Mitofusin 2 is required for neuron specific functions divergent from mitochondrial fusion Rabeah Al-‐Temaimi1,2, Kyle Miller3, John C. Fyfe2 1 Human Genetics Unit, Department of Pathology, Faculty of Medicine, Kuwait University, P.O. Box 24923, Safat 13110 , Kuwait. 2 Laboratory of Comparative Medical Genetics, Department of Microbiology & Molecular
Genetics, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA. 3Department of Zoology, Michigan State University, East Lansing, MI 48824-‐1115, USA
Corresponding Author Rabeah Al-‐Temaimi Head of Human Genetics Unit Dept. Pathology, Faculty of Medicine Kuwait University P.O. Box 24923, Safat 13110 Tel: +965 24636250 Email:
[email protected]
ABSTRACT Mutations in mitofusin 2 (MFN2) gene cause a commonly inherited neuropathy, Charcot Marie Tooth2 type A2 (CMT2 A2). A novel MFN2 mutation (ΔE539) was detected in a canine autosomal recessive fetal onset neuroaxonal dystrophy (FNAD) model. FNAD pathology manifests in the nervous system, causing secondary effects on muscle functions. MFN2 is a nuclear encoded, mitochondrial outer membrane protein involved in maintaining mitochondrial shape, integrity, and dynamics through interaction with its cellular paralogue MFN1, and other protein networks. The aim of this study was to investigate the effects of the novel MFN2 mutation at molecular and cellular levels in vitro. No defects in mitochondrial morphology, distribution, or respiration were detected in FNAD cultured primary cells, despite lost and decreased MFN2ΔE539 protein expression in primary canine fibroblast (PCF) and dorsal root ganglia (DRG) cultures, respectively. Mitochondrial degradation by mitophagy was significantly reduced in FNAD PCFs (p = 0.001). In addition, MFN1 knock-‐down in FNAD PCF resulted in hyperfragmented mitochondria suggesting MFN2ΔE539 is deficient of fusion functions. Whereas MFN1 knockdown in wild-‐type and FNAD DRG cultures resulted in similar hyperfragmented mitochondria indicative of MFN1’s exclusive role in mitochondrial fusion in neurons. In conclusion, MFN2ΔE539 result in protein loss that can be tolerated in non-‐neuronal tissues while pathogenic effects manifest exclusively in neuronal tissues possibly involving neuron specific functions other than mitochondrial fusion. Keywords: Mitofusion 2, Charcot Marie Tooth 2A2, fetal onset neuroaxonal dystrophy, mitochondrial fusion, mitophagy, dorsal root ganglia culture. Footnote Abbreviations: PCF: primary canine fibroblast; PDRG: primary dorsal root ganglia; FNAD: fetal onset neuroaxonal dystrophy
INTRODUCTION Mitofusin 2 (MFN2; Gene ID: 9927) is a nuclear coded mitochondrial outer membrane GTPase protein composed of five domains: a Ras GTPase domain, two coiled coil domains, and two short transmembrane domains. In combination with its cellular paralogue mitofusin 1 (MFN1), it promotes mitochondrial fusion [1,2,3] and is critical for oxidative phosphorylation [4], apoptosis [5,6], autophagy [7], metabolism [8,9], axonal transport [10], and development [11]. Mutations in MFN2 are associated with Charcot Marie Tooth (CMT), one of the most prevalent hereditary sensorimotor neuropathies [12]. The two principal forms of CMT, called CMT1 and CMT2, are both autosomal dominant but differ in that the first is demyelinating and the second primarily is axonal. CMT2A2 (OMIM ID: 609260) is the most common form of axonal CMT [13] and is associated with mutations in MFN2 [14]. Reported MFN2 mutations are mostly missense, though splicing and compound heterozygous mutations are also observed [15]. These mutations typically map to the GTPase domain of the protein, or are in close proximity to it [16]. In addition a unique set of MFN2 mutations, associated with hereditary motor and sensory neuropathy type VI (OMIM ID: 601152) and sporadic severe early onset axonal neuropathy (SEOAN) incidence have also been reported [17,18]. Here we attempt to further define the functional attributes of MFN2 by investigating an MFN2 mutation associated with canine fetal onset neuroaxonal dystrophy (FNAD). FNAD is similar to conatal forms of infantile neuroaxonal dystrophy (INAD, OMIM ID: 256600) [19,20] and is an autosomal recessive neuropathy causing axonal dysfunction prior to birth. The mutation in FNAD is an in frame trinucleotide deletion, predicted