Mitochondrial Membrane Fusion Research Articles

Mitofusin Proteins Tether Proteoliposomes as Shown by Cryo-EM

A balance between mitochondrial membrane fusion and fission is required for normal mitochondrial morphology and function. Shifts in mitochondrial morphology as a result of changes in the balance of membrane fusion and fission accompany cellular differentiation and changes in metabolic state. It is thought that the severe, early-onset peripheral neuropathy Charcot-Marie-Tooth type 2A disease is a manifestation of mitochondrial fusion malfunction as a result of a mutation of one of the two human mitofusins, mitofusin 2. Our aim is to reveal the mechanism by which mitofusins cause membrane fusion. Both mammalian mitofusins contain a large GTPase domain at their N-terminus followed by two transmembrane domains and a short C-terminal domain. It is predicted that GTP hydrolysis and mitofusin conformational changes are coupled to membrane fusion. However, the precise mechanism for mitofusin-mediated membrane fusion remains an open question. In fact, it is still unknown if mitochondrial outer membrane fusion proceeds through the canonical steps of tethering, docking, fusion, and disassembly. To gain insight into the conformational changes that lead to membrane fusion we are examining the structure of mitofusins in a lipid bilayer. As two-pass transmembrane proteins, mitofusins have proven difficult to express and purify in their full-length state and are poor candidates for crystallography. Here we present a purification strategy and the first cryo-em images of full-length mitofusins in a lipid bilayer. In addition, we show that mitofusins tether proteolipsosomes by forming oligomers that interact in trans between synthetic membranes. This implies that mitofusins are sufficient for driving the first step of membrane fusion.

Altered skeletal muscle mitochondrial biogenesis but improved endurance capacity in trained OPA1‐deficient mice

The role of OPA1, a GTPase dynamin protein mainly involved in the fusion of inner mitochondrial membranes, has been studied in many cell types, but only a few studies have been conducted on adult differentiated tissues such as cardiac or skeletal muscle cells. Yet OPA1 is highly expressed in these cells, and could play different roles, especially in response to an environmental stress like exercise. Endurance exercise increases energy demand in skeletal muscle and repeated activity induces mitochondrial biogenesis and activation of fusion-fission cycles for the synthesis of new mitochondria. But currently no study has clearly shown a link between mitochondrial dynamics and biogenesis. Using a mouse model of haploinsufficiency for the Opa1 gene (Opa1(+/-)), we therefore studied the impact of OPA1 deficiency on the adaptation ability of fast skeletal muscles to endurance exercise training. Our results show that, surprisingly, Opa1(+/-) mice were able to perform the same physical activity as control mice. However, the adaptation strategies of both strains after training differed: while in control mice mitochondrial biogenesis was increased as expected, in Opa1(+/-) mice this process was blunted. Instead, training in Opa1(+/-) mice led to an increase in endurance capacity, and a specific adaptive response involving a metabolic remodelling towards enhanced fatty acid utilization. In conclusion, OPA1 appears necessary for the normal adaptive response and mitochondrial biogenesis of skeletal muscle to training. This work opens new perspectives on the role of mitochondrial dynamics in skeletal muscle cells and during adaptation to stress.

Effects of transmembrane potential and pH gradient on the cytochrome c-promoted fusion of mitochondrial mimetic membranes

The present study investigated the effects of ΔΨ and ΔpH (pH gradient) on the interaction of cytochrome c with a mitochondrial mimetic membrane composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cardiolipin (CL) leading to vesicle fusion. ΔpH generated by lowered bulk pH (pH(out)) of PCPECL liposomes, with an internal pH (pH(in)) of 8.0, favored vesicle fusion with a titration sigmoidal profile (pK(a) ~ 6.9). Conversely, ΔpH generated by enhanced pH(in) of PCPECL at a pH(out) of 6.0 favored the fusion of vesicles with a linear profile. We did not observe a significant amount of liposome fusion when ΔpH was generated by lowered pH(in) at a pH(out) of 8.0. At bulk acidic pH, ΔΨ generated by Na⁺ gradient also favored cyt c-promoted vesicle fusion. At acidic and alkaline pH(out), the presence of ΔpH and ΔΨ did not affect cytochrome c binding affinity measured by pyrene quenching. Therefore, cytochrome c-mediated PC/PE/CL vesicle fusion is dependent of ionization of the protein site L (acidic pH) and the presence of transmembrane potential. The effect of transmembrane potential is probably related to the generation of defects on the lipid bilayer. These results are consistent with previous reports showing that cytochrome c release prior to the dissipation of the ΔΨ(M) blocks inner mitochondrial membrane fusion during apoptosis.

