Atosfrcrepnec [email protected];[email protected]) ([email protected]; correspondence for *Authors Memphis, Hospital, USA. Research 38105, Children’s TN Jude St. Neurobiology, Developmental ainlUiest,Deen3574 eulco Korea. of Republic 305-764, Daejeon University, National 71,USA. 27710, o-ogLee Joo-Yong mitochondria and challenged fusion metabolically mitochondrial protects adaptive activates deacetylation MFN1 ARTICLE RESEARCH ß 4954 2014 September 19 Accepted 2014; May 28 Received Hoppel, and to (Lesnefsky 1 chain due transport production electron (ROS) the the species meet in oxygen leakages to however, reactive phosphorylation, order elevates oxidative in in also increase mitochondria An increases need. on energy therefore demands shift functional metabolic to the adaptive phosphorylation This oxidative ATP. and generate (TCA) the through cycle and mitochondria acid in metabolized tricarboxylic (fasting) acids be fatty must glycolysis, acids deprivation Unlike well amino production. as and glucose ATP acids, food for fatty use acids, Upon to amino switch as conditions, can animals. cells and supply, source glucose in main reduced normal cellular and energy to preferred response for the of Under is in glycolysis crucial sources through fuel metabolized survival. is of set environments organismal diverse nutrient a use changing to ability The INTRODUCTION Acetylation HDAC6, ROS, stress, MFN1, Metabolic fusion, Mitochondrial metabolically WORDS: KEY protects fusion mice, mitochondrial mitochondria. HDAC6-knockout findings challenged adaptive These that in degeneration. mitochondrial show abrogated extensive in is resulting fusion acetylation- mitochondrial fasting-induced an Remarkably, fusion. muscle by skeletal dramatic fasting, undergo suppressed to mitochondria subjected species mice defect causes In mutant. oxygen a MFN1 it resistant reactive damage, oxidative instead, mitochondrial affect and of acutely production; production not energy deprivation. does excessive adaptive fusion glucose undergo by mitochondrial MFN1 activation, to or induced failure and HDAC6 fusion Unexpectedly, deacetylation in mitochondrial Deficiency MFN1 prevents fusion. to mitochondrial 1 leads promoting suppress mitofusin HDAC6. interaction starvation, deacetylase to protein glucose This the with to fusion associated becomes response (MFN1) active In metabolically stress. undergo that oxidative evidence mitochondria mitochondrial present of we challenged risk Here, the damage. elevates also oxidative but adaptation supply, metabolic energy This ensures mitochondria. shifts to that production switch energy metabolic a more activate shortage glucose and Fasting ABSTRACT uj Lee Eunji eateto hraooyadCne ilg,Dk nvriy uhm NC Durham, University, Duke Biology, Cancer and Pharmacology of Department 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,45–93doi:10.1242/jcs.157321 4954–4963 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 2 rdaeSho fAayia cec n ehooy Chungnam Technology, and Science Analytical of School Graduate 2 .Pu Taylor Paul J. , 1,2, ,Mga Kapur Meghan *, 3 n s-agYao Tso-Pang and 1 igLi Ming , 3 eatetof Department 1 onCagChoi Moon-Chang , 1, * oee,i sulkl httesl ucino stress-induced of function sole the 2011). al., that physical et unlikely Rambold a is 2011; creating al., it et to by However, (Gomes mitochondria proposed autophagy to of been barrier loss has In excessive orchestrated mystery. network prevent an of highly a mitochondrial formation conditions, remains these inter-connected starvation nutrient cases, severe of most to response in importance changes, The and morphological Detmer 2007). 2005; Shaw, and Chan, (Okamoto conditions oxidative stress various challenged prevent metabolically characterized. to fully the be production to How remains ROS damage 2006). manage al., mitochondria et Sanz 2003; eet Goe l,20;Zage l,20;Lee l,2010b). al., et required Lee be might 2008; HDAC6 al., that possibility et the Zhang raise findings 2007; These al., control. et and (Gao quality defects normal The mitochondrial with an mitochondrial grossly associated commonly 2010a). phenotypes are overt suggests in without mice al., mitophagy (KO) HDAC6 HDAC6-knockout et efficient for However, Lee for role 2008; HDAC6 important al., of by et requirement mitochondria impaired (Narendra of mitophagy elimination disease, Parkinson’s the early-onset E3 facilitates ubiquitin in the HDAC6 gene with causative and conjunction and a In Lee parkin, aggregates 2013). 2010b; ligase al., al., et protein et Lee Lee 2010; 2010a; of Yao, al., et dispose non-selective (Lee selectively damaged defined to ubiquitin-dependent an classically mechanism uses autophagy control Unlike quality autophagy, autophagy. control known. not Why is mtDNA . acquiring mutations to mitochondrial prone the more are of mitochondria fusion-deficient This integrity required the is 2010). maintaining machinery al., fusion for mitochondrial et that indicates (Chen finding in cells observed been double-knockout has MFN1/MFN2 mutations (mtDNA) accumulation DNA aberration, mitochondrial al., morphological and of this et deficiency (Chen to fusion fragmented addition a severely In 2003). are creates that MFN2 mitochondria or in results MFN1 either characterized. of well less Lack is MFN2 and MFN1 of increases proteins regulation pro-fusion The the thereby 2011). al., and et (PKA) (Gomes association connectivity Under mitochondrial A mitochondrial 2007). kinase DRP1 protein Chan, reduces by and as phosphorylation (Detmer starvation, known extreme (also the fission and DRP1 stimulates outer delineate whereas DNM1L) mitochondrial respectively, of to membranes, fusion inner promote crucial coordinately OPA1 are questions The dynamics. mitochondrial function. of these mitochondrial role physiological in to role remodeling regulatory morphological answers dynamic active the an whether plays known Additionally, not autophagy. is against it guard to is fusion mitochondrial iohnraudroatv uinadfsini epneto response in fission and fusion active undergo Mitochondria h rti ectls,HA6 sacmoeto quality of component a is HDAC6, deacetylase, protein The and MFN2 MFN1, GTPases -related the mammals, In 1 ui Choi Sujin , 2 a-ueKim Hak-June , 2 ny Kim Inhye , 2 ,

