RESEARCH ARTICLE

Deficiency of PHB complex impairs respiratory supercomplexformation and activates mitochondrial flashesChongshu Jian, Fengli Xu, Tingting Hou, Tao Sun, Jinghang Li, Heping Cheng and Xianhua Wang*

ABSTRACTProhibitins (PHBs; prohibitin 1, PHB1 or PHB, and prohibitin 2, PHB2)are evolutionarily conserved and ubiquitously expressedmitochondrialproteins. PHBs form multimeric ring complexes acting as scaffolds inthe innermitochondrial membrane.Mitochondrial flashes (mitoflashes)are newly discovered mitochondrial signaling events that reflectelectrical and chemical excitations of the organelle. Here, weinvestigate the possible roles of PHBs in the regulation of mitoflashsignaling. Downregulation of PHBs increases mitoflash frequency byup to 5.4-fold due to elevated basal reactive oxygen species (ROS)production in the mitochondria. Mechanistically, PHB deficiencyimpairs the formation of mitochondrial respiratory supercomplexes(RSCs) without altering the abundance of individual respiratorycomplex subunits. These impairments induced by PHB deficiencyare effectively rescuedby co-expression of PHB1andPHB2, indicatingthat the multimeric PHB complex acts as the functional unit.Furthermore, downregulating other RSC assembly factors, includingSCAFI (also known as COX7A2L), RCF1a (HIGD1A), RCF1b(HIGD2A), UQCC3 and SLP2 (STOML2), all activate mitoflashesthrough elevating mitochondrial ROS production. Our findings identifythe PHB complex as a new regulator of RSC formation and mitoflashsignaling, and delineate a general relationship among RSC formation,basal ROS production and mitoflash biogenesis.

KEY WORDS: Prohibitin, Respiratory supercomplex, Mitochondrialflashes, Reactive oxygen species

INTRODUCTIONProhibitins (PHBs; prohibitin 1, PHB1 or PHB, and prohibitin 2,PHB2) are evolutionarily conserved and ubiquitously expressedmitochondrial proteins that are implicated in diverse cellularprocesses from mitochondrial biogenesis to cell proliferation andembryonic development (Mishra et al., 2006; Nijtmans et al., 2002;Osman et al., 2009b). PHB1 and PHB2 physically interact with eachother to form a multimeric, ∼1.2 MDa ring complex in the innermembrane of mitochondria (IMM) (Tatsuta et al., 2005), and areinterdependent such that the absence of either subunit results indegradation of the other (Osman et al., 2009b). The PHB complexhas been proposed to function as a mitochondrial membranescaffold that might help to recruit membrane proteins to functionalsites of the IMM (Osman et al., 2009b). Moreover, PHBs have beenshown to be associated with complex IV subunits in yeast (Strub

et al., 2011) and with complex I subunits in mammalian cells(Bourges et al., 2004), hinting at the possibility that PHBs mightparticipate in the assembly of mitochondrial electron transportchain (ETC) complexes. Deficiency of PHBs leads to increasedgeneration of reactive oxygen species (ROS) (Kathiria et al., 2012;Schleicher et al., 2008); however, the mechanism underlying PHB-mediated regulation of mitochondrial ROS production is unknown.

Increasing evidence has shown that the mitochondrial ETCcomplexes can form respiratory supercomplexes (RSCs), whichhave been revealed by blue native gel electrophoresis (BNGE)(Schagger and Pfeiffer, 2000; Schägger and von Jagow, 1991) andsingle-particle electron microscopy (Althoff et al., 2011; Dudkinaet al., 2011). Acín-Pérez et al. have shown that isolated RSCs areable to transfer electrons from NADH to O2, revealing therespiratory activity of RSCs (Acín-Pérez et al., 2008). Formationof RSCs has also been shown to facilitate efficient electron transferand reduce ROS production by substrate channeling, which limitsthe amount of electron leakage and protects vulnerable sites ofthe complexes from oxidative damage (Porras and Bai, 2015).Additionally, the formation of the supercomplex stabilizes theassembly of the individual complexes and complex I in particular(Schagger et al., 2004). Several proteins have been identified toparticipate in regulating the assembly of mitochondrial RSCs.Particularly, two related Saccharomyces cerevisiae proteins, Rcf1(which has two human orthologs RCF1a and RCF1b, also known asHIGD1A and HIGD2A , respectively) and Rcf2 (Chen et al., 2012;Strogolova et al., 2012; Vukotic et al., 2012), as well as SCAFI (alsoknown as COX7A2L) (Lapuente-Brun et al., 2013; Pérez-Pérezet al., 2016; Williams et al., 2016), UQCC3 (Desmurs et al., 2015)and SLP2 (also known as STOML2) (Mitsopoulos et al., 2015) havebeen shown to participate in various processes of RSC formation viadifferent mechanisms. Interestingly, PHBs have been found to co-migrate with mitochondrial RSCs in BNGE analysis (Acín-Pérezet al., 2008; Mitsopoulos et al., 2015), suggesting a potential role ofPHBs in RSC formation. However, it remains obscure whether andhow PHBs participate in the formation of RSCs.

