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Tondera Nunnari influenced 2009; al., mitochondrial et cell the Mitra 2008; highly between highlighting between (Du conditions, activity cells, links vary is pathological all functional diameter and whose to architecture physiological and organelles common 2012). polymorphic Mitochondrial length processes Suomalainen, strikingly shape, and in are (Nunnari mitochondria involved induction buffering apoptosis Although calcium supply, play they energy and where including cells functions eukaryotic all crucial in present are Mitochondria Introduction membranes. diameter elongated mitochondrial influences of PMI TMEM11 words: orthologue length, tangle Key human crista dense its controlling and by a PMI that, Since form suggest species. shape. data oxygen cristae tubular our reactive of which morphology, and level mitochondrial in the of in regulators length neurons increases crista are and reduce adult activity to chain sufficient in respiratory is show overexpression exacerbated impaired We PMI function. is that and show of ultrastructure phenotype we Conversely, loss mitochondrial This of that level cells. demonstrates the embryonic morphology at cristae. and inner-membrane the organism, of the whole tips of the the the of at which with scale membrane in the the flies associated that bend at consequences that both are subunits them (PMI) phenotypic ATP-synthase Among I the and identified. Mitochondrion junctions been here crista have production. establish report determinants ATP to crista regulate We required few hence are and and that understood complexes Mitofilins poorly chain the are respiratory morphogenesis concentrate crista that controlling structures Mechanisms inner-membrane mitochondrial are Cristae Summary ( correspondence for *Authors 3 2 1 Macchi diameter Marc mitochondrial and biogenesis crista The 814 -34 asil,Cdx1,France 15, Cedex Marseille, F-13344 uinRoyet Julien R oiooyUi,HdknBidn,P o 3,Uiest fLietr acse od ecse E H,UK 9HN, LE1 Leicester Road, Lancaster Leicester, of University 138, Box PO Universite Building, Aix-Marseille Hodgkin Unit, Toxicology MRC Universite Aix-Marseille 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. iohnrainrmmrnsaecmatetlzdinto compartmentalized are membranes inner Mitochondria Drosophila r ta. 06 emradCa,20;Se tal., et Suen 2007; Chan, and Detmer 2006; al., et ¨rr Cristae, 1, 1 al lFissi El Najla , n hmsRival Thomas and * PMI Drosophila ,CR,Cnr eRcecee erbooi tNuohsooi eMrele M 26 olvr ireDramard, Pierre Boulevard 7286, UMR Marseille, de Neurophysiologie et Neurobiologie en Recherche de Centre CNRS, ´, ,CR,Isiu eBooi uDe du Biologie de Institut CNRS, ´, [email protected] sgntclyiatvtdeprec yatcdfcsadhv eue iesa.Eeto irsoyanalysis microscopy Electron span. life reduced a have and defects synaptic experience inactivated genetically is iohnra opoeei,Oiaiemetabolism Oxidative morphogenesis, Mitochondrial , 1 oet Tufi Roberta , ne-ebaepoenPIcontrols PMI protein inner-membrane 1, ; [email protected] * 2 vlpeetd asil-uiy M 28 -38 asil,France Marseille, F-13288 7288, UMR Marseille-Luminy, de ´veloppement ,Me lneBentobji ´lanie ) PMI nvivo in ucinicessteaeaelnt fmtcodilcitein cristae mitochondrial of length average the increases function edn fcit is(tas ta. 08 ale l,2009). al., et Rabl 2008; positive production al., component the inner-membrane et support of regulation (Strauss su) the tips Finally, e/g g crista (F0F1 and of e ATP-synthase bending subunits F0F1 non-catalytic al., The the et crista 2006). Frezza of of 2006; al., of al., et effector et tightening Meeusen Cipolat key 2006; 2003; the a al., et controls as (Olichon junctions identified also al., originally et fusion, Malsburg OPA1, Rabl der inner-membrane von 2007; 2011). 