Journal of Cell Science nteidctdcl ie n ersnstema f2 esrmns( MEFs deter measurements was KO 20 connectivity, HDAC6 of of measure and mean HDAC6 a MEFs, mito-YFP, the dead of KO represents fraction catalytic HDAC6 mobile and or The in mito-YFP. lines (+HDAC6wt) connectivity of cell wild-type FRAP network by indicated expressing Mitochondrial measured the stably was (D) in HDAC6, MEFs medium. dead KO (–glucose) catalytic HDAC6 or glucose-free wild-type and or expressing MEFs (+glucose) KO complete HDAC6 in of (+HDAC6cd) images mitochondrial Representative (C) DC id n ectltsMN,laigt MFN1 to to leading failure the MFN1, Importantly, fusion. starvation, deacetylates glucose mitochondrial to and and response activation in binds that show HDAC6 We HDAC6-deficient mice. fusion. and in mitochondrial cells failed induces fusion mice metabolic-stress-induced in This fasting and cells cultured under only population mitochondrial conditions. healthy stress a maintaining for ARTICLE RESEARCH rflswe idtp W)adHA6K Eswr nuae nguoepstv r-eaiemdu -) el ihhprue mjrt of (majority connecte hyperfused not with are (mean Cells (majority cells (-G). hyperfragmented of medium and percentage -negative non-connected) as or and presented glucose-positive interconnected and in of scored incubated population were were (mixed mitochondria MEFs normal cytochrome KO interconnected), for HDAC6 are stained and mitochondria and (WT) starvation. h glucose wild-type 5 when under for fusion profiles medium mitochondrial glucose-free for or required (non-treated) is HDAC6 1. Fig. by challenged mouse were KO mice HDAC6 and conditions, (MEFs) stress fibroblasts mitochondrial for under embryonic required is control HDAC6 that quality hypothesis the test To by induced fusion starvation mitochondrial glucose for required is HDAC6 metabolically RESULTS protecting an damage. for oxidative crucial excessive of from is of mitochondria expression part challenged that a is response the HDAC6 and adaptive that MFN1 by by indicate it mitochondrial mediated findings fusion reversed to rather mitochondrial These mutant. leading autophagy; defect MFN1 stress acetylation-resistant by oxidative a metabolic of mitochondria degeneration, acute buildup of to a loss lead caused not or did deficiency fusion mitochondrial undergo nti eot epoieeiec htguoedpiainin deprivation glucose that evidence provide we report, this In 6 .. rmtreidpneteprmns * experiments. independent three from s.d.) c 6 frmtcodi)adwt AI(o uli.()Qatfcto fmitochondrial of Quantification (B) nuclei). (for DAPI with and mitochondria) (for ....** s.e.m.). opeemdu,uo lcs train mitochondrial starvation, glucose upon in fraction) comparable medium, (mobile showed photobleaching lines complete after cell all mito-YFP although of a 1D, recovery Fig. expressing in 2004). MEFs shown al., As et fluorescence in (Szabadkai (mito-YFP) (FRAP) used YFP mitochondria-localized photobleaching we quantitative after connectivity, a recovery mitochondrial network provide of To mitochondrial inactive starvation. estimate catalytic restored glucose the upon in not effectively morphology shown but HDAC6, As wild-type, 2007). stably catalytic-dead of mutant, al., expression MEFs or et 1C, (Gao KO Fig. (+HDAC6wt) mitochondrial (+HDAC6cd) HDAC6 HDAC6 wild-type the in mutant examined with starvation we glucose reconstituted glucose-free 1A,B). phenotype, to in (Fig. this response fragmented fusion visibly confirm undergo became not To they did instead, MEFs mitochondria medium; contrast, KO In HDAC6 1B). (Fig. in by starvation increased glucose network upon mitochondrial of fold in hyperfused percentage a the increase that with net confirmed cells a Quantification 1A). indicating mitochondria (Fig. network, medium fusion that more-connected found glucose-free a we in formed serum, cultured dialyzed 2010). with al., were et supplemented MEFs Romanello 2003; wild-type al., When et mitochondria-dependent (Ogawa increase production to energy fasting or starvation glucose A idtp W)adHA6K Eswr nuae ihcomplete with incubated were MEFs KO HDAC6 and (WT) Wild-type (A) P , 0.01. ora fCl cec 21)17 9446 doi:10.1242/jcs.157321 4954–4963 127, (2014) Science Cell of Journal P , .5 ** 0.05; P , .1(Student’s 0.01 t -test). d) mined 4955 , 2-

