RESEARCH ARTICLE

Cooperative and independent roles of the Drp1 adaptors Mff,MiD49 and MiD51 in mitochondrial fissionLaura D. Osellame1,‡, Abeer P. Singh1,2,‡, David A. Stroud1, Catherine S. Palmer1,*, Diana Stojanovski3,Rajesh Ramachandran4 and Michael T. Ryan1,§

ABSTRACTCytosolic dynamin-related protein 1 (Drp1, also known as DNM1L) isrequired for both mitochondrial and peroxisomal fission. Drp1-dependent division of these organelles is facilitated by a number ofadaptor proteins at mitochondrial and peroxisomal surfaces. Toinvestigate the interplay of these adaptor proteins, we used gene-editing technology to create a suite of cell lines lacking the adaptorsMiD49 (also known as MIEF2), MiD51 (also known as MIEF1), Mffand Fis1. Increased mitochondrial connectivity was observedfollowing loss of individual adaptors, and this was further enhancedfollowing the combined loss of MiD51 and Mff. Moreover, loss ofadaptors also conferred increased resistance of cells to intrinsicapoptotic stimuli, with MiD49 andMiD51 showing themore prominentrole. Using a proximity-based biotin labeling approach, we foundclose associations between MiD51, Mff and Drp1, but not Fis1.Furthermore, we found that MiD51 can suppress Mff-dependentenhancement of Drp1 GTPase activity. Our data indicates that Mffand MiD51 regulate Drp1 in specific ways to promote mitochondrialfission.

KEY WORDS: Mitochondria, Fission, Dynamin-related protein 1,GTPase, Apoptosis

INTRODUCTIONMitochondria are highly dynamic organelles whose movement andshape are governed by cytoskeletal interactions along with balancedfission and fusion events (Labbé et al., 2014; Lackner, 2014; Mishraand Chan, 2014; Richter et al., 2015; Roy et al., 2015).Mitochondrial fission and fusion is tuned to the bioenergetic stateof the cell (Toyama et al., 2016; Wai et al., 2015), and is alsoimportant for quality control and mitochondrial inheritance (Ban-Ishihara et al., 2013; Lackner, 2013). Defects in fission and fusionhave been linked with neurodegenerative diseases such asAlzheimer’s, Huntington’s and Parkinson’s diseases (Chen andChan, 2009). Mitochondrial fission is mediated by dynamin-relatedprotein 1 (Drp1, also known as DNM1L), a large cytosolic GTPase

that is recruited to the surface of the organelle by the adaptors Mff,MiD49 (also known as MIEF2) and MiD51 (also known as MIEF1)(Gandre-Babbe and van der Bliek, 2008; Ingerman et al., 2005;Legesse-Miller et al., 2003; Otera et al., 2010; Palmer et al., 2011;Zhao et al., 2011). Whereas MiD49 and MiD51 are exclusivelyfound on the mitochondrial outer membrane, Mff is additionallylocated on peroxisomes where it can also recruit Drp1 for fission(Koch et al., 2003; Otera et al., 2010; Palmer et al., 2013). Fis1,originally thought to be a Drp1 adaptor in both mammals and yeast(James et al., 2003; Mozdy et al., 2000; Stojanovski et al., 2004),has more recently been proposed to function in mitophagy (Shenet al., 2014; Yamano et al., 2014). However, the role of Fis1 inhomeostatic fission is still controversial.

In addition to Drp1 and adaptors, mitochondrial fission alsoinvolves the endoplasmic reticulum (ER) and actin, which aid in theformation of constriction sites for Drp1 assembly with Mff(Friedman et al., 2011) and MiD proteins (Elgass et al., 2015). Todate, it has not been clear whether these adaptors interact and/orperform separable roles in Drp1-mediated mitochondrial fission.Mitochondria also house and release pro-apoptotic molecules suchas cytochrome c and, as such, are central to the regulation ofthe intrinsic cell death pathway (Martinou and Youle, 2011;Youle and Karbowski, 2005; Youle and Strasser, 2008). WhereasDrp1-dependent mitochondrial fragmentation is known to precedecaspase activation, mitochondrial outer membrane permeabilizationand cytochrome c release, it is not clear whether the adaptors usedare the same as those employed for homeostatic fission (Youle andKarbowski, 2005).

Here, we report on the generation of a suite of Drp1-adaptor-knockout mouse embryonic fibroblasts (MEFs) and a Drp1-knockout MEF cell line to investigate the role of each adaptorprotein and to further understand how these proteins interact andcontribute to Drp1-mediated fission. Our findings indicate that lossof both MiD49 and MiD51, and MiD49 and MiD51 together withMff confer resistance to cell death, with etoposide-treated cellsretaining more cytochrome c in mitochondria than MEFs lackingonly a single adaptor protein. In addition, proximity-based labelingtechniques revealed that MiD51 and Mff are contiguous on themitochondrial surface, suggesting that they interact at sites of Drp1-dependent mitochondrial fission. We also provide evidence thatMiD51 and Mff can act concurrently in Drp1-mediated fissionprocesses by regulating the rate of Drp1 GTP hydrolysis in opposingdirections.

RESULTSCharacterization of gene-edited Drp1 adaptor MEFsTo understand the role played by adaptor proteins in Drp1recruitment and subsequent mitochondrial fission, we generated asuite of single (ΔMiD49, ΔMiD51, ΔMff, ΔFis1 and ΔDrp1), double(ΔMiD49/MiD51), triple (ΔMiD49/MiD51/Fis1) and quadrupleReceived 18 December 2015; Accepted 7 April 2016

1Department of Biochemistry and Molecular Biology, Biomedicine DiscoveryInstitute, Monash University, Melbourne 3800, Australia. 2Department ofBiochemistry, La Trobe Institute for Molecular Science, La Trobe University,Melbourne 3086, Australia. 3Department of Biochemistry and Molecular Biologyand Bio21 Molecular Science and Biotechnology Institute, The University ofMelbourne, Parkville, 3010, Australia. 4Department of Physiology and Biophysics,Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.*Present address: Macfarlane Burnet Institute for Medical Research and PublicHealth, Melbourne 3004, Australia and Monash Micro Imaging, Monash University,Clayton 3800, Australia.‡These authors contributed equally to this work

§Author for correspondence (Michael.Ryan@monash.edu)

M.T.R., 0000-0003-2586-8829

2170

© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 2170-2181 doi:10.1242/jcs.185165

Journal

ofCe

llScience

(ΔMiD49/MiD51/Mff/Fis1) MEF knockouts using gene-editingapproaches. Genomic insertions and deletions (indels) resultingfrom gene-editing clearly showed disruptions to MiD51, Mff andFis1 in cell lines (Fig. S1A–G). When observing the steady-statelevels of morphology proteins in the absence of Drp1 adaptors, itappeared that loss of one or multiple adaptors did not affect thelevels of the other adaptors still present (Fig. 1A). Althoughindividual deletion of MiD49, MiD51, Mff or Fis1 did not appear todrastically change the mitochondrial morphology, or the extent towhich Drp1 localizes to mitochondria, deletion of multiple adaptorsincreased mitochondrial connectivity and reduced Drp1 associationwith the mitochondrial outer membrane (Fig. 1B,C). [ResidualDrp1 signal present on mitochondria lacking all adaptors (ΔMiD49/51/Mff/Fis1) might be due to non-specific signals or Drp1involvement in ER–mitochondria contact-site formation orF-actin contacts at mitochondria.] Mitochondrial morphology andDrp1 correlation at mitochondria was not significantly differentbetween ΔMiD49/51/Mff MEFs and ΔMiD49/51/Mff/Fis1 MEFs,supporting previous findings showing that Fis1 does not contributesignificantly to mitochondrial fission in normal cell homeostasis(Otera et al., 2010).In order to study the connectivity of mitochondrial networks in

the gene-edited MEFs, fluorescence recovery after photobleaching(FRAP) was employed using a matrix-targeted GFP that is fullydiffusible. As can be seen (Fig. 1D,E), mitochondrial connectivitywas most enhanced in ΔMiD49/51/Mff MEFs and ΔMiD49/51/Mff/Fis1 MEFs, reaching similar levels to that seen for ΔDrp1MEFs. Aswell as affecting mitochondrial connectivity, deletion of Mff causedelongation of peroxisomes (Fig. 2A) as reported previously(Gandre-Babbe and van der Bliek, 2008; Otera et al., 2010).Additional deletion of MiD49, MiD51 and Fis1 did not furtherincrease peroxisome length, suggesting that, of the known adaptorproteins, Mff, contributes most to peroxisome division (Fig. 2B;Fig. S2A). Loss of adaptor proteins did not seem to affect ER–mitochondrial contact sites, with mitochondrial constriction sitesand ER tubules observed at similar levels in WT, ΔMff, ΔMiD49/51, ΔMiD49/51/Mff, ΔMiD49/51/Mff/Fis1 and ΔDrp1 MEFs(Fig. S2B,C). In addition, F-actin morphology did not seem to benoticeably affected in ΔMiD49/51/Mff or ΔMiD49/51/Mff/Fis1MEFs (Fig. S2D).To assess the role of the individual adaptors, ΔMiD49/51/Mff/