2011; al., al., al., et et Hoppins et Xie 2011; al., 2005; et al., Harner 2009; et of (John formation into the junctions assembles support crista Mitofilin, to them, complex of MINOS/MitOS/MICOS been the One and have details. 2009). morphogenesis membrane in al., crista molecular et characterized of of Zick determinants inter- cristae-specific 2000; molecular diffusion Mannella, Few the of and a the (Frey consist onto molecules preventing create soluble junctions, cristae by cristae which of evenly environment as opening tubules, to structures The narrow referred 2006b; 2009). tubular space, (Mannella, al., membrane parallel length et mitochondrial and Zick that the invaginations along straight are distributed TIM Cristae as 2006). al., inner-membrane Jakobs, et appear and (Vogel maintained the TOM Wurm are counterpart, 2006; tethering outer-membrane that its links membranes to complex outer physical sites and contact through inner are 2006a; boundaries the Mannella, Inner-membrane between 2009). 2000; al., Mannella, et and Zick (Frey function specific nciaino h ne-ebaepoenPantagruelian protein inner-membrane the of inactivation 1 enCalsLie Jean-Charles , nvivo in ial,teecit eet r soitdwith associated are defects crista these Finally, . ´vens 3 .Mge Martins Miguel L. , eerhArticle Research 2 , Journal of Cell Science iohnra hp aaeeso lcrnmicroscopy of electron Quantification on that 1A). revealed parameters (Fig. sections shape spheres mitochondrial individualised form oigfrcit opnnsidrcl oto mitochondrial control indirectly components crista for 2009a). coding al., content of et Osman loss 2009b; phospholipid whereas al., et inner-membrane 2007), inner-membrane (Osman growth al., crista in affects et inhibits Acehan result that 2006; al., Prohibitin tafazzin et (Xu synthase defects in cardiolipin mutations Consistently, morphogenesis. the crista on impacts also nraeo h rsammrn eghoe ue membrane mitochondria an outer with over correlated control length ( was membrane ratio length crista than length the in of increase cristae increase This 1C,D). longer (Fig. significantly in of morphology mitochondria tubules, crista elongated connected impact form would tested the we inactivation Drosophila 2011), Having in al., 2011). involved its et 2011; protein al., (Rival whether al., shape inner-membrane et mitochondrial et an Malsburg of Harner is regulation der 2009; PMI morphogenesis von inner- al., that 2011; shown et crista al., of Rabl et 2002; mitochondrial importance Hoppins al., the in et (Paumard highlighted proteins have membrane reports Several PMI biogenesis, mitochondria. crista of Results shape tubular controlling why overall explaining the by affects diameter also that, mutation mitochondrial determines suggest PMI of results death our premature and scale, defects organism further decreased the We at production. that with show ROS increased associated and metabolism are oxidative that abnormalities inner-membrane protein in of We inner-membrane disorganization 2011). loss al., that an et show (Rival morphogenesis (PMI), mitochondrial regulates I Mitochondrion an with (Zick polymyocitis and and 2009). 2008) al., al., (Hasegawa et et syndrome Dimmer 2007; Hirschhorn’s Bliek, Alzheimer’s der Wolfs van 2006), 2002), (Chung al., Suh, sclerosis and lateral et amyotrophic inner-membrane and al., 2006), (Celotto (Baloyannis, et neuromuscular abnormal disease syndrome (Acehan many syndrome Leigh’s human, for Barth’s 2007), trait including In 2010; common diseases al., 2011). metabolic a et Mun is al., 2006; and al., organization et 2011) et (Xu 2009), al., level an Head et has organism al., dysfunction the Malsburg their et at and der 2002), impact Rabl al., von et 2005; (Paumard 2005; content al., DNA al., et et (John growth (John metabolism oxidative hnacueo nraelnt Fg C) nadto,the and addition, 1H), (Fig. in In unaffected present 1C5). still was (Fig. were tubules length junctions crista crista