Journal of Cell Science DC id n ectltsMN nrsos to response in starvation MFN1 glucose deacetylates and binds HDAC6 glucose by induced fusion starvation. mitochondrial was promoting are activity for catalytic MEFs its required and HDAC6 that KO+HDAC6cd show KO+HDAC6wt results HDAC6 These and MEFs. in that KO to compared reduced HDAC6 significantly in connectivity ARTICLE RESEARCH olciey hs eut niaeta HDAC6-mediated 4956 that 2F,G). (Fig. indicate activity activity. MFN1 results reduced increases deacetylation showed these again Collectively, the mutant whereas acetylation- fusion, mitochondrial K222Q the promoting than 2B), in active more (Fig. MFN1 significantly wild-type was acetylated MFN1 mutant more K222R resistant MEFs, was KO HDAC6 MFN1 in less where fusion conclusion, is this MFN1 mitochondrial supporting acetylated Further that efficient active. suggesting MFN1 panels), induce 2E, bottom (Fig. acetylation-mimicking to 2E, (Fig. well the failed as mutant contrast, top assay K222Q was In 2E, complementation (Fig. panels). mutant this MEFs middle in MFN1-K222R KO a active acetylation-resistant MFN1 reestablished highly As The in MFN1 S1C). mitochondria wild-type panels). of the Fig. to of network material showed shown expression mutant (supplementary MFN1-K222R been expected, the acetylation and has 2014) reduced in al., Gorovsky, glutamine and et fusion (Ren Wang note, acetylation 2001; mitochondrial lysine Of for restore substitute MEFs. functionally to acetylation-resistant KO mutants and MFN1 MFN1 ability (K222Q), the (K222R) assessed whether acetylation-mimicking we investigate activity, of To MFN1 affects 2009). acetylation al., mass K222 by et identified been to (Choudhary previously spectrometry has response MFN1 of in domain GTPase MFN1 that or only indicate starvation deacetylates findings glucose starvation. and glucose these by binds Collectively, affected HDAC6 2D). not (Fig. is HDAC6 however, acetylation HDAC6; the glucose with is interacts MFN2 to and (MFN2) not acetylation 2 subject to restored subjected mitofusin but MEFs also 3), MFN1-releated KO the lane 2), Notably, HDAC6 2C, deprivation. lane in (Fig. proposition, deacetylation HDAC6 2C, under MFN1 this (Fig. mutant, deacetylation dead wild-type Supporting catalytically MFN1 of conditions. promotes that reintroduction suggest starvation and results 4; to glucose lane These 2B, binds S1B). (Fig. acetylation MEFs HDAC6 Fig. KO S1B), MFN1 HDAC6 material Fig. in material in supplementary observed supplementary not 3; reduction was lane which Panel, marked was AcK complex 2B, a MFN1–HDAC6 (Fig. lane the by 2B, of (Fig. formation accompanied starvation the glucose Notably, medium upon 3). markedly complete HDAC6 became MFN1 with endogenous in associated +glucose), 1, cells lane 2B, in (Fig. 2B, assays by Fig. observed in was co-immunoprecipitation shown complex MFN1–HDAC6 As detectable MFN1. no functionally factor although of pro-fusion HDAC6 the whether independently with investigated interacts fusion therefore mitochondrial indicate We results DRP1. regulates These 2A). HDAC6 by (Fig. elevated that solution clearly MEFs was Hank’s KO al., it with HDAC6 although treatment or et starvation, wild-type glucose in Rambold to induced to of subject not 2011; phosphorylation was inhibitory leading the S637 al., DRP1 that DRP1 PKA, found et we solution), by However, (Gomes 2011). (Hank’s inhibited fusion and starvation mitochondrial phosphorylated nutrient becomes severe Under ctlto facnevdlsn eiu K2)i the in (K222) residue lysine conserved a of Acetylation ossetwt oprbeoye osmto and consumption oxygen comparable with to Consistent able also were wild- MEFs to oxygen increase KO Similar HDAC6 3A). glucose-starved starvation, (Fig. al., MEFs, level MEFs type a wild-type glucose et to in that elevated to (Kamemura to eventually normal comparable MEFs KO report response in HDAC6 in recent in consumption consumption a However, KO oxygen with HDAC6 2012). oxidative basal consistent activity. through lower mitochondrial medium, showed ATP affects whether MEFs status produce investigated fusion next to We the mitochondria. acids, in sources, phosphorylation fuel fatty alternative utilize to including cells forces starvation Glucose under starvation ROS glucose mitochondrial reduces fusion Mitochondrial iohnra uin esbetdwl-yeadHA6KO HDAC6-dependent HDAC6 and of wild-type relevance subjected we physiological fusion, the mitochondrial assess muscle To anterior HDAC6 tibialis in mouse abolished KO is fusion mitochondrial Fasting-induced lvto fmtcodilatvt nrsos oglucose to response in activity the mitochondrial for starvation. required of not is These fusion elevation 3E,F). mitochondrial (Fig. that ATP conditions indicate produce findings starvation to glucose able Similarly, were under starvation. MEFs normally KO acute glucose MFN1 meeting to that fusion-deficient of response the capable indicate in are demands results cells in energetic KO levels These HDAC6 ATP in 3C,D). similar (Fig. mitochondria generated medium also types glucose-free cell both oxidation, ae7 i.4) ae oehr hs idnsidct that indicate starvation. glucose findings upon stress higher these generate ROS together, cells mitochondrial 4C, fusion-deficient Taken An (Fig. mitochondria and 4D). starvation mitochondria HDAC6- 4D). Fig. glucose (Fig. in to 7, of detected subjected starvation lane 4), not MEFs and significant glucose wild-type was 2 from (lanes population upon a cytosol protein the MEFs oxidized not starvation. observed but KO 4C, 8), glucose (Fig. HDAC6 and mitochondria we 6 upon the lanes in ROS compare proteins conclusion, oxidized of ROS of accumulation this buildup can limiting deficiency aberrant Supporting fusion in to mitochondrial 4B). that deacetylation lead (Fig. indicate significantly mitochondria results MFN1 challenged These be metabolically the of in an could not production supporting role but mutant, MEFs (K222R), important MFN1 acetylation-resistant KO (K222Q), the acetylation-mimicking HDAC6 by suppressed in glucose observed after accumulation ROS 4A). (Fig. starvation are mitochondrial displayed which also MEFs, fusion, significant membrane KO inner elevated OPA1 mitochondrial further in 4A). defective (Fig. were starvation which glucose conditions, upon basal under levels have ROS which MEFs, mitochondrial increased KO showed mitochondria, MFN1 fragmented severely Interestingly, 4A). HDAC6 (Fig. in MEFs ROS mitochondrial KO in increase glucose an ROS contrast, in In mitochondrial resulted starvation. starvation glucose of and conditions levels basal similar under produced 4A, Fig. MEFs were in shown wild-type levels As staining. MitoSox normal ROS by in medium cultured glucose-free mitochondrial MEFs or KO end, HDAC6 and wild-type this therefore in mitochondrialdetermined To We affects production. 