The mitochondrial flash (mitoflash) represents a newlydiscovered dynamic activity of the mitochondria, which has beendetected in a range of species from C. elegans to zebrafish and torodents and humans (Hou et al., 2014; Wang et al., 2008). Mitoflashhas been detected by multiple indicators including mt-cpYFP,which dually senses superoxide and pH (Wei-LaPierre et al., 2013),mitoSOX for superoxide and 2,7-dichlorodihydrofluoresceindiacetate (DCF) for total ROS (Zhang et al., 2013), grx1-roGFP2for redox potential (Breckwoldt et al., 2014; Wang et al., 2016),mitoSypHer, pHTomato and SNARF-1 for matrix pH (Santo-Domingo et al., 2013; Wang et al., 2016; Wei-LaPierre et al., 2013),and TMRMor TMRE for mitochondrial membrane potential (Wanget al., 2008; Wei et al., 2011). In addition, label-free imaging ofNADH and FAD autofluorescence also reveals a transient oxidationof NADH and FADH2 (Hou et al., 2014; Wang et al., 2016;Received 12 October 2016; Accepted 14 June 2017

State Key Laboratory of Membrane Biology, Beijing Key Laboratory ofCardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China.

*Author for correspondence (xianhua@pku.edu.cn)

X.W., 0000-0002-2016-9415

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Wei-LaPierre et al., 2013). Thus, individual mitoflashes consist ofmultiple signals including bursting superoxide production, transientmatrix alkalization, oxidative redox shift and mitochondrialmembrane potential depolarization (Hou et al., 2014; Wang et al.,2008, 2016), reflecting electrical and chemical excitation of theorganelle. Importantly, it has been shown that mitoflash generationdepends on intact ETC activity (Wang et al., 2008) and the rate ofmitoflash occurrence is regulated by physiological factors includingmitochondrial basal ROS and matrix Ca2+ (Hou et al., 2013; Jianet al., 2014), as well as matrix protons in the nanodomains of IMM(Wang et al., 2016). It is thus of interest to determine whether PHBsand RSC dynamics can play any significant roles in the regulation ofmitoflash signaling.By investigating potential roles of PHBs in the regulation of

mitochondrial mitoflash activity and RSC formation, we report thatPHB deficiency impairs mitochondrial RSC formation, increasesmitochondrial basal ROS production, and therefore augmentsmitoflash frequency; all these defects are rescued by co-expression of PHB1 and PHB2. Furthermore, we extend our studyto other RSC assembly factors, including SCAFI, RCF1a, RCF1b,UQCC3 and SLP2, to elucidate a general relationship among RSCformation, ROS production and mitoflash signaling.

RESULTSPHB deficiency leads to hyperactivity of mitoflashesIn HeLa cells stably expressing mt-cpYFP (Ma et al., 2011), weknocked down PHB1 or PHB2 through RNA interference. Westernblot analysis revealed that both PHB1 and PHB2 weredownregulated with siRNAs targeting either PHB1 or PHB2(Fig. 1B), consistent with the notion that both PHB subunits areinterdependent and required for the formation of multimeric PHBcomplexes with physiological activity (Osman et al., 2009b).Strikingly, confocal mitoflash imaging showed that downregulationof PHB1 and PHB2 by ∼57–71% increased mitoflash frequency by4.6–5.4-fold (Fig. 1A,C). The kinetics of individual mitoflashes,however, were largely unaltered (Fig. 1D; Fig. S1E–G). However,overexpression of PHB1 and PHB2, individually or simultaneously,exerted only a marginal effect on mitoflash activity (Fig. S1A,C).These results indicate that PHBs, as IMM scaffold proteins, act aspotent regulators of mitoflash activity. That supra-physiologicallevels of PHBs are no longer effective suggests that the endogenousPHBs are presented at saturating levels.Similar to previous reports (Artal-Sanz et al., 2003; Merkwirth

et al., 2008), knockdown of PHB1 or PHB2 resulted inmitochondrial fragmentation, such that more cells containedfragmented rather than tubular mitochondria (Fig. 1A,E). Todiscriminate the effects of mitochondrial morphological changeson mitoflashes, we analyzed the mitoflash activity in subgroups ofcells whose mitochondria displayed tubular, partial tubular orfragmented appearances. We found that the mitoflash response tosiRNA treatment was comparable regardless of mitochondrialmorphology (Fig. 1F). That is, hyperactivation of mitoflashesinduced by loss of PHBs is independent of PHB-dependentmitochondrial morphological changes. This finding is in generalagreement with our recent report showing that the rate of mitoflashoccurrence in a given cell remains constant when the mitochondrialnetwork fragmentizes, hyperfuses or redistributes (Li et al., 2016).To determine whether the effect of PHBs on mitoflashes is cell typespecific, we performed parallel experiments in primary neonatal ratventricular myocytes (NRVMs) and cultured mouse embryonicfibroblasts (MEFs), and showed that knockdown of either PHB1 orPHB2 similarly increased mitoflash frequency (Fig. S2), whereas

overexpression of PHB1 or PHB2 in NRVMs failed to impactsignificantly on mitoflash production (Fig. S1B,D). That is, wefound qualitatively and quantitatively similar results, with PHB1and PHB2 functioning as powerful inhibitors of mitoflash signalingin all three different cell types examined.

