mitochondria are key organelles in intermediate cellular metabolism , energy conversion , and calcium homeostasis ( dimmer and scorrano , 2006 ) . they also integrate and amplify apoptosis induced by intrinsic stimuli , releasing cytochrome c and other proapoptotic factors required for the activation of caspases ( green and kroemer , 2004 ) . cytochrome c release is regulated by proteins of the bcl-2 family that control the permeabilization of the outer membrane ( omm ) ( danial and korsmeyer , 2004 ) . energy conversion occurs at the inner mitochondrial membrane ( imm ) that can be further divided into two subcompartments : the so - called  boundary membrane  and the cristae , separated from the former by narrow tubular junctions ( frey and mannella , 2000 ) . mitochondria become  condensed ,  with an expanded cristae space ( hackenbrock , 1966 ) . during apoptosis , the curvature of the cristae membrane is inverted in a remodeling process required for the complete release of cytochrome c , normally confined in the cristae ( scorrano et  al . , 2002 ; frezza et  al . , 2006 ; yamaguchi et  al . , cristae remodeling occurs in response to proapoptotic bh3-only bcl-2 family members , such as bid , bim - s , and bnip3 , and independently of the outer membrane multidomain bcl-2 family members bax and bak ( scorrano et  al . , 2002 ; cipolat et  al . , 2006 ; whether changes in morphology of the cristae , where respiratory chain complexes ( rccs ) mainly localize ( vogel et  al . , 2006 ) , affect oxidative phosphorylation efficiency , as originally predicted ( hackenbrock , 1966 ) , is unclear . this issue is further complicated by the assembly of rcc in supercomplexes ( rcs ) ( schgger , 1995 ; acn - prez et  al . , 2008 ) , quaternary supramolecular structures that , by channeling electrons among individual rccs , allow the selective use of rcc subsets for nicotine adenine dinucleotide ( nadh)- or flavin adenine dinucleotide - derived electrons ( lapuente - brun et  al . , 2013 ) . such a supramolecular organization is common in cristae : also , the mitochondrial atp synthase is assembled into dimers with greater adenosine triphosphatase ( atpase ) activity ( campanella et  al . interestingly , cristae shape and atpase dimers are linked : in yeast mutants where the atpase can not dimerize , cristae are disorganized ( paumard et  al . , 2002 ; , 2005 ; strauss et  al . , 2008 ) , whereas in mammalian cells , increased cristae density favors atpase dimerization during autophagy ( gomes et  al . , 2011 ) . on the contrary , despite their importance in mitochondrial bioenergetics , the relationship between rcs and cristae shape remains unclear . mitochondrial morphology and ultrastructure depends on  mitochondria - shaping  proteins that regulate organellar fusion and fission ( griparic and van der bliek , 2001 ) . mitofusins ( mfn ) 1 and 2 , highly homologous dynamin - related proteins of the omm , orchestrate fusion ( santel and fuller , 2001 ; legros et  al . , 2002 ; chen et  al . , 2003 ; santel et  al . , mfn1 primarily participates in fusion , cooperating with the imm dynamin - related protein optic atrophy 1 ( opa1 ) ( cipolat et  al . , 2004 ) , whereas mfn2 also tethers mitochondria to the endoplasmic reticulum ( de brito and scorrano , 2008 ) . mitochondrial fission is regulated by the cytoplasmic dynamin - related protein 1 that , upon calcineurin - dependent dephosphorylation , translocates to mitochondria ( yoon et  al . , 2001 ; genetic depletion of opa1 leads to disorganization of the cristae ( frezza et  al . , 2006 ) , and oligomers that contain a soluble and a membrane - bound form of opa1 keep the cristae junctions tight , independently from opa1 role in fusion ( frezza et  al . , 2006 ; during apoptosis , these oligomers are early targets of bid , bim - s , and bnip3 , as well as of intrinsic death stimuli ( frezza et  al . , 2006 ; whereas our knowledge of the molecular determinants of cristae shape and their role in apoptosis is increasing , the relationship between cristae morphology and mitochondrial function remains unexplored . we therefore set out to genetically dissect whether and how cristae shape regulates mitochondrial respiration . we show that cristae morphology determines assembly and stability of rcs and hence optimal mitochondrial respiratory function during life and death of the cell . whether apoptotic cristae remodeling that maximizes cytochrome c release from mitochondria affects mitochondrial function is unclear , mainly because it occurs around the same time as outer membrane permeabilization ( scorrano et  al . , 2002 ) . in order to genetically dissociate the two processes , we inspected the primary structure of the prototypical cristae remodeling inducer bcl-2 family member bid for homology with peptides known to perturb the mitochondrial inner membrane , like mastoparan , a 14 amino acid wasp venom component ( pfeiffer et  al . , 1995 ) . interestingly , bid membrane inserting 6 helix as well as the transmembrane domains of bnip3 and bims that also remodel cristae ( yamaguchi et  al . 