to remove a glutamate residue at position 539 (ΔE539). The mutation maps to a highly conserved region within MFN2 found between the N terminal coiled-‐coil and the transmembrane domains [21]. In this report we investigate the effects of MFN2ΔE539 on mitochondrial dynamics and function in two primary in vitro cell culture systems, FNAD fibroblasts and dorsal root ganglia (DRG). Our findings show mitochondrial morphology and oxidative phosphorylation to be intact, with significant decrease in mitochondrial clearance (mitophagy). Moreover, we found specific knockdown of MFN1 in neuronal mitochondria results in hyperfragmented mitochondria in wild-‐type and MFN2ΔE539 neurons, suggesting a neuron specific function of MFN2 other than mitochondrial fusion is the cause of observed neuropathology in MFN2 related neuropathies. MATERIALS AND METHODS Animals FNAD carrier dogs were maintained in a breeding colony at Michigan State University (MSU) under animal use protocols approved by the Institutional Animal Use and Care Committee (Forms 02/21/2001 and 10/07-‐161-‐00). At birth, stillborn FNAD pups were
directly processed for tissue harvest. Clinically normal littermates were euthanized and their tissues were processed for use as controls. Spinal cord and umbilical cord fragments were collected in sterile DMEM with antibiotics and stored on ice for follow up processing. Derived cells were confirmed as wild-‐type by genotyping, as published previously [20]. Mitochondrial respiration assay Kidneys were isolated for preparation of crude mitochondrial fractions for polarography. Kidneys were immersed in MSH buffer (210 mM mannitol, 750 mM sucrose, 20 mM HEPES, pH 7.4), minced and washed three times. Kidney mince was homogenized in MSH buffer (with 1 mM EDTA, 0.1 mM PMSF) using a glass Teflon homogenizer. Homogenate was centrifuged (600 x g, 10 minutes) and resultant supernatant further centrifuged (7,500 x g, 10 minutes) to yield mitochondrial pellet. Crude mitochondria were washed in MSH buffer without EDTA twice. The final mitochondrial pellet was resuspended in respiration media (220 mM mannitol, 68 mM sucrose, 1.9 mM HEPES, 2.4 mM potassium phosphate buffer, 0.5 mM EDTA, 1.8 mM MgCl2, 5.2 mM succinate, 0.7 g/l BSA, pH 7.4). Mitochondrial respiration was measured as the rate of oxygen consumption from medium using an oxygen polarograph (Gilson Medical Electronics, Middleton, WI) with a 0.5 cm diameter Clark electrode fitted to a 1.75 ml glass reaction chamber maintained at 37°C. Basal respiration was recorded after chamber oxygen saturation, and for active respiration following addition of 0.45 µM ADP. Oxygen consumption was determined and normalized to added protein concentration [22]. Four homozygous MFN2 mutants and four homozygous wildtype MFN2 mitochondrial fractions were assayed in triplicates. All mitochondrial extracts were quantified for protein concentration using Bradford protein assay [23]. Cell Culture Primary canine fibroblast (PCF) cultures were established from excised pup umbilical cord tissue. Excised tissue was washed three times in full PCF growth medium (high glucose DMEM, 10% fetal bovine serum, 0.4 mM L-‐glutamine, 1% penicillin/streptomycin), and minced with sterile scalpels. Tissue fragments were transferred to 60 mm culture dishes containing full PCF growth medium. Primary dorsal root ganglion neuron (PDRG) cultures were established from spinal cord isolated with nerve roots attached. PDRGs were collected and dissociated by incubation (15 minutes at 37°C) with collagenase IA and (30 minutes at 37°C) with trypsin, consecutively. Dissociation was terminated by adding full PDRG growth medium (L15 medium, 10% fetal bovine serum, 2 mM L-‐glutamine, 24 mM NaHCO3, 38 mM glucose, 1% penicillin/streptomycin, 50 ng/ml nerve growth factor (Sigma Aldrich, St. Louis, MO)). Five mechanical triturations were performed with each trituration followed by a brief low speed centrifugation and collection of resultant supernatant into a fresh tube. Resultant pellet was retriturated in PBS. Finally, collected supernatants following triturations were centrifuged (1,000 x g, 5 minutes) and suspended in full PDRG growth medium and plated in 0.1% poly-‐L-‐ornithine coated culture dishes. All cultures were maintained at 37°C and 5% CO2 incubator.