Fis1 MEFs were transfected with plasmids expressing GFP fused toMiD49, MiD51, Mff or Fis1. Re-introduction of MiD49–GFP orMiD51–GFP into ΔMiD49/51/Mff/Fis1 MEFs was sufficient tofragment mitochondria and recruit the cytosolic pool of Drp1 tomitochondria (Fig. 2C). Expression of GFP–Mff rescued both theperoxisomal and mitochondrial elongation associated with theknockout of all adaptor proteins (Fig. 2C). Conversely, re-expression of GFP–Fis1 was not able to shift the morphology ofmitochondria from a fused to a more reticular state, nor was it able torecruit Drp1 in the absence of MiD49, MiD51, Mff and Fis1(Fig. 2C). These results indicate that MiD51, MiD49 and Mff canindependently recruit fission-competent Drp1, whereas Fis1 isincapable of the recruitment.

Loss of Drp1 adaptors confers resistance to cell deathCells harboring defects in mitochondrial fission (phenotypicallypresenting as elongated mitochondria) have been reported to be lesssensitive to CCCP-induced mitochondrial fragmentation,presumably due to the lack of Drp1 recruited to mitochondria(Ishihara et al., 2009; Loson et al., 2013). To determine whether thisis the case in gene-edited MEFs, we assessed their resistance to

CCCP-induced fragmentation (Fig. 3A). Gene-edited MEFswere treated with 20 μM CCCP for 2 h and mitochondrialmorphologies observed and quantified (Fig. 3A,B). ΔDrp1 MEFswere used as a positive control, as they are well documented to resistfragmentation upon uncoupling (Ishihara et al., 2009). ΔMiD49/51,ΔMiD49/51/Mff and ΔMiD49/51/Mff/Fis1 MEFs largely retainedan interconnected mitochondrial network upon CCCP treatment,suggesting that functional Drp1 adaptors are required (Figs 3A,B).Furthermore, gene-edited MEFs were directly tested for sensitivityor resistance to apoptotic stimuli using etoposide (Fig. 3C,D).Imaging analysis of cells treated with etoposide (20 μM etoposide,20 μM QVD, 8 h) revealed that ΔMiD49/51/Mff, ΔMiD49/51/Mff/Fis1 and ΔDrp1 MEFs preferentially retained cytochrome c(Fig. 3C). Although knockout of a single adaptor (MiD49, MiD51or Mff ), conferred some resistance to cell death [fluorescence-activated cell sorting (FACS) analysis; 20 μM etoposide for 16 h],the greatest resistance was observed in cell lines lacking multipleadaptors (ΔMiD49/51, ΔMiD49/51/Mff and ΔMiD49/51/Mff/Fis1;Fig. 3D). These findings are supported by biochemical data in whichcell lines lacking MiD49 and MiD51, MiD49, MiD51 and Mff, andall four adaptors appear to retain more cytochrome c in the heavymembrane fraction than those lacking a single adaptor, withcytochrome c levels in the pellet fraction comparable to levels inΔDrp1 MEFs (Fig. 3E,F).

Proximity-based labeling analysis of MiD51, Mff and Fis1MiD51 and Mff can independently recruit Drp1 to mitochondriaand promote mitochondrial fission (Koirala et al., 2013; Losonet al., 2013; Palmer et al., 2013). To address the involvement ofother proteins in this process, we employed a proximity-basedlabeling technique, BioID (Roux et al., 2013), to determine proteinsin close proximity to the adaptors. MiD51 was tagged with BirA*(MiD51–BirA*–HA) and stably expressed in ΔMiD51/MiD49MEFs (as the MiD proteins are functional redundant, ΔMiD51/MiD49MEFs were selected to ensure MiD49 did not inhibit proteininteractions with MiD51–BirA*) (Fig. 4A). MiD51ΔPEYFP

(MID51ΔPEYFP–BirA*–HA), which lacks the Drp1 recruitmentloop (Richter et al., 2014), was also tagged with BirA* and used as anegative control. Both fusion proteins localized to mitochondria,where MiD51 had a punctate localization and inducedmitochondrial fragmentation, in contrast to MiD51ΔPEYFP. Toensure that the expression of MiD51 construct was not too high asto induce a fission block (Palmer et al., 2013, 2011), we generatedsingle-cell clones and attenuated the expression of MiD51–BirA*through a 4-hydroxytamoxifen (4-OHT)-inducible promoter.Protein levels were analyzed by western blotting to ensure thatexpression of the BirA* fusion proteins was not excessive whencompared to endogenous levels (Fig. S3A). Although the levels ofMiD51–BirA* were greater than endogenous levels, the fusionproteins were not at sufficient levels to cause the dominant-negativephenotype that is associated with MiD51 overexpression (resultingin elongated mitochondrial tubules). In fact, the levels of MiD51–BirA* caused mitochondrial fragmentation (Fig. 4A), which isassociated with low-level MiD51 activity (Palmer et al., 2011).Expression of MiD51ΔPEYFP–BirA* was unable to alter themorphology of ΔMiD49/51 MEFs as it is fission incompetent.Biotin was added to the medium for labeling followed byquantitative label-free liquid-chromatography mass spectrometry(LC-MS) analysis of the purified biotinylated proteins (Table S1).Although many mitochondrial outer membrane proteins weredetected, surprisingly only Drp1 and Mff were significantlyenriched when comparing MiD51 against MiD51ΔPEYFP

2171

RESEARCH ARTICLE Journal of Cell Science (2016) 129, 2170-2181 doi:10.1242/jcs.185165

Journal

ofCe

llScience

(Fig. 4B). This result supports other findings showing that MiD51and Mff might assemble with Drp1 at the same fission foci (Elgasset al., 2015). Interestingly, no ER proteins were found to be enrichedin this analysis.Next, we applied this technique to Mff. We fused BirA*–FLAG

to the N-terminus of Mff (BirA*–FLAG–Mff) and stably expressedit in ΔMff MEFs. A two-repeat mutant of Mff (Mff2RM) that does

not recruit Drp1 (Strack and Cribbs, 2012) was also fused to BirA*and used as a control (BirA*–FLAG–Mff2RM). Both fusion proteinswere localized to mitochondria and peroxisomes, and only the wild-type construct restored (Drp1-mediated) fission activity (Fig. 4C).We verified that the expression of the endogenous protein wascomparable to that of the BirA* fusion proteins (Fig. S3B). Wefound that BirA*–Mff was able to rescue both the mitochondrial

Fig. 1. Characterization of gene-edited MEF cell lines. (A) Gene-edited cell lines were analyzed for steady-state protein levels using antibodies as indicated.The asterisk denotes a non-specific band. (B) Gene-edited MEFs stained with anti-Tom20 (green) and anti-Drp1 (red) antibodies and imaged with confocalmicroscopy. The insets show a magnified view of the boxed region. Scale bars: 20 μm. (C) Pearson correlation (r) analysis showing the linear correlation whereDrp1 colocalizes with Tom20-stained mitochondria. Data are mean±s.e.m. n=15. (D) FRAP analysis. For each indicated cell line, the percentage recoverywas measured over 60 s following photobleaching. (E) Endpoint analysis of FRAP. Fluorescence recovery at 60 s postbleach from E. Data in D and E are mean±s.e.m., n=5. *P<0.05, **P<0.005, ***P<0.0005; n.s, non-significant (Student’s t-test).