Opa1 Is Required for Proper Mitochondrial Metabolism in Early Development

Opa1 catalyzes fusion of inner mitochondrial membranes and formation of the cristae. OPA1 mutations in humans lead to autosomal dominant optic atrophy. OPA1 knockout mice lose viability around embryonic day 9 from unknown reasons, indicating that OPA1 is essential for embryonic development. Zebrafish are an attractive model for studying vertebrate development and have been used for many years to describe developmental events that are difficult or impractical to view in mammalian models. In this study, Opa1 was successfully depleted in zebrafish embryos using antisense morpholinos, which resulted in disrupted mitochondrial morphology. Phenotypically, these embryos exhibited abnormal blood circulation and heart defects, as well as small eyes and small pectoral fin buds. Additionally, startle response was reduced and locomotor activity was impaired. Furthermore, Opa1 depletion caused bioenergetic defects, without impairing mitochondrial efficiency. In response to mitochondrial dysfunction, a transient upregulation of the master regulator of mitochondrial biogenesis, pgc1a, was observed. These results not only reveal a new Opa1-associated phenotype in a vertebrate model system, but also further elucidates the absolute requirement of Opa1 for successful vertebrate development.

Mitochondrial Delivery of Bongkrekic Acid Using a MITO-Porter Prevents the Induction of Apoptosis in Human HeLa Cells

The fact that mitochondrial dysfunction has been implicated in a variety of human diseases suggests that they would be expected as a target organelle for these diseases. Bongkrekic acid (BKA) is a chemical that functions as a ligand of the adenine nucleotide translocator and is known to potently inhibit the mitochondrial permeability transition that is associated with apoptosis. Thus, delivering it to mitochondria would be an innovative therapy for the treatment of mitochondrial diseases that are largely associated with apoptosis. Here, we report on the use of a MITO-Porter, an innovative nanocarrier for mitochondrial delivery via mitochondrial membrane fusion, for delivering BKA to mitochondria. We first constructed a BKA-MITO-Porter, in which BKA is contained in lipid envelopes of a MITO-Porter. We then confirmed that the BKA-MITO-Porter was efficiently internalized into cells and is delivered to mitochondria, similar to a conventional MITO-Porter. Moreover, we evaluated the antiapoptosis effect of the BKA-MITO-Porter in HeLa cells by measuring caspase 3/7 activity. The findings confirmed that the BKA-MITO-Porter showed a strong antiapoptosis effect compared with naked BKA. The results reported here demonstrate its potential for the use in therapies aimed at mitochondrial diseases, as a mitochondrial medicine candidate.

Mitochondrial Dynamism and Cardiac Fate

Defects in mitochondrial biogenesis are well known to contribute to cardiac dysfunction. By contrast, mechanistic details of essential homeostatic mechanisms that maintain mitochondrial health in the heart are only recently being uncovered, and the pathological potential of these processes is largely hypothetical. I will review the role of mitochondrial dynamics, focusing on cyclic organelle fission and fusion, in normal and diseased hearts. Special attention is given to recent insights into the non-canonical functioning of the mitofusin 2 (Mfn2) outer mitochondrial membrane fusion protein as a regulator of sarcoplasmic-reticular calcium crosstalk and a critical determinant of mitophagic culling of damaged mitochondria. Because mitochondrial fusion in normal adult cardiomyocytes occurs so slowly and infrequently, I postulate that the major function of Mfn2 in the heart may not be to redundantly promote mitochondrial fusion with Mfn1, but to centrally orchestrate mitochondrial quality control.