Protein expression analysis PCF and PDRG cultures of wild type normal and FNAD genotypes were grown to confluence. Cultures were trypsinized and collected in full growth media. Suspensions were centrifuged (700xg, 5 minutes) and pellets washed in PBS. Final pellets were resuspended in cold protein extraction buffer (0.3 M mannitol, 0.2 mM EDTA, 10 mM HEPES, 0.1 mM PMSF, pH 7.4). PCF suspensions were homogenized with a glass homogenizer on ice. Suspensions were centrifuged (1,000 x g, 10 minutes, 4°C), and resultant supernatants were centrifuged again (14,000 x g, 15 minutes, 4°C) to produce mitochondrial pellets and post mitochondrial supernatant, respectively. PDRG suspensions were sonicated on ice for 10 seconds and centrifuged (1,000 x g, 10 minutes, 4°C). Resultant supernatant was saved as PDRG total protein lysate and pellets discarded. Western blotting was performed, and antibodies were validated as previously described [24]. Mitochondrial morphology and mitophagy analysis Live cell imaging of mitochondria in in vitro cultures was performed using cover slip bottom culture dishes (MatTek corp., Ashland, MA). Mitochondria were incubated with 0.5 µM Mitotracker green (MTG, Invitrogen Corp., Carlsbad, CA) for 1 hour prior to image capture to analyze mitochondrial morphology and distribution. Mitophagy was analyzed in live cells using consecutive staining with 0.5 µM MTG, and 50 nM lysotracker blue DND 22 (Invitrogen Corp., Carlsbad, CA). Briefly, separate PCF and PDRG cultures were incubated with MTG for 20, and 24 hours under normal culture conditions. Cultures were washed three times with their respective full growth medium and incubated with lysotracker blue for 30 minutes. Cultures were washed two times with full medium, and prepared for live cell imaging. All images were captured using an Olympus FlowView 1500 confocal microscope (Olympus America Inc., Center Valley, PA) equipped with 60X PlanApo objectives at 1.0 or higher digital zoom when required. Colocalization analysis of mitophagy was performed using ImageJ (http://rsb.info.nih.gov/ij/), JACoP plugin [25], and the Excel® program of Microsoft Office for statistical analysis. MFN silencing Twenty seven bp, double stranded RNA interference (dsiRNA) oligomers (Integrated DNA tech., Inc., Coralville, IA) were assessed by qRT PCR for ability to knock down MFN1 and MFN2 transcripts in wild type PCF cultures. Selected dsiRNA with successful knockdown of transcription are listed in table 1. A scrambled noncoding oligomer (NC1) and a TYE563 DS labeled oligomer were used as transfection and cellular uptake controls, respectively. PCF and PDRG cells were seeded at 5 to 10 x 104 cells/ml in 12-‐well culture plates and 35 mm glass bottom culture dishes. PCF and PDRG cultures were incubated in full growth medium without antibiotics overnight at 50% confluence. Lipofectamine 2000 (Invitrogen, Carlsbad, CA) reagent was used for transfection according to manufacturer protocol. All oligomers were used at 20 nM whether in combination with other oligomers or alone, except for the water negative control. All cultures were incubated for 24 hours in oligomer containing OptiMEM (Invitrogen, Carlsbad, CA) before medium was changed into full growth medium and cells were processed for imaging.