2172

RESEARCH ARTICLE Journal of Cell Science (2016) 129, 2170-2181 doi:10.1242/jcs.185165

Journal

ofCe

llScience

and peroxisomal phenotypes associated with the ΔMff MEFs,whereas the inactive mutant BirA*–Mff2RMwas not. Biotin labelingfollowed by quantitative LC-MS analysis of biotinylated proteins(Table S2) revealed that BirA*–Mff significantly biotinylated Drp1compared to the Mff2RM control (Fig. 4D). In this case, endogenousMiD51 was not detected in the samples. This might be due to the

low levels of this protein in cells (Wang et al., 2012) and/or a poorability to detect the protein by mass spectrometry approaches. Totest this, the relative abundance of mitochondrial MiD49, MiD51,Mff and Fis1 was analyzed using recombinant proteins as a standardcurve (Fig. S4A–D). MiD49 (1.5±0.5 ng) and MiD51 (3.2±0.5 ng)were found to exist in relatively low abundance in MEFs compared

Fig. 2. Peroxosimal defects and rescue of knockout MEFs. (A) WT, ΔMff and ΔMiD49/51/Mff/Fis1 MEFs were stained with Hoechst 33258 (blue) andimmunostained with antibodies against Pex14 (green) and cytochrome c (red) and imaged using confocal microscopy. Scale bars: 20 μm. (B) Peroxisome lengthwas quantified from images in A and Fig. S2A and expressed as a fold change (mean±s.e.m.; n≥30,) over WT. *P<0.05 (Student’s t-test). (C) ΔMiD49/51/Mff/Fis1MEFs were transfected with either MiD49–GFP, MiD51–GFP, GFP–Fis1 or GFP–Mff. Cells were stained with Hoechst 33258 (blue) and immunostained forTom20 (top panels) or cytochrome c (bottom panel) (red) and either Drp1 or Pex14 (gray) as indicated and imaged with confocal microscopy. Scale bars: 20 μm.Insets show a magnified view of the boxed region; in C, line-scans (15 µm) from the position indicated in the image are also shown to determine colocalization ofGFP-tagged adaptors (green) with Drp1 (red).

2173

RESEARCH ARTICLE Journal of Cell Science (2016) 129, 2170-2181 doi:10.1242/jcs.185165

Journal

ofCe

llScience

to that of Fis1 (18±8 ng) andMff (386±108 ng) (mean±s.e.m.; n=3).Interestingly, the levels of the adaptors varied between other celllines and tissues, potentially reflecting different regulatoryrequirements.

The role of Fis1 as a Drp1 adaptor has been questioned (Koiralaet al., 2013; Otera et al., 2010). Given that the BioID approachsuccessfully confirmed that Drp1 is in close proximity to Mff andMiD51, we reasoned that this was a valid approach to investigate

Fig. 3. Loss of MiD49 and MiD51 in MEFs confers resistance to cell death. (A) Gene-edited MEFs treated with 20 μM CCCP for 2 h, stained with antibodiesagainst cytochrome c and imaged using confocal microscopy. Scale bars: 20 μm. (B) Cells from A were counted and mitochondrial morphology scored asindicated. Data are mean±s.e.m., n=3. (C) Cells were treated with 20 µM etoposide and 20 µMQVD for 8 h (+Etop.), immunostained for cytochrome c (green) andTom20 (red) and imaged using confocal microscopy. Diffuse cytochrome c staining is observed in etoposide-treatedWTand single-adaptor-knockout cells. Scalebars: 20 μm. (D) Gene-edited cells were treated with 20 μM etoposide for 16 h, stained with annexin-V–FITC and propidium iodide (PI) and analyzed byperforming FACS. Data are mean±s.e.m., n=4. *P<0.05, **P<0.005, ***P<0.0005 (Student’s t-test). (E) Gene-edited cells were treated with 20 μM etoposide for16 h, then the heavy membrane and supernatant fractions were isolated and analyzed by western blotting with antibodies as indicated. (F) The percentage ofcytochrome c released into the supernatant fraction upon etoposide treatment. Data are mean±s.e.m., n=4.

2174

RESEARCH ARTICLE Journal of Cell Science (2016) 129, 2170-2181 doi:10.1242/jcs.185165

Journal

ofCe

llScience

whether Fis1 can do the same. BirA* was fused to the N-terminus ofFis1 (BirA*–FLAG–Fis1) and stably expressed in ΔFis1 MEFs. Ascan be seen (Fig. 4E), the fusion protein was localized tomitochondria. As a negative control, BirA* fused to thetransmembrane domain of Fis1 was employed (BirA*–FLAG–Fis1TMD; here, there is no suitable non-binding mutant control) andwas stably expressed in control MEFs. Although the levels ofBirA*–Fis1 were greater than the endogenous Fis1 levels

(Fig. S3C), the mitochondria remained in reticular form (Fig. 4E),indicating that the levels were not excessive. Biotin labelingfollowed by quantitative LC-MS analysis of biotinylated proteinsrevealed no enrichment of any proteins in Fis1–BirA*-expressingcells relative to control at the threshold levels seen for MiD51 andMff (Fig. 4F; Table S3). One protein showing slight enrichmentwith Fis1–BirA* was TBC1D15, which has been shown to interactwith Fis1 during mitophagy (Shen et al., 2014; Yamano et al.,

Fig. 4. BioID proximity-labeling analysis of MiD51, Mff and Fis1. (A) BirA*-tagged MiD51 and MiD51ΔPEYFP (schematics at top) were expressed in ΔMiD49/51MEFs, stained with Hoechst 33258 (blue) and immunostained using antibodies against the HA tag (green) and Tom20 (red). (B) BioID proximity labeling andenrichment of proteins in BirA*–MiD51 relative to BirA*–MiD51ΔPEYFP. A Student’s t-test was performed on the log-transformed LFQ intensities from threebiological replicates of MiD51 andMiD51ΔPEYFP enrichments, and plotted against the difference between themean log-transformed intensities. The thresholds forenriched were set at P≤0.05 and t-test difference at >1.5 (log10 LFQ intensities). (C) BirA*–Mff and BirA*–Mff2RM (schematics at top) were expressed in ΔMff MEFcells, stained with Hoechst 33258 (blue) and immunostained using anti-Flag (green) and anti-Pex14 (red) antibodies. (D) As for B, comparing relative enrichmentbetween Mff and Mff2RM. (E) BirA*-tagged Fis1 (schematic at top) was expressed in ΔFis1 MEF cells, stained with Hoechst 33258 (blue) and immunostained forFlag (green) and Tom20 (red). (F) As for B, comparing relative enrichment between Fis1 and Fis1TMD. The insets show a magnified view of the boxed region.Scale bars: 20 μm.

2175

RESEARCH ARTICLE Journal of Cell Science (2016) 129, 2170-2181 doi:10.1242/jcs.185165

Journal

ofCe

llScience

2014). We hypothesize that because BioID was not performedunder mitophagy-inducing conditions the association of TBC1D15and Fis1 only occurred at background levels (Table S3). Overall,BioID followed by LC-MS supports that MiD51 and Mff areadaptors for Drp1 and they can exist in close proximity at themitochondrial surface.

MiD51 and Mff exert opposing effects on Drp1 GTPaseactivityAs we could detect MiD51, Mff and Drp1 in close proximity, andthese proteins might exist in the same scission foci (Elgass et al.,2015), we sought to investigate whether the two adaptors regulateDrp1 GTPase activity. To do this we designed a robust in vitrosystem of measuring Drp1 GTPase activity by tethering His-taggedcytosolic domains of adaptor proteins (MiD51ΔN118 and MffΔC20)on artificial liposomes containing a lipid with a charged Ni-chelating head group {6% 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)-iminodiacetic acid)succinyl] (DGS-NTA Ni2+)and 94% di-oleoyl phosphatidylcholine (DOPC)} termed Ni-liposomes. For both MiD51 and Mff, the His tags were located inplace of the transmembrane region, mimicking the orientation of themitochondrial-anchored native forms. In the presence of MiD51,Drp1 GTPase activity was substantially inhibited (Drp1,kcat=21.9 min−1 and Drp1+MiD51, kcat=4.9 min−1; Fig. 5A,B).The inhibition was dependent on the presence of the PEYFP loop ofMiD51 needed for Drp1 recruitment (Richter et al., 2014)(Drp1+MiD51ΔPEYFP: kcat=20.3 min−1; Fig. 5A,B). In contrast toMiD51, and as recently reported (Clinton et al., 2015; Macdonaldet al., 2015), Mff stimulated Drp1 GTPase activity (Drp1+Mff:kcat=40.4 min−1). The opposing effects of MiD51 and Mff on Drp1GTPase activity suggest a divergence in the roles of the adaptors infission foci. To analyze the effect of co-assembly between theadaptors, we monitored the GTPase activity of Drp1 following theaddition of stoichiometric amounts (0.5 μM) of Mff or MiD51, andsubsequently added the second adaptor 4.5 min later. When Mffwas added after MiD51, it was unable to stimulate Drp1 GTPaseactivity (Fig. 5C). However, when MiD51 was added after Mff,stimulation of Drp1 GTPase activity was suppressed (Fig. 5D).Although MiD51 is able to bind ADP (Losón et al., 2014; Richteret al., 2014), addition of ADP to in vitro GTPase assays containingMff and/or MiD51 did not alter the ability of these adaptors topromote or restrict the GTPase ability of Drp1 respectively(Fig. 5D). These results indicate that the two adaptors, MiD51and Mff can regulate Drp1 GTPase activity on the lipid surface inopposing directions, and therefore have distinct and complementaryroles in Drp1-mediated mitochondrial fission.