2010 ) displayed homology to mastoparan ( figures s1a and s1b available online ) . to exploit the role of this homologous sequence in cristae remodeling , we mutagenized the two highly conserved 157 and 158 lys h.  sapiens bid residues to ala ( bid ) ( figure  s1c ) . because this mutation did not impair caspase-8 cleaved recombinant bid ( cbid ) integration in purified mouse liver mitochondria ( mlm ) ( wei et  al . , 2000 ; figure  s1d ) , we could measure its biological activity using an established quantitative , specific cytochrome c release elisa ( scorrano et  al . , 2002 ) . cbid efficiently released cytochrome c from purified mitochondria , whereas a bh3 domain g94e mutant was , as expected , inactive ( wei et  al . , 2000 ) and the cbid mutant released 25%30% more cytochrome c than the baseline ( figure  1a ) , a figure close to the amount of free intermembrane space cytochrome c ( scorrano et  al . , 2002 ) . bak oligomerization was superimposable in cbid or cbid - treated mitochondria ( figure  1b ) ; conversely , two established assays of intramitochondrial cytochrome c redistribution , the cytochrome b5-dependent extramitochondrial nadh oxidation and the ratio of ascorbate - driven over tetramethyl - p - phenylenediamine ( tmpd)-driven respiration ( scorrano et  al . , 2002 ) , indicated that cbid mobilized the cristae cytochrome c pool less efficiently than cbid ( figures 1c and 1d ) . indeed , cbid was unable to remodel mitochondrial cristae , as indicated by morphometric analysis of electron micrographs of mitochondria treated with the bid mutants ( figures 1e and 1f ) ( scorrano et  al . , 2002 ) . cristae remodeling is associated with the disruption of high molecular weight ( hmw ) opa1 oligomers ( frezza et  al . , 2006 ) . western blots of blue native gel electrophoresis ( bnge ) of mitochondrial proteins revealed four major opa1-containing complexes . upon treatment with cbid , opa1 rapidly disappeared from 720  kda hmw complexes ( figures 1 g , s1e , and s1f ) . these hmw forms of opa1 were similarly targeted by cbid but significantly less by cbid , as determined by bnge ( figure  1h , quantification in [ i ] ) . chemical crosslinking experiments ( frezza et  al . , 2006 ) further confirmed that the opa1-containing oligomer is disrupted by the mutants of cbid able to induce cristae remodeling ( figures s1 g and s1h ) . finally , we measured the killing efficiency of these truncated bid ( tbid ) mutants expressed in mouse embryonic fibroblasts ( mefs ) . only tbid efficiently killed mefs : tbid and tbid elicited comparable low levels of cell death , whereas the double tbid mutant appeared completely ineffective ( figure  1j ) , suggesting that both outer membrane permeabilization and mitochondrial cristae remodeling are required for bid - induced apoptosis . in conclusion , bid is deficient in cristae remodeling , cytochrome c release , and induction of apoptosis . the bid mutant dissociates outer membrane permeabilization from cristae remodeling and can be used to investigate the relationship between the latter and mitochondrial function . we therefore measured the effect of the bid mutants on the respiratory control ratio ( rcr ) , an index of respiratory efficiency , of mitochondria incubated with excess exogenous cytochrome c and nadh ( to compensate for the potential effects of inner membrane or outer membrane [ om ] permeabilization ) . cbid reduced rcr only when mitochondria were energized with substrates for complex i ( glutamate / malate ) but not when they were fed with substrates entering the electron transport chain at complex ii ( succinate ) or complex iv ( ascorbate  + tmpd ) ( figure  2a ; data not shown ) . interestingly , these changes were recapitulated by the bh3 domain mutant cbid that does not permeabilize the om but not by the cristae remodeling - deficient mutants cbid and cbid ( figure  2b ) . maximal ( uncoupled ) respiration was similarly affected by the cbid mutants tested , ruling out that bid alters rcr , because it affects atpase or activity or atp / adp exchange ( figure  s2a ) . these experiments suggest that cristae remodeling causes complex i - dependent changes in mitochondrial bioenergetics . complex i is further assembled in quaternary functional rcs with complexes iii and iv ( i  + iii and i  + iii  + iv ) , whereas most complex ii is not found in rcs ( acn - prez et  al . , 2008 ) . thus , the reduction in complex i - supported respiration could be a consequence of specific inhibition of complex i or of issues in rcs function . even after 30  min of acute bid treatment , the specific complex i nadh - ubiquinone reductase activity of purified mitochondria was unaltered ( data not shown ) , prompting us to investigate rcs assembly and stability in  situ . we therefore took advantage of bax , bak ( dko ) mefs , resistant to mitochondrial permeabilization , cytochrome c release , and apoptosis triggered by expression of tbid ( wei et  al . , 2001 ) . upon transduction of metabolically labeled dko mefs with tbid but not with tbid that does not cause cristae remodeling ( figure  s2b ) , the rcs radioactivity signal as well as the rcs / complex v radioactivity ratio were reduced ( figures 2c and 2d ) , and we observed a reduction in the autoradiographic signal of cytochrome b retrieved in rcs compared to that in free complex iii ( figures 2e and 2f ) . whereas this result could suggest that complex iii was incorporated less efficiently into rcs , immunoblotting for the complex i subunit ndufa9 revealed that rcss were also destabilized in dko cells ( figure  2 g ) . functionally , only cbid and cbid that cause cristae remodeling but not cbid reduced glutamate - supported rcr in dko mitochondria ( figure  2h ) . whether rcs disorganization was a general consequence of altered cristae shape , we turned to cells lacking opa1 , a key regulator of cristae morphology ( frezza et  al . , however , chronic opa1 depletion impaired mitochondrial dna ( mtdna ) levels and translation ( figures s3a and s3b ) , complicating the analysis of the relationship between opa1 and rcs and calling for a model of conditional opa1 ablation . we produced , by homologous recombination , c57bl6/j embryonic stem cells with loxp sites inserted in the opa1 gene ( opa1 ) , which were then microinjected in c57bl6/j blastocysts to generate opa1 mice . following cre - mediated recombination , the deletion of exons 2 and 3 resulted in an aberrant exon1exon4 transcript with a stop codon immediately after exon 1 , producing a predicted 10 amino acid ( aa ) residual protein ( figures 3a and 3b ) . chimerism and germ - line transmission of the offspring was tested by pcr , and germ - line transmittants were bred to homozygosity ( figure  3c ) . fibroblasts isolated from the diaphragm of homozygous opa1 7-week - old male mice ( mafs ) were immortalized and used for subsequent analysis . opa1 was completely ablated 24  hr after adenoviral delivery of cre recombinase ( figure  4a ) and , as expected , mitochondria were fragmented ( figures 4b and 4c ) with defects in cristae shape ( figures 4d and 4e ) . four days after cre - mediated opa1 ablation , mtdna copy number ( figure  4f ) and translation ( figures 4 g and 4h ) were unaffected , allowing us to specifically address the role of opa1 and cristae shape in rcs assembly using an assay based on the incorporation of radiolabeled mtdna - encoded proteins into rcc and rcs ( acn - prez et  al . , 2008 ) . upon acute opa1 ablation , the assembly of mtdna - encoded subunits into rcc was not affected ( data not shown ) . we therefore followed the rcs assembly rate ( measured as the ratio between rcs and complex v radioactivity throughout the chase period ) that resulted 8-fold slower when opa1 was ablated from opa1 mafs ( figures 4i and 4j ) . a similar reduction in the rcs assembly rate was observed in opa1 mefs ( figure  s3c ) , suggesting that , in absence of opa1 , rcc are less superassembled , irrespective of their initial levels . to test if acute opa1 ablation altered rcs in  vivo , we tail vein - injected cre - expressing adenoviruses in opa1 animals . after 72  hr , s4a ) , cristae morphology was abnormal ( figure  s4b ) , rcs were reduced ( figure  s4c ) , and glutamate / malate rcr was impaired ( figure  4k ) . these experiments of conditional ablation of opa1 identify a role for cristae shape in rcs assembly in  vitro and in  vivo . the model linking cristae shape to rcs predicts that higher opa1 levels should favor rcs assembly . to verify this hypothesis , we wanted to generate a mouse model of opa1 overexpression . very high opa1 levels are , however , toxic , causing paradoxical mitochondrial fragmentation ( cipolat et  al . , 2004 ) : we therefore targeted , by homologous recombination in the murine x chromosome hprt region , a transgene - carrying mouse variant 1 opa1 under the human -actin promoter ( opa1 ) ( figure  3d ) . the integration into microinjected bpes embryonic stem cells was verified by pcr ( figure  3e ) , and the cells were microinjected into c57bl6/j blastocysts . six generated agouti chimeras with a chimerism exceeding 90% were bred with c57bl6/j mates and tested for germline transmission by fur color and transgene pcr analysis ( figure  3f ) . mice were viable , fertile , grew normally , and resisted to different forms of tissue damage ( t.v . r. menab , m. sandri , f. di lisa , and l.s . , unpublished data ) . immortalized mafs prepared from the diaphragm of hemizygous opa1 7-week - old c57bl6/j male mice displayed an 1.5 increase in opa1 levels compared to age- and sex - matched littermate controls mafs ( wild - type [ wt ] ; figure  5a ) . mitochondria were slightly elongated ( figures 5b and 5c ) and cristae tighter ( figures 5d and 5e ) , without any difference in mtdna levels ( figure  5f ) and translation ( figures 5 g and 5h ) . importantly , in opa1 mafs , rcs assembly ( figures 5i and 5j ) and glutamate - supported rcr ( figure an 50% increase in liver mitochondria opa1 levels ( figure  s5a ) was similarly associated to tighter cristae ( figure  s5b ) and increased rcs levels ( figure  s5c ) . taken together , these results indicate that rcs assembly is facilitated by increased opa1 levels and tighter cristae . we therefore measured the growth of dko cells ( resistant to bid - induced outer membrane permeabilization , caspase - dependent mitochondrial damage , and apoptosis ) in galactose media , where most of cellular atp comes from the respiratory chain ( acn - prez et  al . , 2004 ) . wt and g94e tbid impaired growth in galactose , whereas cells transduced with tbid that does not cause cristae remodeling did not display any defect ( figure  6a ) . growth in galactose - containing media was impaired upon acute ablation of opa1 in mafs , whereas it was normal for fusion - deficient mfn1 , mfn2 mefs ( figures 6b and 6c ) , where mitochondrial fusion is also inhibited . in mfn1 , mfn2 mefs , mtdna copy number was reduced ( figure  s6a ) , but cristae shape ( figures s6b and s6c ) , mtdna translation ( figure  s6d ) , rcs stability ( figure  s6e ) , and assembly ( figure  s6f ) were not affected . thus , the galactose growth defect is not the consequence of impaired fusion but correlates with altered cristae shape and rcs . finally , opa1 mafs grew faster than their wt counterparts in galactose media ( figure  6d ) , further confirming the link between cristae shape , rcs levels , and mitochondria - dependent cellular growth . in conclusion , respiratory chain supercomplexes have been considered bnge artifacts until direct respirometric experiments on purified rcs identified them as the functional mitochondrial respiratory units ( acn - prez et  al . , 2008 ) . since then , rcs have been directly visualized in intact cristae by electron tomography ( davies et  al . , 2011 ) , complex iv assembly factors that favor rcs formation have been identified ( chen et  al . , 2012 ; 2013 ) , and the role of rcs in mitochondrial utilization of reducing equivalents has been demonstrated ( lapuente - brun et  al . , 2013 ) . however , the relationship between cristae shape and rcs , as well as between rcs and mitochondrial function , remained obscure . our results demonstrate that cristae shape regulates respiratory chain supercomplexes stability and assembly , impacting on respiratory efficiency and respiratory cell growth . to dissect the role of cristae shape in rcs structure and function , we genetically ablated the master cristae shape regulator opa1 . individual respiratory chain units associate with opa1 ( zanna et  al . , 2008 ) , and mitochondrial metabolism is deranged in dominant optic atrophy caused by opa1 mutations ( lodi et  al . , 2004 ) . however , the defect in atp production in opa1 haploinsufficient cells was unexplained : opa1 is not essential for assembly of respiratory chain complexes and mtdna levels as well as activities of individual respiratory chain complexes are normal in dominant optic atrophy ( zanna et  al . , 2008 ) . conversely , the reduction in mtdna copy number has been invoked to explain the mitochondrial dysfunction of fusion - deficient  cells from mfn1 , mfn2-deficient mice ( chen et  al . , 2010 ) . our results challenge this hypothesis : upon acute opa1 ablation , mtdna levels are normal , whereas cristae shape , rcs , complex - i - dependent respiration , and respiratory growth are impaired . conversely , in mfn1 , mfn2 cells , mtdna copy number is reduced , but cristae shape , rcs , and respiratory growth are normal . thus , rcs disorganization shall be regarded as a key mechanism of mitochondrial dysfunction accompanying altered organelle morphology . the role of opa1 and cristae shape in rcs organization is further supported by mouse models of opa1 conditional ablation and mild overexpression . the first tool allowed us to dissociate cristae biogenesis from mtdna maintenance : whereas chronic opa1 depletion reduces mtdna copy number and translation , upon acute opa1 ablation , mtdna levels are normal , but cristae are disorganized , impacting on rcs assembly and respiratory function and growth . thus , mtdna reduction appears to be a consequence of chronic fusion inhibition in opa1 ( and double mfn ) cells . we can therefore predict that the opa1 cells will be useful to elucidate how prolonged inhibition of mitochondrial fusion results in mtdna levels reduction . opa1 mild overexpression lends further support to the model linking rcs organization to cristae shape : rcs assembly and respiratory function and growth are increased in opa1 cells without any measurable change in mtdna levels and translation . the opa1 mouse will be instrumental to investigate the role of opa1 and cristae shape in  vivo . the role and mechanisms of cristae remodeling in apoptosis are controversial ( scorrano et  al . , 2002 ; germain et  al . , 2005 ; frezza et  al . , 2006 ; yamaguchi et  al . , 2008 ; 2010 ) , cristae remodeling has been reckoned as a mere feedback mechanism in  situ , occurring after caspase activation ( sun et  al . , 2007 ) . our results suggest that , in addition to its role in cytochrome c release , cristae remodeling also impairs mitochondrial function to precipitate apoptosis . the bid 6 mutant generated here , which does not induce cristae changes and cytochrome c mobilization but permeabilizes the outer membrane , can be a useful tool to dissect in  vivo the involvement of cristae remodeling in developmental and homeostatic apoptosis . we think that cristae remodeling influences rcs by targeting opa1 ( frezza et  al . , 2006 ) , not by altering membrane potential that is normal during cristae remodeling ( scorrano et  al . , 2002 ) or by inhibiting mtdna translation and insertion of mtdna - encoded subunits that similarly appear normal in dko cell - expressing bid ( data not shown ) . our work unravels a role for cristae shape in rcs assembly and stability , mitochondrial respiratory efficiency , and respiratory growth , suggesting that shape of biological membranes can influence membrane protein complexes . moreover , our data highlight the importance of rcs in respiration by complex i - feeding substrates . the pathogenesis of dominant optic atrophy where opa1 is mutated ( alexander et  al . , 2000 ) or of other mitochondrial diseases where opa1 is degraded ( duvezin - caubet et  al . , 2006 ) could also depend on this unexpected opa1 function . in these latter settings , stabilization of opa1 could correct rcs and therefore mitochondrial dysfunction , opening novel therapeutic perspectives for currently intractable diseases . to generate opa1 mice , a mouse bac clone containing the opa1 gene was isolated from the c57bl/6j es bac clone library . an 11 kb hpai dna restriction fragment containing the opa1 fragment was excised with ecorv and xmal to generate blunt ends and inserted into a pko4.4a - loxp cut with xhoi and sali . a loxp site was introduced between intron1 and exon2 of opa1 and a phosphoglycerate kinase ( pgk ) promoter - driven neomycin resistance gene , flanked by two frt sequences and with one loxp sequence downstream , was inserted in intron3 . the targeting vector was linearized and electroporated into c57bl6 embryonic stem cells ( escs ) . three mutated esc lines were microinjected into c57bl6 blastocytes and implanted in host mice to obtain chimeric mice , which were then bred with c57bl6 mates and their offspring tested by pcr for germline transmission . the human -actin promoter was extracted from pdrive - h-actin ( invivogen ) using spei and ncoi and cloned in pentry . the complementary dna of mouse isoform 1 opa1 and polya extracted from pcdna3.1-opa1 ( cipolat et  al . , 2004 ) using nhe1 and ecii was ligated into pentry using quick ligase ( ozyme ) . the transgene was then inserted by homologous recombination in a pdest vector containing part of the human hypoxanthine