Mitochondrial axonal transport Results Mitochondrial respiration is normal in FNAD kidney We have previously shown that FNAD kidney mitochondria do not show any detectable amounts of MFN2ΔE539 protein through Western blotting (Fyfe, et al. 2011). Kidney mitochondrial fractions were used in polarographic measurements of oxygen consumption rate as in [22]. Oxygen consumption rates were adjusted according to total amount of mitochondria added to the reaction chamber. Basal mitochondrial respiration in homozygous wildtype MFN2 and MFN2ΔE539 mitochondrial extracts were similar. Wildtype MFN2 mitochondria’s mean basal respiration was 16.3 (nmoles O2/minute)/μg (SD = 5.2), while in MFN2ΔE539 mitochondria it was 15.3 (nmoles O2/minute)/μg (SD = 7.6). Active mitochondrial respiration was comparable between the two assayed genotypes. Wildtype MFN2 mitochondria mean active respiration was 30.0 (nmoles O2/minute)/μg (SD = 9), while in MFN2ΔE539 mitochondria it was 27.4 (nmoles O2/minute)/μg (SD = 10.2). These findings suggest that ATP production through succinate driven; complex II mitochondrial respiration and maximal respiration through direct ADP conversion is intact in mutant MFN2 mitochondria under controlled experimental conditions. Mitochondrial Morphology In vitro is normal in FNAD PCF and PDRG cells Mitochondrial morphology in FNAD PCF cultures did not show noticeable deviation from wild type cultures (Fig.1a). Mitochondria were mostly tubular in shape of varying lengths, and some spherical mitochondria were also visible. No aberrant distribution, peri-‐nuclear aggregation, or abnormal patterning was noticed. Mitochondrial abundance in FNAD PCF cultures was comparable to wild type PCF cultures. Mitochondrial morphology and distribution in wild type and FNAD PDRG cultures were similar, with tubular and spherical mitochondria randomly spaced within axons and abundant in the cell body (Fig.1b). No abnormal patterns of mitochondrial distribution, shape or abundance were noted in FNAD PDRG cultures. Mitochondrial association with acidic vacuoles is reduced in MFN2ΔE539 PCFs Mitophagy was assessed through colocalization of mitochondria with acidic vacuoles, including lysosomes, in PCF cultures. Qualitative assessments of merged representative images of the two dyes did not show any disparity between mutant and wild type mitochondria. However, Pearson’s coefficient suggested colocalization between channels in both genotypes. Mander’s coefficient of overlap was also suggestive and the fraction of mitochondrial overlap with acidic vacuoles (M2) was calculated for each genotype. There were fewer mitochondria associated with acidic vacuoles in the MFN2ΔE539 genotype cells (11%) compared to wild type (20%) after 20 hrs of incubation with MTG (p= 0.003).
Similarly, the fraction of acidic vacuoles colocalized with mitochondria (M1) at 20 hours of MTG incubation was less in MFN2ΔE539 PCFs (21%) than in the wild type cells (47%, p= 0.001). After 24 hours of MTG incubation the differences in colocalization of the two dyes between the two genotypes were lost. In PDRG cultures of both genotypes acidic vacuole and mitochondrial colocalization was present. However, no significant differences were found between wild type and mutant genotypes after 20 hours incubation with MTG and the fractions of mitochondria and acidic vacuoles contributing to colocalization were comparable. MFN2 Protein expression is reduced and MFN1 expression is increased in primary cell cultures MFN2 expression was not detected by Western blot (Fig. 2a) in the total protein or subcellular fractions of MFN2ΔE539 fibroblasts. Findings were concordant with FNAD affected dog kidney, brain, and brainstem tissues assayed previously (Fyfe, et al. 2011). In contrast, FNAD PDRG cultures showed detectable amounts of MFN2ΔE539 protein, albeit ~ 60% less than in wild type PDRG cultures, in total protein lysates (Fig. 2b). MFN1 expression detected by western blot was increased by ~40% in FNAD PDRG compared to wild type PDRG cultures. This finding was consistent with the previously shown increase of MFN1 in FNAD brainstem tissue. MFN Silencing In vitro MFN1 knockdown in wild type PCF cultures did not impart any morphological changes to mitochondria (Fig. 3a). However, in FNAD PCF cultures, MFN1 knockdown resulted in hyperfragmented, punctate mitochondria (Fig. 3a, right). Similar mitochondrial morphology was observed when wild type and FNAD PCF cultures were subjected to double MFNs knockdown (Fig. 3b). In contrast, MFN1 knockdown in wild type and FNAD PDRG cultures showed pronounced mitochondrial fragmentation (Fig. 4a). Similarly, double MFNs knockdown resulted in analogous mitochondrial morphology (Fig. 4b). No changes in mitochondrial morphology were observed in wild type or FNAD PCF or PDRG cells treated with the NC1 oligo.