PHB deficiency activates mitoflashes through increasingmitochondrial basal ROS productionNext, we sought to investigate the mechanisms whereby PHBdeficiency activates mitoflashes. Since loss of PHBs leads toaugmented ROS production (Kathiria et al., 2012; Schleicher et al.,2008), and since mitochondrial ROS constitute a robust mitoflashactivator (Hou et al., 2013), we hypothesized that knockdown ofPHBs activates mitoflashes through elevating mitochondrial basalROS. To test this hypothesis, we measured the mitochondrialROS level in normal cells and in cells deficient for PHBs.Because mitochondria constitute the major site for cytosolic ROSproduction, the cytosolic ROS level was also measured in parallelexperiments to reflect mitochondrial ROS production. Our resultsshowed that knockdown of PHBs enhanced both cytosolic andmitochondrial ROS levels, as evidenced by the augmented DCFfluorescence signal, which responds to multiple ROS (Fig. 2A,C),and the mitoSOX fluorescence signal, which is relatively selectivefor superoxide and targeted in mitochondria (Fig. 2A,D; Fig. S3).Furthermore, in permeabilized HeLa cells with succinate as thesubstrate of respiration, the H2O2 production was determinedfluorimetrically utilizing the Amplex® Red–horseradish peroxidasemethod (Muller et al., 2004) with modifications. Consistent with theabove, deficiency of PHB1 or PHB2 increased the H2O2 productionby 55% and 36%, respectively (Fig. 2B). These results underline acrucial role of PHBs in repressing mitochondrial ROS production,in agreement with previous reports (Kathiria et al., 2012; Schleicheret al., 2008).

To determine the link between the elevation of ROS and thehyperactivity of mitoflashes induced by PHB deficiency, we treatedHeLa cells with the mitochondria-targeted ROS scavengermitoTEMPO. This mitochondria enriched antioxidant attenuated theH2O2 production in permeabilized cells regardless of PHB knockdown(Fig. 2B). In intact cells, both cytosolic and mitochondrial ROS levels,measured with DCF and mitoSOX, respectively, were decreased in allgroups (Fig. 2C,D). At the same time, mitoflash activity wassignificantly mitigated (Fig. 2E). Quantitatively, when all data areconsidered, linear regression analysis revealed a strong positivecorrelation between mitoflash frequency and mitochondrial ROSlevels as indexed by mitoSOX (r=0.91, P=0.01) (Fig. 2F). Thus, weconclude that PHBs regulate mitoflashes primarily through alteringmitochondrial ROS production. It is noteworthy that oxidativestimulants, H2O2 and menadione, elicited greater mitoflash activityin both control and PHB-deficient cells (Fig. 2G), supporting the ideathat basal ROS act as the mitoflash trigger.

PHB complex deficiency impairs mitochondrial RSCformationHow do PHBs regulate mitochondrial ROS levels? The major sitesfor mitochondrial ROS production reside at complexes of themitochondrial ETC (Murphy, 2009). Moreover, individualmitochondrial ETC complexes are able to form RSCs thatfacilitate electron transfer and substrate tunneling, thus limitingbasal ROS production and resulting in more efficient electrontransfer and proton pumping during oxidative phosphorylation(Porras and Bai, 2015). Since PHBs have been found to co-migratewith mitochondrial RSCs in BNGE analysis (Acín-Pérez et al.,

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2008; Mitsopoulos et al., 2015), we hypothesized that PHBs mightmodulate mitochondrial ROS production through regulating RSCformation.

To test this hypothesis, we determined the effect of PHBknockdown on RSC formation by performing BNGE followed byimmunoblotting. As shown in Fig. 3A, immunoblot analysis using

Fig. 1. Hyperactivity ofmitoflashes in PHB-deficient HeLacells. (A) Confocal images of HeLa cells stably expressingmt-cpYFP.Mitoflash regions aremaskedin red, and raster plots beneath the corresponding image show the timing of individual events, denoted by vertical ticks, during the 100 s recording period. Notethat knockdown of PHB1 or PHB2 (siPHB1 or siPHB2) induced mitochondrial fragmentation as compared to the negative control (NC). Scale bar: 20 µm.(B) Western blots for PHB expression. Two siRNAs were used to target PHB1 (siPHB1-1 and siPHB1-2) or PHB2 (siPHB2-1 and siPHB2-2). n=3 independentexperiments for each group. *P<0.05 versus NC group. (C) Effects of PHB1 or PHB2 knockdown on mitoflash frequency. n=58–234 cells for each group.***P<0.001 versus NC group. (D) Averaged time courses of mitoflashes aligned by onset. n=54–70 mitoflashes for each trace. (E) Mitochondrial morphologicalchanges in PHB-deficient cells. Tubular, cells with primarily tubular mitochondria; partial tubular, cells containing punctuated mitochondrial fragments andat least three clearly tubular mitochondria; fragmented, cells containing punctuated mitochondrial fragments and less than three tubular mitochondria. n=15–68image files for each group. *P<0.05; **P<0.01 versus NC. (F) Mitoflash frequency in cells with different mitochondrial morphology. n=5-97 cells. All quantitativedata are expressed as mean±s.e.m.

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antibodies against complex I (NDUFB8), complex II (SDHA),complex III (UQCRC1), complex IV (COX IV), and complex V(ATP5A) showed that complexes I, III, and IV were assembled intoRSCs with high molecular mass, while complexes II and V mainlyremained as individual entities in HeLa cells. Knockdown of eitherPHB1 or PHB2 significantly reduced the RSC contents as detectedby anti-NDUFB8, anti-UQCRC1 or anti-COX IV staining(Fig. 3A). Quantitative analysis with complex V as the referencerevealed that, in PHB-deficient cells, the contents of complex I-,

complex III-, and complex IV-containing RSCs were reduced by upto 40%, 42% and 59%, respectively (Fig. 3B). Meanwhile, theIII2–IV supercomplex assembly was also decreased as revealed byboth anti-UQCRC1 and anti-COX IV immunoblots (Fig. 3A,B).However, no significant changes were detected for the complex IIIdimmers and complex IV monomers (Fig. 3A,B). Moreover,western blot analysis following SDS-PAGE showed thatknockdown of PHB1 or PHB2 did not affect the expression ofindividual respiratory complexes, as indexed with NDUFB8