2006). fusion al., chain ROS mitochondrial transport et electron whether Sanz the determined 2003; in Hoppel, leakages and to (Lesnefsky due production ROS nraei xdtv hshrlto ol oetal edto lead potentially could phosphorylation oxidative in Increase motnl,teaern iohnra O production ROS mitochondrial aberrant the Importantly, ora fCl cec 21)17 9446 doi:10.1242/jcs.157321 4954–4963 127, (2014) Science Cell of Journal b oiainaduiieplii cd(i.3B). (Fig. acid palmitic utilize and -oxidation b -

Journal of Cell Science 22 rK2QMN vrxrsini DC OMF.Cnrlo F-oiieclswr aeoie nohprue,nra n fragmented and mean normal as hyperfused, presented into are categorized and were category cells each CFP-positive in or cells Control of MEFs. percentage KO as HDAC6 scored in mitochondria, overexpression MFN1 K222Q or K222R anti-cytochrome- and (green) anti-CFP with immunostaining 25 bars: Scale antibodies. EERHARTICLE RESEARCH bevdi idtp,btntHA6K,mc fe fasting after mice KO, HDAC6 indeed was not muscle but starvation, skeletal wild-type, first in in general glucose acetylation a observed MFN1 We variable, lower to more of Although trend similar phosphorylation. deacetylation. MFN1 fasting, caused oxidative whether energy more investigated generate mitochondrial to muscle skeletal from forcing fasting, to mice anti-cytochrome- and (green) anti-CFP expr with an plasmid immunostained using a immunoprecipitation were with to cells transfected transfection, subjected were after and MEFs a h h KO as 18 MFN1 5 used (E) At for antibodies. was MFN1. medium MFN2 actin glucose-free K222Q and and and or w lysine anti-GFP normal K222R anti-acetyl blotted using wild-type, in using and confirmed CFP-tagged cultured analysis antibody, was blotting were anti-MFN1 expression western MEFs an HDAC6 and (CD) KO with Reconstituted antibody dead HDAC6 (IP) anti-MFN2 catalytic indicated. immunoprecipitation and its as to Wild-type WTor MFN1 subjected (D) HDAC6 and expressing h, control. (AcK) KO loading 5 HDAC6 lysine for (C) acetyl or medium HDAC6, MEFs glucose-free for KO or HDAC6 antibodies complete and Wild-type with (B) incubated GAPDH. were and HDAC6 mutant, DRP1, S616), or starvation. S637 (at glucose (p-DRP1) under MFN1 ( deacetylates glucose-free and or with (control) interacts HDAC6 2. Fig. 2 m lcs)mdu,o aksslto o n ujce owsenbotn nlssuigatbde gis hshrltdDRP1 phosphorylated against antibodies using analysis blotting western to subjected and h 5 for solution Hank’s or medium, glucose) .()HA6K Eswr rnfce ihapamdepesn F-agdwl-ye 22 rK2QMN,floe by followed MFN1, K222Q or K222R wild-type, CFP-tagged expressing plasmid a with transfected were MEFs KO HDAC6 (F) m. c rd nioy cl as 10 bars: Scale antibody. (red) ic(i.5,fd o aes.Uo atn,mtcodi in mitochondria fasting, Upon Z- panels). the of top side fed, either 5A, on (Fig. state, pairs anterior disc fed in tibialis KO stereotypically the by analyzed HDAC6 resided under and muscle muscles 5A, wild-type next Fig. anterior both in in tibialis shown mitochondria We As the microscopy. S1D–F). in electron morphology Fig. mitochondrial material (supplementary A idtp W)adHA6K Eswr nuae ihcomplete with incubated were MEFs KO HDAC6 and (WT) Wild-type (A) 6 m ..fo he needn xeiet.** experiments. independent three from s.d. ora fCl cec 21)17 9446 doi:10.1242/jcs.157321 4954–4963 127, (2014) Science Cell of Journal .()Qatfcto fmtcodilpoie fe F1wild-type, MFN1 after profiles mitochondrial of Quantification (G) m. P , 0.01. c (red) ith essing 4957

Journal of Cell Science nuae ihguoepstv E n ngtv eim()fr5hadsbetdt T nlss eaieAPlvl eedtrie ycmaigt comparing by determined were levels ATP Relative analysis. ATP to subjected mean and are h Results 5 MEFs. for wild-type (F) in medium level -negative ATP and average (E) glucose-positive with incubated aayi edHA6(HA6d eeicbtdwt lcs-oiie()ad-eaiemdu D o n ujce oAPaayi.Rltv ATP Relative analysis. ATP to subjected and h 5 for (D) medium ( -negative mean and the (C) comparing glucose-positive by with determined incubated were were levels (+HDAC6cd) HDAC6 dead catalytic * EERHARTICLE RESEARCH ucnt eyrgns SH ope I le ciiy As activity. 