Mgm1 Alters Membrane Topology and Promotes Local Membrane Bending to Drive Mitochondrial Membrane Fusion

Cellular membrane remodeling events such as mitochondrial dynamics, vesicle budding and cell division rely on the large GTPases of the dynamin superfamily. Dynamins have long been characterized as fission molecules; however, how they mediate membrane fusion is largely unknown. Recently, we showed that the mitochondrial dynamin Mgm1 can mediate fusion by first tethering opposing membranes and undergoing nucleotide-dependent structural transition. However, it is still unclear how Mgm1 could act directly on the membrane to drive the fusion of tethered membranes. Here, we show that Mgm1 binding to the membrane could alter membrane topology and promote local membrane bending. By atomic force microscopy, we showed that Mgm1 created roughness and a tubulated structure on a supported lipid bilayer, and by monitoring giant liposomes Mgm1 recruited on and deformed the liposomes. These data suggest Mgm1 lipid interactions and changes in the protein/lipid complexes could apply forces onto the membrane to possibly promote fusion of opposing membrane. Together our data provide a possible mechanism of how Mgm1 mediates mitochondrial fusion and shed light onto how dynamins function as fusion molecules.

A uvs-5 Strain Is Deficient for a Mitofusin Gene Homologue, fzo1 , Involved in Maintenance of Long Life Span in Neurospora crassa

Mitochondria are highly dynamic organelles that continuously fuse and divide. To maintain mitochondria, cells establish an equilibrium of fusion and fission events, which are mediated by dynamin-like GTPases. We previously showed that an mus-10 strain, a mutagen-sensitive strain of the filamentous fungus Neurospora crassa, is defective in an F-box protein that is essential for the maintenance of mitochondrial DNA (mtDNA), long life span, and mitochondrial morphology. Similarly, a uvs-5 mutant accumulates deletions within its mtDNA, has a shortened life span, and harbors fragmented mitochondria, the latter of which is indicative of an imbalance between mitochondrial fission and fusion. Since the uvs-5 mutation maps very close to the locus of fzo1, encoding a mitofusin homologue thought to mediate mitochondrial outer membrane fusion, we determined the sequence of the fzo1 gene in the uvs-5 mutant. A single amino acid substitution (Q368R) was found in the GTPase domain of the FZO1 protein. Expression of wild-type FZO1 in the uvs-5 strain rescued the mutant phenotypes, while expression of a mutant FZO1 protein did not. Moreover, when knock-in of the Q368R mutation was performed on a wild-type strain, the resulting mutant displayed phenotypes identical to those of the uvs-5 mutant. Therefore, we concluded that the previously unidentified uvs-5 gene is fzo1. Furthermore, we used immunoprecipitation analysis to show that the FZO1 protein interacts with MUS-10, which suggests that these two proteins may function together to maintain mitochondrial morphology.

Membrane Tethering and Nucleotide-dependent Conformational Changes Drive Mitochondrial Genome Maintenance (Mgm1) Protein-mediated Membrane Fusion

Cellular membrane remodeling events such as mitochondrial dynamics, vesicle budding, and cell division rely on the large GTPases of the dynamin superfamily. Dynamins have long been characterized as fission molecules; however, how they mediate membrane fusion is largely unknown. Here we have characterized by cryo-electron microscopy and in vitro liposome fusion assays how the mitochondrial dynamin Mgm1 may mediate membrane fusion. Using cryo-EM, we first demonstrate that the Mgm1 complex is able to tether opposing membranes to a gap of ∼15 nm, the size of mitochondrial cristae folds. We further show that the Mgm1 oligomer undergoes a dramatic GTP-dependent conformational change suggesting that s-Mgm1 interactions could overcome repelling forces at fusion sites and that ultrastructural changes could promote the fusion of opposing membranes. Together our findings provide mechanistic details of the two known in vivo functions of Mgm1, membrane fusion and cristae maintenance, and more generally shed light onto how dynamins may function as fusion proteins.