Fig. 2. PHB deficiency activatesmitoflashes through increasing basalmitochondrial ROS production.(A) Representative confocal images ofDCF and mitoSOX fluorescence in HeLacells. NC, negative control; siPHB1,knockdown of PHB1; siPHB2, knockdownof PHB2. Scale bar: 100 µm. (B) Amplex®

Red-measured H2O2 production inpermeabilized HeLa cells with succinateas the substrate. Mitochondrial ROSscavenger mitoTEMPO (10 µM) was usedto pretreat the cells for 1 h. n=3independent experiments. *P<0.05;**P<0.01 for siPHB1 or siPHB2 versusnegative control (NC) group in the absenceor presence of mitoTEMPO. #P<0.05;##P<0.01 for mitoTEMPO-treated versusuntreated group. (C,D) MitoTEMPO(10 µM) decreased ROS levels asmeasured by using DCF (C) and mitoSOX(D). a.u., arbitrary units. n=35–141 cells foreach group. **P<0.01; ***P<0.001 forsiPHB1 or siPHB2 versus NC group in theabsence or presence of mitoTEMPO.###P<0.001 for mitoTEMPO-treatedversus untreated group. (E) Effect ofmitoTEMPO on mitoflash frequency.n=33–104 cells for each group.***P<0.001 for siPHB1 or siPHB2 versusNC group in the absence or presence ofmitoTEMPO. ###P<0.001 mitoTEMPO-treated versus untreated group. (F)Correlation between mitochondrial ROSlevel and mitoflash frequency. The dashedline shows a linear regression with r=0.91,and P=0.01. The data were from D and E.(G) Mitoflash activation by oxidantsmenadione (200 µM) or H2O2 (50 µM) inNC and PHB-deficient cells. n=22–87 cellsfor each group. **P<0.01 for menadione-or H2O2-treated versus untreated group.#P<0.05; ##P<0.01 for siPHB1 or siPHB2versus NC. All quantitative data areexpressed as mean±s.e.m.

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(complex I), SDHA (complex II), UQCRC1 (complex III), MTCO2(complex IV), and ATP5B (complex V) (Fig. 3C,D). This resultindicates that the impaired RSC formation is not due to a diminutionin the expression of individual subunits of ETC complexes. Takentogether, our results provide direct evidence for PHBs as new andimportant mitochondrial RSC regulators.Parallel functional assays showed that the mitochondrial

respiration was significantly repressed in PHB-deficient cells. In

particular, the basal, ATP-coupled and maximal oxygenconsumption rates (OCRs) were all suppressed by knockdown ofPHBs (Fig. 3E,F), while expression of individual respiratorycomplexes in the PHB-deficient cells was unchanged (Fig. 3C,D),indicating that the overall reduction in respiration in PHB-deficientcells was not due to a decrease in mitochondrial mass. In otherwords, PHB knockdown exerted opposing effects on mitoflashactivity and mitochondrial respiration. This finding is in contrast to

Fig. 3. PHB deficiency impairsmitochondrial RSC formation.(A) Representative BNGEimmunoblots of mitochondrial RSCsfrom HeLa cells with or withoutknockdown of PHB1 or PHB2. RSCswere visualized by antibodies againstcomplexes III (CIII, UQCRC1) andcomplex IV (CIV, COX IV), and thenstripped and reprobed for complex I(CI, NDUFB8) and complex V (CV,ATP5A). The membranes were thenblotted for complex II (CII, SDHA).NC, negative control; siPHB1,knockdown of PHB1; siPHB2,knockdown of PHB2. (B) Quantitativeresults for the level of RSCs, III2-IV,III2, and IV complexes as quantifiedwith indicated antibodies. Anti-ATP5A immunoblots were used asthe reference. n=4–6 independentexperiments. *P<0.05 versus NC.(C) Representative western blots ofindividual respiratory complexes I to Vwith the indicated antibodies. GAPDHand TIM23 were used as cytosol andmitochondrial-loading controls,respectively. (D) Quantitative resultsfor experiments shown in C. n=6independent experiments. *P<0.05versus NC. (E) Whole-cell oxygenconsumption rate (OCR) in differentgroups. The dotted lines indicate thetime of adding oligomycin (1 µM),FCCP (0.5 µM) and rotenone andantimycin A (Rot&AA, 1 µM each).(F) Quantitative results for basal,ATP-coupled, maximal and proton-leak associated OCR. n=12measurements from threeindependent experiments foreach group. *P<0.05; **P<0.01versus NC. All quantitative data areexpressed as mean±s.e.m.

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the report that mitoflash frequency is positively correlated withmitochondrial respiration under certain experimental conditions(Gong et al., 2015), indicating that mitoflash biogenesis is notalways a unique function of the mitochondrial metabolic rate.To validate the aforementioned roles of PHBs, we performed

rescue experiments. To avoid possible siRNA attack, the rat PHB1and PHB2 tagged with 3×Flag were re-expressed in HeLa cellswhose endogenous PHB1 and PHB2 were downregulated by RNAinterference (Fig. 4A). Only co-expression of PHB1 and PHB2, butnot expression of PHB1 or PHB2, was able to restore the complex I,III and IV contents in RSCs (Fig. 4B). Moreover, co-expression ofPHB1 and PHB2 largely lowered both cytosolic and mitochondrialROS and the mitoflash frequency to their control levels, whereasre-expression of PHB1 or PHB2 alone was ineffective (Fig. 4C–E).These results substantiate the notion that the multimeric PHBcomplex acts as the functional unit that is essential for regulatingRSC formation, ROS production and mitoflash activity.

