damaged organelles from fusing with the healthy mitochondrial pool, facilitating mitochondrial clearance by autophagy. Drosophila clueless (clu) was found to interact genetically with PINK1 and parkin to regulate mitochondrial clustering in germ cells. However, whether Clu acts in MQC has not been investigated. This study shows that overexpression of Drosophila Clu complements PINK1, but not parkin, mutant muscles. Loss of clu leads to the recruitment of Parkin, VCP/p97, p62/Ref(2)P and Atg8a to depolarized swollen mitochondria. However, clearance of damaged mitochondria is impeded. This paradox is resolved by the findings that excessive mitochondrial fission or inhibition of fusion alleviates mitochondrial defects and impaired mitophagy caused by clu depletion. Furthermore, Clu is upstream of and binds to VCP in vivo and promotes VCP-dependent Marf degradation in vitro. Marf accumulates in whole muscle lysates of clu-deficient flies and is destabilized upon Clu overexpression. Thus, Clu is essential for mitochondrial homeostasis and functions in concert with Parkin and VCP for Marf degradation to promote damaged mitochondrial clearance.

Celardo, I., Costa, A. C., Lehmann, S., Jones, C., Wood, N., Mencacci, N. E., Mallucci, G. R., Loh, S. H. and Martins, L. M. (2016). Mitofusin-mediated ER stress triggers neurodegeneration in pink1/parkin models of Parkinson's disease. Cell Death Dis 7: e2271. PubMed ID: 27336715
Summary:
Mutations in PINK1 and PARKIN cause early-onset Parkinson's disease (PD), thought to be due to mitochondrial toxicity. This study shows that in Drosophila pink1 and parkin mutants, defective mitochondria also give rise to endoplasmic reticulum (ER) stress signalling, specifically to the activation of the protein kinase R-like endoplasmic reticulum kinase (PERK) branch of the unfolded protein response (UPR). Enhanced ER stress signalling in pink1 and parkin mutants is mediated by mitofusin bridges, which occur between defective mitochondria and the ER. Reducing mitofusin contacts with the ER is neuroprotective, through suppression of PERK signalling, while mitochondrial dysfunction remains unchanged. Further, both genetic inhibition of dPerk-dependent ER stress signalling and pharmacological inhibition using the PERK inhibitor GSK2606414 were neuroprotective in both pink1 and parkin mutants. It is concluded that activation of ER stress by defective mitochondria is neurotoxic in pink1 and parkin flies and that the reduction of this signalling is neuroprotective, independently of defective mitochondria.
Kandul, N. P., Zhang, T., Hay, B. A. and Guo, M. (2016). Selective removal of deletion-bearing mitochondrial DNA in heteroplasmic Drosophila. Nat Commun 7: 13100. PubMed ID: 27841259
Summary:
Mitochondrial DNA (mtDNA) often exists in a state of heteroplasmy, in which mutant mtDNA co-exists in cells with wild-type mtDNA. High frequencies of pathogenic mtDNA result in maternally inherited diseases; maternally and somatically acquired mutations also accumulate over time and contribute to diseases of ageing. Reducing heteroplasmy is therefore a therapeutic goal and in vivo models in post-mitotic tissues are needed to facilitate these studies. This study describes a transgene-based model of a heteroplasmic lethal mtDNA deletion (mtDNAΔ) in adult Drosophila muscle. Stimulation of autophagy, activation of the PINK1/parkin pathway or decreased levels of mitofusin result in a selective decrease in mtDNAΔ. Decreased levels of mitofusin and increased levels of ATPIF1, an inhibitor of ATP synthase reversal-dependent mitochondrial repolarization, result in a further decrease in mtDNAΔ levels. These results show that an adult post-mitotic tissue can be cleansed of a deleterious genome, suggesting that therapeutic removal of mutant mtDNA can be achieved.
Zhang, T., Mishra, P., Hay, B. A., Chan, D. and Guo, M. (2017). Valosin-containing protein (VCP/p97) inhibitors relieve Mitofusin-dependent mitochondrial defects due to VCP disease mutants. Elife 6. PubMed ID: 28322724
Summary:
Missense mutations of valosin-containing protein (VCP; see Drosophila TER94 ) cause an autosomal dominant disease known as inclusion body myopathy, Paget disease with frontotemporal dementia (IBMPFD) and other neurodegenerative disorders. The pathological mechanism of IBMPFD is not clear and there is no treatment. This study shows that endogenous VCP negatively regulates Mitofusin (see Drosophila Marf), which is required for outer mitochondrial membrane fusion. Because 90% of IBMPFD patients have myopathy, an in vivo IBMPFD model was generated in adult Drosophila muscle, which recapitulates disease pathologies. Common VCP disease mutants were shown to act as hyperactive alleles with respect to regulation of Mitofusin. Importantly, VCP inhibitors suppress mitochondrial defects, muscle tissue damage and cell death associated with IBMPFD models in Drosophila. These inhibitors also suppress mitochondrial fusion and respiratory defects in IBMPFD patient fibroblasts. These results suggest that VCP disease mutants cause IBMPFD through a gain-of-function mechanism, and that VCP inhibitors have therapeutic value.
Edenharter, O., Schneuwly, S. and Navarro, J. A. (2018). Mitofusin-dependent ER stress triggers glial dysfunction and nervous system degeneration in a Drosophila model of Friedreich's ataxia. Front Mol Neurosci 11: 38. PubMed ID: 29563863
Summary:
Friedreich's ataxia (FRDA) is the most important recessive ataxia in the Caucasian population. It is caused by a deficit of the mitochondrial<.a> protein frataxin. A forward genetic screen was carried out to analyze genetic interactions between genes controlling mitochondrial homeostasis and Drosophila frataxin. The screen has identified silencing of Drosophila mitofusin (Marf) as a suppressor of FRDA phenotypes in glia. Drosophila Marf is known to play crucial roles in mitochondrial fusion, mitochondrial degradation and in the interface between mitochondria and endoplasmic reticulum (ER). The results indicated that frataxin-deficiency had a small impact on mitochondrial morphology but enhanced mitochondrial clearance and altered the ER stress response in Drosophila. Remarkably, it was demonstrated that downregulation of Marf suppresses ER stress in frataxin-deficient cells and this is sufficient to improve locomotor dysfunction, brain degeneration and lipid dyshomeostasis in the FRDA model. In agreement, chemical reduction of ER stress by means of two different compounds was sufficient to ameliorate the effects of frataxin deficiency in three different fly FRDA models. Altogether, these results strongly suggest that the protection mediated by Marf knockdown in glia is mainly linked to its role in the mitochondrial-ER tethering and not to mitochondrial dynamics or mitochondrial degradation and that ER stress is a novel and pivotal player in the progression and etiology of FRDA. This work might define a new pathological mechanism in FRDA, linking mitochondrial dysfunction due to frataxin deficiency and mitofusin-mediated ER stress, which might be responsible for characteristic cellular features of the disease and also suggests ER stress as a therapeutic target.
Trevisan, T., Pendin, D., Montagna, A., Bova, S., Ghelli, A. M. and Daga, A. (2018). Manipulation of mitochondria dynamics reveals separate roles for form and function in mitochondria distribution. Cell Rep 23(6): 1742-1753. Pubmed ID: 29742430
Summary:
Mitochondria shape is controlled by membrane fusion and fission mediated by mitofusins, Opa1, and Drp1, whereas mitochondrial motility relies on microtubule motors. These processes govern mitochondria subcellular distribution, whose defects are emphasized in neurons because of their polarized structure. This study examined how perturbation of the fusion/fission balance affects mitochondria distribution in Drosophila axons. Knockdown of Marf or Opa1 resulted in progressive loss of distal mitochondria and in a distinct oxidative phosphorylation and membrane potential deficit. Downregulation of Drp1 rescued the lethality and bioenergetic defect caused by neuronal Marf RNAi, but induced only a modest restoration of axonal mitochondria distribution. Surprisingly, Drp1 knockdown rescued fragmentation and fully restored aberrant distribution of axonal mitochondria produced by Opa1 RNAi; however, Drp1 knockdown did not improve viability or mitochondria function. These data show that proper morphology is critical for proper axonal mitochondria distribution independent of bioenergetic efficiency. The health of neurons largely depends on mitochondria function, but does not depend on shape or distribution.
BIOLOGICAL OVERVIEW

Mitochondrial fusion and fission affect the distribution and quality control of mitochondria. This study shows that Marf (Mitochondrial associated regulatory factor, also known as Drosophila Mitofusin), is required for mitochondrial fusion and transport in long axons. Moreover, loss of Marf leads to a severe depletion of mitochondria in neuromuscular junctions (NMJs). Marf mutants also fail to maintain proper synaptic transmission at NMJs upon repetitive stimulation, similar to Drp1 fission mutants. However, unlike Drp1, loss of Marf leads to NMJ morphology defects and extended larval lifespan. Marf is required to form contacts between the endoplasmic reticulum and/or lipid droplets (LDs) and for proper storage of cholesterol and ecdysone synthesis in ring glands. Interestingly, human Mitofusin-2 rescues the loss of LD but both Mitofusin-1 and Mitofusin-2 are required for steroid-hormone synthesis. These data show that Marf and Mitofusins share an evolutionarily conserved role in mitochondrial transport, cholesterol ester storage and steroid-hormone synthesis (Sandoval, 2014).

Mitochondrial dynamics plays a critical role in the control of organelle shape, size, number, function and quality control of mitochondria from yeast to mammals. It consists of fusion and fission of mitochondria, which are regulated by several GTPases. Mitochondrial fusion requires the fusion of the outer membrane followed by inner membrane fusion. In mammals, Mitofusin 1 (Mfn1) and Mitofusin 2 (Mfn2) regulate outer mitochondrial fusion whereas inner membrane fusion is controlled by Optic atrophy protein 1 (Opa1). Mitochondrial fission is regulated by Dynamin related protein 1 (Drp1). Decreased fusion results in fragmented round mitochondria, while defective fission leads to fused and enlarged mitochondria (Sandoval, 2014).