increase PMI of consequence of display a diameter cause rather mitochondria is a of curvature than that 10% suggesting cristae although curved 1C4,G), overall (Fig. type, by parallel the wild unaffected biogenesis, for was cristae the As increased the of this of decrease morphology a Despite and 1C,D,F). length (Fig. crista average the ( overexpression PMI nti aucit edsrbdtecneune associated consequences the described we manuscript, this In proteins organization, crista in role their with Consistent otostemrhgnsso cristae of morphogenesis the controls nvivo in mro.I otatt oto iohnrathat mitochondria control to contrast In embryos. c.m./o.m. Drosophila PMI PMI nciainof inactivation aGL UAS-PMI da-GAL4 i.1) ovrey esoe that showed we Conversely, 1F). Fig. ; ucinidcscit lnainand elongation crista induces function uatcitewr ut tagtand straight quite were cristae mutant PMI el.W eosrt htthese that demonstrate We cells. PMI uatmtcodi contain mitochondria mutant Drosophila uainpooe synaptic provokes mutation Drosophila PMI rgesardcinof reduction a triggers ) uatmitochondria mutant dls Altogether adults. PMI c.m./o.m. Pantagruelian PMI inactivation. mutants ratio PMI rnucd iia lnainadbnigo crista of less of bending bending Although flight whether adult determine from To 2A,B). and mitochondria 3). (Fig. (Fig. in muscles present elongation controls also were membranes similar in were pronounced, that observed swirls concentric never form often mitochondria periphery, invaginations mitochondrion control membrane the in At those 2A,B). than (Fig. longer and bent dramatically M otosteeogto n h rhtcueo crista of architecture the and elongation the membranes. Taken protein controls 1A). inner-membrane (Fig. the PMI that cells demonstrate wild-type within results in distributed our together as regularly and matrix are mitochondrial that size the cristae same parallel of and Although straight 2011). as al., shape of et those masses, (Rival from giant mitochondria indistinguishable form morphologically neurons are fly which adult of from mitochondria size mutant increased overall for analysed the reported of we consequence simple mitochondria, a not was i.3 upeetr aeilFg 3.Ti otatdwith contrasted This S3). 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PMI diameter direct (Fig. stage mitochondrial a developmental and early highlighted this length quantifications crista between These correlation embryos. mutant parameters these in quantified we diameter, mitochondrial increased in seen biogenesis crista uncontrolled In cristae, diameter of mitochondrial size the regulating By erndslydsrih,prle n vnysae cristae spaced from evenly cristae and wild-type parallel of 2A,B), mitochondria (Fig. straight, While S1A,B; 2011). displayed Fig. al., material neuron et (supplementary (Rival tubules type S2A) thin wild Fig. the of the instead in which neurons observed in in mitochondria brains giant unique adult in obtained o.m. to contrast In gene 1A). drp1 (Fig. mitochondria pro-fusion entities the spherical tubular small of long invalidation the opa1 of Conversely, 1A). hanks (Fig. interconnected Consistent fusion fusion. display unbalanced of inner-membrane consequence the and with fission mitochondrial of compared then that We 1I). (Fig. h aultdadhgl iognsdcitefrom cristae disorganised highly the and with vacuolated contrasted from this the in observed Again crista cristae in 3). Cristae wild-type decreased (Fig. a biogenesis with than S3). membrane consistent packed mitochondria, 3; less wild-type (Fig. and Fig. in shorter diameter appeared myocytes the material overexpressing affecting without supplementary length mitochondrial not is that phenotype a in controls, observed to compared increased strongly PMI nciaini dl ri ern Fg A supplementary 2A; (Fig. neurons brain adult in inactivation and eut ntefamnaino iohnrait numerous