phosphoribosyltransferase locus . the resulting vector was linearized using pvui and electroporated into c57bl6 bpes cells by nucleis ( france ) . three positive esc recombinant clones were microinjected into c57bl6 blastocysts and implanted into host pseudopregnant female c57bl6 to obtain chimeric mice . six chimeras ( identified by fur agouti color ) were bred with c57bl6 mates , and germline transmission was verified by fur color and pcr . mitochondria ( 10  mg / ml ) were suspended in buffer d ( 1  m 6-aminohexanoic acid , 1.25% v / v digitonin , 50  mm bis - tris - hcl , ph 7 ) and centrifuged . the supernatant was collected , and 5% serva blue g dye in 1  m 6-aminohexanoic acid was added to 1/3 of the final sample volume . equal amounts ( 100  g ) of mitochondrial proteins were separated by 3%13% gradient bnge ( schgger , 1995 ) . for rcs detection , the concentration of digitonin in buffer d was 4% ( v / v ) . for two - dimensional ( 2d ) blue native ( bn)/bnge , the lane cut from the first - dimension bnge was casted on top of a native 3%14% gradient gel in 1% ( v / v ) dodecyl maltoside . for 2d bn / sds page , the lane cut from the first - dimension bnge was incubated for 1  hr at 25c in 1% sds and 1% -mercaptoethanol and then casted on top of an 8% or a 16.5% denaturing gel . after electrophoresis , the complexes were electroblotted on a polyvinylidene fluoride ( pvdf ) membrane and probed with the indicated antibodies . to detect rcs from radiolabelled cells , samples were treated as described above , and after electrophoresis , the gels were dried and the signal was detected following exposure for 36  days . labeling of mtdna - encoded proteins was performed with [ s]-methionine and cysteine ( express protein labeling mix , perkin elmer life sciences ) . cells were preincubated for 12  hr in the presence of 40  g / ml chloramphenicol in uridine - supplemented medium and then exposed for 2  hr to the [ s ] protein labeling mix ( pulse ) in the presence of 50  g / ml cycloheximide . cells were washed for four times with pbs and cold dulbecco s modified eagle s medium ( dmem ) and then cultured for the indicated time ( chase ) prior to lysis and protein separation by bnge . to assay basal mtdna translation , equal protein amounts ( 40  g ) from cells metabolically labeled as described above treated for 30  min with 50  g / ml emetine were separated by sds - page . mitochondria from liver of mice and from the indicated cell lines were isolated as in frezza et  al . cytochrome c release , ascorbate / tmpd - driven respiration and cytochrome b5-dependent nadh oxidation were determined as described ( scorrano et  al . , crosslinking , mitochondria treated as indicated were incubated with 1  mm 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride ( edc ) or 1  mm bismaleimidohexane ( bmh ) , as previously described ( frezza et  al . , 2006 ) . carbonate extraction was performed as previously described ( dimmer et  al . , 2008 ) . details and procedures for sds - page and immunoblotting can be found in the extended experimental procedures . the retroviral vector pmig - tbid was described previously ( cheng et  al . , 2001 ) . kkaa , g94e , and g94ekkaa mutants were generated by site - direct mutagenesis using kod polymerase ( biolabs ) . to measure mtdna copy number , total cellular dna was amplified using specific oligodeoxynucleotides for mt - co2 and sdha by real - time pcr . mefs and human embryonic kidney 293 cells ( hek293 ) cells were cultured as described ( gomes et  al . , 2011 ) . when indicated , in dmem , glucose was substituted with 0.9  mg / ml galactose . ecotropic viruses were generated as described ( cheng et  al . , 2001 ) . wt , opa1 , opa1 , and mafs sv40 transformed cell lines were generated from the diaphragm of the respective 7-week - old mouse killed by cervical dislocation . acute opa1 ablation in opa1 mafs was obtained by infection with adenoviruses expressing cytomegalovirus ( cmv)-cre - gfp ( ad - cre ; 300 pfu / cell ; vector biolabs ) . opa1 mafs were infected and dko mefs were transduced 24 or 16  hr before the growth was assessed . apoptosis was measured by flow cytometric detection ( facscalibur ) of the annexin - v - pe positive events in the gfp - positive population . details can be found in the extended experimental procedures . for mitochondrial imaging , cells stained with a rabbit polyclonal anti - tom20 ( santa cruz ; 1:200 ) , as previously described ( frezza et  al . , 2006 ) electron microscopy ( em ) was performed as described ( scorrano et  al . , 2002 ) . details can be found in the extended experimental procedures . extended experimental proceduresmouse handlingfor genotyping of the opa1 mice , the following primers were used : primer ck1 5-cag tgt tga tga cag ctc ag-3 ; primer ck2 5-cat cac aca cta gct tac att tgc-3. for genotyping of the opa1 mice , the following primers were used : primer tgf 5-gca atg acg tgg tcc tgt tttg-3 ; primer tgr 5-gat agg tca ggt aag caa gca ac-3 ; primer wtr 5-gag gga gaa aaa tgc gga gtg-3 ; primer wtf 5-ctc cgg aaa gca gtg agg taa g-3.all mice procedures were performed according to protocols approved by the local ethic committees ( protocol 32/2011 ceasa university of padova , venetian institute of molecular medicine ; license 1034/3703/02 cantonal veterinarian authority , university of geneva ) . opa1 mice were tail injected with adenoviruses ( 2x10 pfu ) ( vector biolabs ) . after 3  days , they were killed by cervical dislocation and the liver was extracted.mitochondrial assaysmitochondria from mouse liver and from the indicated cell lines were isolated as described ( frezza et  al . , 2007 ) . mitochondria were isolated from opa1 mice 3  days after the tail - vein injection