Expression of the Alternative Oxidase Influences JNK Signaling and Cell Migration

Exp ession o he Al e na i e Oxidase Influences Jun N-Te minal Kinase Signaling and Cell Mig a ion Ana Andjelko ic´, a,b Amelia Mo das, a,b *Lyon B uinsma, a,b *Annika Ke ola, a,b *Giuseppe Cannino, a,b *Luca Gio dano, a,b * P a een K. Dhandapani, a,b,c Ma en Szibo , a,b,c E ic Du ou , a,b Howa d T. Jacobs a,b,c a Facul y o Medicine and Li e Sciences, Uni e si y o Tampe e, Tampe e, Finland b BioMediTech Ins i u e, Uni e si y o Tampe e, Tampe e, Finland c Ins i u e o Bio echnology, Uni e si y o Helsinki, Helsinki, Finland ABSTRACT Down egula ion o Jun N- e minal kinase (JNK) signaling inhibi s cell mig a ion in di e se model sys ems. In D osophila pupal de elopmen , a enua ed JNK signaling in he ho acic do sal epi helium leads o de ec i e midline closu e, e- sul ing in cle ho ax. He e we epo ha concomi an exp ession o he Ciona in- es inalis al e na i e oxidase (AOX) was able o compensa e o JNK pa hway down- egula ion, subs an ially co ec ing he cle ho ax pheno ype. AOX exp ession also p omo ed wound-healing beha io and single-cell mig a ion in immo alized mouse emb yonic fib oblas s (iMEFs), coun e ac ing he e ec o JNK pa hway inhibi ion. Howe e , AOX was no able o escue de elopmen al pheno ypes esul ing om knockdown o he AP-1 ansc ip ion ac o , he canonical a ge o JNK, no i s a - ge s and had no e ec on AP-1-dependen ansc ip ion. The mig a ion o AOX- exp essing iMEFs in he wound-healing assay was di e en ially s imula ed by an i- mycin A, which edi ec s espi a o y elec on flow h ough AOX, al e ing he balance be ween mi ochond ial ATP and hea p oduc ion. Since o he ea men s a ec ing mi ochond ial ATP did no s imula e wound healing, we p opose inc eased mi o- chond ial hea p oduc ion as he mos likely p ima y mechanism o ac ion o AOX in p omo ing cell mig a ion in hese a ious con ex s. KEYWORDS AP-1, Jun N- e minal kinase, al e na i e oxidase, ansc ip ion, wound healing Cell mig a ion is an essen ial p ocess in animal de elopmen , as well as in issue epai . I has been widely s udied in model sys ems, whe e he ocus has been la gely on mechanosensa ion and mechano ansduc ion (1, 2). The ansc ip ional and cy oskele al egula ion o cell mig a ion ensu es coo dina ion and an abili y o espond o ex insic and in insic cues (2). A he cellula le el, he mos s udied mammalian model is he sc a ch o wound-healing assay, in which a linea sc a ch is made in a confluen monolaye o cells, which hen mig a e o close he gap a a measu able a e (3). In D osophila de elopmen , cell mig a ion has been s udied in emb yogenesis, in he p ocess o do sal closu e (4, 5), and la e on du ing me amo phosis, when many o he same genes a e in ol ed in ho acic closu e (6). This p ocess in ol es cells e e ing om he wing imaginal discs, which sp ead o e he p eexis ing la al epide mis (7). These mig a ing cell shee s e en ually use a he midline o c ea e a closed epi helial laye ha gi es ise o he cu icula s uc u es o he do sal ho ax. In an ea lie s udy (8), we epo ed ha he p ocess o do sal ho acic closu e is dis up ed by he exp ession o a commonly used, inducible d i e o ansgene exp es- sion, GeneSwi ch, in he p esence o he inducing s e oid RU486. GeneSwi ch is a modified e sion o he Saccha omyces ce e isiae ansc