Loss of these mitochondrial GTPases results in lethality in worms, flies and mice (Chen, 2003; Westermann, 2009; Debattisti, 2013). Mutations in the human DRP1 gene causes a dominant fatal infantile encephalopathy associated with defective mitochondrial and peroxisomal fission. On the other hand, missense mutations in OPA1 lead to a dominant optic atrophy. Depending on the severity of the mutation, patients may also suffer from ataxia and neuropathy. Also, missense mutations in MFN2 cause Charcot-Marie-Tooth type 2A, a common autosomal dominant peripheral neuropathy associated with axon degeneration. Finally, aberrant levels of mitochondrial GTPases have been associated with Parkinson's, Huntington's and Alzheimers' diseases. These observations in model organisms and human patients suggest that mitochondrial dynamics affects neuronal maintenance in many different contexts (Sandoval and references therein, 2014).

A significant imbalance of mitochondrial fission and fusion may affect the subcellular distribution of mitochondria, especially in neurons since they need to efficiently traffic from the soma to the synapses. Loss of Drosophila Drp1 impairs the delivery of mitochondria to neuromuscular junctions (NMJs), likely because they are large and interconnected. This defect is also associated with a severe depletion of mitochondria in NMJs, which affects local ATP production. This in turn affects the trafficking of synaptic vesicles upon endocytosis during prolonged stimulation. Similarly, in vertebrates, loss of Drp1 leads to an accumulation of mitochondria in the soma and reduced mitochondrial density in dendrites of hippocampal neurons. The Drp1 data in flies and vertebrates indicate that the expanded size of mitochondria affects their mobility (Sandoval, 2014).

Mitochondrial trafficking may also be affected by the physical interaction between the mitochondria and the transport machinery. Recent studies have documented a direct interaction between Mfn2 and a motor adaptor complex for mitochondrial transport, Miro2 (Misko, 2010; see Drosophila Miro). Moreover, loss of MFN2 in Purkinje cells displayed reduced mitochondrial motility in cerebellar dendrites (Chen, 2007) and reduced mitochondrial transport in axons in cultured dorsal root ganglion neurons (Misko, 2010). These data suggest that an interaction of Mfn2 with Miro2 may be important for its role in trafficking (Misko, 2010). Although loss of both Drp1 and MFN2 impair mitochondrial trafficking, a careful comparison of the phenotypes associated with loss of Drosophila Drp1, Mitofusin or Marf, would be useful as the suggested mechanisms by which they impair transport seem very different (Sandoval, 2014).

In addition to their roles in fission and fusion, Drp1, Mfns and Opa1 have been implicated in a variety of other processes. For example, Drp1 has been shown to facilitate the induction of apoptosis whereas Opa1 was shown to affect the stability of cristae junction in inner mitochondrial membrane. Finally, Mfn2 also tethers mitochondria to the endoplasmic reticulum (ER) to mediate Ca2+ uptake (de Brito, 2008). However, the molecular mechanisms underlying these non-canonical functions are less well studied (Sandoval, 2014).

In an unbiased screen designed to identify essential genes that affect neuronal function (Yamamoto, 2014), the first mutant allelic series was identified of Marf in Drosophila. This study exploits these mutants to determine how loss of Marf affects mitochondrial transport when compared to Drp1 loss. Surprisingly, NMJ defects were identified only in Marf mutants but not in Drp1 mutants. These defects are regulated non-cell autonomously by steroid-hormones produced in ring glands (RG), a major endocrine organ in insects. Through expression of human MFN1 or MFN2 in Marf mutant RG, it was shown that MFN1 and MFN2 have both distinct and complementary roles (Sandoval, 2014).

How does loss of fission or fusion affect mitochondrial function? In the absence of fusion mixing of mitochondrial DNA and proteins may be severely impaired. Given that mitochondrial proteins are in an environment rich in oxygen radicals, lack of fusion may cause more damage than when fission is impaired (Chen, 2012). Simply stated, loss of fusion proteins like Marf, MFN1 or MFN2 may cause more severe phenotypes than the loss of a fission protein like Drp1. Moreover, proteins like Marf and Drp1 may perform other functions that are not directly related to fusion or fission, and hence affect other processes. Based on a careful phenotypic comparison of loss of Marf and Drp1 in Drosophila many similarities and differences were found (Sandoval, 2014).

Marf mutants display small mitochondria whereas Drp1 mutants exhibit large fused mitochondria. Interestingly, both mutants accumulate mitochondria in the cell body of the neurons and the proximal axonal segments. In Drp1 mutants, the mitochondria seem to be severely elongated in axons where they fail to reach the NMJs, as previously described. The impairment in axonal transport is thought to be due to the fact that the mitochondria are hyperfused and cannot easily be transported. Indeed, loss of Marf in Drp1 mutants can restore mitochondrial trafficking proximally but distal axonal trafficking is still impaired. In Marf mutants, even though mitochondria are small and can enter the axons, the numbers of mitochondria that travel distally toward the NMJs are dramatically reduced. Hence, loss of Marf impairs mitochondrial trafficking and longer axons are more severely affected than shorter axons. Since longer axons are more severely affected in CMT2A patients, defects in mitochondrial trafficking may be at the root of some of the phenotypes associated with the disease (Sandoval, 2014).

Mfn2 has been implicated in axonal transport via binding to Miro2. Indeed, knockdown of MIRO2 in cultured vertebrate neurons affects mitochondrial transport in an identical fashion as loss of MFN2 (Misko, 2010). However, the severity of mitochondrial transport that observed in Marf mutants is much less pronounced than what has been described in dmiro mutants and what was observe when dmiro is lost. Moreover, removal of dmiro in Marf mutants dramatically enhances the Marf phenotype and almost abolishes axonal localization of mitochondria, arguing that Marf cannot be solely responsible for mitochondrial transport in Drosophila (Sandoval, 2014).

A comparison of the presence of mitochondria at NMJ synapses shows that Marf mutants have fewer mitochondria than Drp1 mutants. Moreover, Marf mutants but not Drp1 mutants display a severe increase in small clustered boutons. The small and clustered boutons have also been observed in other mutants like endophilin, synaptojanin, eps15, dap 160, flower and dmiro. However, unlike in Marf mutants, the bouton phenotypes are fully rescued by neuronal expression of the cognate protein within MN in the above mentioned mutants. Moreover, knockdown of Marf in neuron, muscle or glia does not recapitulate the bouton phenotype observe in Marf mutants, suggesting a unique cell non-autonomous requirement of Marf for proper NMJ morphology (Sandoval, 2014).

Marf mutants exhibit two obvious phenotypes at NMJs: a severe depletion of mitochondria and a doubling of the number of boutons combined with a severe reduction in size whereas Drp1 mutants only exhibit a severe reduction in mitochondria. However, electrophysiological studies show that loss of Marf does not affect basal synaptic transmission similar to what is observed in Drp1 mutants. Both respond similarly to wild type NMJs when stimulated at 0.2 Hz and both show a progressive run down at 10 Hz when compared to controls. Moreover, endocytosis using FM1-43 and 60 mM K+ is not impaired in Marf and Drp1 mutants, suggesting a defect in reserve pool mobilization in both mutants. The data also show that the bouton defects observed in Marf mutants do not contribute to the run down in synaptic transmission since Drp1 boutons are normal in number and size yet also have a run down in synaptic transmission (Sandoval, 2014).

Loss of Marf in ring glands (RG) recapitulates the bouton phenotype observed in Marf mutants and expression of Marf in RG fully rescues this phenotype. Interestingly, both Marf and Opa1 are required for steroid hormone production and both lead to extended larval lifespan when knocked down in the RG only (8-10 days), whereas Drp1 mutations do not affect steroid hormone synthesis. Reduction of ecdysone production by knockdown of the prothoracicotropic hormone receptor (torso) in the RG also leads to an extended larval lifespan (9 days) and an increased growth of NMJs. Interestingly, knockdown of Drosophila SUMO (dsmt3) in RG lead to a defect in cholesterol import in the RG, reduced 20E levels and an extended larval lifespan (19 days). Hence, the severe reduction in ecdysone synthesis in Marf mutant RG underlies the prolonged larva stages and NMJ morphological defects (Sandoval, 2014).

The reduction in the number of LDs in RGs when Marf is lost suggests that these RGs are unable to store cholesterol. This storage of cholesterol esters probably permits the RG to produce large amounts of ecdysone when needed, especially at the larval stage and larval to pupal transitions. Cholesterol storage and steroid hormone biosynthesis requires both the ER and mitochondria in vertebrates but loss of MFN1 or MFN2 have not been shown to affect LD synthesis. Defects of anchoring mitochondria to the ER and LDs in Marf RGs argue that these defects lead to the loss of LD and production of ecdysone. In agreement with this hypothesis, expression of human MFN2, which tethers ER to mitochondria (de Brito, 2008), in Marf mutants restores LD synthesis and organelle contacts. Moreover, expression of human MFN2 in RNAi mediated Marf knockdown in neurons and muscles rescues ER morphology and stress (Debattisti, 2014). However, MFN2 expression alone in Marf mutant RG did not restore ecydsone synthesis, arguing that there are other mitochondrial defects associated with the loss of Marf (Sandoval, 2014).

The current data show that co-expression of human MFN1 and MFN2 fully rescue the observed phenotypes in Marf mutants. Although RG-specific expression of MFN1 in Marf mutants did not restore LD numbers or organelle contacts, MFN1 is still necessary for ecdysone synthesis together with MFN2, suggesting a role downstream of cholesterol ester storage for both proteins. Moreover, knockdown of Opa1 in RG did not alter LD numbers but causes reduced 20E levels and aberrant NMJs. Opa1 resides within the inner mitochondrial membrane, suggesting its role in ecdysone synthesis is within the mitochondria. Ecdysone synthesis within the mitochondria requires two cytochrome p450 enzymes encoded by disembodied and shadow. Hence, it is likely that impairment in fusion but not fission affects the function of these enzymes (see Model of Marf dual function in steroid synthesis in the ring glands) (Sandoval, 2014).