of the indicated adenoviruses . cytochrome c release , ascorbate / tmpd - driven respiration and cytochrome b5-dependent nadh oxidation were determined as described ( scorrano et  al . , 2002 ) . to measure respiration , mitochondria ( 1 mg / ml ) were incubated in experimental buffer ( eb : 150  mm kcl , 10  mm tris mops , 10  m egta - tris , 10m atp ) . to determine bid effect of rcr , eb was supplemented with 5  m cytochrome c , 2  mm nadh and 3.2 pmol  mg cbid . when indicated , mitochondria were transferred into a clark s type oxygen electrode chamber and 5  mm glutamate /2.5  mm malate or 2  m rotenone/10  mm succinate were added . basal o2 consumption was recorded ( state 2 ) and after 2  min 100  m adp was added ( state 3 ) , followed by 2.5  g / ml oligomycin ( state 4 ) and 200  m fccp ( state 3u ) . for complex iv dependent - o2 consumption , mitochondria ( 1  mg / ml ) were incubated for the indicated time in eb supplemented with 5  m cytochrome c. mitochondria were treated with 1  nmol / mg antimycin a and 2  nm rotenone and they were transferred into a clark s type oxygen electrode chamber . after 2  min 6  mm ascorbate and 300  m tmpd were added and the complex iv - dependent o2 consumption rate was measured.the enzymatic activity of complex i and citrate synthase was determined as described ( spinazzi et  al . , 2012).transmission electron microscopyem of cells and isolated mitochondria were performed as described ( scorrano et  al . , 2002 ) and thin sections were imaged on a tecnai-20 electron microscope ( philips - fei ) . for morphometric analysis of mitochondrial cristae , cristae width was measured using the image j multimeasure plug-in.measurement of mitochondrial dna copy numbertotal cellular dna was isolated using phenol / chlorophorm 24:25 ( v / v ) and was amplified using specific oligodeoxynucleotides for mt - co2 and sdha by real - time pcr using platinum sybr green qpcr supermix ( invitrogen ) following manufacturer s indications . the mtdna copy number per cell was calculated using sdha amplification as a reference for nuclear dna content.imaging4x10 cells seeded on 13  mm round glass coverslips were fixed , permeabilized and blocked as previously described ( frezza et  al . , 2006 ) and then stained with a rabbit polyclonal anti - tom20 ( santa cruz , 1:200 ) overnight at 4c . after washing with pbs , cells were incubated with texasred conjugated goat anti - rabbit ( 1:500 , invitrogen ) for 1  hr at room temperature and coverslips were mounted on slides using prolong gold antifade reagent ( invitrogen ) and analyzed by confocal microscopy . adenovirus infected cells were identified by expression of gfp.confocal images were acquired using a nikon eclipse te300 inverted microscope equipped with a spinning - disk perkinelmer ultraview lci confocal system , a piezoelectric z axis motorized stage ( pifoc , physik instrumente ) , and an orca er 12-bit ccd camera ( hamamatsu photonics ) . for detection of gfp or texasred fluorescence , samples were excited using the 488  nm or the 543  nm lines of a he  ne laser ( perkinelmer , waltham , ma , usa ) with exposure times of 100 - 200  ms using a 60x , 1.4 na plan apo objective ( nikon ) . measurement of mitochondrial major axis length was performed in at least five mitochondria per cell , in a minimum of 50 cells / experiment ( 250 mitochondria / experiment ) . mitochondrial length was then quantified using the roi manager plug - in of imagej software.biochemistryfor protein crosslinking , mitochondria treated as indicated were incubated with 1  mm edc or 1  mm bmh as previously described ( frezza et  al . , 2006 ) . for sds - page , mitochondrial proteins ( 20  g ) were separated on 3%8% tris - acetate or 4%12% tris - glycine ( nupage , invitrogen ) polyacrilamide gels , transferred onto pvdf membranes ( biorad ) and probed using the indicated primary antibodies and isotype matched secondary antibodies conjugated to horseradish peroxidase whose signal was detected with ecl ( amersham ) . the following primary antibodies were employed : monoclonal anti - opa1 ( 1:1000 bd pharmingen ) , rabbit polyclonal anti - bak - nt ( 1:1000  upstate ) , mouse monoclonal anti - fp subunit ( 1:5000 ) , mouse monoclonal anti - ndufa9 subunit ( 1:1000 ) , mouse monoclonal anti - core2 subunit ( 1:1000 ) , mouse monoclonal anti - co1subunit ( 1:1000 ) ( mitoscience ) , mouse monoclonal anti - vdac1 ( 1:1000 abcam ) , rabbit polyclonal anti - tom20 ( 1:1000 santa cruz ) , mouse monoclonal anti - tim23 ( 1:1000 bd pharmingen ) ,  rabbit polyclonal anti - bid ( 1:1,000 , a kind gift of a. gross , weizmann institute , rehovot ) . densitometry was performed by analyzing optical density using gel pro analyzer.carbonate extraction was performed as previously described ( dimmer et  al . , 2008).molecular biologythe retroviral vector pmig - tbid was described previously ( cheng et  al . , 2001 ) . kkaa , g94e and g94ekkaa mutants were generated by site - direct mutagenesis using kod polymerase ( biolabs ) and the following primers : for bid forward 5-aca atg ctg ttg gcc gcc gcc gtg gcc agt cac-3 and reverse 5-gtg act ggc cac ggc ggc ggc caa cag cat tgt-3 ; for bid forward 5-ctc gcc caa ata gaa gat gag atg gac cac aac-3 and reverse 5-gtt gtg gtc cat ctc atc ttc tat ttg ggc gag-3.recombinant protein expressionp7/p15 recombinant bid was produced , purified and cleaved with caspase-8 as described ( frezza et  al . , 2006).assays of cell growth and deaththe growth of wt , opa1 mafs was determined by counting viable cells ( as determined by trypan blue exclusion ) daily for 5  days . 1.5x10 