DISCUSSION Mutations in MFN2 cause CMT2A2, while MFN2 is ubiquitously expressed the predominant pathology occurs in neurons. The reason neurons are selectively affected is unknown. The mutations in MFN2 that cause CMT2A2 are typically autosomal dominant. While many of MFN2 alleles that cause CMT2A2 disrupt mitochondrial morphology, some do not [26,27]. This could be because the alleles disrupt some function of MFN2 unrelated to mitochondrial fusion, yet important for neuronal function, or because MFN1 in cell types assayed can competently complement for MFN2 fusion functions [3,28]. In order to better understand why disruption of MFN2 leads to CMT2A2 it would be helpful to assess the effects of MFN2 loss of function in an animal model, yet mice null for MFN2 die mid
gestation due to placental defects [11]. In a previously described canine FNAD phenotype [19], a homozygous MFN2ΔE539 mutation was associated with pathology confined to the nervous system, and consequent impairment of innervated muscles. The mutation affects MFN2 protein expression but does not affect gene transcription, as MFN2 transcripts were detected in FNAD tissues [20]. Protein analysis showed complete loss of MFN2 expression in kidney, brain, brainstem, and fibroblasts. This provides an excellent system that complements previous studies. We began with the working hypothesis that loss of MFN2 expression would generate pathology as a result of defects in mitochondrial fusion. In our current study we assayed for mutant MFN2 protein expression in primary culture systems and found it to be lost in PCF but detectable in PDRG cells that are coupled to the neuronal pathology observed in FNAD. The deleted glutamate residue in MFN2ΔE539 is a constitutively conserved residue in all bilateria species catalogued in HomoloGene database (http://www.ncbi.nlm.nih.gov/homologene/8915). Building on our prior studies, we hypothesized that MFN2 loss of function would disrupt mitochondrial function and morphology. To test this, we first examined FNAD embryonic fibroblasts. In primary FNAD fibroblast cultures lacking MFN2 expression, measures of mitochondrial morphology, distribution, respiration, and membrane potential (data not shown) showed no significant differences from wild type fibroblast cultures. Furthermore, mitochondrial dynamics were intact in FNAD PCF despite the lack of MFN2 expression, most likely through MFN1 expression that has been shown to complement the mitochondrial fusion function of MFN2 [26]. We confirmed this assumption through MFN1 knockdown in FNAD PCF that resulted in hyperfragmented mitochondria, indicating decreased fusion potential due to lack of both MFNs. A similar hyper-‐fragmented mitochondria phenotype was generated by double knockdown of MFNs in wild type PCF mitochondria. These findings further demonstrate that MFN1 is efficient in complementing MFN2 fusion function in FNAD PCF whereas MFN2ΔE539, if expressed at all, does not. These results were similar to reports that showed mitochondrial morphology and functions to be intact in CMT2A2 patients’ fibroblasts [27,29]. In contrast, our findings disagree with findings in murine embryonic fibroblasts deficient of MFN2 [11], possibly due to species-‐ specific dependence on MFN1 for mitochondrial fusion. Or perhaps MFN2 null MEFs which were produced by introducing a premature stop codon might still be producing partial MFN2 proteins competitively forming heterodimers with MFN1 and thus reducing its fusion complementation property, or still partial MFN2 with maintained outer membrane insertion properties. In addition, MFN2 null MEFs were presumed null by the assurance that an intact GTPase domain is essential for MFN2 function which is debatable since MFN2 lacking a GTPase domain has normal mitochondrial morphology [21], and since most CMT2A2 MFN2 mutants that have mutations in the GTPase domain have normal mitochondrial morphology and fusion, presumably through interacting with MFN1. Inaddition, our MFN2ΔE539 mitochondrial morphology and fusion results replicate previous reported findings for CMT2A2 MFN2 mutants that are within highly conserved regions when introduced homozygously in mouse embryonic fibroblasts [26]. A hyper-‐fragmented phenotype was only visible when these MFN2 mutants were introduced in an MFN1 null