Opa1 and MFN2 but not Drp1 have been implicated in vertebrate steroidogenesis. Interestingly, in placental trophoblast cells (BeWO) in culture the loss of OPA-1 promotes progesterone production by 70% whereas loss of MFN2 has been reported to lead to a 20% decrease in progesterone production (Wasilewski, 2012). In contrast, testosterone production in MA-10 Leydig cells was unaffected by loss of OPA1 whereas loss of MFN2 did affect testosterone production by 40% in MA-10 Leydig cells. Hence, in both vertebrate endocrine cells, loss of MFN2 or OPA-1 affected steroids very differently as was observe very similar phenotypes associated with the loss of either protein in the current study. This study also suggests that MFN2 functions upstream of cholesterol entry into the mitochondria at the cholesterol storage stage, since MFN2 restores LD synthesis in Drosophila RG. However, rescuing LD production is not sufficient to restore ecdysone synthesis, suggesting a secondary defect (see Model of Marf dual function in steroid synthesis in the ring glands). In summary, these data indicate that MFN1 and MFN2 have separate functions in vivo that are integrated in a single protein in fly Marf (Sandoval, 2014).

Dissociation of mitochondrial from sarcoplasmic reticular stress in Drosophila cardiomyopathy induced by molecularly distinct mitochondrial fusion defects

Mitochondrial dynamism (fusion and fission) is responsible for remodeling interconnected mitochondrial networks in some cell types. Adult cardiac myocytes lack mitochondrial networks, and their mitochondria are inherently 'fragmented'. Mitochondrial fusion/fission is so infrequent in cardiomyocytes as to not be observable under normal conditions, suggesting that mitochondrial dynamism may be dispensable in this cell type. However, it has been previously reported that cardiomyocyte-specific genetic suppression of mitochondrial fusion factors Optic atrophy 1 (Opa1) and Mitofusin (Marf) evokes cardiomyopathy in Drosophila hearts. Fusion-mediated remodeling of mitochondria may be critical for cardiac homeostasis, although never directly observed. Alternately, inner membrane Opa1 and outer membrane mitofusin/MARF might have other as-yet poorly described roles that affect mitochondrial and cardiac function. This study compared heart tube function in three models of mitochondrial fragmentation in Drosophila cardiomyocytes: Drp1 expression, Opa1 RNAi, and mitofusin MARF RNA1. Mitochondrial fragmentation evoked by enhanced Drp1-mediated fission did not adversely impact heart tube function. In contrast, RNAi-mediated suppression of either Opa1 or mitofusin/MARF induced cardiac dysfunction associated with mitochondrial depolarization and ROS production. Inhibiting ROS by overexpressing superoxide dismutase (SOD) or suppressing ROMO1 prevented mitochondrial and heart tube dysfunction provoked by Opa1 RNAi, but not by mitofusin/MARF RNAi. In contrast, enhancing the ability of endoplasmic/sarcoplasmic reticulum to handle stress by expressing Xbp1 rescued the cardiomyopathy of mitofusin/MARF insufficiency without improving that caused by Opa1 deficiency. The study concludes that decreased mitochondrial size is not inherently detrimental to cardiomyocytes. Rather, preservation of mitochondrial function by Opa1 located on the inner mitochondrial membrane, and prevention of ER stress by mitofusin/MARF located on the outer mitochondrial membrane, are central functions of these “mitochondrial fusion proteins” (Bhandari, 2015).

By performing a side-by-side detailed comparison of cardiomyopathies provoked by interrupting fusion of either the outer or inner mitochondrial membranes, this study identified distinct cellular mechanisms for the different molecular lesions. RNAi-mediated suppression of outer mitochondrial membrane mitofusin/MARF and inner mitochondrial membrane Opa1 provokes similar overt phenotypes: in both models mitochondrial size is approximately halved, the proportion of depolarized (sick) mitochondria increases to ~ 40%, mitochondrial ROS production is comparably greater (~ 50%), and the heart tubes exhibit similar reductions in fractional shortening. However, the cardiac defect caused by Opa1 deficiency is readily corrected by attacking the disease at the level of mitochondrial ROS production, through SOD expression or ROMO1 suppression. Indeed, mitochondrial structural and functional abnormalities are also improved by these genetic maneuvers, suggesting that they interrupt a vicious cycle of ROS-induced mitochondrial degeneration provoked by Opa1 insufficiency. Thus, interrupting endogenous mitochondrial ROS production greatly abrogates both the mitotoxicity and the cytotoxicity evoked by Opa1 suppression. This suggests a central role for mitochondrial degeneration in the Opa1-deficient fly heart model and, by extension, other heart diseases caused by defective inner mitochondrial membrane fusion proteins (Bhandari, 2015).

Whereas mitochondrial size and polarization status are similarly impaired in mitofusin/MARF insufficient heart tubes, neither of the mitochondrial-targeted interventions directed at reducing ROS, both of which rescue Opa1 deficient hearts, improve the cardiomyopathy induced by mitofusin/MARF deficiency. Indeed, whereas transgenic expression of SOD1 or SOD2 normalizes both ROS and heart tube function in Parkin-deficient heart tubes and Opa1-deficient heart tubes, it is remarkable that SOD fails to improve ROS levels in the mitofusin/MARF deficient heart tubes. Together with the original observation of transient heart tube functional improvement with SOD1, these observations suggest that there is 'ROS escape' in the mitochondrial fusion impaired model that confers resistance to SOD expression or ROMO1 suppression. Instead, heart tube function is normalized without improving either mitochondrial structural or functional abnormalities by genetically enhancing the cardiomyocytes' ability to handle ER stress through XBP1 expression. These results, although surprising, are consistent with an essential role for mitofusin-mediated mitochondrial-ER/SR cross-talk in managing the ER stress response as proposed earlier in heart and in other tissues (Bhandari, 2015).

Previously, the consequences of Opa1 deficiency on Drosophila eye phenotypes have been found to be rescuable with SOD1, which is in accordance with findings of this study. Another study in Drosophila neurons and skeletal muscle also supports an important role for mitofusins, but not Opa1, as modulators of ER stress. However, only human Mfn2, and not human Mfn1, can correct abnormalities induced by mitofusin/MARF suppression in flies. This contrasts with findings in this study that cardiac-specific expression of either human Mfn1 or Mfn2 will fully correct cardiomyopathy induced by cardiomyocyte-specific MARF RNAi. Furthermore, ER dysmorphology has been found in MARF-deficient fly tissues, which was not detected in MARF-deficient heart tubes. It is likely either that the heart has different requirements for mitofusins and ER/SR morphology, or that the powerful tinman gene promoter used for cardiomyocyte-specific gene manipulation confers different expression characteristics to the heart models. Either way, the overall conclusions regarding a role of ER stress in mitofusin deficiency are in agreement (Bhandari, 2015).

Cardiomyocyte mitochondria are the Oompa-Loompas of the heart (with apologies to Roald Dahl): They are diminutive, structurally homogenous, and frequently overlooked despite toiling endlessly behind the scenes to keep the place running. Research has tended to focus on the mitochondrial work product (cardiac metabolism) and the means by which the general mitochondrial population is sustained (through biogenesis), rather than the fate of individual organelles. Indeed, individual cardiomyocyte mitochondria seem hardly worthy of observation, being monotonously similar in appearance and lacking the morphometric remodeling or intra-cellular mobility that has sparked detailed investigations (and visually engaging movie clips) in other cell types. The data presented in this study emphasize that (for mitochondria as well as Oompa-Loompas) size is not the critical determinant of function; it is literally what is inside that counts. Accordingly, one should eschew generalizations and extrapolations of mitochondrial status and dysfunction based strictly on morphometry (Bhandari, 2015).

Reduction of endoplasmic reticulum stress attenuates the defects caused by Drosophila mitofusin depletion

Ablation of the mitochondrial fusion and endoplasmic reticulum (ER)-tethering protein Mfn2 causes ER stress, but whether this is just an epiphenomenon of mitochondrial dysfunction or a contributor to the phenotypes in mitofusin (Mfn)-depleted Drosophila melanogaster is unclear. This paper shows that reduction of ER dysfunction ameliorates the functional and developmental defects of flies lacking the single Mfn mitochondrial assembly regulatory factor (Marf). Ubiquitous or neuron- and muscle-specific Marf ablation is lethal, altering mitochondrial and ER morphology and triggering ER stress that was conversely absent in flies lacking the fusion protein Optic atrophy 1. Expression of Mfn2 and ER stress reduction in flies lacking Marf corrects ER shape, attenuating the developmental and motor defects. Thus, ER stress is a targetable pathogenetic component of the phenotypes caused by Drosophila Mfn ablation (Debattisti, 2014).

To explore the relative role of ER and mitochondria in the Mfn ablation phenotype, Drosophila would be a suitable model only if the ER-modulating function of Mfns did not emerge in evolution later than Marf. Therefore this study set out to address whether Marf can shape both mitochondria and ER. Expression of comparable Marf levels in mouse embryonic fibroblasts (MEFs) lacking Mfn1 and/or Mfn2 restored the elongated mitochondrial morphology as efficiently as human Mfns (hMfns). The altered Mfn2-/- ER morphology is selectively recovered by hMfn2, independently of its ability to correct mitochondrial shape. By inspecting 3D-reconstructed, volume-rendered images from confocal stacks of ER-targeted YFP (ER-YFP), it was noted that Marf-V5 recovered Mfn2-/- ER morphology as efficiently as hMfn2. Accordingly, effective Marf knockdown in Drosophila S2R+ cells caused mitochondrial fragmentation and ER fragmentation and clumping, which were complemented by the reexpression of an RNAi-resistant mutant of Marf, substantiating that the phenotypes observed are specifically caused by Marf loss. Thus, Drosophila Mfn modulates both mitochondrial and ER morphology (Debattisti, 2014).