into mitochondria of fragmentation the in results ai a oml(i.1,,) iia eut were results Similar 1C,D,F). (Fig. normal was ratio vrxrsino ncdw of knockdown or overexpression uatebys h vrg imtro iohnrais mitochondria of diameter average the embryos, mutant drp1 opa1 PMI drp1 drp1 and mitochondria, uatmtcodi h rsalnt and length crista the mitochondria mutant M otoscit lnain815 elongation crista controls PMI or ncotmue(siaae l,2009), al., et (Ishihara mouse knockout opa1 opa1 uat Fg B.T etwehrthe whether test To 1B). (Fig. mutants htae epciey fetr of effectors respectively, are, that PMI drp1 PMI drp1 drp1 oeepesn el,o with or cells, -overexpressing iohnra hntp to phenotype mitochondrial PMI uatnuosappeared neurons mutant iohnrasilcontains still mitochondria PMI uatmtcodi.As mitochondria. mutant PMI e2GL UAS-PMI mef2-GAL4 influences nciainrslsin results inactivation opa1 uat a eae to related was mutants drp1 PMI hc reduced which , PMI uatembryos mutant uatinner- mutant PMI uat,in mutants, cristae PMI opa1 c.m./ drp1 PMI - ; Journal of Cell Science h iohnra imtr( diameter mitochondrial the faltecitewr de oehradmlile y2t siaetettllnt ftecit ebaeprmtcodin edvd h oa len total the divide We mitochondrion. per membrane crista the of length ( ratio total membrane the membrane/outer estimate wild-type, crista to a with 2 obtain compared by to perimeter multiplied mitochondrion and the together by added membrane were cristae the all of olwdb ue HSD Tukey by followed ( embryos mutant ucin c) m rsa ebae m ue ebae m,inrmmrn onay a iohnra matrix. mitochondrial ma, boundary; membrane inner imb, membrane; outer om, membrane; cristae Cm, (cj). junctions a eemndfrec rsab esrn h eito ewe h culcit eghadtelnt fasrih ielnigec xrmt ft of extremity each linking line straight a of ( length genotype). the and each length for crista analysed actual embryos between the between observed deviation was the difference measuring significant by crista ( each for ANOVA determined by was analysed was ( significance background Statistical wild-type genotypes. a in PMI Overexpressing PMI ifrnewsosre in observed was difference cells. wild-type Conversely in seen normally tubules elongation. a crista expressing controls PMI 1. 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(ventral embryos fly 12–13 type, uatmtcodi r ie n aeeogtdcite(,6.Wti aeeby,citegnrlyapa tagtadprle 5,atog t although (5), parallel and straight appear generally cristae embryo, same a wild-type Within the containing 6). fragment (5, cristae elongated have (sinuosity bent and wider are mitochondria mutant P es ak,wihmk cuaemaueet ifcl.Saitclsgiiac a nlsdb n-a NV frdiameter (for ANOVA one-way for by (nd) analysed determined was not was significance Length Statistical condition. difficult. each measurements for accurate analysed make were which embryos hanks, independent dense three least at from mitochondria wild-type, from mitochondria Whereas , .01 olwdb ue HSD Tukey by followed 0.0001) ; drp1 n 5 5 infcnl eue rsalnt oprdt wild-type, to compared length crista reduced significantly 75) , ora fCl cec 2 (3) 126 Science Cell of Journal opa1 opa1 mit::YFP . .)i 0 ftemtcodi 6.Cnitnl,toemtcodilanraiisaersudi h control the in rescued are abnormalities mitochondrial those Consistently, (6). mitochondria the of 10% in 1.1) PMI+ uatmtcodi r rgetdit ueossalshrclette.( entities. spherical small numerous into fragmented are mitochondria mutant and PMI uinpoentree otemtcodilmatrix. mitochondrial the to targeted protein fusion .Oeepesn M nawl-yebcgon ytema ftwo of mean the by background wild-type a in PMI Overexpressing ). othoc post drp1 uatebys(eta pdri) h egho iohnrai eue nboth in reduced is mitochondria of length The epidermis). 