cell system. Those MFN2 mutations overlap with the R1 and R2 highly conserved regions of unknown function and the GTPase domain, whereas our mutation is the first reported in the R5 conserved region of unknown function. These findings suggest that MFN2ΔE539 pathogenesis in nervous tissues is possibly through a mechanism other than dysfunctional mitochondrial fusion. In contrast to PCF, protein expression of MFN2ΔE539 was detectable in cultured PDRG cells derived from FNAD pups. FNAD PDRG mitochondria showed no defects in mitochondrial morphology. MFN1 knockdown in PDRG generated hyperfragmented mitochondria analogous to PCF, demonstrating that MFN2ΔE539 cannot rescue mitochondrial fusion in this cell type, at least at the demonstrated expression level. The same phenotype was observed when MFN1 was knocked down in wild type PDRG indicating that in these cultured sensory neurons, wild type MFN2 contributes little if anything to mitochondrial fusion. To our knowledge, this is the first report of wild type MFN2 failing to complement MFN1 mitochondrial fusion functions in neuronal cultures. Most of the reported studies in MFN1 null cells focused on fibroblast culture systems, the few that have used neuron cell systems focused on creating MFN2 null neurons to introduce mutant MFN2 forms [10,30], or failed to provide evidence of MFN1 loss in an MFN1 null neuron system [31]. Our finding here also suggest that the pathological events in CMT2A2 related MFN2 loss of function mutations affect neurons for reasons other than loss of mitochondrial fusion. Mfn2 has been proposed to be involved in extramitochondrial functions that include mitochondrial transport in neurons and ER-‐mitochondrial interaction. In addition, in our study, we found significant dysregulation of mitophagy in FNAD fibroblast cultures. Decreased mitophagy seen in mutant cells could be due to loss of MFN2 expression [32]. Mitophagy is regulated by MFN2 ubiquitination through the PINK1/Parkin pathway [7,33]. At this point it is unclear whether this MFN2 mutation would alter a structural or a functional property of MFN2 that leads to defects in mitophagy. However, it is possible that the mutation itself might target MFN2 for earlier degradation and absolve its role in mitophagic regulation. Degradation of MFN2ΔE539 either by targeted early protein degradation would be protective in FNAD non neuronal tissues, but does not fully explain their lack of detectable pathology or the fatal pathology found in nervous tissues. MFN2 function in neurons has been the subject of vigorous research to determine its role in the pathogenesis of a number of related neurodegenerative diseases [34,35,36]. An MFN2 function seemingly unique to neurons is the axonal transport of mitochondria [10]. We have attempted to investigate this function in a sampled time lapse imaging experiment and in both wild type and MFN2ΔE539 PDRG cells movement was evident (data not shown). However, quantitative and qualitative measurements need to be assessed for a conclusive result. Moreover, we have shown that MFN2ΔE539 is expressed in PDRG cells, albeit at decreased levels. It is plausible that reduced stability of MFN2ΔE539 is briefly tolerated by interacting with the axonal transport machinery. However, such association might be inefficient in maintaining mitochondrial axonal transport in long motor and sensory
neurons, which are sites of the most evident pathology in the FNAD model. It is also probable that additional deficits in expressed MFN2ΔE539 regulatory mitochondrial functions including energy production, endoplasmic reticulum tethering, and mitophagy contribute to the death signals that result in neuronal degeneration. In contrast to previous studies, we found very mild defects in mitochondrial morphology as the result of MFN2 loss of function. Because MFN1 complements, MFN2, this suggests that one of the toxic gain of function effects of MFN2 CTM2A may be the disruption of MFN1 function. Together our results suggest that defects in mitochondrial fusion caused by mutations in MFN2 may be something of a red herring and that instead the pathology in CMT2A may be primarily the result of “extra-‐mitochondrial” functions of MFN2 that are particular important for neuronal health. Likewise, this helps to explain why some alleles of MFN2 lead to pathology without significant effects on mitochondrial fusion. One implication of our work is that simply increasing the expression or activity of MFN1 in neurons, may not rescue normal function. Altered axonal transport and trafficking of mitochondria has been reported when selected CMT2A2 MFN2 mutants were expressed in DRG cultures [37]. Therefore, we suggest that axonal transport of mitochondria should be investigated in neuron culture systems expressing different MFN2 mutations. Such experiments would provide causative evidence for MFN2 mutations that failed to generate defects in non-‐neuronal tissues. And possibly point out the pathogenic mechanism contributing to variable phenotype severities. Such studies might provide targets for better pharmaceutical treatment and management of MFN2 related neuronal disorders.