ip ion ac o GAL4 inco po a - ing he ligand-binding domain o he p oges e one ecep o so as o place i unde Recei ed 5 Ma ch 2018 Re u ned o modifica ion 11 Ap il 2018 Accep ed 11 Sep embe 2018 Accep ed manusc ip pos ed online 17 Sep embe 2018 Ci a ion Andjelko ic´ A, Mo das A, B uinsma L, Ke ola A, Cannino G, Gio dano L, Dhandapani PK, Szibo M, Du ou E, Jacobs HT. 2018. Exp ession o he al e na i e oxidase influences Jun N- e minal kinase signaling and cell mig a ion. Mol Cell Biol 38:e00110-18. h ps:// doi.o g/10.1128/MCB.00110-18. Copy igh © 2018 Andjelko ic´ e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional license. Add ess co espondence o Howa d T. Jacobs, howa d. .jacobs@u a.fi. *P esen add ess: Amelia Mo das, Ins i u e o Molecula , Cell and Sys ems Biology, Uni e si y o Glasgow, Glasgow, Sco land, Uni ed Kingdom; Lyon B uinsma, Labo a o y o Sys ems and Syn he ic Biology, Wageningen Uni e si y & Resea ch, Wageningen, The Ne he lands; Annika Ke ola, VTT Technical Resea ch Cen e o Finland L d., Espoo, Finland; Giuseppe Cannino, CNR Ins i u e o Neu oscience and Depa men o Biomedical Sciences, Uni e si y o Pado a, Padua, I aly; Luca Gio dano, Uni e si y o Pi sbu gh School o Medicine, Di ision o Ca diology, Pi sbu gh, Pennsyl ania, USA. E.D. and H.T.J. con ibu ed equally o his a icle. RESEARCH ARTICLE c ossm Decembe 2018 Volume 38 Issue 24 e00110-18 mcb.asm.o g 1Molecula and Cellula Biology on Janua y 3, 2019 by gues h p://mcb.asm.o g/Downloaded om s e oid con ol (9, 10). Since p oges e one o i s analogues a e no ound in D osophila, i had been assumed ha GeneSwi ch plus RU486 would be pheno ypically ine in o he wise wild- ype flies, which is indeed he case in adul s. Al hough cle ho ax was he mos d ama ic and equen pheno ype obse ed in GeneSwi ch-exp essing flies ea ed h oughou de elopmen on RU486-con aining medium, o he de elopmen al dysmo phologies we e also obse ed, including wings wi h apop o ic egions, abno - mal o missing b is les, and cle abdomen. In he cou se o hese s udies, we obse ed ha coexp ession o he mi ochon- d ial al e na i e oxidase (AOX) om Ciona in es inalis was able o e e he cle ho ax and o he dysmo phological pheno ypes b ough abou by GeneSwi ch plus RU486 (8). Exp ession o an o he wise ine ansgene, such as g een fluo escen p o ein (GFP), he al e na i e NADH dehyd ogenase Ndi1 om yeas , o e en a ca aly ically inac i e a ian o AOX, was unable o co ec GeneSwi ch-plus-RU486- induced cle ho ax (8). AOX ep esen s an accesso y componen o he mi ochond ial espi a o y chain (RC), which is ound in mic obes, plan s, and some me azoan phyla bu no insec s o e eb a es (11). AOX p o ides a non-p o on-mo i e bypass o complexes III (cIII) and IV (cIV) o he s anda d RC. In a ious con ex s, i is able o elie e me abolically dele e ious s esses a ising om damage, oxic inhibi ion, o o e load o he RC (11, 12). Fu he mo e, when exp essed in human cells, flies, o mice, Ciona AOX can alle ia e he damaging pheno ypes associa ed wi h RC inhibi ion (13–19). Howe e , he link be- ween espi a o y homeos asis and dysmo phologies esul ing om GeneSwi ch plus RU486 is unknown. These findings p omp ed us o es whe he AOX could e e he cle ho ax pheno ype b ough abou by gene ic manipula ions in he signaling ne wo k ha main ains he mig a o y beha io o he cell shee