DISCUSSIONAlthough it has been established that Mff, MiD49, and MiD51recruit Drp1, how these adaptors interact with each other and otherproposed molecules (including Fis1) in mitochondrial fission isnot well understood. By creating MEFs that lack Drp1 adaptors,we were able to more precisely probe the importance of theseproteins in Drp1-mediated fission. The levels of these proteins inMEFs appear to vary greatly, with Mff being the predominantadaptor at mitochondria. However, the concentrations of adaptorswithin actual fission foci has yet to be determined. Indeed, loss ofany single adaptor (MiD49, MiD51 or Mff ) or Fis1 did notappreciably affect the amount of Drp1 found at the mitochondrialsurface. This is consistent with the redundant nature of the Drp1adaptors regardless of the relative amount of endogenous adaptorpresent on mitochondria (Koirala et al., 2013; Otera et al., 2010;

Palmer et al., 2013). Several studies have concluded that Mff is themajor receptor for Drp1 (Liu and Chan, 2015; Loson et al., 2013;Otera et al., 2010); however, the degree of mitochondrialelongation in ΔMff MEFs was similar to that seen in ΔMiD51 orΔMiD49/51 MEFs. Recently, it has been found that mice lackingMff can live for up to 13 weeks and eventually die fromcardiomyopathy due to mitochondrial fission defects (Chenet al., 2015). This phenotype is significantly milder than theembryonic lethality seen for Drp1-knockout mice (Ishihara et al.,2009; Wakabayashi et al., 2009), suggesting that mitochondrialfission can take place in other tissues lacking Mff (Chen et al.,2015). Mff might also play an important role in neurons given itsability to efficiently stimulate the GTPase activity of the brain-specific Drp1 isoform compared to the ubiquitously expressedisoform (Macdonald et al., 2015).

Recent data indicates that Mff and the MiDs can exist in the samefission foci that also contact the ER (Elgass et al., 2015; Friedmanet al., 2011). In our BioID experiment using MiD51 fused withBirA*, we were able to confirm that both MiD51 and Mff are inclose proximity to Drp1 under basal cellular conditions.Surprisingly, we did not detect members of the mitochondrialconstriction machinery, including ER scaffolding proteins such asSyn17, INF2 and Spire1 in close proximity to MiD51 or Mff(Arasaki et al., 2015; Korobova et al., 2013; Manor et al., 2015). Apotential explanation for this is that the criteria for enrichment in ourBioID analysis was quite stringent because any proximity labelingseen inMiD51 orMff mutants defective in Drp1 binding was treatedas background. Thus, we might have biased against any interactionswith adaptors that are independent of Drp1. Given this, whencompared against the BioID experiments using Fis1 or BirA* thatwas associated with the mitochondrial outer membrane (Fis1TMD),we saw an enrichment of cytoskeletal components (includingmyosin and actin) in the Mff and MiD51 proximity-labelingexperiments (data not shown). Current models suggest that actin–myosin fibers might assemble from the ER platform to engage atmitochondrial constriction sites (Ji et al., 2015; Korobova et al.,2013; Li et al., 2015). Thus, the relative distance of adaptor proteinsfrom the ERmachinery and the crowding of cytoskeletal proteins in-between might provide a substantial barrier to prevent activatedbiotin from cross-linking to ER components. Attempts to enhancebiotinylation of other potential fission components by adding themitochondrial uncoupler CCCP and shortening the time-frame ofthe biotin addition did not yield additional proteins to thosepreviously detected (data not shown).

During apoptosis, Drp1 binding to mitochondria is increased andoccurs concurrently with Bax translocation, upstream of caspaseactivation and cytochrome c release (Hoppins and Nunnari, 2012;Karbowski et al., 2002). What remains unclear, however, is whetherthe adaptors that mediate Drp1 recruitment under normal cellhomeostasis perform the same role upon the initiation of apoptoticsignaling (Scorrano, 2009). MiD49 and MiD51 appear to beimportant in the apoptotic recruitment of Drp1 to the mitochondrialsurface, although apoptosis can still proceed in their absence. LikeDrp1-knockout cells (Ishihara et al., 2009), cell lines lacking allknown adaptors retained significantly more cytochrome c withinmitochondria following apoptotic induction than MEFs lackingonly a single adaptor. Recently, McBride and colleagues found thatDrp1 oligomer formation facilitates cristae remodeling and Bax-and Bak-mediated cytochrome c release following apoptoticactivation (Prudent et al., 2015). The apoptotic protectionafforded by loss of adaptor proteins in cells might result fromimpaired formation of the Drp1 oligomer.

2176

RESEARCH ARTICLE Journal of Cell Science (2016) 129, 2170-2181 doi:10.1242/jcs.185165

Journal

ofCe

llScience

During manuscript revisions, Mihara and colleaguesindependently detailed the apoptotic resistance of Mff, andMiD49 and MiD51 gene-edited MEFs (Otera et al., 2016).Similar to results obtained here, MiD49 and MiD51 double-knockout MEFs retained more cytochrome c within mitochondriathan either wild-type or single MiD-knockout MEFs (Otera et al.,2016). In contrast, MEFs lacking Mff were not resistant tocytochrome c release following actinomycin D treatment (Oteraet al., 2016). Otera and colleagues (2016) concluded that loss ofMiD49 and MiD51 impairs Opa1-dependent cristae remodelingduring apoptosis. Although we also observed some apoptoticresistance in the ΔMiD49/51 MEFs, we saw greater resistance inΔMiD49/51/Mff and ΔMiD49/51/Mff/Fis1 MEFs, suggesting anadditive effect of Mff in apoptotic resistance and/or variableresponses to different apoptotic stimuli.The role of Fis1 as a bona fide Drp1 adaptor has been

controversial, with recent reports downplaying the role of Fis1 inhomeostatic fission, and assigning it a more pronounced role instress or chemical-induced mitochondrial division and mitophagy(Shen et al., 2014; Yamano et al., 2014). Our results support Fis1 asnot being intrinsically involved in homeostatic fission. Loss of Fis1in MEFs did not induce obvious morphological changes to themitochondrial or peroxisomal networks and unlike Mff or MiD51,the BioID approach showed no indication that Fis1 was in proximityto Drp1. However, ΔFis1MEFs displayed some apoptotic resistance

and the possibility exists that Fis1 acts during stress- or apoptotic-mediated fragmentation (Gomes and Scorrano, 2008; Shen et al.,2014; Wang et al., 2012).

It is not clear how the adaptors engage with Drp1 in fission andthis is exacerbated by different conclusions made regarding theoligomeric state at which Drp1 is recognized. For example, Clintonand colleagues recently concluded that Mff recruits dimeric Drp1for subsequent assembly (Clinton et al., 2015), whereas Liu andChan concluded the opposite, namely that Drp1 oligomers areselectively recruited by Mff (Liu and Chan, 2015). Likewise, onestudy has found that MiD49 and MiD51 could not sequesterassembly-defective forms of Drp1 (Palmer et al., 2011) whereasanother study has shown that MiD49 and MiD51 could recruitassembly-deficient, dimeric Drp1 (Liu and Chan, 2015). Disparityin function between adaptors also lies in the opposing effects onDrp1 GTPase activity at the membrane – Drp1 GTPase activity isinhibited by MiD proteins whereas Mff stimulates it. This isconsistent with other recent studies (Clinton et al., 2015; Macdonaldet al., 2015). Taken together, these results present intriguing rolesforMiD49,MiD51 andMff – although they can both recruit Drp1 tomitochondria, they have functional divergence in regulating Drp1GTPase activity. Although the inhibitory effect that MiD51 exertson the GTPase activity of Drp1 was significantly stronger than thestimulatory effect seen byMff, the stoichiometry of these moleculesat mitochondrial fission sites and their mode of regulation (Chen

Fig. 5. MiD51 and Mff have opposingeffects on Drp1 GTPase activity. Drp1GTPase activity stimulated by Ni-liposomes in the presence or absence ofthe indicated Drp1 adaptor variants wasplotted by (A) linear regression, and themean kcat values were calculated (B).(C) Drp1 GTPase activity stimulated byNi-liposomes with the incorporation ofMff at 4.5 min as indicated. (D) Drp1GTPase activity stimulated by Ni-liposomes with indicated adaptorsadded at 4.5 min and where indicated,20 µM ADP (hatched lines) added at8.5 min. Data mean±s.d. for n≥3.