The analysis of mitochondrial and ER shape next moved to in vivo, taking advantage of upstream activation sequence (UAS)-MarfRNAi transgenic flies, in which it was possible to control expression of Marf RNAi in space and time using the Gal4-UAS system. Because the RNAi was specific and efficiently ablated Marf in vivo, whether Marf loss altered development was examined. Ubiquitous, tubulin-Gal4-driven Marf down-regulation was lethal, arresting development at the second instar larvae stage. Taking advantage of crosses between the UAS-MarfRNAi flies and lines carrying both a UAS-mitochondrial GFP (mtGFP) and the Gal4 transgene under the control of different tissue-specific promoters, it was noticed that in ubiquitous MarfRNAi larvae, mitochondria were clustered and fragmented in ventral ganglion neuronal cell bodies and small and clumped in ventral ganglion axons. Likewise, in the smaller MarfRNAi muscles, mitochondria appeared clustered in the perinuclear region. When MarfRNAi expression was spatially restricted using the neuronal Elav-Gal4 and the muscular Mef2-Gal4 driver lines, different degrees of lethality were observed but very similar mitochondrial morphological alterations. In the nervous system and in the muscle, Marf ablation was lethal at the pupa stage; however, some escapers (~10%) reached adulthood when it was ablated in the muscle. Mitochondria were fragmented and clustered in neuronal cell bodies and lost their sarcomeric organization to clump around the nuclei in the muscle. Thus, Marf is essential for normal mitochondrial morphology in all the tissues in which it was ablated (Debattisti, 2014).

To explore also whether ER shape was altered by Marf ablation in vivo, MarfRNAi lines carrying an ER fluorescent reporter (ER-GFP) were generated. In control ventral ganglion neuronal cell bodies, the ER appeared to be interconnected throughout the whole cytoplasm, and in the muscle, the sarcoplasmic reticulum (SR) displayed a typical sarcomeric organization with interconnected cisternae. Upon neuron-specific Marf ablation, fragmented ER cisternae accumulated in the cell cortex. In muscles from ubiquitous and muscle-specific MarfRNAi larvae, the sarcomeric SR pattern was lost with punctiform and individual cisternae. The ER derangement was further supported by electron micrographs of Marf-ablated neuronal cell bodies showing dilated, fragmented ER cisternae and by assays of fluorescence loss in photobleaching (FLIP) in which ER-GFP diffusion was slower in MarfRNAi SR, indicative of a loss of SR cisternae continuity. The in vivo morphological analysis demonstrates that Marf ablation affects mitochondrial as well as ER shape. The role of lower, nonmetazoan Mfns in ER shape is not explored, but it appears that Fzo1p, the Saccharomyces cerevisiae Mfn is not involved in ER-mitochondria tethering, performed instead by a specialized complex. Perhaps, in chordates, the requirement to separate ER-mitochondria tethering and cross talk from mitochondrial fusion in tissues pressured for the emergence of two Mfns: for example, in the heart, both Mfns are abundantly expressed, but although mitochondrial fusion is not apparent, Mfn2-dependent SR-mitochondrial juxtaposition is crucial for mitochondrial Ca2+ uptake, consistent with the impact of Mfn2 ablation on cardiomyocyte development. The data show that the primordial metazoan Mfn regulates mitochondrial as well as ER shape (and ER-mitochondria tethering in S2 cells), indicating in Marf the potential ancestor of the two functionally different chordate Mfns (Debattisti, 2014).

The relative contribution of the coexisting mitochondrial and ER alterations to the phenotypes of MarfRNAi flies were assessed. Whether hMfn 1 and 2, which differ in their ability to modulate ER morphology and cross talk with mitochondria (de Brito, 2008; Cerqua, 2010; De Stefani, 2011; Chen, 2012; Ngoh, 2012), were equally efficient in complementing MarfRNAi flies was tested. UAS transgenic lines were generated for the ectopic expression of Myc-tagged hMfn1, hMfn2, or hMfn2R94Q, a mutation associated with CMTIIA neuropathy that complements Mfn2-/- mitochondrial, but not ER, defects. Because neuronal- and muscle-specific hMfn expression was compatible with life, neuronal or muscle expression was driven of UAS-MarfRNAi and UAS-hMfns-Myc. Although the three hMfns were expressed at comparable levels in the transgenic larvae brain, only hMfn2 partially rescued the MarfRNAi lethality, allowing survival to adulthood of ~50% of the individuals. hMfn2 efficacy was not increased by coexpression with hMfn1, which alone or with hMfn2R94Q (a combination that fully complements Mfn2-/- mitochondrial, but not ER, morphology) was unable to rescue MarfRNAi lethality. In hMfn2-expressing MarfRNAi flies, neuronal cell body mitochondria were fragmented, but ER morphology was restored. The picture was similar in muscle-specific MarfRNAi flies: only hMfn2 could partially rescue the developmental defect and restored SR, but not mitochondrial, morphology (Debattisti, 2014).

These data were extended by addressing whether hMfn2 could also functionally rescue MarfRNAi flies. Using the weak myosin heavy chain (MHC)-Gal4 promoter, it was possible to obtain muscle-specific MarfRNAi adult individuals. In these muscle-specific MarfRNAi flies, hMfn2 selectively corrected the SR dysmorphology and, in a routinely used test for Drosophila locomotor performance, rescued the impaired climbing performance of MarfRNAi flies. Thus, hMfn2 complements MarfRNAi fly development and function, correcting their ER shape. This model of weak muscle Mfn ablation appears useful to screen for genetic interactors that ameliorate the climbing performance, impossible in the available mouse models of CMTIIa, and to screen for drugs that might ameliorate the locomotor phenotype (Debattisti, 2014).

Having established that ER shape defects correlate with MarfRNAi fly climbing defects, it was asked whether ER dysmorphology was accompanied by ER stress. The prototypical ER stress inducer DTT switched on a genetically encoded GFP reporter of the ER stress transcription factor Xbp1 in numerous neurons of treated larvae. Likewise, GFP-positive neurons were found when this reporter line was crossed with the MarfRNAi flies. Another marker, the ER stress-induced chaperone binding Ig protein (BiP), accumulated in muscles and neurons upon ubiquitous Marf ablation. ER stress was not, however, a common response to mitochondrial fusion ablation because, in flies lacking Opa1, the GFP Xbp1 reporter was not activated, and the levels of BiP were normal. To assess the contribution of ER stress to the MarfRNAi phenotype, flies were examined expressing a constitutively spliced Xbp1 variant (mXbp1s) that ameliorates phenotypes caused by ER stress in flies. Expression of mXbp1s partially rescued the developmental defect of MarfRNAi flies, ameliorating ER and SR, but not mitochondrial, morphology in ubiquitous and muscle-specific MarfRNAi individuals. Most importantly, once expressed in muscle-specific MarfRNAi flies, Xbp1s corrected the impaired climbing performance. The conclusions from these genetic experiments were further corroborated when it was observed that the chemical chaperones tauroursodeoxycholic acid (TUDCA) and 4-phenylbutyric acid (PBA), which reduce ER stress in vitro and in vivo, reduced ER stress, ameliorated development, and improved climbing of MarfRNAi flies. Collectively, these data indicate that genetic and pharmacological ER stress manipulation is sufficient to restore ER morphology and locomotor function in MarfRNAi Drosophila (Debattisti, 2014).

In principle, the ER stress and fragmentation observed in Marf-deficient tissues could be secondary to mitochondrial dysfunction as, for example, it was reported for a mouse model of myopathy. However, multiple evidence support a specific role for Marf in ER function: (a) Opa1 ablation does not cause ER stress; (b) hMfn2 selectively complements ER, but not mitochondrial, shape in MarfRNAi flies; and (c) Marf ablation resembles the ER stress caused by Mfn2 ablation in mammals (Ngoh, 2012), with far-reaching implications for insulin production and neuronal control of metabolism (Debattisti, 2014).

This analysis substantiates a model in which Mfn2 regulates ER shape and function also in the fruit fly. Despite the numerous functional differences between Mfn1 and Mfn2, they are believed to be functionally interchangeable. The current data show in vivo that the two Mfns are functionally different and indicate that ER shape and function are regulated only by Mfn2. This genetic analysis indicates that the metazoan Marf harbors both mitochondria- and ER-shaping functions of mammalian Mfns (Debattisti, 2014).

ER stress attenuation emerges as a new potential pharmacological target for CMTIIa in which Mfn2 is mutated and for other conditions in which it is malfunctioning. In a proof-of-principle experiment, the locomotor defects of flies lacking Marf in the muscle were corrected by two ER chemical chaperones, including TUDCA, an already FDA-approved drug that holds potential to be further tested in CMTIIa models (Debattisti, 2014).

Control of mitochondrial structure and function by the Yorkie/YAP oncogenic pathway

Mitochondrial structure and function are highly dynamic, but the potential roles for cell signaling pathways in influencing these properties are not fully understood. Reduced mitochondrial function has been shown to cause cell cycle arrest, and a direct role of signaling pathways in controlling mitochondrial function during development and disease is an active area of investigation. This study shows that the conserved Yorkie/YAP signaling pathway implicated in the control of organ size also functions in the regulation of mitochondria in Drosophila as well as human cells. In Drosophila, activation of Yorkie causes direct transcriptional up-regulation of genes that regulate mitochondrial fusion, such as opa1-like (opa1) and mitochondria assembly regulatory factor (Marf), and results in fused mitochondria with dramatic reduction in reactive oxygen species (ROS) levels. When mitochondrial fusion is genetically attenuated, the Yorkie-induced cell proliferation and tissue overgrowth are significantly suppressed. The function of Yorkie is conserved across evolution, as activation of YAP2 in human cell lines causes increased mitochondrial fusion. Thus, mitochondrial fusion is an essential and direct target of the Yorkie/YAP pathway in the regulation of organ size control during development and could play a similar role in the genesis of cancer (Nagaraj, 2012).