4958 blue) that II, complex staining (SDH, histological dehydrogenase by succinate tibialis evaluated in cytochrome were assessed complexes white activities chain muscle the 5A, transport anterior (Fig. packed electron damage, mitochondrial damage densely mitochondrial of confirm less mitochondrial To contained of arrowheads). and indicative for swollen cristae, required frequently mice, is fuse were HDAC6 to KO mice. Thus, in failed fusion HDAC6 arrowheads). black but mitochondrial fasting-induced white aggregated fasted, fasted 5A, were (Fig. 5A, dramatic in (Fig. mitochondria that sarcomeres individual underwent Remarkably, mitochondria multiple elongated arrowheads). muscles large spanned into often anterior fused and realignment tibialis wild-type ( rate consumption oxygen as presented are Data (Student’s analyzer. significant flux not ( activities. extracellular n.s., medium mitochondrial XF24 glucose-free for Seahorse or essential a +glucose) not using (full, is complete fusion with mitochondrial incubated starvation-induced Glucose 3. Fig. P , neetnl,ufsdmtcodi nfse DC Omice KO HDAC6 fasted in mitochondria unfused Interestingly, .5 .. o infcn (Student’s significant not n.s., 0.05; c xds CX ope V rw)and brown) IV, complex (COX, oxidase t ts) B amtt xdto nlss aapeetda h mean the as presented Data analysis. oxidation Palmitate (B) -test). t ts) CD idtp n DC OMF,adHA6K Essal xrsigwl-ye(HA6t or (+HDAC6wt) wild-type expressing stably MEFs KO HDAC6 and MEFs, KO HDAC6 and Wild-type (C,D) -test). 6 .. fAPlvli idtp Es hc a e s10.(,)Wl-yeadMN OMF were MEFs KO MFN1 and Wild-type (E,F) 100%. as set was which MEFs, wild-type in level ATP of s.d.) 6 ..fo he needn experiments. independent three from s.d. 2 lcs)fr5o 8ha niae n ujce ooye osmto aeanalysis rate consumption oxygen to subjected and indicated as h 18 or 5 for glucose) eurdt rmt iohnra uinadprevent and fusion is fasting. HDAC6 mitochondrial by challenged that muscle show skeletal promote in results damage al., These mitochondrial to et 2010). (Lee al., dysfunction required et mitochondrial SDH of Chen in hallmark 1998; increase a compensatory activity was a is genome, in with activity SDH decrease nuclear activity A IV the muscle. in complex KO by COX HDAC6 encoded fasted increase in is detected which specifically an blue), Conversely, 5C, tibialis (Fig. wild-type muscle. in not mitochondrial but KO the anterior types. HDAC6 IV by in encoded complex observed fiber is was COX which genome, in muscle brown), decrease 5B, similar different (Fig. marked activity a showed the fasting, and upon reflects muscle in However, which wild-type activities anterior appearances, condition, checkerboard mitochondrial tibialis fed light-blue and the mice brown under KO 5B,C, Fig. HDAC6 in shown ora fCl cec 21)17 9446 doi:10.1242/jcs.157321 4954–4963 127, (2014) Science Cell of Journal 6 ..o he needn xeiet.Ex Etomoxir. Etx, experiments. independent three of s.d. 6 ... rmtreidpnetasywls * wells. assay independent three from s.e.m.) A idtp W)adHA6K Eswere MEFs KO HDAC6 and (WT) Wild-type (A) P , 0.05; he

Journal of Cell Science needn xeiet.* experiments. independent EERHARTICLE RESEARCH iohnrafo en erddb uohg ne more under autophagy by degraded being active response from stress mitochondria identify the of findings mitochondria. part challenged mitochondrial Our metabolically integral protects an that excessive damage. as fusion and binds, in mitochondrial stress resulting which oxidative fusion, prevents HDAC6, and HDAC6 deacetylation mitochondrial of MFN1 induced deacetylase loss fasting- The and glucose-starvation- MFN1. fusion protein activates mitochondrial and deacetylates adaptive the from metabolically orchestrated that requires highly oxidative evidence limit production The to presented fusion stress. damage. active have undergo oxidative energy we mitochondria mitochondrial challenged report, of this elevates risk In the and simultaneously mitochondria reprogramming metabolic activate mean that shortage the glucose are Data and 100%). Fasting at (set sample qu +glucose to MEF DISCUSSION program Hsp90 wild-type ImageJ reaction. the the derivatization with to the analyzed relative were after intensity images moiety of w Oxyblot dinitrophenyl fractions percentage (D) to respectively. mitochondrial as antibody fractions, and meter presented mitochondrial Cytosolic an fluorescence are mean h. and by a and cytosolic as 5 detected with the intensity for presented were analyzed for medium band are proteins makers glucose-free were Data or as oxidized and assay. complete used where staining the in were assay MitoSox Tom20 incubated after Oxyblot to were concentration transfectio an MEFs subjected protein capillary KO to were the Neon HDAC6 subjected h, a to and using 5 Wild-type normalized mutants (C) for was K222Q wells. medium MFN1 value independent and glucose-free fluorescence K222R or The MFN1 complete for Promega). in plasmids OP (GloMax, KO, expression cultured ( MFN1 or were wild-type, (pcDNA), medium immortalized Cells plasmid glucose-free T control system. large or a SV40 with (+glucose) and transfected KO complete were HDAC6 in MEFs (WT), incubated starvation. wild-type glucose immortalized were 3T3 upon MEFs MitoSox. cells KO with fusion-deficient ROS mitochondrial mitochondrial in of accumulation analysis ROS cytometry mitochondrial Aberrant 4. Fig. ciefso a eetybe rpsdt prevent to proposed been recently has fusion Active P , .5(Student’s 0.05 t -test). 2 lcs)fr5h tie ihMtSxadaaye yFC.()HA6KO HDAC6 (B) FACS. by analyzed and MitoSox with stained h, 5 for glucose) w tescniin satvtdb ifrn mechanisms: different by activated mitophagy. is these excessive protects conditions under fusion from stress mitochondrial former proposal, mitochondria two this the with shields Consistent whereas latter adaptation: stress oxidative the from physiological by mitochondria active represents elicited metabolically starvation distinct 5). extreme fusion and and mitochondrial a fasting 4 or that (Figs deprivation damage suggest glucose mitochondrial findings and accompanied These stress is challenge oxidative undergo to metabolic by failure upon a fusion that in found defect mitochondrial we prominent complexes (supplementary Instead, a fusion. respiratory despite mitochondria mitochondrial S2E) mitochondrial Fig. of or glucose- material (supplementary S2A–D) of loss Fig. not analysis significant did material Our however, a 2011). mice, (Gomes HDAC6-deficient al., reveal solution) fasted et or Hank’s cells Rambold starved in 2011; (e.g. al., et starvation nutrient extreme ora fCl cec 21)17 9446 doi:10.1242/jcs.157321 4954–4963 127, (2014) Science Cell of Journal A ersnaiehsormo flow of histogram Representative (A) 6 ..o three of s.d. 6 ..o three of s.d. and 4959 antify ere A1 n