RSC formation and mitoflash signalingAs RSC formation represents a physiological and intrinsic activityof mitochondria that limits ROS production and facilitates substratetunneling, this process is richly regulated by numerous assemblyfactors. We therefore extended our study to delineate whether ourconclusion can be generalized to RSC factors other than PHBs. Forthis purpose, five reported assembly factors were chosen, namely,SCAFI, RCF1a, RCF1b, UQCC3 and SLP2, which have beenshown to participate in different processes of RSC formation viadifferent mechanisms (Chen et al., 2012; Desmurs et al., 2015;Lapuente-Brun et al., 2013; Mitsopoulos et al., 2015; Pérez-Pérezet al., 2016; Strogolova et al., 2012; Vukotic et al., 2012). Each ofthe aforementioned five RSC regulators was knocked down by twodifferent siRNAs (Table S1, Fig. S4) and the one with higherknockdown efficiency was used for functional assays. BNGEfollowed by immunoblot analysis showed that the complex I, III andIV contents in RSCs were all significantly reduced with knockdownof one of the five regulators in HeLa cells (Fig. 5A,B). Particularly,SCAFI knockdown showed the most dramatic effect, with a strongimpairment of III2–IV supercomplex (Fig. 5A,C), in agreement withthe proposed role of SCAFI in stabilizing III2–IV arrangement(Pérez-Pérez et al., 2016) or assembling complex IV into thesupercomplexes (Lapuente-Brun et al., 2013). Knockdown ofRCF1b, UQCC3, or SLP2 also reduced the amount of the III2–IVsupercomplex (Fig. 5A,C). Measuring the cytosolic andmitochondrial ROS showed that disruption of the mitochondrialRSCs through downregulating the above assembly factors allaugmented ROS production (Fig. 6A,B). Meanwhile, the mitoflashfrequency was markedly increased in each group (Fig. 6C),accompanied by only minor or negligible changes in mitoflashkinetics (Fig. S5). Importantly, mitoTEMPO treatment largelyreduced mitoflash frequency in all groups, wherein bothmitochondrial and cytosolic ROS levels were decreased (Fig. 6A–C).Linear regression analysis revealed a positive correlation betweenmitoflash frequency and mitochondrial ROS level in these cellsdeficient in RSC assembly factors with or without mitoTEMPOtreatment (r=0.97, P<0.0001) (Fig. 6D). These results indicate that,irrespective of the specific RSC regulators, impairing RSCformation always activates mitoflashes, presumably via enhancingbasal mitochondrial ROS production. As for respiration, however,these RSC regulators displayed differing effects: knockdown ofSCAFI, RCF1a or SLP2 decreased the basal, ATP-coupled andmaximal OCR, whereas knockdown of RCF1b or UQCC3exhibited little effects (Fig. 6E). This result substantiates the

notion that, while the RSC regulation of mitoflash is mainlyattributable to a basal ROS-dependent mechanism, additionalmechanisms might be invoked to contribute to the RSC-mediatedregulation of mitochondrial metabolism. Collectively, our resultsnot only unveil RSC formation as a novel mitoflash-regulatorymechanism, but also underscore a complex relationship betweenmitochondrial respiration and mitoflash biogenesis.

DISCUSSIONPHB complex as a novel regulator of RSC formationWhile the mitochondrial PHB1 and PHB2 complex is not anobligatory component of the mitochondrial ETC and oxidativephosphorylation, we have provided strong evidence that PHBcomplex acts as bona fide regulator of mitochondrial RSCformation. Knockdown of either PHB1 or PHB2, which causedcomparable reduction of both PHB1 and PHB2, decreased thecontents of complex I, III and IV in RSCs, without altering theexpression of individual respiratory complex subunits examined.Co-expression of PHB1 and PHB2, but not individual expression ofeither, effectively rescued the RSC formation in the PHB-deficientcells, indicating that the multimeric PHB complex acts as thefunctional unit. Consistent with this, various roles for the functionalPHB complex have been reported. For example, it has beenproposed to function as a mitochondrial membrane scaffold thatmight recruit membrane proteins to functional sites of the IMM(Osman et al., 2009b). It has also been implicated in regulatingmitochondrial morphogenesis (Merkwirth et al., 2008), stabilizingthe mitochondrial genome (Kasashima et al., 2008), and modulatingmembrane protein degradation via the mitochondrial m-AAAprotease (Steglich et al., 1999).

Several possible mechanisms can be considered for PHBcomplex-mediated RSC formation. First of all, as a scaffold in theIMM, PHB complex might directly facilitate the RSC assemblythrough interacting with the subunits of respiratory complexes, forinstance, through the subunits of complexes I and IV (Bourges et al.,2004; Strub et al., 2011). Second, because the PHB complexinteracts with cardiolipin and regulates its abundance in differentcontexts (Osman et al., 2009a; Richter-Dennerlein et al., 2014), it ispossible that the PHB complex regulates the stability of RSCsthrough interacting with cardiolipin, which is required for thestability of RSCs (Zhang et al., 2002, 2005). Third, the multimericring complex of PHBs could help to establish a functionalmicrodomain in the IMM with microdomain-specific protein andlipid constituents (Osman et al., 2009b) that facilitate the assemblyof RSCs. Regardless of the specific mechanism, the identification ofPHB complex as a RSC regulator sheds new light not only ondynamic regulation of RSCs but also on a possible role of theIMM-enriched PHBs.