s e e ing om he wing discs. Th ee such classes o mu an s ha e been s udied. Fi s , cle ho ax is mani es ed by specific, ecessi e alleles o he gene encoding he D osophila RXR homologue, ul aspi acle (usp), which ac s as a dime iza ion pa ne o he ecdysone ecep o (20). Second, compound he e ozygo es o ano he essen ial ansc ip ion ac o , he GATA ac o pannie (pn ), also gi e ise o his pheno ype (21). One pn allele used in hese s udies is pn MD237 , a hypomo ph c ea ed by inse ion o GAL4 in o he p omo e egion o one o he wo an agonis ic pn iso o ms. This allele was o iginally isola ed in an enhance - ap sc een and has p o en use ul as a d i e o ansgene exp ession in he specific domain o pn exp ession in he do sal epi he- lium; hus, i is o en e e ed o as pn -GAL4. Thi d, cle ho ax esul s om mu a ions in he Jun N- e minal kinase (JNK) signaling pa hway (4)(Fig. 1A). JNK (22) is a membe o he mi ogen-ac i a ed p o ein (MAP) kinase amily ha ac i a es he AP-1 ansc ip ion ac o by phos- pho yla ing i s c-Jun subuni (23). AP-1 has a ple ho a o cellula oles, which include he egula ion o cell mig a ion bo h in de elopmen (24) and in pa hology, e.g., umo in asion (25). I is also subjec o many ypes o egula ion (26). JNK is i sel ac i a ed by a a ie y o s esses h ough a classic kinase cascade (27,28). In he con ex o ho acic closu e, he ini ia ing s imulus appea s o be he engage- men o ecep o y osine kinase p (29) (PDGF [pla ele -de i ed g ow h ac o ] and VEGF [ ascula endo helial g ow h ac o ecep o ] ecep o ela ed). Cle ho ax is p oduced by mu an alleles o he JNK kinase (JNKK) hemip e ous (hep)( 30)o o he AP-1 subuni kayak (kay; he D osophila o holog o mammalian c-Fos)( 31). The use o pn -GAL4 o o he d i e s o b ing abou he local down egula ion o JNK a ge s, such as sca ace (se ine p o ease) (32), o o e exp ession o he AP-1 a ge pucke ed (puc;a phospha ase egula o o JNK ia a nega i e eedback loop) (33)o he issue inhibi o o me allop o eases (Timp)( 34) can also p oduce cle ho ax, while down- egula ion o puc can escue cle ho ax caused by mu a ions o hep (30). One key a ge o JNK in do sal closu e (35,36) is he ans o ming g ow h ac o ␤ amily membe decapen aplegic (dpp). In ho acic closu e, dpp p omo es he mig a ion o cells a he imaginal leading edge (7), bu i ac s in a pa allel pa hway a he han Andjelko ic´ e al. Molecula and Cellula Biology Decembe 2018 Volume 38 Issue 24 e00110-18 mcb.asm.o g 2 on Janua y 3, 2019 by gues h p://mcb.asm.o g/Downloaded om downs eam o JNK (30). One key a ge o dpp in ho acic closu e is pn (37). A dpp homologue in mammals is simila ly in ol ed in pala al closu e (38). We he e o e se ou o es whe he AOX could escue cle ho ax when induced by manipula ions o he JNK pa hway and he associa ed gene ne wo k desc ibed abo e. RESULTS AOX exp ession mi iga es cle ho ax due o down egula ion o JNK signaling. We fi s confi med, using RNA in e e ence (RNAi) and he pn -GAL4 (pn MD237 ) d i e , ha he down egula ion o key componen s o he JNK signaling cascade (Fig. 1A)(22)in he mediodo sal egion du ing D osophila de elopmen esul ed in a pheno ype o cle ho ax. A e e i ying he exp ession pa e n con e ed by pn -GAL4, using he GFP epo e al eady p esen in he pn MD237 s ock (Fig. 1B; see Table S1 in he supplemen al ma e ial), we combined i wi h RNAi inse ions a ge