C.O.2 DNM2 mutations cause multiple mtDNA deletions in muscle: A novel disorder of mtDNA maintenance

Centronuclear myopathy (CNM) is a congenital myopathy characterised by delayed motor milestones, progressive facial weakness, ptosis, ophthalmoplegia and centrally-located myonuclei caused by autosomal dominant mutations in the dynamin-2 gene, DNM2. Dnm2 is a large GTPase protein, one of three classical dynamins that is ubiquitously expressed, and key to regulating cytoskeleton and membrane trafficking within cells. Recently, a DNM2 (KI-Dnm2R465W) animal model was shown to develop phenotypic and morphological abnormalities similar to those observed in the human disease, with abnormal central accumulation of mitochondria in the muscle fibres of the heterozygous mice. Furthermore, mutations in OPA1 and MFN2 – also dynamin-like GTPase proteins which are involved in mitochondrial membrane fusion – have recently been shown to be associated with impaired mitochondrial DNA (mtDNA) stability, inferring a crucial role for these proteins in regulating mitochondrial networks and mtDNA maintenance. We investigated five patients with dominant DNM2 mutations. In addition to the typical features of CNM, the muscle biopsies demonstrated variable amount of cytochrome c oxidase (COX)-deficient muscle fibres indicative of mitochondrial dysfunction. We show here that these focal mitochondrial biochemical defects are due to clonally-expanded mtDNA deletions, characteristic of mtDNA maintenance abnormality. Confocal microscopy studies of patient fibroblasts reveal a quantitative disruption of the dynamic mitochondrial network associated with the p.R369W DNM2 mutation, which interestingly corresponded with the most severe mitochondrial defect in muscle. Similar results were observed in HeLa cells following Dnm2 siRNA knockdown, whilst transfection of NIH3T3 cells with mutant p.R369W Dnm2 led to a significant decrease in mitochondrial objects compared to controls. Together, our data suggest an important and emerging role for Dnm2 in mtDNA maintenance and stability.

Mitofusin 2 mutations affect mitochondrial function by mitochondrial DNA depletion

Charcot-Marie-Tooth neuropathy type 2A (CMT2A) is associated with heterozygous mutations in the mitochondrial protein mitofusin 2 (Mfn2) that is intimately involved with the outer mitochondrial membrane fusion machinery. The precise consequences of these mutations on oxidative phosphorylation are still a matter of dispute. Here, we investigate the functional effects of MFN2 mutations in skeletal muscle and cultured fibroblasts of four CMT2A patients applying high-resolution respirometry. While maximal activities of respiration of saponin-permeabilized muscle fibers and digitonin-permeabilized fibroblasts were only slightly affected by the MFN2 mutations, the sensitivity of active state oxygen consumption to azide, a cytochrome c oxidase (COX) inhibitor, was increased. The observed dysfunction of the mitochondrial respiratory chain can be explained by a twofold decrease in mitochondrial DNA (mtDNA) copy numbers. The only patient without detectable alterations of respiratory chain in skeletal muscle also had a normal mtDNA copy number. We detected higher levels of mtDNA deletions in CMT2A patients, which were more pronounced in the patient without mtDNA depletion. Detailed analysis of mtDNA deletion breakpoints showed that many deleted molecules were lacking essential parts of mtDNA required for replication. This is in line with the lack of clonal expansion for the majority of observed mtDNA deletions. In contrast to the copy number reduction, deletions are unlikely to contribute to the detected respiratory impairment because of their minor overall amounts in the patients. Taken together, our findings corroborate the hypothesis that MFN2 mutations alter mitochondrial oxidative phosphorylation by affecting mtDNA replication.

Mitofusin 2-Containing Mitochondrial-Reticular Microdomains Direct Rapid Cardiomyocyte Bioenergetic Responses Via Interorganelle Ca 2+ Crosstalk