Regulation of mitoflash activity by RSC formationMitoflash represents a new form of mitochondrial functional andsignaling activity (Hou et al., 2014; Wang et al., 2008, 2012). Weand others have shown that mitoflash generation is functionally andmechanically interlinked with mitochondrial ETC activity. Itrequires the activity of the ETC to be intact (Shen et al., 2014;Wang et al., 2008), and is thought to act as a biomarker ofmitochondrial respiration under certain conditions (Gong et al.,2015). Furthermore, it can be regulated by factors that participate inmitochondrial metabolism indirectly, such as mitochondrial Ca2+

uptake (Jian et al., 2014), basal ROS production (Hou et al., 2013)and H+ nanodomains (Wang et al., 2016). Here, we revealed a novelform of mitoflash regulation involving not specific molecular

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Fig. 4. Co-expression of rat PHB1 and PHB2 rescues the impairments in PHB-deficient HeLa cells. (A) Western blots for PHB1 and PHB2 expression.siRNAs targeting human PHB1 (siPHB1), PHB2 (siPHB2), or negative control siRNA (NC) were transfected into HeLa cells for 12 h and then the rat PHB1(rPHB1), PHB2 (rPHB2) or both (rPHB1+rPHB2) were expressed in the PHB-deficient or NC cells. GAPDH was used as internal control. (B) RepresentativeBNGE immunoblot analysis of mitochondrial RSC formation after re-expression of PHB1 and PHB2. RSCs were visualized by antibodies against complex III(CIII, UQCRC1) and complex IV (CIV, COX IV), and then stripped and reprobed for complex I (CI, NDUFB8). The membranes were subsequently blotted forcomplexes II (CII, SDHA) and V (CV, ATP5A). It is notable that the RSC formation was rescued only after co-expression of rPHB1 and rPHB2 in the PHB-deficientHeLa cells. (C,D) Measurement of cytosolic ROS with DCF (C) or mitochondrial ROS with mitoSOX (D) after re-expression of PHB1 and/or PHB2. a.u.,arbitrary units. n=30–129 cells for each group. ***P<0.001 versus NC group. n.s., no significance versus NC. (E) Effect of PHB1 and PHB2 re-expression onmitoflash frequency. n=74–131 cells for each group. ***P<0.001 versus NC group. n.s., no significance versus NC. All quantitative data are expressed asmean±s.e.m.

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entities but rather a subtle arrangement of mitochondrial respiratorycomplexes, that is the formation of RSCs. We demonstrated agenerally inverse relation between mitoflash activity and RSCformation: lower RSC formation leads to greater mitoflash activity.These findings indicate that regulating mitoflash activity is acommon event downstream of RSC remodeling.We have provided several lines of evidence that link RSC

regulation of basal ROS production and mitoflash activation.Downregulation of seven RSCs regulators, namely, PHB1, PHB2,SCAFI, RCF1a, RCF1b, UQCC3, SLP2, which impairs RSCformation via overlapping but different mechanisms, all increasedmitochondrial basal ROS production in conjunction with aconcomitant increase in mitoflash activity. Importantly, loweringmitochondrial ROS level with mitoTEMPO reduced mitoflashes inthese RSC assembly factor-deficient cells. This result reinforces theidea that the RSC disruption causes leaky ETC and heightens basalROS production, and that basal ROS constitute an endogenousphysiological trigger of mitoflashes. Interestingly, this hyperactivemitoflash response occurred with little changes in metabolicparameters (as for knockdown of RCF1b and UQCC3) or evenwith a significantly repressed metabolic activity (as for knockdownof PHBs, SCAFI, RCF1a and SLP2). Moreover, previous reportshave also shown cell type-dependent effects of RSC disruption onmitochondrial respiration. For instance, SCAFI deficiency causessignificantly reduced mitochondrial respiration in MEFs (Ikeda

et al., 2013), but only has a mild effect in 143B cells (Pérez-Pérezet al., 2016), and even enhanced the respiration in isolated livermitochondria (Lapuente-Brun et al., 2013). In T cells, impairingRSCs through SLP2 knockout increased uncoupled respiration butdid not alter the basal respiration (Mitsopoulos et al., 2015). Thesefindings with manipulations of RSCs highlight that, while deeplyinterlinked, mitoflash signaling and metabolic activity can bedifferentially regulated and, consequentially, mitoflash biogenesisis not a unique function of mitochondrial respiration.

In summary, we have identified that PHB1 and PHB2 constitutebona fide regulators of mitochondrial RSC formation and mitoflashproduction. Through manipulating PHBs and a panel of otherRSC regulators, we have further demonstrated that RSC formationnegatively regulates mitoflash activity by limiting basal ROSproduction, while its effects on mitochondrial respiration varyamong different regulators examined. These findings not onlyidentify a novel function of PHBs as the IMM scaffold proteins, butalso underscore the profound implications of RSC formation in theregulation of mitochondrial ROS and mitoflash signaling as well asrespiratory functions. Future investigations are needed to examinethe contribution of PHB-regulated mitoflash signaling in PHB-regulated diverse processes from mitochondrial biogenesis to cellproliferation and embryonic development. Furthermore, wespeculate that alteration of RSC formation and hyper-activation ofmitoflashes might represent the organelle-level etiology of certain

Fig. 5. Impaired RSC formation bydownregulation of RSC assembly factors.(A) BNGE immunoblot analysis of RSCs afterknocking down SCAFI, RCF1a, RCF1b,UQCC3 or SLP2. NC, negative control siRNA.RSCs were visualized by antibodies againstcomplex III (CIII, UQCRC1) and complex IV(CIV, COX IV), and then stripped and reprobedfor complex I (CI, NDUFB8). The membraneswere subsequently blotted for complexes II (CII,SDHA) and V (CV, ATP5A). (B,C) Quantitativeresults for experiments shown in A. The anti-ATP5A immunoblot was used as the reference.Data are mean±s.e.m. for n=4 independentexperiments. *P<0.05 versus NC group.