ed agains baske (bsk; encoding JNK), hemip e ous (hep; encoding JNKK), misshapen (msn; JNKKKK), and PDGF- and VEGF- ecep o ela ed (p ; encoding he ecep o y osine kinase a he op o he cascade [Table S2]). These all p oduced a cle ho ax pheno ype o a ious se e i ies (see Fig. 1C o examples), acco ding o he es ed cons uc /inse ion and empe a u e. The wo isola es o he pn -GAL4 d i e ga e indis inguishable mo phological pheno- ypes and we e he e o e used in e changeably in he emainde o he s udy. Unde condi ions p oducing he clea es pheno ypes, bu a oiding subs an ial le hali y (ex- cep in he case o p , whe e i was una oidable), we hen combined hese wi h exp ession cons uc s o AOX o o a con ol ansgene, he GFP gene (Fig. 2 and 3). Fo bsk and hep we es ed mul iple RNAi lines (Table S1), each p oducing cle ho ax when combined wi h he pn -GAL4 d i e . Al hough he se e i y o cle ho ax a ied FIG 1 Cle ho ax p oduced by down egula ion o JNK signaling. (A) Summa y o he main s eps in he JNK signaling cascade in D osophila ho acic de elopmen indica ing D osophila genes by hei s anda d symbols and hei unc ional assignmen s in ed ex . The do ed line o dpp ep esen s i s ac i a ion by AP-1 in emb yonic do sal closu e bu no in pupal ho acic closu e. pn is ac i a ed by dpp o egula e he do sal pheno ype. The s eps indica ed wi h a g een backg ound a e he ones ha we e clea ly influenced by AOX, based on he da a p esen ed la e in he pape . TGF- ␤ , ans o ming g ow h ac o ␤ . (B) Li e-cell imaging o a 13- o 15-h-old emb yo (i), an L3-s age la a (ii), and a pupa (iii) o flies exp essing GFP unde he con ol o he pn -GAL4 d i e (o iginal pn MD237 s ain). (C) Examples o ho acic pheno ypes sco ed as no mal, mild, o se e e, wi h a ows indica ing he end wi hin each class owa d mo e se e e cle ho ax pheno ypes. AOX and Cell Mig a ion Molecula and Cellula Biology Decembe 2018 Volume 38 Issue 24 e00110-18 mcb.asm.o g 3 on Janua y 3, 2019 by gues h p://mcb.asm.o g/Downloaded om sligh ly be ween expe imen s, exp ession o AOX (Fig. 2A and 3A and B) bu no ha o GFP (Fig. 2B) led o a significan and subs an ial shi owa d a wild- ype pheno ype in he p ogeny o bsk o hep knockdown flies. Two di e en RNAi lines o each gene showed he same e ec (Fig. 3E o G). Any con ibu ion o he alle ia ion o he pheno ype om p omo e dilu ion was excluded by measu ing he amoun o AOX RNA d i en by pn -GAL4 in pupae wi h and wi hou one o he double-s anded RNA (dsRNA) cons uc s o hep, which showed no significan di e ence (Fig. 2D). FIG 2 AOX escues cle ho ax p oduced by down egula ion o JNK signaling. (A, B) E ec s o coex- p essing AOX (A) o GFP (B) on he p opo ion o di e en pheno ypic classes esul ing om knockdown o bsk and hep, using he pn -GAL4 d i e and RNAi lines KK 104569 (bsk) and GD 47507 (hep). Fo de ails o he c osses, see Table S2 in he supplemen al ma e ial. The da a ep esen he means ⫾SEM o nine eplica e ials in each expe imen , wi h nindica ing he o al numbe o flies analyzed in each case. S a is ically significan di e ences be ween he p opo ions o AOX- o GFP-exp essing and -nonexp essing flies o di e en pheno ypic classes a e shown. P alues, as indica ed, we e de e - mined by pai ed, wo- ailed S uden ’s es wi h Bon e oni co ec ion. (C) E ec o coexp essing AOX o GFP on pupal semile hali y caused by knockdown