Mitochondrial Ca(2+) uptake is essential for the bioenergetic feedback response through stimulation of Krebs cycle dehydrogenases. Close association of mitochondria to the sarcoplasmic reticulum (SR) may explain efficient mitochondrial Ca(2+) uptake despite low Ca(2+) affinity of the mitochondrial Ca(2+) uniporter. However, the existence of such mitochondrial Ca(2+) microdomains and their functional role are presently unresolved. Mitofusin (Mfn) 1 and 2 mediate mitochondrial outer membrane fusion, whereas Mfn2 but not Mfn1 tethers endoplasmic reticulum to mitochondria in noncardiac cells. To elucidate roles for Mfn1 and 2 in SR-mitochondrial tethering, Ca(2+) signaling, and bioenergetic regulation in cardiac myocytes. Fruit fly heart tubes deficient of the Drosophila Mfn ortholog MARF had increased contraction-associated and caffeine-sensitive Ca(2+) release, suggesting a role for Mfn in SR Ca(2+) handling. Whereas cardiac-specific Mfn1 ablation had no effects on murine heart function or Ca(2+) cycling, Mfn2 deficiency decreased cardiomyocyte SR-mitochondrial contact length by 30% and reduced the content of SR-associated proteins in mitochondria-associated membranes. This was associated with decreased mitochondrial Ca(2+) uptake (despite unchanged mitochondrial membrane potential) but increased steady-state and caffeine-induced SR Ca(2+) release. Accordingly, Ca(2+)-induced stimulation of Krebs cycle dehydrogenases during β-adrenergic stimulation was hampered in Mfn2-KO but not Mfn1-KO myocytes, evidenced by oxidation of the redox states of NAD(P)H/NAD(P)(+) and FADH(2)/FAD. Physical tethering of SR and mitochondria via Mfn2 is essential for normal interorganelle Ca(2+) signaling in the myocardium, consistent with a requirement for SR-mitochondrial Ca(2+) signaling through microdomains in the cardiomyocyte bioenergetic feedback response to physiological stress.

Stress-Induced Phosphorylation and Proteasomal Degradation of Mitofusin 2 Facilitates Mitochondrial Fragmentation and Apoptosis

Mitochondria play central roles in integrating pro- and antiapoptotic stimuli, and JNK is well known to have roles in activating apoptotic pathways. We establish a critical link between stress-induced JNK activation, mitofusin 2, which is an essential component of the mitochondrial outer membrane fusion apparatus, and the ubiquitin-proteasome system (UPS). JNK phosphorylation of mitofusin 2 in response to cellular stress leads to recruitment of the ubiquitin ligase (E3) Huwe1/Mule/ARF-BP1/HectH9/E3Histone/Lasu1 to mitofusin 2, with the BH3 domain of Huwe1 implicated in this interaction. This results in ubiquitin-mediated proteasomal degradation of mitofusin 2, leading to mitochondrial fragmentation and enhanced apoptotic cell death. The stability of a nonphosphorylatable mitofusin 2 mutant is unaffected by stress and protective against apoptosis. Conversely, a mitofusin 2 phosphomimic is more rapidly degraded without cellular stress. These findings demonstrate how proximal signaling events can influence both mitochondrial dynamics and apoptosis through phosphorylation-stimulated degradation of the mitochondrial fusion machinery.

Functional characterization of mitofusin‐like protein from Dictyostelium discoideum

The balance between mitochondrial fission and fusion maintains mitochondrial morphology and function. In mammals, three large GTPases ‐ Opa1 and the mitofusins Mfn1 and Mfn2 ‐ are required for the fusion of inner and outer mitochondrial membranes. The Dictyostelium mitofusin MfnA GTPase domain shares only 24% identity with the corresponding region of human Mfn1 and lacks a typical Fzo‐mitofusin family region. To define the cellular function of the Dictyostelium protein, we used molecular genetic approaches to manipulate the production level of MfnA and to follow the localization of the fluorophore‐tagged protein. In cells producing MfnA with a C‐terminal eYFP‐tag, the protein is targeted to the outer mitochondrial membrane. Overproduction of wild type MfnA and GTPase deficient mutants K132A or S133N leads to peri‐nuclear mitochondrial clustering and the phenotype is not due to any cytoskeletal defect. Unlike mammalian Mfn1/2, both N‐terminal and C‐terminal MfnA domains are required for mitochondrial targeting. N‐terminal deleted construct shows partial co‐localization with the ER, while double deletion of N‐terminal and transmembrane domain induces association with plasma membrane and contractile vacuoles. MfnA null cells have fewer and more dispersed mitochondria but appear otherwise normal. Thus our results reveal that MfnA alters mitochondrial morphology by fusing outer mitochondrial membranes.