2177

RESEARCH ARTICLE Journal of Cell Science (2016) 129, 2170-2181 doi:10.1242/jcs.185165

Journal

ofCe

llScience

et al., 2015; Loson et al., 2013; Palmer et al., 2013, 2011) is unclear.Based on these and other studies, a possible scenario is that MiD49and MiD51 engages with, and facilitates, Drp1 assembly at themitochondrial outer membrane by inhibiting GTPase activity.Given that Mff stimulates Drp1 GTPase activity, this points to itsrole in the constriction phase of fission (Francy et al., 2015).Because MiD51, Mff and Drp1 are present in close proximity,possibly in the same fission complex, the adaptors might alsofacilitate Drp1 cycling at the membrane. The transition fromMiD51-dependent inhibition to Mff-dependent stimulation of Drp1GTPase activity might be dependent on stoichiometric levels of theadaptors at the membrane. The presence of additional signals tomediate the transition is also likely. Although much detail is still tobe worked out, the redundant nature of the adaptors indicates thatDrp1 can function independently of either regulatory step or thatadditional proteins play similar roles. For example, actin hasrecently been shown to assemble with Drp1, and like Mff, stimulatethe GTPase activity of Drp1 (Ji et al., 2015). Furthermore,sequestration of Drp1 at mitochondria upon MiD49 or MiD51overexpression also recruits actin (Palmer et al., 2011). Additionalproteins (including ER components) might substitute for the role ofMiD49 and MiD51 in regulating Drp1 activity under somesituations, including in non-chordate metazoans that only containMff.

MATERIALS AND METHODSGeneration of gene-edited MEFsMouse embryonic stem cell (ESC) lines containing a gene-trap disruption ofsmcr7 (MiD49) were obtained from the Knockout Mouse Project (KOMP)Repository (project ID: VG10452). The ESCs were injected into blastocyststo generate mouse lines in C57Bl/6N mice (Australian PhenomicsNetwork). MEFs lacking MiD49 were generated from mid-gestation(E10.5 day) embryos from heterozygotes mating pairs. All experimentswere performed in accordance with Animal Ethics Committee approval(Monash and La Trobe Universities).

TALEN pairs were generated against the following genes in the indicatedgenomic regions: Mff target site, exon 1, 5′-GCTGAGATGGCAGAA-3′;and Fis1 target site, exon 2, 5′-AGAATTTTGAAAGGAAATTTC-3′.ΔDrp1 MEFs were generated using the CRISPR/Cas9 system (Ran et al.,2013). The target site was determined through gene analysis usingCHOPCHOP (Montague et al., 2014). The Drp1 target site was exon 1,5′-GCAGGACGTCTTCAACACAG-3. TALEN pairs for MiD51 were asreported previously (Richter et al., 2014). TALEN and CRISPR/Cas9 celllines were sorted through fluorescence (GFP andmCherry for TALEN pairs,GFP for CRISPR/Cas9) flow cytometry to give single cells to ensureclonality. Genomic verification of all cell lines was performed as reportedpreviously (Stroud et al., 2013). Control MEFs were used as controls. Theindels generated are shown in Fig. S1.

Cell culture, transfections and drug treatmentsAll MEF lines were cultured in Dulbecco’s modified Eagle’s medium(DMEM) containing 10% (v/v) fetal bovine serum (FBS) and penicillin–streptomycin, and were maintained at 37°C in an atmosphere of 5% CO2.Transient transfections of MEF cells were performed using LipofectamineLTX with Plus Reagent or Lipofectamine 2000 (Invitrogen) according tomanufacturer’s instructions. Cell lines were routinely tested for mycoplasmacontamination using PlasmaTest (InvivoGen).

Antibodies, chemicals and plasmidsCommercial antibodies used were anti-Drp1 (1:500, catalogue number:611738, BD Biosciences), anti-Opa1 (1:500, catalogue number: 612606,BD Biosciences), anti-cytochrome c (1:500, native epitope: 556432,denatured epitope: 556433, BD Biosciences), anti-MiD51 (1:500,catalogue number 20164-1-AP, Proteintech), anti-Tom20 (1:500,catalogue number 11415, Santa Cruz Biotechnology) and anti-SDHA

(1:500, catalogue number 14715, Abcam) antibodies. These antibodies havebeen validated previously (Palmer et al., 2013; Richter et al., 2014).Polyclonal antibodies against Fis1, Hsc70, Mff and MiD49 were raised in-house and have been reported previously (Richter et al., 2014; Stojanovskiet al., 2004). Polyclonal antibodies were raised against Bak (lacking thetransmembrane domain) and used at a dilution of 1:1000.

Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (Sigma) was usedat a final concentration of 20 μM for 2 h at 37°C. Etoposide (Sigma) wasused at a final concentration of 20 μM for 16 h at 37°C for FACS andwestern blot analysis, and for 8 h for mitochondrial morphology analysis.QVD (Sigma) was used at a final concentration of 20 µM.

MiD49–GFP, MiD51–GFP, mt-GFP, GFP–Mff, GFP–Fis1 and GFP–Sec61 plasmids have been previously reported (Elgass et al., 2015; Palmeret al., 2011; Stojanovski et al., 2004). MiD51 constructs were first clonedinto pcDNA3.1 MCS-BirA(R118G)-HA (a gift from Kyle Roux, SanfordChildren's Health Center, Sanford Research, Sioux Falls, SD; Addgeneplasmid #36047). For lentiviral expression, open reading frames (ORFs) ofmouse MiD51–BirA*–HA and mouse MiD51ΔPEYFP–BirA*–HA werecloned into the pF 5xUAS MCS SV40 vector using BglII and XbaI. Forretroviral expression, ORFs of BirA*–Flag–human-Mff (isoform 8),BirA*–Flag–human-Mff 2RM (isoform 8) and BirA*–Flag–human-Fis1were cloned into pMX-IRES-GFP vector using the EcoRI and SalI sites. Forbacterial expression and purification, ORFs of MiD51 (119–463) andMiD51 (119–463)ΔPEYFP were cloned in pET10N vector using the NotI andBamHI sites, whereas ORFs of MffΔC20 were cloned into pET28a(+) usingthe NcoI and XhoI sites.

Proximity-labeling experiments using BioIDGeneration, selection and induction of 4-hydroxy-tamoxifen-inducible MEFcell lines (for MiD51 constructs) were performed as previously described(Richter et al., 2014). Retrovirally transduced cells (for Mff and Fis1constructs) were generated as previously described (Lazarou et al., 2015).Transduced cells expressing BirA*-tagged proteins were grown in two 15-cmtissue culture plates at 70–90% confluency in DMEM containing 10% (v/v)FBS and 50 µM biotin (Sigma-Aldrich) for the labeling time period. For eachsample, three replicates were cultured. The cells were then harvested, and totalprotein levels of all replicates equalized by protein estimation using a BCAprotein assay kit (Pierce). The cell pellet was frozen at −80°C. Pellets wereresuspended and solubilized in 1 ml RIPA lysis buffer (50 mM Tris-HCl pH7.5, 150 mMNaCl, 1% NP-40, 1 mM EDTA, 1 mM EGTA and 0.1% SDS),supplemented with 1× protease cocktail inhibitor (Roche), 0.5% sodiumdeoxycholate and 250 U of benzonase (Sigma-Aldrich). Lysis was performedat 4°C for 1 h with shaking. The lysates were sonicated on ice at 30%amplitude (3×10 s bursts with 2 s rest in between). The lysates werecentrifuged for 30 min at 20,000 g for 30 min at 4°C. The cleared lysate wasthen incubated with Streptavidin-mag beads (Pierce) for 3 h at 4°C. The beadswere separated using a magnet and washed two times with RIPA lysis bufferfollowed by twowashes with TAP lysis buffer (50 mMHEPES-KOH pH 8.0,100 mM KCl, 10% glycerol, 2 mM EDTA, 0.1% NP-40) and three 1 mlwashes with 50 mM ammonium bicarbonate pH 8.0. Proteins bound to thebeads were digested with 5 µg/ml trypsin at 37°C overnight. The supernatantwas harvested and beads were washed two times with 50 mM ammoniumbicarbonate pH 8.0, and washes were pooled together with the supernatant.The pooled tryptic digest was lyophilized and resuspended in 8 M urea,50 mM ammonium bicarbonate and acidified with 1% (v/v) trifluoroaceticacid (TFA) prior to desalting on SDB-XC (Empore) StageTips as previouslydescribed (Rappsilber et al., 2007).