o p (RNAi line KK 105353) using he pn -GAL4 d i e . Fo de ails o he c osses, see Table S2 in he supplemen al ma e ial. The da a ep esen he means ⫾SEM o nine eplica e ials in each expe imen , wi h nindica ing he o al numbe o flies analyzed in each case. S a is ically significan di e ences be ween classes a e indica ed, wi h P alues being de e mined by analysis o a iance wi h he Tukey pos hoc hones ly significan di e ence (HSD) es . No e ha con e sion o pe cen ages o each ial co ec s o di e en ial le hali y and o o he ial-specific anomalies. (D) qRT-PCR analysis o AOX RNA (means ⫾SD; n⫽3) in hemizygous UAS-AOX F6 ansgenic flies ha we e also hemizygous o pn MD237 (pn -GAL4), wi h o wi hou he hep RNAi cons uc o line GD 47509. Values we e no malized agains hose o RpL32 and hen agains he mean alue o flies exp essing AOX only, o gene a e he ela i e alues shown. KD, knockdown. Andjelko ic´ e al. Molecula and Cellula Biology Decembe 2018 Volume 38 Issue 24 e00110-18 mcb.asm.o g 4 on Janua y 3, 2019 by gues h p://mcb.asm.o g/Downloaded om Fo msn knockdown, e y ew flies eclosed using he a ailable RNAi line, and AOX o GFP exp ession p oduced no significan change in pheno ype, despi e a end owa d he wild ype o AOX (Fig. 3C) and inc eased se e i y in he case o GFP (Fig. 3F). A p knockdown line was pupal semile hal when combined wi h he pn -GAL4 d i e , e en a 18°C. As a esul , he numbe o eclosing p ogeny was insu ficien o enable a s a is ically meaning ul analysis o he ho acic pheno ype acco ding o se e i y, bu he mean p opo ion o p ogeny wi h cle ho ax was abou 80% in his and pa allel p knockdown expe imen s. Coexp ession o AOX, bu no GFP, ga e subs an ial escue o semile hali y (Fig. 2C), wi h 71% (75/105) o he eclosing flies ha ing a no mal ho ax. AOX exp ession can influence mammalian cell mig a ion. The ailu e o ho acic do sal closu e du ing D osophila de elopmen indica es a de ec in cell mig a ion, which AOX exp ession was able o co ec . To es he gene ali y o his finding, we conduc ed cell mig a ion assays in mammalian cells. Mouse emb yonic fib oblas s (MEFs) we e isola ed om AOX hemizygous mice and wild- ype li e ma es and im- mo alized using a s anda d e o i al ansduc ion p ocedu e wi h i uses encoding human papilloma i us 16 (HPV16) oncop o eins E6 and E7 (39). AOX-endowed immo - alized MEFs (iMEFs) showed an inc eased speed o wound closu e in he s anda d FIG 3 Confi ma ion o AOX escue o cle ho ax caused by JNK knockdown. E ec s o coexp essing AOX o GFP on he p opo ion o di e en pheno ypic classes esul ing om knockdown o bsk,hep, and msn, using he pn -GAL4 d i e . (A, B) Repea s o expe imen s whose esul s a e shown in Fig. 2A. (C) Resul s o assays wi h RNAi line KK 101517 (msn) wi h coexp ession o AOX. (D, E, G) Repea s o he expe imen s whose esul s a e shown in Fig. 2A, using al e na e RNAi lines, GD 38138 (bsk) and GD 47509 (hep). (F) Resul s o assays wi h RNAi line KK 101517 (msn) wi h coexp ession o GFP. Fo de ails o he c osses, see Table S2 in he supplemen al ma e ial. The da a ep esen he means ⫾SEM o nine eplica e ials in each expe imen , wi h nindica ing he o al numbe o flies analyzed in each case. S a is ically significan di e ences be ween he p opo ions o AOX- o GFP-exp essing and -nonexp essing flies o di e en pheno ypic classes a e shown. P