Regulation of acid-base transporters by reactive oxygen species following mitochondrial fragmentation

Mitochondrial morphology is determined by the balance between the opposing processes of fission and fusion, each of which is regulated by a distinct set of proteins. Abnormalities in mitochondrial dynamics have been associated with a variety of diseases, including neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, and dominant optic atrophy. Although the genetic determinants of fission and fusion are well recognized, less is known about the mechanism(s) whereby altered morphology contributes to the underlying pathophysiology of these disease states. Previous work from our laboratory identified a role for mitochondrial dynamics in intracellular pH homeostasis in both mammalian cell culture and in the genetic model organism Caenorhabditis elegans. Here we show that the acidification seen in mutant animals that have lost the ability to fuse their mitochondrial inner membrane occurs through a reactive oxygen species (ROS)-dependent mechanism and can be suppressed through the use of pharmacological antioxidants targeted specifically at the mitochondrial matrix. Physiological approaches examining the activity of endogenous mammalian acid-base transport proteins in rat liver Clone 9 cells support the idea that ROS signaling to sodium-proton exchangers contributes to acidification. Because maintaining pH homeostasis is essential for cell function and viability, the results of this work provide new insight into the pathophysiology associated with the loss of inner mitochondrial membrane fusion.

Visualization of Supported Lipid Bilayer Remodelling by s-mgm1 using Correlated Confocal Fluorescence and Atomic Force Microscopy

In yeast, the GTPase s-mgm1 is responsible for the fusion of inner mitochondrial membranes, a process essential for maintenance of normal mitochondrial morphology and function. Direct, real-time visualization of the effects of s-mgm1 upon mitochondrial mimic membranes is particularly relevant to elucidating the mechanism by which it acts. Here, we utilize both confocal microscopy and AFM to demonstrate that s-mgm1 spontaneously induces GTP-independent pinching and tubulation of lipids in the gel phase. Subsequent addition of GTP causes further remodelling of the membrane. Similar experiments using ATR-FTIR suggest that the membrane induces increased order in protein conformation. Our data is consistent with a model by which s-mgm1 promotes fusion of opposing membranes by pinching and tubulation.

Less than perfect divorces: dysregulated mitochondrial fission and neurodegeneration

Research efforts during the last decade have deciphered the basic molecular mechanisms governing mitochondrial fusion and fission. We now know that in mammalian cells mitochondrial fission is mediated by the large GTPase dynamin-related protein 1 (Drp1) acting in concert with outer mitochondrial membrane (OMM) proteins such as Fis1, Mff, and Mief1. It is also generally accepted that organelle fusion depends on the action of three large GTPases: mitofusins (Mfn1, Mfn2) mediating membrane fusion on the OMM level, and Opa1 which is essential for inner mitochondrial membrane fusion. Significantly, mutations in Drp1, Mfn2, and Opa1 have causally been linked to neurodegenerative conditions. Despite this knowledge, crucial questions such as to how fission of the inner and outer mitochondrial membranes are coordinated and how these processes are integrated into basic physiological processes such as apoptosis and autophagy remain to be answered in detail. In this review, we will focus on what is currently known about the mechanism of mitochondrial fission and explore the pathophysiological consequences of dysregulated organelle fission with a special focus on neurodegenerative conditions, including Alzheimer's, Huntington's and Parkinson's disease, as well as ischemic brain damage.