Mass spectrometryPeptides were analyzed by online nano-high-pressure liquidchromatography (HPLC) electrospray ionization-tandem massspectromery (MS/MS) on an LTQ-Orbitrap Elite Instrument (MiD51) orQ Exactive Plus (Mff and Fis1) connected to an Ultimate 3000 HPLC(Thermo-Fisher Scientific). For the MiD51 experiments, chromatographyand mass spectrometry acquisition parameters were as previously described(Formosa et al., 2015). For Mff and Fis1 experiments, peptides reconstitutedin 0.1% TFA and 2% acetonitrile (ACN) were loaded onto a trap column(Acclaim C18 PepMap nano Trap×2 cm, 100 μm I.D., 5-μmparticle size and

2178

RESEARCH ARTICLE Journal of Cell Science (2016) 129, 2170-2181 doi:10.1242/jcs.185165

Journal

ofCe

llScience

300-Å pore size; Thermo-Fisher Scientific) at 15 μl/min for 3 min beforeswitching the precolumn in line with the analytical column (Acclaim RSLCC18 PepMap Acclaim RSLC nanocolumn 75 μm×50 cm, PepMap100 C18,3-μm particle size, 100-Å pore size; Thermo-Fisher Scientific). Theseparation of peptides was performed at 250 nl/min using a non-linearACN gradient of buffer A (0.1% formic acid, 2% ACN) and buffer B (0.1%formic acid, 80% ACN), starting at 2.5% buffer B to 42.5% over 60 min.Data were collected in positive mode using a Data Dependent Acquisitionm/z of 375–1800 as the scan range, and higher-energy collisionaldissociation (HCD) for MS/MS of the 12 most intense ions with z ≥2.Other instrument parameters were: MS1 scan at 70,000 resolution (at200 m/z), MS maximum injection time 50 ms, AGC target 3E6, normalizedcollision energy at 27% energy, isolation window of 1.8 Da, MS/MSresolution of 17,500, MS/MS AGC target of 1E5, MS/MS maximuminjection time of 100 ms, minimum intensity set at 1E3 and dynamicexclusion set to 20 s. For data analysis, Thermo raw files were analyzedusing the MaxQuant platform (version 1.5.3.17) searching against theUniprot mouse database containing reviewed canonical isoforms in FASTAformat (June 2015; 16,725 entries), the human sequences for bait proteinsMff and Fis1, and a database containing common contaminants, by theAndromeda search engine (Cox et al., 2011). Default settings for label-freequantification (LFQ) were used with modifications. Briefly, N-terminalacetylation and methionine oxidation were used as variable modifications.False discovery rates of 1% for proteins and peptides were applied bysearching a reverse database, and ‘Re-quantify’ and ‘Match between runs’options were enabled. Unique and razor peptides were used forquantification, using a minimum ratio count of one. ‘Label-freequantification’ was turned on, with the ‘LFQ min. ratio count’ set to one.Data analysis was performed using the Perseus software (version 1.5.2.6).Only proteins with a sequence coverage of >5%, more than one uniquepeptide and valid values in at least two out of three replicates (perexperimental group) were considered for data analysis. A one-tailedStudent’s t-test was performed between control (MiD51PEYFP–BirA*,BirA*–Mff2RM or BirA*–Fis1TMD) and bait (MiD51–BirA*, BirA*–Mff orBirA*–Fis1) experimental groups.

Bacterial expression and purificationDrp1Drp1 was purified with an N-terminal CBP tag and expressed in BL-21CodonPlus expression cells (Agilent) following IPTG induction. Cells werelysed and affinity purified using calmodulin-agarose beads (G-Biosciences).Drp1 was further purified by ion-exchange chromatography using DEAE-Sepharose™ Fast Flow beads (GE Healthcare Life Sciences) to yield high-purity Drp1.

MiD51 variantspET10N-MiD51 and pET10N-MiD51ΔPEYFP were expressed in BL-21CodonPlus expression cells (Agilent) following IPTG induction. Cells werelysed and purified by Immobilized Metal Affinity Chromatography (IMAC)using Ni-NTA agarose beads (Qiagen) followed by removal ofcontaminating proteins by size-exclusion chromatography (Superdex™200 16/60 HiLoad; GE Healthcare Life Sciences) to yield high-purityMiD51 and MiD51ΔPEYFP.

MiD49pQE-30-mouse MiD49ΔN50 was expressed in XL-1 Blue E. coli cellsfollowing IPTG induction. Cells were lysed and purified by IMAC using Ni-NTA agarose beads followed by removal of contaminating proteins by size-exclusion chromatography (Superdex™ 200 16/60 HiLoad; GE HealthcareLife Sciences) to yield high-purity MiD49.

MffpET28a(+)-Mff (isoform 8) was expressed in BL-21 Codon Plus E. coli cellsfollowing IPTG induction. Cells were lysed and purified by ImmobilizedMetal Affinity Chromatography (IMAC) using Ni-NTA agarose beads(Qiagen) followed by removal of contaminating ∼70-kDa migrating speciesby ion-exchange chromatography (mono Q 5/50 GL; GE Healthcare LifeSciences), to yield high-purity Mff.

GST–Fis1pGEX-4T-1-Fis1ΔTMD was expressed in BL-21-CodonPlus expression cellsfollowing IPTG induction. Cells were lysed and purified by affinitypurification using glutathione-agarose resin to yield high-purityGST–Fis1TMD.

GTPase assaysStoichiometric amounts of Drp1 adaptors (0.5 µM) were pre-incubated onNi-liposomes [6% DGS-NTA (Ni2+), 94% DOPC; Avanti Polar Lipids]liposomes for 15 min followed by Drp1 incubation for a further 15 min atroom temperature before GTP was added to initiate the reaction at 37°C overa defined time using the GTPase assay previously described (Macdonaldet al., 2014). Additional adaptors and variants were added at 4.5 min and20 µm ADP added at 8.5 min when required.

Mitochondrial isolation and western blottingMitochondrial isolation and separation of proteins by Tris-Tricine SDS-PAGE was performed as previously described (Johnston et al., 2002).Signals were detected on PVDF membrane using ECL chemiluminescentsubstrate (BioRad) on a BioRadXRS+ ChemiDoc system with Image Labsoftware (BioRad).

Immunofluorescence assaysCells were fixed with 4% (w/v) paraformaldehyde in PBS (pH 7.4),permeabilised with 0.2% (w/v) Triton X-100 in PBS and incubated withprimary antibodies for 60 min at room temperature. Primary antibodies werelabeled with either Alexa-Fluor-488, Alexa-Fluor-568- or Alexa-Fluor-647-conjugated anti-mouse-IgG or anti-rabbit-IgG secondary antibodies(Molecular Probes). F-actin was stained using a phalloidin–FITCconjugate (Thermo-Fisher). Hoechst 33258 (1 μg/ml) was used to stainnuclei.

MicroscopyConfocal microscopy was performed on either a Leica TCS SP5 5 channelconfocal microscope or a Leica TCS SP8 equipped with HyD detectorsusing a 63× oil immersion objective. All images were processed usingImageJ (Schneider et al., 2012), MetaMorph software (Visitron Systems) orLeica AF processing software (Leica). Colocalization statistics wereobtained using the image in its entirety (single planes) and are presentedas Pearson correlation units (r). Images in all experimental groups wereobtained using the same settings, except for detector gain adjustments on theLeica TCS SP5 microscope and accumulation time on the Leica TCS SP8microscope. When required, z-sectioning was performed using 1-μm slices.Colocalization of GFP and Alexa Fluor 568 signals were determined using15-μm linescans using Metamorph software (Visitron Systems).

Live-cell imagingTo detect ER–mitochondrial contact sites, cells were transfected with GFP–Sec61, stained with 25 nM Mito-Tracker Red (Thermo-Fisher) and imagedin DMEM containing 10% (v/v) FBS and maintained at 37°C under anatmosphere of 5% CO2. Fluorescence recovery after photobleaching(FRAP) was performed using the FRAP wizard on the Leica TCS SP5confocal microscope. Cells were imaged in DMEM containing 10% (v/v)FBS and maintained at 37°C under an atmosphere of 5% CO2.Photobleaching of the GFP signal was achieved using a 488 nm laser at100% power for 200 ms (sufficient to achieve 10–20% of the startingfluorescence signal). Subsequent recovery of the GFP signal was monitoredover 60 s at 15% laser power.

Fluorescence-activated cell sortingMEFs were harvested and resuspended in annexin-binding buffer (10 mMHEPES pH 7.4, 140 mMNaCl2, 25 mMCaCl2) and stained for 30 min with250 μg/ml annexin-V–FITC and 50 μg/ml propidium iodide prior toanalysis on a FACS Calibur (Becton Dickinson).

Statistical analysisData were generated from a minimum of three independent experiments.Statistical analysis was performed using Microsoft Excel or Prism. For all

2179

RESEARCH ARTICLE Journal of Cell Science (2016) 129, 2170-2181 doi:10.1242/jcs.185165

Journal

ofCe

llScience

graphs and traces in Figs 1–3, Figs S2 and S3, error bars represent themean±s.e.m. For Fig. 5, error bars represent the mean±s.d. All other datawhere possible and appropriate were analyzed using a Student’s t-test.Significance is expressed as *P<0.05, **P<0.005, ***P<0.0005.