alues, as indica ed, we e de e mined by pai ed, wo- ailed S uden ’s es wi h Bon e oni co ec ion. No e ha con e sion o pe cen ages o each ial co ec s o di e en ial le hali y and o o he ial-specific anomalies. AOX and Cell Mig a ion Molecula and Cellula Biology Decembe 2018 Volume 38 Issue 24 e00110-18 mcb.asm.o g 5 on Janua y 3, 2019 by gues h p://mcb.asm.o g/Downloaded om sc a ch assay (Fig. 4A), which was main ained in he p esence o a ious d ugs, no ably, pho bol my is a e ace a e (PMA), an indi ec ac i a o o AP-1-dependen ansc ip ion (ac ing ia p o ein kinase C), and he JNK inhibi o SP600125. Howe e , JNK inhibi o V dec eased he a e o wound closu e o AOX-endowed iMEFs o he same le el as wild- ype iMEFs. The sc a ch assay conduc ed on p ima y MEFs (a passage 6) e ealed no di e ence in mig a ion a e be ween AOX-endowed and con ol MEFs (Fig. 4B). All p ima y lines mig a ed much mo e slowly han iMEFs, wi h inhibi o V also p oducing subs an ial cell dea h. The a e o single-cell mig a ion o AOX-endowed iMEFs was also significan ly g ea e han ha o con ol iMEFs (Fig. 4C). AOX exp ession has no sys ema ic e ec on c-Jun phospho yla ion. We nex es ed he same se o JNK modula o s o hei e ec s on c-Jun phospho yla ion a JNK a ge si es (40) Se 63 and Se 73, which has been shown o p omo e wound healing in he sc a ch assay (41). This was done in iMEFs (Fig. 5A), as well as in wo o he cell lines, he HEK293-de i ed AP-1 ansc ip ional epo e line used la e in he s udy (HEK-AP1) (Fig. 5B) and human fib oblas line BJ-5 a (Fig. 5C). Only SP600125 dec eased he amoun o phospho yla ed c-Jun, whe eas JNK inhibi o V ins ead inc eased i , as did PMA. The p esence o AOX did no influence c-Jun phospho yla ion a hese si es in iMEFs (Fig. 5A), al hough i did appea o po en ia e he e ec o inhibi o V in an AOX-exp essing BJ-5 a cell clone (Fig. 5C). AOX does no escue cle ho ax caused by manipula ion o AP-1 exp ession o o he a ge s. We easoned ha di ec ly down egula ing c-Jun o i s dime iza ion pa ne , c-Fos, encoded in D osophila by Jun- ela ed an igen (J a) and kayak (kay), espec i ely, should p oduce e ec s ha la gely o e ide i s egula ion by JNK and he beneficial e ec s o AOX. Acco dingly, coexp ession o AOX had only a sligh e ec on he se e i y o cle ho ax induced by knockdown o kay o J a using he pn -GAL4 FIG 4 E ec s o AOX on mammalian cell mig a ion. (A, B) Ra e o wound closu e in sc a ch assay o cul u ed wild- ype iMEFs (con ol) and AOX hemizygous iMEFs (A) and p ima y MEFs (B) a passage 6, as indica ed, ei he un ea ed (un ), ea ed wi h only 0.2% DMSO, wi h PMA (20 mM), wi h SP600125 (20 ␮ M in 0.2% DMSO), o wi h JNK inhibi o V (inh V; 20 ␮ M), as shown. As e isks abo e he ba s indica e a s a is ically significan di e ence (de e mined by one-way analysis o a iance wi h he Tukey pos hoc HSD es ) om un ea ed cells o he gi en geno ype. As e isks joining he ba s indica e s a is ically significan di e ences be ween he geno ypes o a gi en ea men , based on he same s a is ical analysis. Fo cla i y, o he significan di e ences a e no shown. All da a poin s a e based on h ee biological eplica es, each analyzed in iplica e, excep o DMSO only, which used only wo biological eplica es. Fo he p ima y MEFs in panel B, he means ⫾SD a e o pooled da a om wo cell lines o each geno ype analyzed in