Mitochondrial Fusion is Essential for Organelle Function and Cardiac Homeostasis

Mitochondria constitute 30% of myocardial mass. Mitochondrial fusion and fission appear essential for health of most tissues. Mitochondrial fission occurs in neonatal cardiomycyte and is implicated in cardiomyocyte death. Mitochondrial fusion has not been observed in postmitotic myocytes of adult hearts, and its occurrence and function in this context are controversial. Determine the consequences on organelle and organ function of disrupting cardiomyocyte mitochondrial fusion in vivo. The murine mfn1 and mfn2 genes, encoding mitofusins (Mfn) 1 and 2 that mediate mitochondrial tethering and outer mitochondrial membrane fusion, were interrupted by Cre-mediated excision of essential exons in neonatal (Nkx2.5-Cre) and adult (MYH6 modified estrogen receptor-Cre-modified estrogen receptor plus tamoxifen or Raloxifene) hearts. Embryonic combined Mfn1/Mfn2 ablation was lethal after e9.5. Conditional combined Mfn1/Mfn2 ablation in adult hearts induced mitochondrial fragmentation, cardiomyocyte and mitochondrial respiratory dysfunction, and rapidly progressive and lethal dilated cardiomyopathy. Before heart failure developed, cardiomyocyte shortening and calcium cycling were unaffected by absence of Mfn1 and Mfn2. Based on the time course over which fusion-defective mitochondrial size decreases, a mitochondrial fusion/fission cycle in adult mouse hearts occurs approximately every 16 days. Mitochondrial fusion in adult cardiac myocytes is necessary to maintain normal mitochondrial morphology and is essential for normal cardiac respiratory and contractile function. Interruption of mitochondrial fusion causes lethal cardiac failure at a time corresponding to 3 or 4 cycles of unopposed mitochondrial fission.

Mitofusins are required for angiogenic function and modulate different signaling pathways in cultured endothelial cells

The mitofusin proteins MFN1 and MFN2 function to maintain mitochondrial networks by binding one another and initiating outer mitochondrial membrane fusion. While it has recently been recognized that vascular endothelial cells rely upon mitochondria as signaling rather than energy-producing moieties, the role of mitochondrial dynamics in endothelial cell function has not been addressed. To begin to understand what role mitochondrial dynamics play in this context, we examined the regulation of MFN1 and MFN2 and the consequences of siRNA-mediated knockdown of these proteins in cultured endothelial cells. Treatment with VEGF-A led to the upregulation of MFN2 and, to a lesser extent, MFN1. Knockdown of either MFN led to disrupted mitochondrial networks and diminished mitochondrial membrane potential. Knockdown of either MFN decreased VEGF-mediated migration and differentiation into network structures. MFN ablation also diminished endothelial cell viability and increased apoptosis under low mitogen conditions. Knockdown of MFN2 uniquely resulted in a decrease in the generation of reactive oxygen species as well as the blunting of the gene expression of components of the respiratory chain and transcription factors associated with oxidative metabolism. In contrast, ablation of MFN1 led to the selective reduction of VEGF-stimulated Akt-eNOS signaling. Taken together, our data indicate that mitochondrial dynamics, particularly those mediated by the mitofusins, play a role in endothelial cell function and viability.

Sequential requirements for the GTPase domain of the mitofusin Fzo1 and the ubiquitin ligase SCFMdm30 in mitochondrial outer membrane fusion

The ability of cells to respire requires that mitochondria undergo fusion and fission of their outer and inner membranes. The means by which levels of fusion 'machinery' components are regulated and the molecular details of how fusion occurs are largely unknown. In Saccharomyces cerevisiae, a central component of the mitochondrial outer membrane (MOM) fusion machinery is the mitofusin Fzo1, a dynamin-like GTPase. We demonstrate that an early step in fusion, mitochondrial tethering, is dependent on the Fzo1 GTPase domain. Furthermore, the ubiquitin ligase SCF(Mdm30) (a SKP1-cullin-1-F-box complex that contains Mdm30 as the F-box protein), which targets Fzo1 for ubiquitylation and proteasomal degradation, is recruited to Fzo1 as a consequence of a GTPase-domain-dependent alteration in the mitofusin. Moreover, evidence is provided that neither Mdm30 nor proteasome activity are necessary for tethering of mitochondria. However, both Mdm30 and proteasomes are critical for MOM fusion. To better understand the requirement for the ubiquitin-proteasome system in mitochondrial fusion, we used the N-end rule system of degrons and determined that ongoing degradation of Fzo1 is important for mitochondrial morphology and respiration. These findings suggest a sequence of events in early mitochondrial fusion where Fzo1 GTPase-domain-dependent tethering leads to recruitment of SCF(Mdm30) and ubiquitin-mediated degradation of Fzo1, which facilitates mitochondrial fusion.