Journal of Cell Science EERHARTICLE RESEARCH 4960 mitochondrial active and activity HDAC6 more the MFN1 by of fine-tuning is regulated whereas for acetylation allow MFN1 reversible might fusion Thus, mutant 2E–G). mitochondrial severely (Fig. K222R is promoting MFN1 acetylation-resistant in mutant Indeed, 2003). K222Q defective al., domain et acetylation-mimicking The (Santel GTPase the fusion activity HDAC6 the mitochondrial GTPase S1A). MFN1 for within require the important inhibit Fig. K222 might not acetylation acetylatable that material suggests does the of (supplementary interaction location this activating activity and note, promotes catalytic was deacetylating HDAC6 Of binding, that mice MFN1. by HDAC6-deficient indicate findings fusion in in These 2B; mitochondrial impaired mice. (Fig. were and fasting acetylation cells fusion deacetylation MFN1 MFN1 and mitochondrial Both in and S1D–F). Fig. cells reduction material a supplementary cultured by accompanied in mitochondrial starvation prevent non-specifically its and not connectivity fusion. mitochondrial does for stress-induced required deficiency not of supplementary is forms HDAC6 and that 1 all indicating (Fig. wild- S3C,D), starvation to Fig. glucose material similar Hank’s under DRP1 S3A,B), with MEFs Fig. treatment cells type inhibitory material KO upon (supplementary HDAC6 networks and view, solution 2011; mitochondrial this al., form Supporting study), et 2011). can al., (Gomes (this et starvation Rambold extreme fasting IV upon complex glucose COX phosphorylation in or to decrease in response but activity (blue) (blue) in SDH starvation SDH in (C) deacetylation increase and the MFN1 (brown) Note IV HDAC6-dependent indicated. 100 complex as bars: COX mice Scale of KO muscle. Analysis HDAC6 arrowhea KO (B) White or HDAC6 WT mice. vacuolated. fasted WT fasted arrowhea are fasted Yellow in or in which indicated. staining fed mitochondria of as from (brown) elongated many and sections condition mice, fused anterior each mark KO tibialis from arrowheads HDAC6 Black transverse muscle fasted Z-disc. anterior the in tibialis of mitochondria side of either unfused sections on pairs longitudinal muscle. in on residing in performed mitochondria fusion was mitochondrial microscopy starvation-induced for Electron required fasting. is HDAC6 5. Fig. efudta iohnra uinidcdb glucose by induced fusion mitochondrial that found We m m. 09 az n ar 09 uk ta. 02.Tu,w ao a favor we Thus, al., 2012). et al., elevated et (Anderson Suski production 2009; Nair, ROS of and subsequent Lanza backflow and 2009; electron degrees the I further increase complex to some to proposed awaits been Fig. has showed material which that S3E), (supplementary and potential all issue membrane MFN1- mitochondrial HDAC6-, crucial MEFs that noticed 2006; a OPA1-KO we al., is However, et affects investigation. connectivity (Yu accumulation to mitochondrial models How contribute ROS 2011). other to al., in et shown Nakamura production been ROS has excessive fission oxidative less mitochondrial an generate with stress, mitochondria Consistent or from networked deprivation. acid that glucose proposal fatty arise the production upon from oxidation could production acid ROS energy amino which mitochondrial limiting fusion in accumulation, mitochondria increase in active ROS that indicate deacetylation suppresses results These MFN1 4B). crucial (Fig. mitochondrial a of supporting reverse starvation, could role glucose MEFs upon KO glucose accumulation but HDAC6 ROS to 3). K222R, in (Fig. MFN1 subject of K222Q, MEFs expression not MEFs Notably, OPA1-KO 4A). in and (Fig. detected deprivation was MFN1- adaptation however, HDAC6-, ROS, in energetic mitochondrial of acute buildup Aberrant for 2013). al., et involves required Tailor and 2012; al., HDAC6 et to of conditions. (Lee shown DRP1 independent non-stressed of is been down-modulation activity under have this is inhibitors fusion However, It HDAC stresses. mitochondrial that or increase noting challenges metabolic worth to also response in dynamics u nlssidctsta dpiemtcodilfso snot is fusion mitochondrial adaptive that indicates analysis Our A idtp W)o DC Omc eefdo ujce o4 of h 48 to subjected or fed were mice KO HDAC6 or (WT) Wild-type (A) ora fCl cec 21)17 9446 doi:10.1242/jcs.157321 4954–4963 127, (2014) Science Cell of Journal sshow ds smark ds