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types of mitochondrial diseases, and if so, preserving RSCs tonormalize mitoflash signaling might open a new way to theprevention and treatment of such diseases.

MATERIALS AND METHODSReagentsDulbecco’s modified Eagle’s medium (DMEM), Lipofectamine RNAiMax,Lipofectamin 2000, penicillin, streptomycin, and the Amplex® Red hydrogenperoxide kit were from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS)was from Gibco (Carlsbad, CA). Menadione, H2O2, mitoTEMPO, and anti-tubulin (1:2000, T6199) monoclonal antibody were from Sigma (St Louis,MO). MitoSOX and 2,7-dichlorodihydrofluorescein diacetate (DCF) werefromMolecular Probes (Eugene, OR). Anti-PHB1 (1:1000, 603101) and anti-PHB2 (1:1000, 611801) antibodies were from BioLegend (San Diego, CA).Antibodies against SLP2 (1:2000, 60052-1-Ig), GAPDH (1:1000, 10494-1-AP), NDUFB8 (1:1000, 14794-I-AP), or COX IV (1:1000, 11242-I-AP) were

from Proteintech (Chicago, IL). Antibodies against SCAFI (1:1000, ab66107),SDHA (1:2000, ab14715), UQCRC1 (1:2000, ab110252) and ATP5A(1:2000, ab176569) were from Abcam (Cambridge, UK). Antibody againstTIM23 (1:2000, 611223) was from BD Biosciences (Franklin Lakes, NJ).

Cell culture, RNA interference and adenovirus infectionHeLa cells stably expressing mt-cpYFP (Ma et al., 2011), mouse embryofibroblasts (MEFs, from ATCC, Manassas, VA) and primary isolatedneonatal rat ventricular myocytes (NRVMs; all animal experiments wereperformed according to approved guidelines) were grown in DMEMsupplemented with 10% FBS and 1% penicillin and streptomycin at 37°Cunder 5% CO2. For RNA interference, 100 nM siRNA or 2 µg shRNAplasmids were transiently transfected into the cpYFP-expressing HeLa cellsor MEFs with RNAiMax or Lipofectamine 2000 according to themanufacturer’s instructions. The siRNAs used in this study are listed inTable S1. Knockdown efficiency was determined by western blotting or

Fig. 6. Effects of impairing RSC formation on mitochondrial ROS, mitoflash and respiration. (A,B). Measurement of cytosolic ROS with DCF (A) ormitochondrial ROS with mitoSOX (B) in HeLa cells lacking the indicated RSC assembly factors (siRNA-mediated knockdown) with or without mitoTEMPO(10 µM) treatment. NC, negative control siRNA. n=35–123 cells for each group. ***P<0.001 versus NC group in the absence of mitoTEMPO; ###P<0.001 formitoTEMPO-treated versus untreated group. (C) Mitoflash frequencies in different groups. n=134–258 cells for each group. ***P<0.001 versus NC in the absenceof mitoTEMPO. ###P<0.001 mitoTEMPO-treated versus untreated group. (D) Correlation between mitochondrial ROS level and mitoflash frequency. The dashedline shows a linear regression with r=0.97 and P<0.0001. The data were from B and C. (E) Quantitative results for basal, ATP-coupled, maximal and proton-leakassociated oxygen consumption rate (OCR) in different groups. n=12–18 measurements from three independent experiments for each group. **P<0.01;***P<0.001 versus NC. All quantitative data are expressed as mean±s.e.m.

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real-time PCR, and the primers used are listed in Table S2. For adenovirus-mediated gene transfer, the cpYFP-expressing HeLa cells were infected withadenoviruses carrying the Flag-tagged PHB1 or PHB2 gene at a multiplicityof infection (m.o.i.) of 20. For studies in MEFs or NRVMs, the cells wereinfected with PHB1- or PHB2-carrying adenoviruses or transfected withsiRNAs with simultaneous mt-cpYFP adenovirus infection. For the rescueexperiments, the adenovirus carrying rat PHB1 or PHB2 gene tagged with3×Flag was added to the medium at an m.o.i. of 20 after 24 h of siRNAtransfection. Confocal imaging or western blot analysis was performed 72 hafter siRNA transfection and 48 h after virus infection.

Confocal microscopy and image processingA Zeiss LSM710 inverted confocal microscope with a 40×1.3 NA oil-immersion objective was used for acquiring images. Cells were in Tyrode’ssolution consisting (in mM) of: 137 NaCl, 5.4 KCl, 1.2MgCl2, 1.2 NaH2PO4,10 D-glucose, and 20 HEPES (pH 7.35, adjusted with NaOH) duringconfocal imaging. For mitoflash detection, raster scan images were taken byexciting the cpYFP fluorescence alternately at 405 and 488 nm and collectingthe emission at >505 nm. 100 frames of 512×512 pixels were collected at 1 s/frame in bidirectional scanning mode. For measuring cytosolic andmitochondrial ROS levels, HeLa cells were loaded with DCF (5 µM) ormitoSOX (5 µM) in Tyrode’s solution at 37°C for 20 min and washed threetimes before imaging. DCF fluorescence was taken by excitation at 488 nmand emission collection at >500 nm. MitoSOX fluorescence was taken byexcitation at 514 nm and emission collection at 580–740 nm.