iplica e a passage 6. (C) Ra e o mig a ion o single iMEFs o he indica ed geno ypes. As e isks deno e s a is ical significance, as shown (S uden ’s es , unpai ed; n⫽31 o con ol iMEFs; n⫽21 o AOX-endowed iMEFs). Andjelko ic´ e al. Molecula and Cellula Biology Decembe 2018 Volume 38 Issue 24 e00110-18 mcb.asm.o g 6 on Janua y 3, 2019 by gues h p://mcb.asm.o g/Downloaded om d i e (Fig. 6A and B). Simila ly, AOX was unable o escue he le hali y caused by o e ex- p ession o he AP-1 a ge , puc, which also an agonizes he ac ion o bsk (Fig. 6C). No e, howe e , ha his esul may be i ial, since puc o e exp ession gene a es a se e e emb yonic pheno ype, due o he inhibi ion o do sal closu e. As discussed ea lie , pn is conside ed o ac in ho acic closu e ia a pa hway pa allel o he JNK pa hway. Since he pn -GAL4 line pn MD237 is also a pn hypomo ph, we combined i wi h he pn D1 mu an as a compound he e ozygo e, p oducing, as expec ed, a pheno ype o se e e cle ho ax (Fig. 6D). This was no alle ia ed by AOX, whe he i was supplied using a cons i u i e o a GAL4-dependen ansgene (Fig. 6D). Ou findings a e consis en wi h he in e ence ha AOX ac s on JNK signaling ups eam o AP-1 bu canno compensa e o a deficiency o AP-1 i sel no o a pa allel pa hway also equi ed o ho acic closu e. FIG 5 Phospho yla ion s a us o JNK a ge esidues in c-Jun. Wes e n blo s o whole-cell p o ein ex ac s om con ol and AOX-exp essing iMEFs (A), HEK-AP1 cells (B), and AOX-exp essing o con ol human BJ-5 a fib oblas s (C) un ea ed (un ) o ea ed wi h JNK modula o s, as shown: 0.2% DMSO, 20 ␮ M SP600125 (SP) in 0.2% DMSO, 20 ␮ M JNK inhibi o V (V o inh V), o 8 nM PMA. The molecula weigh s o he majo bands de ec ed by each an ibody, in e ed om size ma ke s un on all gels, we e as expec ed (100 kDa o ␣ -ac inin [ ␣ -ac ], 47 kDa o c-Jun phospho yla ed a esidue Se 73 [pSe 73] o Se 63 [pSe 63]). Sepa a e blo s we e ini ially p obed o pSe 73 o pSe 63, and hen in bo h cases he blo s we e ep obed o ␣ -ac inin as a loading con ol. D ug concen a ions we e based ei he on dose- esponse cu es ob ained using he HEK-AP1 cell ansc ip ional epo e sys em ( o PMA and JNK inhibi o V; see Table S3 in he supplemen al ma e ial) o on ials o de e mine he highes concen a ion a which he e was no e idence o subs an ial cell dea h ( o SP600125). Blo images we e op imized o b igh ness and con as , o a ed, and c opped wi h he addi ion o whi e ames o di ide s o cla i y, bu wi h no o he manipula ions. AOX and Cell Mig a ion Molecula and Cellula Biology Decembe 2018 Volume 38 Issue 24 e00110-18 mcb.asm.o g 7 on Janua y 3, 2019 by gues h p://mcb.asm.o g/Downloaded om AOX does no influence AP1-dependen ansc ip ion in cul u ed cells. To in es iga e he mechanism by which AOX impac s he ou come o JNK signaling, we es ed whe he i influences ansc ip ion di ec ed by AP-1. Using a well-es ablished luci e ase-based AP-1 epo e sys em (42) and a a ie y o di e en exp ession con- FIG 6 AOX does no escue cle ho ax p oduced by al e ed exp ession o AP-1 o o he a ge s. (A, B) E ec s o coexp essing AOX on he p opo ion o di e en pheno ypic classes esul ing om knockdown o kay (a 25°C) and J a (a 18°C), using he pn -GAL4 d i e and RNAi lines GD 6212 (kay) and KK 107997 (J a) (A) and al e na e RNAi lines GD 19512 (kay) and GD 10835 (J a) (B). Fo de ails o he