Journal of Cell Science neuiyo traini nvra tescniini nature, in condition stress universal food demands a that is energetic Given starvation damage. or increased oxidative insecurity excessive meet incurring mitochondria without would network that mitochondrial a of ensure suggest formation findings the Fig. whereby our model material mechanism, a specific supplementary the thereby of 4A; Regardless (Fig. and S3E). accumulation potential, ROS trans-membrane stress limit under lower connectivity might mitochondrial conditions increased where model ARTICLE RESEARCH eeosgf rmRcadYue(ainlIsiue fHealth, of Institutes (National Youle a Richard were CFP–MFN1 from and Lee Mito-YFP gift 2007; expressing al., HDAC6 Plasmids et generous (Gao 2010b). various previously al., with described et reconstituted as prepared MEFs were KO constructs HDAC6 and Wild-type plasmids and lines Cell the METHODS AND MATERIALS preventing by damage that, ROS. mitochondrial of suggest buildup data suppress Our could fusion 2010). counterparts mitochondrial actively al., mechanism, dilution et healthy passive Chen a their beyond 2009; al., with complementing et mtDNA by (Nakada mitochondrial function pathogenic the that mitochondrial proposed recessive target maintain support previously relevant can been a phenotypes has fusion is It machinery mitochondrial HDAC6. fusion of MFN1 (Chen These the that 2010). mice hypothesis al., double-knockout skeletal- et in MFN1/MFN2 reported analogous that previously out muscle-specific been point to have al., important defects is et not it mitochondrial (Chen 2010a), al., could mitophagy et HDAC6- Lee we and 2010; as transport such Although mechanisms, mitochondrial 5). other dependent (Fig. of involvement activity the exclude SDH degenerative activity IV increased by complex and COX characterized mitochondrial decreased defects morphology, mitochondrial demands. showed metabolic that great events future. of the be changing signaling enables in would interest response that and mitochondrial to program adaptive components this elaborate constitute the an adapt of of Characterization part to a is mitochondria it context, that mitochondrial suggests this for remodeling dispensable morphological In mitochondrial but dramatic The fusion, mitochondria. movement. for required of is concentration HDAC6 active by and preceded that likely transport indicate is findings were fusion These mitochondrial they 5A). KO fasting-induced properly, (Fig. fuse HDAC6 congregated to and in failed clustered muscles mitochondria anterior although tibialis fasted Interestingly, S4). similar a materialFig. (supplementary tissues suggesting other conditions, in mitochondria fasting in of regulation mitochondria under fragmented brain and KO smaller HDAC6 observed also KO HDAC6 We in re- disrupted mice. clearly spatial process extensive a to fusion, undergo and muscles organization response indicates fasted mitochondria in in giant mitochondria of that remodeling appearance The morphological 5A). (Fig. striking fasting as a tumors revealed of fitness metabolic the medium for the remodeling important well. mitochondrial more in be is active galactose also Thus, be phosphorylation could 2004). with al., oxidative to glucose et when (Rossignol replacing appear cells by also tumor elevated the in in mitochondria survival connected and Furthermore, fitness a individual wild. plays maintaining although likely in conditions, role fusion, crucial laboratory standard mitochondrial under dispensable adaptive HDAC6-dependent h lcrnmcocp nlsso kltlmtcodi has mitochondria skeletal of analysis microscopy electron The h nue iohnrai atdHA6K muscles KO HDAC6 fasted in mitochondria unfused The n ujce ofursec-ciae elsrig(AS analysis (FACS) sorting cell 2007). al., fluorescence-activated et (Mukhopadhyay to subjected and ehsa D.LpfcaieLX(nirgn a sdfor used was (Invitrogen) LTX protocol. manufacturer’s to Lipofectamine according transfection MD). Bethesda, eeaqie yaLiaS5cnoa irsoe(ec DMI6000C). (Leica microscope confocal Images SP5 immunostaining. Leica for a processed by then acquired and Cells were PBS h. followed with 5 coverslips, for washed medium glass -negative were on or glucose-positive cultured with were incubation al., Cells the et by 2004). (Hubbert al., et previously Lee described 2002; as performed was Immunostaining microscopy Immunofluorescence h. 5 or for dialyzed 11995) F0392) 10% (Sigma with number 11966) serum number product product (Invitrogen, (Invitrogen, incubated DMEM -negative and DMEM times medium glucose-positive three were (PBS) with Eagle’s confluence saline 70% serum phosphate-buffered at calf modified with cells fetal washed experiments, 10% starvation Dulbecco’s glucose with For 11995) (FCS). in number product maintained Invitrogen, (DMEM; were starvation MEFs glucose and 611113). culture Cell (BD, DRP1 DRP1 and phosphorylated 3455) 2118), and Signaling, 4867 Signaling, Biotechnology, (Cell (Cell GAPDH Scientific, Cruz 556432), cytochrome Thermo sc11415), (Santa (BD, and Biotechnology, Cruz 9441 (Santa MFN1 Tom20 Signaling, the MA1-2021), used: against (Cell Antibodies acetyl-lysine were 2007). sc100561), al., proteins et to (Gao 991 following previously acids described amino against as generated 1149 was antibody HDAC6 Anti-mouse reagents and Antibodies rprdb dig40 adding was mixture by The medium. prepared of fresh h for exchanged 10 was After medium penicillin-streptomycin. the incubation, and glucose-negative serum FBS or glucose-positive dialyzed with either medium using dishes plated 6-cm and counted duplicate were in Cells 2006). al., et (DeBerardinis assays described b b or glucose-positive (5 with MitoSOX with incubated loaded PBS, h, 5 with Probes). for medium washed (Molecular -negative were indicator using cells determined superoxide was The mitochondrial starvation glucose red to MitoSOX response in generation ROS analysis 2001). ROS Neefjes, Mitochondrial were and fractions (Reits Mobile methods software. published Images AF2.0 to ROIs LAS according imaged laser. in calculated Leica 514-nm intensity with fluorescence (ROIs) the the quantified and of was interest intervals 1-s iterations of in taken two were regions with to Circular photobleaching after used. according was