AcknowledgementsWe thank Grant Dewson (Walter and Eliza Hall Institute, Melbourne, Australia), GiaVoeltz (University of Colorado, Boulder, CO) and Michael Lazarou (MonashUniversity, Melbourne, Australia) for plasmids, Jason Mears (Case WesternReserve University, Cleveland, OH) for the pCAL-N-EK-hDrp1-3 plasmid, StefanStrack (University of Iowa, Iowa City, IA) for Mff2RM cDNA and Heidi McBride foradvice. We also thank the Monash Micro Imaging, FlowCore and Proteomicsplatforms at Monash University.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsL.D.O. generated and characterized gene-edited MEFs, A.P.S. performed BioIDpulldowns and in vitro GTPase experiments, D.A.S. performed and analyzed massspectrometry experiments, C.S.P. made gene-edited MEFs. M.T.R. conceivedexperiments. All authors analyzed results and wrote the manuscript.

FundingThis work was supported with funding from the National Health and MedicalResearch Council (NHMRC) [grant number 1049968]; and Australian ResearchCouncil [grant number DP160102176]. D.A.S. is a NHMRC Peter Doherty Fellow.R.R. was supported by an American Heart Association (AHA) BeginningGrant-in-Aid [grant number 13BGIA14810010].

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.185165/-/DC1

ReferencesArasaki, K., Shimizu, H., Mogari, H., Nishida, N., Hirota, N., Furuno, A., Kudo, Y.,Baba, M., Baba, N., Cheng, J. et al. (2015). A role for the ancient SNAREsyntaxin 17 in regulating mitochondrial division. Dev. Cell 32, 304-317.

Ban-Ishihara, R., Ishihara, T., Sasaki, N., Mihara, K. and Ishihara, N. (2013).Dynamics of nucleoid structure regulated by mitochondrial fission contributes tocristae reformation and release of cytochrome c. Proc. Natl. Acad. Sci. USA 110,11863-11868.

Chen, H. and Chan, D. C. (2009). Mitochondrial dynamics-fusion, fission,movement, and mitophagy-in neurodegenerative diseases. Hum. Mol. Genet.18, R169-R176.

Chen, H., Ren, S., Clish, C., Jain, M., Mootha, V., McCaffery, J. M. and Chan,D. C. (2015). Titration of mitochondrial fusion rescues Mff-deficientcardiomyopathy. J. Cell Biol. 211, 795-805.

Clinton, R. W., Francy, C. A., Ramachandran, R., Qi, X. and Mears, J. A. (2015).Dynamin-related protein 1 oligomerization in solution impairs functionalinteractions with membrane-anchored mitochondrial fission factor. J. Biol.Chem. 291, 478-492.

Cox, J., Neuhauser, N., Michalski, A., Scheltema, R. A., Olsen, J. V. and Mann,M. (2011). Andromeda: a peptide search engine integrated into the MaxQuantenvironment. J. Proteome Res. 10, 1794-1805.

Elgass, K. D., Smith, E. A., LeGros, M. A., Larabell, C. A. and Ryan, M. T. (2015).Analysis of ER-mitochondria contacts using correlative fluorescence microscopyand soft X-ray tomography of mammalian cells. J. Cell Sci. 128, 2795-2804.

Formosa, L. E., Mimaki, M., Frazier, A. E., McKenzie, M., Stait, T. L., Thorburn,D. R., Stroud, D. A. and Ryan, M. T. (2015). Characterization of mitochondrialFOXRED1 in the assembly of respiratory chain complex I. Hum. Mol. Genet. 24,2952-2965.

Francy, C. A., Alvarez, F. J. D., Zhou, L., Ramachandran, R. and Mears, J. A.(2015). The mechanoenzymatic core of dynamin-related protein 1 comprises theminimal machinery required for membrane constriction. J. Biol. Chem. 290,11692-11703.

Friedman, J. R., Lackner, L. L., West, M., DiBenedetto, J. R., Nunnari, J. andVoeltz, G. K. (2011). ER tubules mark sites of mitochondrial division. Science334, 358-362.

Gandre-Babbe, S. and van der Bliek, A. M. (2008). The novel tail-anchoredmembrane protein Mff controls mitochondrial and peroxisomal fission inmammalian cells. Mol. Biol. Cell 19, 2402-2412.

Gomes, L. C. and Scorrano, L. (2008). High levels of Fis1, a pro-fissionmitochondrial protein, trigger autophagy. Biochim. Biophys. Acta 1777, 860-866.

Hoppins, S. and Nunnari, J. (2012). Mitochondrial dynamics and apoptosis–theER connection. Science 337, 1052-1054.

Ingerman, E., Perkins, E. M., Marino, M., Mears, J. A., McCaffery, J. M.,Hinshaw, J. E. and Nunnari, J. (2005). Dnm1 forms spirals that are structurallytailored to fit mitochondria. J. Cell Biol. 170, 1021-1027.

Ishihara, N., Nomura, M., Jofuku, A., Kato, H., Suzuki, S. O., Masuda, K., Otera,H., Nakanishi, Y., Nonaka, I., Goto, Y.-i. et al. (2009). Mitochondrial fission factorDrp1 is essential for embryonic development and synapse formation in mice. Nat.Cell Biol. 11, 958-966.

James, D. I., Parone, P. A., Mattenberger, Y. and Martinou, J.-C. (2003). hFis1, anovel component of the mammalian mitochondrial fission machinery. J. Biol.Chem. 278, 36373-36379.

Ji, W.-k., Hatch, A. L., Merrill, R. A., Strack, S. and Higgs, H. N. (2015). Actinfilaments target the oligomeric maturation of the dynamin GTPase Drp1 tomitochondrial fission sites. eLife 4, e11553.

Johnston, A. J., Hoogenraad, J., Dougan, D. A., Truscott, K. N., Yano, M., Mori,M., Hoogenraad, N. J. and Ryan, M. T. (2002). Insertion and assembly of humantom7 into the preprotein translocase complex of the outer mitochondrialmembrane. J. Biol. Chem. 277, 42197-42204.

Karbowski, M., Lee, Y.-J., Gaume, B., Jeong, S.-Y., Frank, S., Nechushtan, A.,Santel, A., Fuller, M., Smith, C. L. and Youle, R. J. (2002). Spatial and temporalassociation of Bax with mitochondrial fission sites, Drp1, and Mfn2 duringapoptosis. J. Cell Biol. 159, 931-938.

Koch, A., Thiemann, M., Grabenbauer, M., Yoon, Y., McNiven, M. A. andSchrader, M. (2003). Dynamin-like protein 1 is involved in peroxisomal fission.J. Biol. Chem. 278, 8597-8605.

Koirala, S., Guo, Q., Kalia, R., Bui, H. T., Eckert, D. M., Frost, A. and Shaw, J. M.(2013). Interchangeable adaptors regulate mitochondrial dynamin assembly formembrane scission. Proc. Natl. Acad. Sci. USA 110, E1342-E1351.

Korobova, F., Ramabhadran, V. and Higgs, H. N. (2013). An actin-dependent stepin mitochondrial fission mediated by the ER-associated formin INF2.Science 339,464-467.

Labbe, K., Murley, A. and Nunnari, J. (2014). Determinants and functions ofmitochondrial behavior. Annu. Rev. Cell Dev. Biol. 30, 357-391.

Lackner, L. L. (2013). Determining the shape and cellular distribution ofmitochondria: the integration of multiple activities. Curr. Opin. Cell Biol. 25,471-476.

Lackner, L. L. (2014). Shaping the dynamic mitochondrial network. BMC Biol. 12,35.

Lazarou, M., Sliter, D. A., Kane, L. A., Sarraf, S. A., Wang, C., Burman, J. L.,Sideris, D. P., Fogel, A. I. and Youle, R. J. (2015). The ubiquitin kinase PINK1recruits autophagy receptors to induce mitophagy. Nature 524, 309-314.

Legesse-Miller, A., Massol, R. H. and Kirchhausen, T. (2003). Constriction andDnm1p recruitment are distinct processes in mitochondrial fission. Mol. Biol. Cell14, 1953-1963.

Li, S., Xu, S., Roelofs, B. A., Boyman, L., Lederer, W. J., Sesaki, H. andKarbowski, M. (2015). Transient assembly of F-actin on the outer mitochondrialmembrane contributes to mitochondrial fission. J. Cell Biol. 208, 109-123.

Liu, R. and Chan, D. C. (2015). The mitochondrial fission receptor Mff selectivelyrecruits oligomerized Drp1. Mol. Biol. Cell 26, 4466-4477.

Loson, O. C., Song, Z., Chen, H. and Chan, D. C. (2013). Fis1, Mff, MiD49, andMiD51 mediate Drp1 recruitment in mitochondrial fission. Mol. Biol. Cell 24,659-667.