 opa1 mafs were infected with the indicated adenoviruses and after 24  hr the growth was determined by counting viable cells ( as determined by trypan blue exclusion ) daily for 4  days . 1.5x10 dko mefs were transduced with the indicated retroviruses and after 16  hr the growth was determined by counting viable gfp positive cells by flow cytometry.for measurements of apoptosis , 1.5x10 cells of the indicated genotype grown in 6 well - plates were transfected with the indicated vector . after 48  hr apoptosis was measured by flow cytometric detection ( facscalibur ) of the annexin - v - pe positive events in the gfp - positive population.cell culture , transfection , virus production , and transductiondko mefs and hek293 cells were cultured as previously described ( gomes et  al . , 2011 ) . when indicated , glucose in dmem was substituted with 0.9  mg / ml galactose . cells were transfected using transfectin ( biorad ) following manufacturer s instruction.ecotropic viruses were generated by cotransfecting the hek293 packaging cell lines with the packaging vector pik and the appropriate pmig as previously described ( cheng et  al . , 2001 ) . viral supernatant were retrieved and used to transduce dko mefs in the presence of 4  g / ml of hexadimethrine bromide ( sigma ) . following an over - night transduction , the rate of gfp expression was typically around 70% as determined by flow cytometry.wt , opa1 , opa1 , mafs sv40 transformed cell lines were generated respectively from wt , opa1 , opa1 mice . male 7  weeks old mice were killed by cervical dislocation and their diaphragm were carefully dissected , washed in dmem , minced and then enzymatically digested with a collagenase 0.5% ( sigma ) solution for 10  min at 37c . cell were spun at 200  g for 3  min , resuspended in dmem containing 4.5  mg / ml glucose , 10% fetal bovine serum ( fbs ) , 50  u / ml penicillin , 50  g / ml streptomycin , 100  m nonessential aminoacids ( gibco ) , 50  g / ml uridine , 25  mm hepes ( gibco ) , 2.5  g / ml fungizone ( gibco ) and seeded on a 0.1% gelatin ( sigma ) precoated plate . after 15  min the supernatant was removed and the adherent fibroblasts were left growing in complete dmem . mafs were transformed using an sv40 large t antigen expressing plasmid as previously described ( cipolat et  al . , 2006 ) and for genotyping of the opa1 mice , the following primers were used : primer ck1 5-cag tgt tga tga cag ctc ag-3 ; primer ck2 5-cat cac aca cta gct tac att tgc-3. for genotyping of the opa1 mice , the following primers were used : primer tgf 5-gca atg acg tgg tcc tgt tttg-3 ; primer tgr 5-gat agg tca ggt aag caa gca ac-3 ; primer wtr 5-gag gga gaa aaa tgc gga gtg-3 ; primer wtf 5-ctc cgg aaa gca gtg agg taa g-3. all mice procedures were performed according to protocols approved by the local ethic committees ( protocol 32/2011 ceasa university of padova , venetian institute of molecular medicine ; license 1034/3703/02 cantonal veterinarian authority , university of geneva ) . opa1 mice were tail injected with adenoviruses ( 2x10 pfu ) ( vector biolabs ) . after 3  days , they were killed by cervical dislocation and the liver was extracted . mitochondria from mouse liver and from the indicated cell lines were isolated as described ( frezza et  al . , 2007 ) . mitochondria were isolated from opa1 mice 3  days after the tail - vein injection of the indicated adenoviruses . cytochrome c release , ascorbate / tmpd - driven respiration and cytochrome b5-dependent nadh oxidation were determined as described ( scorrano et  al . , 2002 ) . to measure respiration , mitochondria ( 1  mg / ml ) were incubated in experimental buffer ( eb : 150  mm kcl , 10  mm tris mops , 10  m egta - tris , 10m atp ) . to determine bid effect of rcr , eb was supplemented with 5  m cytochrome c , 2  mm nadh and 3.2 pmol  mg cbid . when indicated , mitochondria were transferred into a clark s type oxygen electrode chamber and 5  mm glutamate /2.5  mm malate or 2  m rotenone/10  mm succinate were added . basal o2 consumption was recorded ( state 2 ) and after 2  min 100  m adp was added ( state 3 ) , followed by 2.5  g / ml oligomycin ( state 4 ) and 200  m fccp ( state 3u ) . for complex iv dependent - o2 consumption , mitochondria ( 1  mg / ml ) were incubated for the indicated time in eb supplemented with 5  m cytochrome c. mitochondria were treated with 1  nmol / mg antimycin a and 2  nm rotenone and they were transferred into a clark s type oxygen electrode chamber . after 2  min 6  mm ascorbate and 300  m tmpd were added and the complex iv - dependent o2 consumption rate was measured . the enzymatic activity of complex i and citrate synthase was determined as described ( spinazzi et  al . em of cells and isolated mitochondria were performed as described ( scorrano et  al . , 2002 ) and thin sections were imaged on a tecnai-20 electron microscope ( philips - fei ) . for morphometric analysis of mitochondrial cristae , cristae width was measured using the image j multimeasure plug - in . total cellular dna was isolated using phenol / chlorophorm 24:25 ( v / v ) and was amplified using specific oligodeoxynucleotides for mt - co2 and sdha by real - time pcr using platinum sybr green qpcr supermix ( invitrogen ) following manufacturer s indications . the mtdna copy number per cell was calculated using sdha amplification as a reference for nuclear dna content . 