Leica Leica (2.5 software a analysis, (Invitrogen) FRAP with AF2.0 For imaged DMI6000C). LAS LTX (Leica were microscope confocal cells SP5 Lipofectamine Transfected protocol. mito-YFP-expressing manufacturer’s using 2004). the al., with et transfected plasmid (Szabadkai transiently previously were described cells Briefly, as performed was FRAP assay FRAP connectivity ( cells Mitochondrial number 100 cell of category than mean each (more as of in presented experiments and independent cells analyzed) majority three of were from number hyperfragmented, counted The and are connected. was ‘3’, interconnected not mitochondria of are or of mitochondria population mitochondria; majority mixed hyperfused, non-connected normal, ‘1’, ‘2’, according criteria: interconnected; categorized following are cells to analysis, morphology mitochondrial For S.Temxuewsvree o tlat1mnadte 20 then and min 1 least at fatty-acid-free for essentially vortexed 10% was of mixture solution The a BSA. in stock palmitate unlabeled odtriennseii ons h ape eeicbtdovernight incubated were samples The (200 counts. 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Journal of Cell Science im,272) n ltdwt ae.Teeun a ujc to subject was eluent The water. with quantification. for eluted counts scintillation and 217425), Sigma, ehd(T ilmnsec sa i SI,Rce emn)in Germany) Roche, protocol. II, manufacturer’s the HS to according kit lysates cellular prepared assay freshly Bioluminescence (ATP bioluminescence luciferase-driven method the by quantified was ATP Intracellular assay ATP (1 Resin Dowex to conjugated spun, were 400 and h) (12–13 ARTICLE RESEARCH 4962 Chan, and E. S. Fraser, E., E. Griffin, J., A. Ewald, A., S. Detmer, H., Chen, T., C. Lin, A., D. Kane, L., T. Woodlief, E., K. Boyle, E., M. Lustig, J., E. Anderson, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.157321/-/DC1 online available material Supplementary material Supplementary months. 12 manuscript. after the release of design, preparation for study or PMC in publish in to role Deposited decision no analysis, had 2R01- and funders numbers collection T.-P.Y. The [grant data to Health AR055613] of and Institutes NS054022 National National and Chungnam number J.-Y.L; 2012 [grant to Korea of University of fund Foundation research Research and National NRF-2012M3A9C6050087]; by supported is work This Funding performed E.L. and I.K. H.-J.K., S.C., experiments. the M.-C.C., T.-P.Y. wrote M.L., and M.K., J.-Y.L. J.-Y.L., project; manuscript; the T.-P.Y. designed and J.P.T. J.-Y.L., contributions Author interests. competing no declare authors The interests Competing manuscript. (Duke Rao the Yanhua reading and critically NC) University, for (Duke NC) OPA1 McClure University, and Allie MFN1 thank for We CA) Technology, MEFs. of KO Institute (California Chan David thank We Acknowledgements neuromuscular.wustl.edu/pathol/histol/cox.htm). http:// were Center and staining Disease (http://neuromuscular.wustl.edu/pathol/histol/sdh.htm Neuromuscular SDH website University and Washington the G COX from Fluoromount for obtained in protocol mounted and distilled Detailed washed, with (SouthernBiotech). activity, washed SDH activity, for COX stained for water, stained and nitrogen. cutting prepared liquid optimal in were frozen in Slides and embedded (Tissue-Tek) and compound (OCT) dissected temperature was muscle anterior staining analysis. Tibialis SDH further and to COX subjected Histological were and Duke performed supply Mice the water IACUC. with at were the h housed with 48 Duke experiments accordance were for at in fasted mice animal facilities All facility mouse guidelines. EM All University approved by to imaging. processed according and in for and fixed wild-type immediately solution University from were muscles h glutaraldehyde collected 48 anterior 4% were Tibialis After mice. microscopy. muscles KO electron HDAC6 anterior for tibialis used was fasting, muscle anterior Tibialis microscopy Electron to according S7510) protocol. using (Chemicon, manufacturer’s detected kit the were detection fractions oxidation mitochondrial protein Oxidized and Oxyblot analysis. cytosol protein oxidized both the in to proteins subjected were fraction 2 each protocol. manufacturer’s of the to according 89874) Mitochondrial (Pierce, the kit isolation using prepared, were fractions mitochondrial and Cytosolic analysis protein Oxidized uinadaeesnilfrebyncdevelopment. embryonic for essential al. are and et fusion H. C. to H. intake D. Szeto, fat excess humans. S., and link P. rodents state both Rabinovitch, redox in resistance cellular L., insulin and Kang, emission H2O2 III, Mitochondrial W., J. Price, 20) iouisMn n f2codntl euaemitochondrial regulate coordinately Mfn2 and Mfn1 Mitofusins (2003). m fmdu a olce rmec ih h samples The dish. each from collected was medium of l 6 .Ci.Invest. Clin. J. 020inecag resin, ion-exchange 80–200 .Cl Biol. Cell J. 119 160 573-581. , 189-200. , (2009). m g az,I .adNi,K S. K. Nair, and R. I. Lanza, T., Komiya, A., Y., Shitara, Nixon, Y., Ohtsuka, A., S., Ohkubo, Ito, M., Y., Ogawa, K., Kawaguchi, Kamemura, R., Shao, A., Guardiola, C., L. Hubbert, Scorrano, and P. G. T. Benedetto, Yao, Di and Y. C., J. L. Lee, S., Gomes, Y. Lee, J., Lu, C., C. Hubbert, S., Y. Gao, Detmer,S.A.andChan,D.C. e,C . sns .E,Cug .S,Widuh .adAkn .M. J. Aiken, and R. Weindruch, S., S. Chung, E., L. Aspnes, M., C. Lee, P. T. Yao, and Y. J. Lee, B. C. Thompson, and J. J. Lum, J., R. DeBerardinis, C., T. Walther, M., Rehman, L., M. 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