Previous studies have shown that the Hippo pathway functions in flies as well as vertebrates to control organ size. Furthermore, mutations in components of this pathway have been implicated in multiple forms of cancer. This pathway has been shown to directly promote cell proliferation and repress apoptosis. This study shows that the mitochondrion is an important additional target of the Hippo pathway. An increase in Yki activity causes an increase in mitochondrial fusion due to direct transcriptional activation of major mitochondrial fusion genes. Increased mitochondrial fusion has been linked to regulation of the G1-S checkpoint and Cyclin E activity, and cells harboring fused mitochondria are resistant to stress-induced apoptosis. Interestingly, these are precisely the phenotypes seen upon activation of the Hpo/Yki pathway. It seems likely that this pathway independently activates the cell cycle, represses apoptosis, and promotes mitochondrial and metabolic changes described in this study that together cause tumor growth. However, since mitochondrial fusion plays a role in S-phase entry and stress resistance (Mitra, 2009), it is attractive to speculate that the mitochondrial effects could indirectly affect Yki's up-regulation of Cyclin E and DIAP. This is supported by-observation that in hpo, opa1 double-mutant clones, Cyclin E expression is suppressed when compared with that seen in hpo mutant clones. The increase in fusion gene levels upon Yki activation is modest. This may be important in producing the observed phenotype of fused but functional mitochondria. Gross overexpression of fusion genes leads to abnormal and globular mitochondria. Moreover, the modification of mitochondrial structure by Yki is accompanied by an up-regulation of antioxidant enzymes and subunits of complex I of the electron transport chain and a dramatic reduction in intracellular ROS. It has recently been demonstrated that other oncogenes (Ras, Raf, and Myc) also reduce ROS, but in the current system, these oncogenes do not up-regulate mitochondrial fusion as seen with activation of the Yki pathway, suggesting that different oncogenic pathways could alter ROS by distinct mechanisms. The importance of mitochondrial fusion in Yki signaling is further highlighted by the observation that a reduction in mitochondrial fusion suppresses Yki-mediated growth phenotypes. This observed link between the Hpo/Yki pathway and mitochondrial fusion is of significance to both normal development and cancer biology. This study reveals that increased mitochondrial fusion and expansion by Yki is evolutionarily conserved and that this function of the Hippo pathway is relevant for both normal and patho-physiological situations (Nagaraj, 2012).

DRP1-dependent mitochondrial fission initiates follicle cell differentiation during Drosophila oogenesis

Exit from the cell cycle is essential for cells to initiate a terminal differentiation program during development, but what controls this transition is incompletely understood. This paper demonstrates a regulatory link between mitochondrial fission activity and cell cycle exit in follicle cell layer development during Drosophila melanogaster oogenesis. Posterior-localized clonal cells in the follicle cell layer of developing ovarioles with down-regulated expression of the major mitochondrial fission protein DRP1 had mitochondrial elements extensively fused instead of being dispersed. These cells did not exit the cell cycle. Instead, they excessively proliferated, failed to activate Notch for differentiation, and exhibited downstream developmental defects. Reintroduction of mitochondrial fission activity or inhibition of the mitochondrial fusion protein Marf-1 in posterior-localized DRP1-null clones reversed the block in Notch-dependent differentiation. When DRP1-driven mitochondrial fission activity was unopposed by fusion activity in Marf-1-depleted clones, premature cell differentiation of follicle cells occurred in mitotic stages. Thus, DRP1-dependent mitochondrial fission activity is a novel regulator of the onset of follicle cell differentiation during Drosophila oogenesis (Mitra, 2012).

The Drosophila follicle cell layer encapsulates egg chambers containing 15 nurse cells and one oocyte. The follicle cells comprising this cell layer progress through different developmental stages. During stages 1-5 (S1-5), most follicle cells undergo mitotic divisions, with a few cells exiting the mitotic cycle under Notch activation to form stalk cells separating consecutive egg chambers. During S6-8, all follicle cells exit the mitotic cycle in response to Notch activation and differentiate into an endocycling, polarized epithelium patterned into posterior follicle cells (PFCs), main body cells (MBCs), and anterior follicle cells (AFCs). To examine the effect of inhibiting mitochondrial fission activity in this system, Drosophila follicle cell clones where generated mozygous for a functionally null allele of DRP1 called drp1KG. Clones were identified by lack of a ubiquitin promoter-GFP (UbiGFP) label in their nucleus. The potentiometric dye tetramethylrhodamine ethyl ester (TMRE), which incorporates into the mitochondrial matrix, was used to label mitochondria (Mitra, 2012).

In an S10 egg chamber, nonclonal cells containing a nuclear UbiGFP label have mitochondrial elements widely distributed. Microirradiation at a single point within mitochondria of these cells triggers depolarization (i.e., loss of fluroescent TMRE signal) only at the irradiated site, with little loss of TMRE outside the microirradiated site. This suggested the mitochondrial network of these cells is discontinuous. In drp1KG clones (no UbiGFP label), mitochondria were tightly clustered in a small region of each cell. Single-point microirradiation of mitochondria in a drp1KG clone depolarizes the cell's entire mitochondrial cluster, with complete loss of TMRE signal in 5 s. This indicated that mitochondria in drp1KG clones are highly fused. Reduced mitochondrial fission in drp1KG clones, therefore, causes normally fragmented mitochondrial elements in follicle cells to hyperfuse into a tight cluster (Mitra, 2012).

Next, weather presence of drp1KG clones affects follicle epithelial layer organization was examined. In S6-8 egg chambers, follicle cells normally form a single epithelial monolayer. The presence of drp1KG clones, however, disrupts this monolayer arrangement. The effect is most striking in the PFC region, in which drp1KG clones massively overproliferate. The overpopulated clones undergo mitotic cycling even at S10 or later: they incorporate BrdU, demonstrating that they synthesize DNA, and stain with pH3 antibody, indicating that they transit through mitosis. Surrounding heterozygous tissue and drp1KG MBC clones, in contrast, are postmitotic: they neither incorporate BrdU nor stain for pH3. DRP1 depletion thus prevents cell cycle exit primarily in drp1KG PFC clones, leading to their overpopulation in postmitotic egg chambers (Mitra, 2012).

As cell cycle exit is a prerequisite for initiating differentiation, whether the drp1KG PFCs are prevented from differentiating was examined. Follicle cells in S6-8 egg chambers normally undergo cell cycle exit to differentiate under the influence of the homeodomain gene Hindsight (Hnt). Notably, clones of drp1KG in the PFC region marked by CD8GFP fail to express Hnt, unlike surrounding nonclonal cells. 95% of drp1KG PFC clones show this phenotype, whereas no drp1KG MBC clonal cells do. Thus, drp1KG PFC clones fail to differentiate (Mitra, 2012).

Hnt expression is rescued in all drp1KG PFC clones generated in the background of HA-DRP1 and in 43% of drp1KG PFC clones with DRP1 reintroduced into them. In both conditions, DRP1 expression prevented the clustered mitochondrial phenotype. Lack of differentiation in drp1KG PFC clones, therefore, results from loss of DRP1 activity (Mitra, 2012).

Down-regulation of Marf-1, the Drosophila homologue of mitofusins (Deng, 2008), combined with DRP1 down-regulation in drp1KG PFC clones causes 22% of the clones to now partially express Hnt. Because Marf-1 RNAi expression causes mitochondrial fragmentation when expressed alone or in drp1KG PFC clones, it was concluded that fragmentation of mitochondria reverses the differentiation block in drp1KG PFCs. Therefore, DRP1-driven mitochondrial fission is required for PFCs to differentiate. Loss of function of the inner mitochondrial membrane fusion protein OPA1 caused cell death in this system (Mitra, 2012).

Differentiation of Drosophila follicle cells requires Notch receptor activation. Upon ligand binding, the Notch receptor is cleaved to release the Notch intracellular domain (NICD), which redistributes into the nucleus to activate genes required for differentiation. To investigate whether DRP1-driven mitochondrial fission activity acts upstream or downstream of Notch activation in driving PFC differentiation, whether NICD is cleaved and released from the plasma membrane was examined in drp1KG PFC clones. Significant NICD levels are retained on the plasma membrane in drp1KG PFC clones marked by CD8GFP relative to nonclonal cells in S6-8 egg chambers. The Notch extracellular domain (NECD) is also retained on the plasma membrane in these clones, confirming that Notch is inactive. In addition, Cut down-regulation, which occurs in response to Notch activation, does not occur in drp1KG PFC clones. DRP1-driven mitochondrial fission activity thus acts upstream of Notch activation to drive PFC differentiation (Mitra, 2012).

NICD loss from the membrane (indicative of Notch activation) increases by 28.2% in drp1KG PFC clones after Marf-1 down-regulation. This suggested that Notch inactivation in drp1KG PFC clones is related to mitochondria being highly fused, with mitochondrial fission a prerequisite for Notch receptor activation in the PFCs. Importantly, expression of an activated Notch (N-Act) domain in drp1KG PFC clones partially overrides the differentiation block in 53% of drp1KG PFC clones, resulting in Hnt expression in these clones. As this occurs without the fused mitochondrial morphology of drp1KG PFC clones changing, the data confirmed that DRP1's role in triggering PFC differentiation is upstream of Notch (Mitra, 2012).

Why is DRP1's role in triggering follicle cell differentiation specific to PFCs? Indeed, drp1KG MBC clones show no differentiation block, as Notch activation still occurs in drp1KG MBC clones. Higher levels of bound DRP1 was found in PFCs compared with MBCs after cell permeabilization with digitonin, which may reflect different mitochondrial morphology between PFCs and MBCs. Supporting this, in S6-8 ovarioles it was found that mitochondria in PFCs exist as dispersed fragments both apically and basolaterally, whereas mitochondria in MBCs are tightly clustered at the lateral side of the nucleus. After S9, no observable differences were seen in mitochondrial morphology (Mitra, 2012).