4x10 cells seeded on 13  mm round glass coverslips were fixed , permeabilized and blocked as previously described ( frezza et  al . , 2006 ) and then stained with a rabbit polyclonal anti - tom20 ( santa cruz , 1:200 ) overnight at 4c . after washing with pbs , cells were incubated with texasred conjugated goat anti - rabbit ( 1:500 , invitrogen ) for 1  hr at room temperature and coverslips were mounted on slides using prolong gold antifade reagent ( invitrogen ) and analyzed by confocal microscopy . confocal images were acquired using a nikon eclipse te300 inverted microscope equipped with a spinning - disk perkinelmer ultraview lci confocal system , a piezoelectric z axis motorized stage ( pifoc , physik instrumente ) , and an orca er 12-bit ccd camera ( hamamatsu photonics ) . for detection of gfp or texasred fluorescence , samples were excited using the 488  nm or the 543  nm lines of a he  ne laser ( perkinelmer , waltham , ma , usa ) with exposure times of 100 - 200  ms using a 60x , 1.4 na plan apo objective ( nikon ) . measurement of mitochondrial major axis length was performed in at least five mitochondria per cell , in a minimum of 50 cells / experiment ( 250 mitochondria / experiment ) . mitochondrial length was then quantified using the roi manager plug - in of imagej software . for protein crosslinking , mitochondria treated as indicated were incubated with 1  mm edc or 1  mm bmh as previously described ( frezza et  al . , 2006 ) . for sds - page , mitochondrial proteins ( 20  g ) were separated on 3%8% tris - acetate or 4%12% tris - glycine ( nupage , invitrogen ) polyacrilamide gels , transferred onto pvdf membranes ( biorad ) and probed using the indicated primary antibodies and isotype matched secondary antibodies conjugated to horseradish peroxidase whose signal was detected with ecl ( amersham ) . the following primary antibodies were employed : monoclonal anti - opa1 ( 1:1000 bd pharmingen ) , rabbit polyclonal anti - bak - nt ( 1:1000  upstate ) , mouse monoclonal anti - fp subunit ( 1:5000 ) , mouse monoclonal anti - ndufa9 subunit ( 1:1000 ) , mouse monoclonal anti - core2 subunit ( 1:1000 ) , mouse monoclonal anti - co1subunit ( 1:1000 ) ( mitoscience ) , mouse monoclonal anti - vdac1 ( 1:1000 abcam ) , rabbit polyclonal anti - tom20 ( 1:1000 santa cruz ) , mouse monoclonal anti - tim23 ( 1:1000 bd pharmingen ) ,  rabbit polyclonal anti - bid ( 1:1,000 , a kind gift of a. gross , weizmann institute , rehovot ) . carbonate extraction was performed as previously described ( dimmer et  al . , 2008 ) . the retroviral vector pmig - tbid was described previously ( cheng et  al . , 2001 ) . kkaa , g94e and g94ekkaa mutants were generated by site - direct mutagenesis using kod polymerase ( biolabs ) and the following primers : for bid forward 5-aca atg ctg ttg gcc gcc gcc gtg gcc agt cac-3 and reverse 5-gtg act ggc cac ggc ggc ggc caa cag cat tgt-3 ; for bid forward 5-ctc gcc caa ata gaa gat gag atg gac cac aac-3 and reverse 5-gtt gtg gtc cat ctc atc ttc tat ttg ggc gag-3. p7/p15 recombinant bid was produced , purified and cleaved with caspase-8 as described ( frezza et  al . , 2006 ) . the growth of wt , opa1 mafs was determined by counting viable cells ( as determined by trypan blue exclusion ) daily for 5  days . 1.5x10 
 opa1 mafs were infected with the indicated adenoviruses and after 24  hr the growth was determined by counting viable cells ( as determined by trypan blue exclusion ) daily for 4  days . 1.5x10 dko mefs were transduced with the indicated retroviruses and after 16  hr the growth was determined by counting viable gfp positive cells by flow cytometry . for measurements of apoptosis , 1.5x10 cells of the indicated genotype grown in 6 well - plates were transfected with the indicated vector . after 48  hr apoptosis was measured by flow cytometric detection ( facscalibur ) of the annexin - v - pe positive events in the gfp - positive population . dko mefs and hek293 cells were cultured as previously described ( gomes et  al . , 2011 ) . when indicated , glucose in dmem was substituted with 0.9  mg / ml galactose . ecotropic viruses were generated by cotransfecting the hek293 packaging cell lines with the packaging vector pik and the appropriate pmig as previously described ( cheng et  al . , 2001 ) . viral supernatant were retrieved and used to transduce dko mefs in the presence of 4  g / ml of hexadimethrine bromide ( sigma ) . following an over - night transduction , wt , opa1 , opa1 , mafs sv40 transformed cell lines were generated respectively from wt , opa1 , opa1 mice . male 7  weeks old mice were killed by cervical dislocation and their diaphragm were carefully dissected , washed in dmem , minced and then enzymatically digested with a collagenase 0.5% ( sigma ) solution for 10  min at 37c . cell were spun at 200  g for 3  min , resuspended in dmem containing 4.5  mg / ml glucose , 10% fetal bovine serum ( fbs ) , 50  u / ml penicillin , 50  g / ml streptomycin , 100  m nonessential aminoacids ( gibco ) , 50  g / ml uridine , 25  mm hepes ( gibco ) , 2.5  g / ml fungizone ( gibco ) and seeded on a 0.1% gelatin ( sigma ) precoated plate . after 15  min the supernatant was removed and the adherent fibroblasts were left growing in complete dmem . mafs were transformed using an sv40 large t antigen expressing plasmid as previously described ( cipolat et  al . , 2006 ) and
summaryrespiratory chain complexes assemble into functional quaternary structures called supercomplexes ( rcs ) within the folds of the inner mitochondrial membrane , or cristae . here 
 , we investigate the relationship between respiratory function and mitochondrial ultrastructure and provide evidence that cristae shape determines the assembly and stability of rcs and hence mitochondrial respiratory efficiency . 
 genetic and apoptotic manipulations of cristae structure affect assembly and activity of rcs in  vitro and in  vivo , independently of changes to mitochondrial protein synthesis or apoptotic outer mitochondrial membrane permeabilization . we demonstrate that , accordingly , the efficiency of mitochondria - dependent cell growth depends on cristae shape . 
 thus , rcs assembly emerges as a link between membrane morphology and function .