Acknowledgements
We would like to acknowledge Prof. Shelagh M. Ferguson-‐Miller for providing access to her laboratory facility, and Carrie Hiser and Neil Bowlby for sharing their experience. We would like to thank Philip Lamoureux at Kyle Miller laboratory, and Melinda Kay-‐Frame at the laser Capture Microscopy (LCM) unit, MSU.
References
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Table 1. Control and selected knock-‐down dsiRNA sense (S) and anti-‐sense (A) sequences. DsiRNA name
Duplex sequence
NC1 negative control
S 5’- CGU UAA UCG CGU AUA AUA CGC GUA T -3’ A 5’- AUA CGC GUA UUA UAC GCG AUU AAC GAC -3’
TYE 563 transfection
S 5’- TCC UUC CUC UCU UUC UCU CCC UUG UGA -3’
control
A 5’- TCA CAA GGG AGA GAA AGA GAG GAA -3’
MFN1 Duplex 1
S 5’- AGA AUA UAU GGA AGA CGU ACG CAG A -3’ A 5’- UCU GCG UAC GUC UUC CAU AUA UUC UGG -3’
MFN2 Duplex 1
S1 5’- CCC GGU UAC GAC AGA AGA ACA GGT T -3’ A1 5’- AAC CUG UUC UUC UGU CGU AAC CGG GUC -3’
Duplex 2
S2 5’- CGA GCA ACG GGA AGA GCA CUG UAA T -3’ A2 5’- AUU ACA GUG CUC UUC CCG UUG CUC GUC -3’
Figure 1. Mitochondrial morphology in (A) Wildtype MFN2 PCF (left), and MFN2ΔE539 PCF (right); (B) wildtype MFN2 in PDRG (left), and MFN2ΔE539 PDRG (right). (bar = 5µm)
Figure 2. MFN2 protein expression in MFN2wt and MFN2ΔE539 cell cultures. (A) PCF cells subcellular fractions consistently showed complete loss of MFN2ΔE539 compared to MFN2wt PCF cells. (B) PDRG cells total lysate MFN2ΔE539 expression was detectable albeit in decreased amounts when compared to MFN2wt PDRG lysate. MFN1 expression was increased in MFN2ΔE539 PDRG lysate when compared to MFN2wt PDRG lysate.
Figure 3. MFN1 knockdown in MFN2wt and MFN2ΔE539 PCF cells resulted in different phenotypes (A), while MFN2wt PCF (left) mitochondria retained normal morphology MFN2ΔE539 PCF cells (center, right) showed hyperfragmented mitochondria. In MFNs double knockdown experiment (B), both MFN2wt (left) and MFN2ΔE539 (right) PCF mitochondria displayed similar hyperfragmented morphology.
Figure 4. MFN1 knockdown in MFN2wt (A) and MFN2ΔE539 (B) PDRG cells resulted in similar phenotypes of hyperfragmented mitochondria indicating that MFN2wt is incapable of complementing MFN1 fusion function in PDRG cells.