Fluorescence loss in photobleaching (FLIP) experiments in follicle cells of S6-8 egg chambers revealed that the dispersed mitochondria of PFCs have less matrix continuity relative to the fused mitochondrial cluster of MBCs. Furthermore, single-point microirradiation caused a 44% loss in TMRE mitochondrial signal per MBC compared with a 12% loss per PFC. The rapid loss of mitochondrial TMRE signal in MBCs was similar to drp1KG clonal cells, with mitochondrial morphology in wild-type MBCs indistinguishable from that of drp1KG MBC clones. Together, the observed differences in mitochondrial organization and bound DRP1 levels in PFCs and MBCs suggested greater DRP1-driven mitochondrial fission activity occurs in PFCs relative to MBCs. This corroborates findings that PFCs, unlike MBCs, differentiate under the influence of DRP1 (Mitra, 2012).

PFCs are known to be specified by EGF receptor (EGFR) signaling. In egfrt1/egfrt1 egg chambers (hypomorphic allele of EGFR), mitochondria in PFCs are primarily clustered to one side of the nucleus, in contrast to those in wild-type or egfrt1/+ egg chambers, in which mitochondria are dispersed throughout cells. A similar clustering of mitochondria occurs when a dominant-negative (DN) form of EGFR (EGFR-DN) is clonally expressed in the PFC population. Because PFC mitochondria cluster/fuse in the absence of EGFR signaling, the data suggest that EGFR activation in PFCs promotes mitochondria fragmentation in these cells. This could explain why MBCs, which do not receive the EGFR signal, have fused mitochondria. The underlying basis for how EGFR signaling influences mitochondrial dynamics (by altering fission or fusion components) requires further investigation (Mitra, 2012).

Interestingly, PFCs expressing EGFR-DN did not escape differentiation in spite of having clustered mitochondria. This may imply that a highly fused mitochondrial cluster may only allow escape from differentiation in the context of activated EGFR signaling. Indeed, EGFR-DN expression in drp1KG PFC clones (with fused mitochondria) partially induces differentiation (i.e., Hnt expression) in 40% of the clonal cells compared with no Hnt expression in drp1KG PFC clone. Expression of an activated form of EGFR (EGFR-Act) did not induce differentiation in drp1KG PFC clones. This explains why MBCs, which are not exposed to the EGFR ligand, do not proliferate under DRP1 down-regulation. Thus, cross talk exists between mitochondria and the EGFR signaling pathway in postmitotic PFCs, which helps cells decide whether to differentiate or continue in the mitotic cycle (Mitra, 2012).

Whether DRP1 activity is important for regulation of cell cycle exit of mitotic follicle cells to allow onset of differentiation was investigated. The majority of follicle cells in S1-5 (during which all cells are mitotic) have fragmented mitochondria, suggesting that DRP1-dependent fission activity is high. drp1KG follicle cell clones introduced into the mitotic follicle cell layer and lacking UbiGFP harbor characteristic mitochondrial clusters. Clones also contain more pH3-positive cells and have qualitatively greater incorporation of BrdU relative to nonclonal tissue, with Cut expression unaltered. Without DRP1, therefore, S1-5 follicle cells undergo faster mitotic cycling (Mitra, 2012).

To test whether DRP1 activity is necessary for mitotic cells to differentiate, Marf-1 RNAi was expressed to allow unopposed DRP1 activity in S1-5 egg chambers. Strikingly, Marf-1 RNAi expressing follicle cell clones (marked by CD8GFP) show premature expression of Hnt, whereas neighboring nonclonal mitotic follicle cells do not. The effect is not restricted to any stage or region of the mitotic follicle cell layer. The Marf-1 RNAi follicle cell clones exhibit increased mitochondrial mass as assessed by HSP-60 staining and MitoTracker loading, similar to that reported previously from mitofusin knockout mice . Importantly, drp1KG follicle cell clones expressing Marf-1 RNAi do not show premature differentiation; Hnt and HSP-60 expression levels are comparable with wild-type cells. Therefore, the premature differentiation of Marf-1 RNAi clones is dependent on DRP1. This indicates that DRP1-driven mitochondrial fission activity is required for mitotic follicle cells to exit the cell cycle and initiate their differentiation regimen (Mitra, 2012).

Because of DRP1's role in differentiation, lack of DRP1 should generate developmental defects. Consistently, DRP1 down-regulation in early follicle cells in the germarium inhibits stalk cell formation, required to separate consecutive egg chambers. The missing stalk cells in egg chambers, encapsulated by early drp1KG follicle cell clones, leads to fused egg chambers containing pH3-labeled drp1KG clonal cells that lack UbiGFP. FasIII-enriched polar cells, known to induce stalk cells, are seen in wild-type ovarioles but are absent in the drp1KG clonal follicle cell population. Lack of polar cells is not the basis of cell proliferation of drp1KG PFCs because FasIII-positive polar cells appear in the surrounding heterozygous tissue. In addition, compound egg chambers with drp1KG follicle stem cell clones frequently arise, including egg chambers with 30 nurse cells and two oocytes (Mitra, 2012).

Down-regulation of DRP1 also causes developmental defects in the postmitotic follicle cell layer. There, in 22% of the cases, drp1KG PFC clones fail to trigger migration of the oocyte nucleus toward the anterior. The postmitotic stage drp1KG phenotypes resemble loss of function of the Hippo-Salvador-Warts pathway, which has tumor suppressor effects in higher organisms, including mice (Mitra, 2012).

The observed link between cell differentiation and mitochondrial fission state during oogenesis could relate to cyclin E, which controls S-phase entry. Indeed, inhibition of mitochondrial ATP synthesis in a cytochrome oxidase mutant promotes specific degradation of cyclin E (but not other cyclins) and blocks S-phase entry in Drosophila. In fibroblasts, cyclin E levels increase under conditions of DRP1 inhibition. In Drosophila follicle cells cyclin E levels were found to increase when DRP1 is down-regulated and decrease when Marf-1 is down-regulated. This suggests that DRP1-driven mitochondrial fission activity may cause cell cycle exit by lowering cyclin E levels to allow differentiation (Mitra, 2012).

The results support a model in which mitochondrial fission/fusion dynamics regulates cell differentiation across the follicle cell layer of the Drosophila ovariole (see A model for mitochondria's role in cell fate determination). In mitotic stages, increased DRP1-driven mitochondrial fission is required for cell cycle exit as noted in premature DRP1-dependent differentiation of Marf-1 RNAi clones and enhanced proliferation of drp1KG clones. During postmitotic transition, activation of EGFR in the posterior region causes mitochondrial fragmentation. This, in turn, permits cell cycle exit and Notch activation, which drives PFC differentiation. In drp1KG PFC clones with fused mitochondria, therefore, Notch remains inactive, and cells proliferate. In the main body region, not exposed to the EGFR ligand, postmitotic differentiation and patterning occur in the absence of DRP1. Thus, cell proliferation/differentiation mechanisms have an intimate relationship to mitochondrial morphology and function during follicle layer development (Mitra, 2012).

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. This study reports that Drosophila Reaper can induce mitochondrial fragmentation by binding to and inhibiting the pro-fusion protein MFN2 and its Drosophila counterpart dMFN/Marf. These in vitro and in vivo analyses reveal that dMFN overexpression can inhibit cell death induced by Reaper or gamma-irradiation. In addition, knockdown of dMFN causes a striking loss of adult wing tissue and significant apoptosis in the developing wing discs. These 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 (Thomenius, 2011).

MARF and Opa1 control mitochondrial and cardiac function in Drosophila

Mitochondria interact via actions of outer and inner membrane fusion proteins. The role of mitochondrial fusion in functioning of the heart, where mitochondria comprise approximately 30% of cardiomyocyte volume and their intermyofilament spatial arrangement with other mitochondria is highly ordered, is unknown. This study modeled and analyzed mitochondrial fusion defects in Drosophila melanogaster heart tubes with tincΔ4Gal4-directed expression of RNA interference (RNAi) for mitochondrial assembly regulatory factor (MARF) and optic atrophy (Opa)1. Live imaging analysis revealed that heart tube-specific knockdown of MARF or Opa1 increases mitochondrial morphometric heterogeneity and induces heart tube dilation with profound contractile impairment. Sarcoplasmic reticular structure was unaffected. Cardiomyocyte expression of either human mitofusin (mfn)1 or -2 rescued MARF RNAi cardiomyopathy, demonstrating functional homology between Drosophila MARF and human mitofusins. Suppressing mitochondrial fusion increased compensatory expression of nuclear-encoded mitochondrial genes, indicating mitochondrial biogenesis. The MARF RNAi cardiomyopathy was prevented by transgenic expression of superoxide dismutase. It is concluded that mitochondrial fusion is essential to cardiomyocyte mitochondrial function and regeneration. Reactive oxygen species are key mediators of cardiomyopathy in mitochondrial fusion-defective cardiomyocytes. Postulated mitochondrial-endoplasmic reticulum interactions mediated uniquely by mfn2 appear dispensable to functioning of the fly heart (Dorn, 2011).