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PMI PMI etrsanormal a restores ) drp1 PMI+ ai shge in higher is ratio uatcmae owl type. wild to compared mutant uatebysa tg 13 stage at embryos mutant da-GAL4 m G )addaee ( diameter and m) h vrg rsasinuosity crista average The ) uatbcuetblsform tubules because mutant ie()i hc genomic a which in (7) line P , m .01 o length for 0.0001, )wspotdagainst plotted was m) rvr( driver drp1 n 5 6mitochondria) 66 PMI c.m/o.m. aGL UAS- da-GAL4 and m ecit.No crista. he uatflies mutant )i wild- in m) P e a be can hey opa1 , t fthe of gth 0.0001) ratio. PMI PMI Journal of Cell Science oto le,apeoyeta ol ersudb genetically by the rescued be were in could levels that crista place ATP from phenotype a normal 4, extracts takes Fig. flies, in control for in that lower shown required metabolism significantly As is cristae. would oxidative inactivation mitochondrial PMI the PMI whether that affect investigated showed we organisation, we As function chain mitochondria. 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PMI a mitochondrial inactivating organism was on of the impact specifically at the analysed function more we structure, and and shape level the subcellular of consequences the the characterised Having PMI nrae O ees hshptei a aiae by validated was hypothesis the to This secondary be levels. could lethality adult ROS that or S1A–C), suspect increased nuclei) Fig. to us apoptotic material led (supplementary (vacuolization, damages degeneration mitochondrial of sign aged addition, from In muscles any 5A). and brains (Fig. that lethality fact adult the premature was such death in result since premature size not mitochondria This increased 5A). to due (Fig. not expression PMI restoring uatfishv eue lifespan reduced a have flies mutant ros.I otat vrxrsino M ymaso the of means by PMI of GAL4 overexpression panel, (bottom contrast, swirls In concentric crista arrows). form elongated and have bend can panels), which (top membranes, contrast, enlarged In are panel). which (bottom mitochondria, stack membranes dense a parallel form wavy and of spaced evenly are wild-type cristae In mitochondria, images). muscle magnification crista high and (bottom, images) showing ultrastructure magnification (DLM) low muscles (top, flight morphology adult mitochondrial diameter. of mitochondrial images influences TEM level Representative expression PMI 3. 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PMI+ n 5 PMI 10 Journal of Cell Science Discussion behaviour. normal for consequence, in mitochondria is and of synapses, transport morphogenesis to PMI efficient activity, targeting neuronal In mitochondrial for S6B,D). required by Fig. PMI-dependent restored material material (supplementary conclusion, were supplementary neurons phenotypes 6D; in (Fig. These expression of mutants S6). in PMI layers increased Fig. and of neuropil neuropils bodies the in Whereas and reduced cell synapses. in was cortical density and enriched their of bodies controls, highly cell succession were of regular up mitochondria a made contain layers imaged neuronal therefore neuropil that We impaired 2005). lobes al., to and optic et transported sensitivity (Verstreken be 6A) bang (Fig. to PMI activity in massive resulting too whether to are synapses, mitochondria asked mitochondria of their we levels, transport although the observed, Indeed, affect synapses. defects also could synaptic inactivation the to xed h iepnof lifespan the extends pCasper[SOD2] PMI, opooy(upeetr aeilFg 2.Atog reduced mitochondrial by Although production of ATP by S2). bang Fig. restoration material rescued Both 6A,C), tissue-specific (supplementary (Fig. morphology were the 6B,C). neurons (Fig. with into activity expression consistent flies neuronal PMI these targeting impaired specifically in and EPSP detected sensitivity of rate was higher a failure (EPSP), potential post-synaptic excitatory nciaino iepni oeae( moderate is lifespan on inactivation yaLgrn et(*** test rank Log a by the of mean the