Loson, O. C., Liu, R., Rome, M. E., Meng, S., Kaiser, J. T., Shan, S.-o. and Chan,D. C. (2014). The mitochondrial fission receptor MiD51 requires ADP as acofactor. Structure 22, 367-377.

Macdonald, P. J., Stepanyants, N., Mehrotra, N., Mears, J. A., Qi, X., Sesaki, H.and Ramachandran, R. (2014). A dimeric equilibrium intermediate nucleatesDrp1 reassembly on mitochondrial membranes for fission. Mol. Biol. Cell 25,1905-1915.

Macdonald, P. J., Francy, C. A., Stepanyants, N., Lehman, L., Baglio, A., Mears,J. A., Qi, X. and Ramachandran, R. (2015). Distinct splice variants of dynamin-related protein 1 differentially utilize mitochondrial fission factor as an effector ofcooperative GTPase activity. J. Biol. Chem., 291, 493-507.

Manor, U., Bartholomew, S., Golani, G., Christenson, E., Kozlov, M., Higgs, H.,Spudich, J. and Lippincott-Schwartz, J. (2015). A mitochondria-anchoredisoform of the actin-nucleating spire protein regulates mitochondrial division. eLife4, e08828.

Martinou, J.-C. and Youle, R. J. (2011). Mitochondria in apoptosis: Bcl-2 familymembers and mitochondrial dynamics. Dev. Cell 21, 92-101.

Mishra, P. and Chan, D. C. (2014). Mitochondrial dynamics and inheritance duringcell division, development and disease. Nat. Rev. Mol. Cell Biol. 15, 634-646.

Montague, T. G., Cruz, J. M., Gagnon, J. A., Church, G. M. and Valen, E. (2014).CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. NucleicAcids Res. 42, W401-W407.

Mozdy, A. D., McCaffery, J. M. and Shaw, J. M. (2000). Dnm1p GTPase-mediatedmitochondrial fission is a multi-step process requiring the novel integralmembrane component Fis1p. J. Cell Biol. 151, 367-380.

Otera, H., Wang, C., Cleland, M. M., Setoguchi, K., Yokota, S., Youle, R. J. andMihara, K. (2010). Mff is an essential factor for mitochondrial recruitment of Drp1during mitochondrial fission in mammalian cells. J. Cell Biol. 191, 1141-1158.

2180

RESEARCH ARTICLE Journal of Cell Science (2016) 129, 2170-2181 doi:10.1242/jcs.185165

Journal

ofCe

llScience

Otera, H., Miyata, N., Kuge, O. and Mihara, K. (2016). Drp1-dependentmitochondrial fission via MiD49/51 is essential for apoptotic cristae remodeling.J. Cell Biol. 212, 531-544.

Palmer, C. S., Osellame, L. D., Laine, D., Koutsopoulos, O. S., Frazier, A. E. andRyan, M. T. (2011). MiD49 and MiD51, new components of the mitochondrialfission machinery. EMBO Rep. 12, 565-573.

Palmer, C. S., Elgass, K. D., Parton, R. G., Osellame, L. D., Stojanovski, D. andRyan, M. T. (2013). MiD49 and MiD51 can act independently of Mff and Fis1 inDrp1 recruitment and are specific for mitochondrial fission. J. Biol. Chem. 288,27584-27593.

Prudent, J., Zunino, R., Sugiura, A., Mattie, S., Shore, G. C. and McBride, H. M.(2015). MAPL SUMOylation of Drp1 stabilizes an ER/mitochondrial platformrequired for cell death. Mol. Cell 59, 941-955.

Ran, F. A., Hsu, P. D., Lin, C.-Y., Gootenberg, J. S., Konermann, S., Trevino,A. E., Scott, D. A., Inoue, A., Matoba, S., Zhang, Y. et al. (2013). Double nickingby RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154,1380-1389.

Rappsilber, J., Mann, M. and Ishihama, Y. (2007). Protocol for micro-purification,enrichment, pre-fractionation and storage of peptides for proteomics usingStageTips. Nat. Protoc. 2, 1896-1906.

Richter, V., Palmer, C. S., Osellame, L. D., Singh, A. P., Elgass, K., Stroud, D. A.,Sesaki, H., Kvansakul, M. and Ryan, M. T. (2014). Structural and functionalanalysis of MiD51, a dynamin receptor required for mitochondrial fission. J. CellBiol. 204, 477-486.

Richter, V., Singh, A. P., Kvansakul, M., Ryan, M. T. and Osellame, L. D. (2015).Splitting up the powerhouse: structural insights into the mechanism ofmitochondrial fission. Cell. Mol. Life Sci. 72, 3695-3707.

Roux, K. J., Kim, D. I. and Burke, B. (2013). BioID: a screen for protein-proteininteractions. Curr. Protoc. Protein Sci. 74, Unit 19 23.

Roy, M., Reddy, P. H., Iijima, M. and Sesaki, H. (2015). Mitochondrial division andfusion in metabolism. Curr. Opin. Cell Biol. 33, 111-118.

Schneider, C. A., Rasband,W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ:25 years of image analysis. Nat. Methods 9, 671-675.

Scorrano, L. (2009). Opening the doors to cytochrome c: changes in mitochondrialshape and apoptosis. Int. J. Biochem. Cell Biol. 41, 1875-1883.

Shen, Q., Yamano, K., Head, B. P., Kawajiri, S., Cheung, J. T. M., Wang, C., Cho,J.-H., Hattori, N., Youle, R. J. and van der Bliek, A. M. (2014). Mutations in Fis1disrupt orderly disposal of defective mitochondria. Mol. Biol. Cell 25, 145-159.

Stojanovski, D., Koutsopoulos, O. S., Okamoto, K. and Ryan, M. T. (2004).Levels of human Fis1 at themitochondrial outer membrane regulate mitochondrialmorphology. J. Cell Sci. 117, 1201-1210.

Strack, S. and Cribbs, J. T. (2012). Allosteric modulation of Drp1 mechanoenzymeassembly and mitochondrial fission by the variable domain. J. Biol. Chem. 287,10990-11001.

Stroud, D. A., Formosa, L. E., Wijeyeratne, X. W., Nguyen, T. N. and Ryan, M. T.(2013). Gene knockout using transcription activator-like effector nucleases(TALENs) reveals that human NDUFA9 protein is essential for stabilizing thejunction between membrane and matrix arms of complex I. J Biol. Chem. 288,1685-1690.

Toyama, E. Q., Herzig, S., Courchet, J., Lewis, T. L., Jr., Loson, O. C., Hellberg,K., Young, N. P., Chen, H., Polleux, F., Chan, D. C. et al. (2016). AMP-activatedprotein kinase mediates mitochondrial fission in response to energy stress.Science 351, 275-281.

Wai, T., Garcia-Prieto, J., Baker, M. J., Merkwirth, C., Benit, P., Rustin, P.,Ruperez, F. J., Barbas, C., Iban ez, B. and Langer, T. (2015). Imbalanced OPA1processing and mitochondrial fragmentation cause heart failure in mice. Science350, aad0116-1-11.

Wakabayashi, J., Zhang, Z., Wakabayashi, N., Tamura, Y., Fukaya, M., Kensler,T. W., Iijima, M. and Sesaki, H. (2009). The dynamin-related GTPase Drp1 isrequired for embryonic and brain development in mice. J. Cell Biol. 186, 805-816.

Wang, K., Long, B., Jiao, J.-Q., Wang, J.-X., Liu, J.-P., Li, Q. and Li, P.-F. (2012).miR-484 regulates mitochondrial network through targeting Fis1. Nat. Commun.3, 781.

Yamano, K., Fogel, A. I., Wang, C., van der Bliek, A. M. and Youle, R. J. (2014).Mitochondrial Rab GAPs govern autophagosome biogenesis during mitophagy.eLife 3, e01612.

Youle, R. J. and Karbowski, M. (2005). Mitochondrial fission in apoptosis. Nat.Rev. Mol. Cell Biol. 6, 657-663.

Youle, R. J. and Strasser, A. (2008). The BCL-2 protein family: opposing activitiesthat mediate cell death. Nat. Rev. Mol. Cell Biol. 9, 47-59.

Zhao, J., Liu, T., Jin, S., Wang, X., Qu, M., Uhlen, P., Tomilin, N., Shupliakov, O.,Lendahl, U. and Nister, M. (2011). Human MIEF1 recruits Drp1 to mitochondrialouter membranes and promotesmitochondrial fusion rather than fission.EMBO J.30, 2762-2778.

2181

RESEARCH ARTICLE Journal of Cell Science (2016) 129, 2170-2181 doi:10.1242/jcs.185165

Journal

ofCe

llScience