The mitochondrial fusion-promoting factor mitofusin is a substrate of the PINK1/parkin pathway

Loss-of-function mutations in the PINK1 or parkin genes result in recessive heritable forms of parkinsonism. Genetic studies of Drosophila orthologs of PINK1 and parkin indicate that PINK1, a mitochondrially targeted serine/threonine kinase, acts upstream of Parkin, a cytosolic ubiquitin-protein ligase, to promote mitochondrial fragmentation, although the molecular mechanisms by which the PINK1/Parkin pathway promotes mitochondrial fragmentation are unknown. This study tested the hypothesis that PINK1 and Parkin promote mitochondrial fragmentation by targeting core components of the mitochondrial morphogenesis machinery for ubiquitination. The steady-state abundance of the mitochondrial fusion-promoting factor Drosophila Mitofusin (Mitochondrial assembly regulatory factor, Marf or dMfn) is inversely correlated with the activity of PINK1 and Parkin in Drosophila. dMfn is ubiquitinated in a PINK1- and Parkin-dependent fashion and dMfn co-immunoprecipitates with Parkin. By contrast, perturbations of PINK1 or Parkin did not influence the steady-state abundance of the mitochondrial fission-promoting factor Drp1 or the mitochondrial fusion-promoting factor Opa1, or the subcellular distribution of Drp1. These findings suggest that dMfn is a direct substrate of the PINK1/Parkin pathway and that the mitochondrial morphological alterations and tissue degeneration phenotypes that derive from mutations in PINK1 and parkin result at least in part from reduced ubiquitin-mediated turnover of dMfn (Poole, 2010).

In previous work, it has been shown that genetic manipulations that promote mitochondrial fragmentation, including increased drp1 gene dosage and decreased opa1 or dmfn gene dosage, dramatically suppress the PINK1 and parkin mutant phenotypes in Drosophila. These findings, coupled with previous work demonstrating that PINK1 acts upstream of Parkin in a common pathway, led to a hypothesis that PINK1 and Parkin influence mitochondrial integrity by regulating core components of the mitochondrial morphogenesis machinery through ubiquitination. The current results provide direct support for this hypothesis by demonstrating that dMfn is ubiquitinated in a PINK1- and Parkin-dependent fashion and that the steady-state abundance of dMfn is increased in PINK1 and parkin mutants and decreased in PINK1- and Parkin-overexpressing flies. These findings suggest a model in which PINK1 phosphorylates either dMfn or Parkin and thereby increases the efficiency with which Parkin is able to ubiquitinate dMfn. The subsequent ubiquitin-mediated turnover of dMfn would then inhibit mitochondrial fusion, and thus promote mitochondrial fragmentation. While the current findings were in review, another study primarily using cultured Drosophila S2 cells (Ziviani, 2010) also reported that dMfn is a substrate of the PINK1/Parkin pathway, thus providing additional support for these conclusions (Poole, 2010).

The finding that the PINK1/Parkin pathway promotes mitochondrial fragmentation led to a proposal that this pathway may act to segregate damaged portions of the mitochondrial reticulum for turnover through an autophagic mechanism . Several recent studies provide compelling support for this hypothesis by demonstrating that treatment of cultured vertebrate cells with mitochondrial damaging agents triggers PINK1 to selectively recruit Parkin to damaged mitochondria, where Parkin acts to promote the autophagic turnover of these mitochondria, presumably by ubiquitinating particular mitochondrial. These studies, together with the current findings raise the possibility that the selective Parkin-mediated ubiquitination and subsequent degradation of dMfn on damaged portions of the mitochondrial reticulum, coupled with ongoing mitochondrial fission serves to sequester the mitochondrial damage to small fusion-incompetent mitochondria that are subsequently eliminated through autophagy. However, the size of ubiquitinated dMfn suggests that it is triply ubiquitinated and previous work indicates that a chain of four or more ubiquitins is required for efficient targeting to the proteasome. Thus, alternative interpretations of the findings, although not mutually exclusive, are that ubiquitination of dMfn inactivates the fusion-promoting activity of dMfn, or serves as a tag marking the damaged mitochondria for destruction by autophagy. The finding that the ubiquitination of a peroxisomal surface protein is sufficient to signal the autophagic degradation of this organelle is consistent with the latter model. Experiments are currently underway to distinguish these possibilities (Poole, 2010).

While a model in which the PINK1/Parkin pathway promotes mitochondrial fragmentation through the ubiquitination of dMfn is completely consistent with previous work on PINK1 and Parkin in Drosophila, recent findings from vertebrate cell culture studies challenge this model. In particular, several of the studies of PINK1 in vertebrate systems have found that reduced PINK1 activity results in mitochondrial fragmentation, suggesting that PINK1 may promote mitochondrial fusion -- exactly the opposite of the conclusion drawn from studies of the PINK1/Parkin pathway in flies. While additional work will be required to resolve these apparent conflicts, it is important to point out that the findings from studies of PINK1 and Parkin in flies have involved tissues that are profoundly affected by loss of PINK1 and Parkin activity, whereas the tissue sources of the cells that have been used in at least some of the conflicting vertebrate studies are largely unaffected by mutations in PINK1 and parkin. Thus, a possible explanation for these apparently discordant findings is that the mitochondrial fragmentation resulting from reduced PINK1 activity that has been observed in vertebrate systems involves a compensatory induction of mitochondrial fragmentation in these cells, which perhaps also explains their relative insensitivity to the loss of PINK1 activity. In potential support of this model is the finding that while the mitochondrial fragmentation seen in PINK1-deficient vertebrate cells can be rescued by inactivating Drp1, this manipulation enhances the cell death associated with PINK1 deficiency, a finding that is entirely consistent with work in flies. Future work should resolve these apparent conflicts and further clarify the influence of PINK1- and Parkin-dependent ubiquitination of dMfn on mitochondrial integrity (Poole, 2010).

Drosophila parkin requires PINK1 for mitochondrial translocation and ubiquitinates mitofusin

Loss of the E3 ubiquitin ligase Parkin causes early onset Parkinson's disease, a neurodegenerative disorder of unknown etiology. Parkin has been linked to multiple cellular processes including protein degradation, mitochondrial homeostasis, and autophagy; however, its precise role in pathogenesis is unclear. Recent evidence suggests that Parkin is recruited to damaged mitochondria, possibly affecting mitochondrial fission and/or fusion, to mediate their autophagic turnover. The precise mechanism of recruitment and the ubiquitination target are unclear. This study shows in Drosophila cells that PINK1 is required to recruit Parkin to dysfunctional mitochondria and promote their degradation. Furthermore, PINK1 and Parkin mediate the ubiquitination of the profusion factor Mfn on the outer surface of mitochondria. Loss of Drosophila PINK1 or parkin causes an increase in Mfn abundance in vivo and concomitant elongation of mitochondria. These findings provide a molecular mechanism by which the PINK1/Parkin pathway affects mitochondrial fission/fusion as suggested by previous genetic interaction studies. It is hypothesized that Mfn ubiquitination may provide a mechanism by which terminally damaged mitochondria are labeled and sequestered for degradation by autophagy (Ziviani, 2010).

Maintenance of mitochondrial homeostasis appears to be an important function of the PINK1/Parkin pathway in multiple model systems and is likely a key factor in mediating neurodegeneration. Recent studies have begun to shed light on the potential mechanism by which this pathway maintains a healthy mitochondrial population. Emerging evidence indicates that PINK1 is required to recruit Parkin to damaged or dysfunctional mitochondria, whereupon it promotes mitophagy. Regulated mitochondrial fission and fusion events are thought to contribute to a quality control mechanism to help 'sort out' terminally damaged mitochondria for degradation. Importantly, PINK1 and parkin have previously been shown to genetically interact with components of the mitochondrial fission/fusion machinery and to affect mitochondrial morphology; however, the molecular mechanisms are not known. This study provides further evidence that PINK1 is required for Parkin translocation to damaged mitochondria and that this pathway affects mitochondrial morphology. Evidence is also provided that the PINK1/Parkin pathway promotes the ubiquitination and regulates the levels of the profusion protein Mfn, thus providing a potential molecular mechanism by which PINK1/Parkin may modulate mitochondrial dynamics (Ziviani, 2010).

Consistent with recent reports, this study found that the translocation of Parkin to damaged mitochondria and their subsequent autophagy is dependent on PINK1. However, the molecular mechanisms that promote Parkin's recruitment to mitochondria are still unclear. PINK1's kinase activity, but not mitochondrial localization, appears to be necessary for Parkin translocation. Because PINK1 can be found extramitochondrially and may directly phosphorylate Parkin, this may be a mechanism to stimulate its translocation. Alternatively, it may phosphorylate a Parkin substrate, e.g., Mfn, and thereby provide a recruitment signal. Interestingly, this study found that loss of Mfn reduces but does not eliminate Parkin translocation. Recent evidence indicates that Parkin also ubiqutinates VDAC on the outer mitochondrial surface (Geisler, 2010), suggesting that there may be multiple recruitment substrates. Although further work is required to elucidate these mechanisms, these findings suggest a molecular basis for the genetic hierarchy in which PINK1 acts upstream of Parkin (Ziviani, 2010).

To understand the role of Parkin translocation, this study took a candidate approach to identifying putative substrates. Because the function of Parkin and PINK1 has been linked with mitochondrial dynamics, key components of the mitochondrial fission and fusion machinery were surveyed for ubiquitin modification. Mfn, which localizes to the outer surface of mitochondria, was found to be ubiquitinated in a PINK1/Parkin-dependent manner and accumulates upon loss of PINK1 or parkin. Interestingly, the ubiquitinated isoforms do not show a typical ubiquitination 'ladder' but instead appear to reflect a pattern of one and three or four ubiquitin adducts. Although it remains to be shown that Parkin directly mediates this ubiquitination, there is evidence that Parkin can mediate monoubiquitination and K27 and K63 linkages. These modes of ubiquitination are not typically linked to proteasome degradation, and there is growing speculation that important pathogenic functions of Parkin may be proteasome independent (Ziviani, 2010).