actin-GAL4 (by ubiquitously (SOD2) dismutase superoxide mitochondrial u eut hwta h iohnrainrmmrn protein inner-membrane mitochondria the PMI that show organisation results and Our length cristae controls PMI Although 2012). Dupont al., 1990; Ganetzky, and et Elkins 1980; Wyman, and (Tanouye wild-type ( a rate of survival Insertion controls. of in survival days median The flies. in adult lifespan Decreased 5. Fig. 820 ly eta oei euaigcit opooy Could morphology. crista regulating in role central a plays ora fCl cec 2 (3) 126 Science Cell of Journal driver: PMI+ M,atGL UAS-SOD2 act-GAL4 PMI, .I otatt the to contrast In ). ,o nyi ern ( neurons in only or ), PMI PMI P , 0.001). uatfis ttsia infcnewsanalysed was significance Statistical flies. mutant uatmtcodi rbbycontributes probably mitochondria mutant PMI PMI drp1 PMI uatflies. mutant drp1 uat eeal ogenerate to able were mutants uatfishv omlATP normal have flies mutant PMI uat rp o1 asvru 50 versus days 13 to drops mutants PMI 1 /drp1 M,ea-A4UAS-SOD2 elav-GAL4 PMI, uain h matof impact the mutation, eoi osrc restores construct genomic ro eoi transgene: genomic a of or 2 .( ). 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PMI zygotic and maternal homozygous from obtained were and sqh-mit::YFP eeaeaspecific a generate a netda h aecrmsmllcto ocet oto eceln ( line rescue control a create to location chromosomal same Df(PMI_PGRP-LD the at inserted was mef2-GAL4 uatmtcodi rbbytigr h nrae width increased the triggers probably mitochondria mutant uatmtcodi oetertblrsaeadbecome and shape tubular their lose mitochondria mutant uatmtcodi omlrespheres, large form mitochondria mutant .I the In ). rmD .Go and Guo, M. Dr from tidcrmsm)tasee htwr hncosdt A4fis(Rival flies GAL4 to crossed then were that transgenes chromosome) (third ora fCl cec 2 (3) 126 Science Cell of Journal PMI tcsadculture and stocks PMI PMI eae to females , rmPo.J Phillips, J. Prof. from elav-GAL4 ie eefo h loigo tc Center. Stock Bloomington the from were lines PMI+ uatfisadcontrol and flies mutant nc u le tthe at flies out knock uat.Teedt upr h data different that idea the support data These mutants. PMI p[w+] ˚ nwihol the only which in , nasadr onelaa diet. agar cornmeal standard a on C drp1 ;FT0,drp1 FRT40A, w; ie wild-type a line, ,Pac[PMI_PGRP-LD]attp2 ), uat( mutant , ˚ nasadr onelaa it l ue eekept were tubes All diet. agar cornmeal standard a on C act5c-GAL4 and UAS-opa1 drp1 PMI ;Df(PMI_PGRP-LD w; A-M attp40 UAS-PMI PMI opa1 1 ,hp0FP R4A Ovo FRT40A, hsp70-FLP; w, Pcasper[SOD2] uatfmlsbigteeoedvi fboth of devoid therefore being females mutant and , hsp-FLP Chung. J. Prof. from PMI+ 1 uatmtcodi okmuch look mitochondria mutant Pac[PMI_PGRP-LD] drp1 attp2 sqh-mit::YFP ; PMI PMI PMI nue rgetto of fragmentation induces PGRP-LD 2 lee eefo rP Verstreken, P. 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Baloyannis, sae yhmgnsn w le sn etei i0 eprto buffer respiration MiR05 in was KH pestle respiration mM a 10 III and using HEPES, State sensor flies flux. mM oxygen two (20 the oxygen homogenising of by background time assayed instrumental response and concentration, for the oxygen intervals) the on correction time of dependent derivative (2-second deconvolution time acquisition the signal data of calculation for including used analysis, was Austria) Innsbruck, chn . u . tks .L n clm,M. Schlame, and L. D. Stokes, Y., Xu, D., Acehan, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.115675/-/DC1 online available material Supplementary Recherche (ANR-blanc); la Recherche de la Franc National Association de the Centre Nationale and the Agence by the supported Scientifique; was work This images. L. EM Funding on of quantification Dr and analysis measurement and for for Nguyen Belenguer C. (CRN2M) and P. for (IBDML) Devaux Prof. Richard Arnaune J. 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