Custom-developed programs written in Interactive Data Language(Boulder, CO) image were used for image processing (available from thecorresponding author on request). Mitoflash identification and parametermeasurement were aided with the custom-devised computer algorithmFlashSniper (Li et al., 2012; https://github.com/ljhis007/flashsniper).Amplitude (ΔF/F0, maximum fluorescence increase over baseline) and thekinetic properties of mitoflash, including time to peak (Tp, time from onsetto peak), and duration (T50, time from onset to 50% decay) were measuredautomatically with FlashSniper. The mitoSOX fluorescence was quantifiedby averaging over the mitochondrial regions identified with an iterativethreshold algorithm using the IDL Software Research Systems, exclusive ofthe nucleus (Fig. S3).

Whole-cell respiration analysisThe mitochondrial respiration was measured using XF24 Extracellular FluxAnalyzer (Seahorse Bioscience, North Billerica, MA) according to themanufacturer’s instructions. Briefly, HeLa cells were seeded into the XF24microplate at a density of 4×104 per well at 48 h after siRNA transfection.The cellular oxygen consumption rate (OCR) was monitored the next day inunbuffered assay medium (Sigma D5030) supplemented with 2 mMGlutaMAX (Gibco), 2.5 mM sodium pyruvate and 25 mM glucose (pH7.4 at 37°C), following the sequential addition of oligomycin (1 μM), FCCP(500 nM), and rotenone (1 μM) and antimycin A (1 μM). Basal OCR refersto the respiration rate measured prior to the addition of oligomycin.ATP-coupled OCR was calculated by subtracting the OCR in the presenceof oligomycin from basal OCR. Maximal OCR was calculated bysubtracting the OCR in the presence of rotenone and antimycin A fromthose in the presence of FCCP. Proton leak-coupled OCR was calculated bysubtracting the OCR in the presence rotenone and antimycin A from those inthe presence of oligomycin.

Measurement of H2O2 productionH2O2 production in permeabilized HeLa cells was measured by using theAmplex® Red hydrogen peroxide kit (Molecular Probes) as previouslydescribed (Muller et al., 2004) with modifications. Briefly, cells weretrypsinized, washed, counted and then permeabilized in the assay medium(containing 110 mMKCl, 0.5 mMK2HPO4, 20 mMHEPES, 1 mMMgCl2,10 μM EGTA, pH 7.4) with 50 μg/ml saponin for 30 s. 4×104 cells wereincubated in the assay medium supplied with 10 mM succinate, 50 mMAmplex® Red, with 5 units/ml hydrogen peroxide in each well. Increase inAmplex® Red fluorescence was followed over 60 min at room temperaturewith excitation at 530 nm and emission at >590 nm in a 96-well microplate

reader (Biotek, Winooski, VT). H2O2 production was indexed by theincrease of Amplex® Red fluorescence per minute.

Analysis of mitochondrial RSCs by BNGEMitochondria were isolated with a Dounce homogenizer using theMitochondrial Isolation Kit for Cultured Cells (ThermoFisher, Waltham,MA) according to the manufacturer’s instructions. BNGE was conductedusing the NativePAGE™ system (Invitrogen). Briefly, mitochondria weresolubilized with digitonin (4 g per g of protein) for 30 min on ice. After a30 min centrifugation at 13,000 g, the supernatant was collected and theprotein concentration was determined through BCA analysis (ThermoFisher).50 μg proteins were loaded per lane in the 4–16% Bis-Tris gel. Afterelectrophoresis, proteins were transferred to a PVDF membrane and thensequentially probed with specific antibodies against complex I (NDUFB8),complex II (SDHA), complex III (UQCRC1), complex IV (COX IV), andcomplex V (ATP5A). Blots were visualized using secondary antibodiesconjugated with IRDye (LI-COR, Lincoln, NE, USA) and an Odysseyimaging system (LI-COR). The anti-NDUFB8, anti-UQCRC1, or anti-COXIV immunoblot bands with high molecular mass were used to reflect thecomplex I-, complex III-, or complex IV-containing RSCs.

StatisticsData are expressed as mean±s.e.m. When appropriate, Student’s t-test wasapplied to determine the statistical significance, and a linear regressionmodel was used to investigate correlation between mitoflash activity andmitoSOX-indexed ROS level. The coefficient of determination (R2) wasused to evaluate the goodness of fit of the model. Prism linear regressionanalysis was used for the statistical analysis. P<0.05 was consideredstatistically significant.

AcknowledgementsWe thank Dr Z. Y. Song at Wuhan University for the generous gift of sh-mPHB1 andsh-mPHB2 plasmids.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: C.J., H.C., X.W.; Methodology: C.J., F.X., T.H.; Software: T.S.,J.L.; Validation: F.X.; Formal analysis: Investigation: C.J., F.X., T.H.; Data curation:T.S., J.L.; Writing - original draft: C.J.; Writing - review & editing: H.C., X.W.;Supervision: H.C., X.W.; Project administration: H.C., X.W.; Funding acquisition:H.C., X.W.

FundingThis work was supported by the National Key Basic Research Program of China(2013CB531200 and 2016YFA0500403) and the National Science Foundation ofChina (31130067, 31470811, 31670039, and 31521062).

Data availabilityAll custom software used in this paper are available from the corresponding authorupon request.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.198523.supplemental

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