Numerous elegant studies have demonstrated that the mitochondrial network is extremely dynamic and responds rapidly and reversibly to many physiological changes including potentially toxic challenges such as oxidative stress and calcium flux. Although mitochondrial remodeling can contribute to promoting cell death, it can also act in a protective manner by contributing to a quality control process that likely involves degradation by autophagy/lysosomes. Recent work has reported observations that, following a fission event, regulated fusion of daughter mitochondria can determine whether they rejoin the network or are sequestered for degradation. Refusion appears to be dependent upon the recovery of mitochondrial membrane potential after division and likely represents a mechanism to sort out terminally dysfunctional mitochondria. Because Mitofusins mediate the tethering and fusion of mitochondria via homo- and heterotypic interaction of their HR2 domains, it is hypothesized that Parkin-mediated Mfn ubiquitination may interfere with intermolecular interactions preventing fusion. Alternatively, Mfn ubiquitination may lead to a selective removal of Mfn from damaged mitochondria and thus reduce the refusion capacity of those mitochondria. Consistent with this, it was found that loss of parkin or PINK1, and hence loss of ubiquitination, leads to increased Mfn levels and mitochondrial elongation, presumably due to excess fusion. Thus, Mfn ubiquitination may provide a signal that simultaneously prevents the refusion of terminally damaged mitochondria and labels them for safe degradation by autophagy (Ziviani, 2010).

It is reasonable to suppose that under normal conditions the majority of mitochondria are relatively healthy, and thus mitochondrial turnover is an infrequent event. This is supported by the observation that Complex Vα levels are not significantly altered by decreased mitophagy. However, this rationale implies that Mfn accumulates and is selectively ubiquitinated on mitochondria targeted for degradation although this remains to be shown. Interestingly, the current findings provide a molecular mechanism that can explain the previously reported genetic interactions between PINK1 and parkin and the fission/fusion factors -- in particular, that promoting mitochondrial fragmentation by overexpression of Drp1 or by reduction of Mfn and Opa1 is able to partially suppress the locomotor deficits, muscle degeneration, and mitochondrial abnormalities. Together these findings suggest that aberrant accumulation of Mfn may mediate the loss of mitochondrial homeostasis caused by loss of PINK1 or parkin. Although further work will be needed to determine whether this contributes to PD pathogenesis, these results support the emerging hypothesis that the PINK1/Parkin pathway acts to regulate the safe degradation of terminally damaged mitochondria as a quality control mechanism (Ziviani, 2010).

The Parkinson's disease genes pink1 and parkin promote mitochondrial fission and/or inhibit fusion in Drosophila

Mutations in PTEN-induced kinase 1 (pink1) or parkin cause autosomal-recessive and some sporadic forms of Parkinson's disease. pink1 acts upstream of parkin in a common genetic pathway to regulate mitochondrial integrity in Drosophila. Mitochondrial morphology is maintained by a dynamic balance between the opposing actions of mitochondrial fusion, controlled by Mitofusin (mfn) and Optic atrophy 1 (opa1), and mitochondrial fission, controlled by drp1. This study explored interactions between pink1/parkin and the mitochondrial fusion/fission machinery. Muscle-specific knockdown of the fly homologue of Mfn (Marf) or opa1, or overexpression of drp1, results in significant mitochondrial fragmentation. Mfn-knockdown flies also display altered cristae morphology. Interestingly, knockdown of Mfn or opa1 or overexpression of drp1, rescues the phenotypes of muscle degeneration, cell death, and mitochondrial abnormalities in pink1 or parkin mutants. In the male germline, genetic interactions were observed between pink1 and the testes-specific mfn homologue fuzzy onion, and between pink1 and drp1. These data suggest that the pink1/parkin pathway promotes mitochondrial fission and/or inhibits fusion by negatively regulating mfn and opa1 function, and/or positively regulating drp1. However, pink1 and parkin mutant flies show distinct mitochondrial phenotypes from drp1 mutant flies, and flies carrying a heterozygous mutation in drp1 enhance the pink1-null phenotype, resulting in lethality. These results suggest that pink1 and parkin are likely not core components of the drp1-mediated mitochondrial fission machinery. Modification of fusion and fission may represent a novel therapeutic strategy for Parkinson's disease (Deng, 2008; full text of article).


REFERENCES

Search PubMed for articles about Drosophila Marf

Bhandari, P., Song, M. and Dorn, G.W. (2015). Dissociation of mitochondrial from sarcoplasmic reticular stress in Drosophila cardiomyopathy induced by molecularly distinct mitochondrial fusion defects. J Mol Cell Cardiol 80: 71-80. PubMed ID: 25555803

Cerqua, C., Anesti, V., Pyakurel, A., Liu, D., Naon, D., Wiche, G., Baffa, R., Dimmer, K. S. and Scorrano, L. (2010). Trichoplein/mitostatin regulates endoplasmic reticulum-mitochondria juxtaposition. EMBO Rep 11: 854-860. PubMed ID: 20930847

Chen, H., Detmer, S. A., Ewald, A. J., Griffin, E. E., Fraser, S. E. and Chan, D. C. (2003). Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development. J Cell Biol 160: 189-200. PubMed ID: 12527753

Chen, H., McCaffery, J. M. and Chan, D. C. (2007). Mitochondrial fusion protects against neurodegeneration in the cerebellum. Cell 130: 548-562. PubMed ID: 17693261

Chen, Y., Csordas, G., Jowdy, C., Schneider, T. G., Csordas, N., Wang, W., Liu, Y., Kohlhaas, M., Meiser, M., Bergem, S., Nerbonne, J. M., Dorn, G. W., 2nd and Maack, C. (2012). Mitofusin 2-containing mitochondrial-reticular microdomains direct rapid cardiomyocyte bioenergetic responses via interorganelle Ca(2+) crosstalk. Circ Res 111: 863-875. PubMed ID: 22777004

Debattisti, V. and Scorrano, L. (2013). D. melanogaster, mitochondria and neurodegeneration: small model organism, big discoveries. Mol Cell Neurosci 55: 77-86. PubMed ID: 22940086

Debattisti, V., Pendin, D., Ziviani, E., Daga, A. and Scorrano, L. (2014). Reduction of endoplasmic reticulum stress attenuates the defects caused by Drosophila mitofusin depletion. J Cell Biol 204: 303-312. PubMed ID: 24469638

de Brito, O. M. and Scorrano, L. (2008). Mitofusin 2 tethers endoplasmic reticulum to mitochondria. Nature 456: 605-610. PubMed ID: 19052620

Deng, H., Dodson, M. W., Huang, H. and Guo, M. (2008). The Parkinson's disease genes pink1 and parkin promote mitochondrial fission and/or inhibit fusion in Drosophila. Proc Natl Acad Sci U S A 105: 14503-14508. PubMed ID: 18799731

De Stefani, D., Raffaello, A., Teardo, E., Szabo, I. and Rizzuto, R. (2011). A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature 476: 336-340. PubMed ID: 21685888

Dorn, G. W., 2nd, Clark, C. F., Eschenbacher, W. H., Kang, M. Y., Engelhard, J. T., Warner, S. J., Matkovich, S. J. and Jowdy, C. C. (2011). MARF and Opa1 control mitochondrial and cardiac function in Drosophila. Circ Res 108: 12-17. PubMed ID: 21148429

Misko, A., Jiang, S., Wegorzewska, I., Milbrandt, J. and Baloh, R. H. (2010). Mitofusin 2 is necessary for transport of axonal mitochondria and interacts with the Miro/Milton complex. J Neurosci 30: 4232-4240. PubMed ID: 20335458

Mitra, K., Wunder, C., Roysam, B., Lin, G. and Lippincott-Schwartz, J. (2009). A hyperfused mitochondrial state achieved at G1-S regulates cyclin E buildup and entry into S phase. Proc Natl Acad Sci U S A 106: 11960-11965. PubMed ID: 19617534 Mitochondria undergo fission-fusion events that render these

Mitra, K., Rikhy, R., Lilly, M., Lippincott-Schwartz, J. (2012) DRP1-dependent mitochondrial fission initiates follicle cell differentiation during Drosophila oogenesis. J Cell Biol 197: 487-497. PubMed ID: 22584906

Nagaraj, R., Gururaja-Rao, S., Jones, K. T., Slattery, M., Negre, N., Braas, D., Christofk, H., White, K. P., Mann, R. and Banerjee, U. (2012). Control of mitochondrial structure and function by the Yorkie/YAP oncogenic pathway. Genes Dev 26: 2027-2037. PubMed ID: 22925885

Ngoh, G. A., Papanicolaou, K. N. and Walsh, K. (2012). Loss of mitofusin 2 promotes endoplasmic reticulum stress. J Biol Chem 287: 20321-20332. PubMed ID: 22511781

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Sandoval, H., Yao, C. K., Chen, K., Jaiswal, M., Donti, T., Lin, Y. Q., Bayat, V., Xiong, B., Zhang, K., David, G., Charng, W. L., Yamamoto, S., Duraine, L., Graham, B. H. and Bellen, H. J. (2014). Mitochondrial fusion but not fission regulates larval growth and synaptic development through steroid hormone production. Elife 3. PubMed ID: 25313867

Thomenius, M., Freel, C. D., Horn, S., Krieser, R., Abdelwahid, E., Cannon, R., Balasundaram, S., White, K. and Kornbluth, S. (2011). Mitochondrial fusion is regulated by Reaper to modulate Drosophila programmed cell death. Cell Death Differ 18: 1640-1650. PubMed ID: 21475305

Wasilewski, M., Semenzato, M., Rafelski, S. M., Robbins, J., Bakardjiev, A. I. and Scorrano, L. (2012). Optic atrophy 1-dependent mitochondrial remodeling controls steroidogenesis in trophoblasts. Curr Biol 22: 1228-1234. PubMed ID: 22658590

Westermann, B. (2010). Mitochondrial dynamics in model organisms: what yeasts, worms and flies have taught us about fusion and fission of mitochondria. Semin Cell Dev Biol 21: 542-549. PubMed ID: 20006727

Yamamoto, S., et al. (2014). A Drosophila genetic resource of mutants to study mechanisms underlying human genetic diseases. Cell 159: 200-214. PubMed ID: 25259927

Ziviani, E., Tao, R. N. and Whitworth, A. J. (2010). Drosophila parkin requires PINK1 for mitochondrial translocation and ubiquitinates mitofusin. Proc. Natl. Acad. Sci. 107(11): 5018-23. PubMed ID: 20194754


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date revised: 26 January, 2016

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