c osses, see Table S2 in he supplemen al ma e ial. Because he GD 10835 (J a) cons uc is ca ied on ch omosome X, wo pa allel c osses we e equi ed o es he e ec s o AOX exp ession in each sex, and s a is ical analysis was no meaning ul in his case. The da a ep esen he means ⫾SEM o nine eplica e ials in each expe imen , wi h nindica ing he o al numbe o flies analyzed in each case. S a is ically significan di e ences be ween he p opo ions o AOX-exp essing and -nonexp essing flies o di e en pheno ypic classes a e shown. P alues, as indica ed, we e de e mined by pai ed, wo- ailed S uden ’s es wi h Bon e oni co ec ion. No e ha J a knockdown using RNAi line KK 107997 (J a) was le hal a 25°C and ha AOX did no escue his le hali y. (C) E ec o coexp essing AOX o GFP on pupal le hali y caused by o e exp ession o puc unde he con ol o he pn -GAL4 d i e . P ogeny classes a e as indica ed, and all con ained, in addi ion, he UAS-puc o e exp ession cons uc . Fo de ails o he c osses, see Table S2 in he supplemen al ma e ial. The da a ep esen he means ⫾SEM o nine eplica e ials in each expe imen , wi h nindica ing he o al numbe o flies analyzed in each case. No e ha con e sion o pe cen ages o each ial co ec s o di e en ial le hali y and o he ial-specific anomalies. (D) Pheno- ypes o pn MD237 /pn D1 compound he e ozygo es wi h and wi hou he p esence o AOX ansgenes, as indica ed. Nei he he GAL4-d i en UAS-AOX F6 ansgene no homozygosi y o he ub-AOX ansgenes on ch omosomes 2 and X p oduced he escue o he s ong cle ho ax pheno ype. No e ha because p ogeny pheno ypes we e essen ially uni o m o a gi en geno ype, no meaning ul a iances could be calcula ed. Andjelko ic´ e al. Molecula and Cellula Biology Decembe 2018 Volume 38 Issue 24 e00110-18 mcb.asm.o g 8 on Janua y 3, 2019 by gues h p://mcb.asm.o g/Downloaded om s uc s o AOX, we es ed whe he AOX exp ession in D osophila S2 cells was able o al e AP-1-dependen ansc ip ion unde di e en condi ions o JNK pa hway ac i a- ion. Fi s , we compa ed he ansc ip ional eadou in cells co ans ec ed wi h he epo e plasmids and wi h AOX cloned in o he coppe -inducible exp ession ec o pMT/V5-His B, wi h i s na u al s op codon, wi h ha in cells ans ec ed wi h he emp y ec o . JNK pa hway ac i a ion was achie ed using he pUAST-Hep ac plasmid, included in all ans ec ions in combina ion wi h pAc -Gal4, which p omo es pUAST-Hep ac ansc ip ion by cons i u i e exp ession o Gal4. T ans ec ion e ficiency was con olled by he inclusion o a cons i u i ely exp essed plasmid encoding enilla luci e ase, which can be expe imen ally dis inguished om he fi efly luci e ase o he epo e cons uc . Finally, o measu e backg ound ansc ip ion independen ly o AP-1, he sys em in- cludes a mu a ed e sion o he epo e (which was used as an al e na i e in ans- ec ions), o which AP-1 does no bind. Despi e he complexi y o his sys em, i ga e clea -cu esul s. AOX p oduced no significan change in AP-1-dependen luci e ase exp ession unde bo h basal and JNK-ac i a ed condi ions (Fig. 7A; Table S4). Nex , we es ed epo e cells co ans ec ed wi h a plasmid (pAC/AOX) (43) di ec ing cons i u i e AOX exp ession unde he con ol o a ␤ -ac in p omo e e sus cells co ans ec ed wi h he emp y ec o . Again, AOX exp ession had no e ec on he ansc ip ional eadou (Fig. 7B; Table S4). Using a sys em in which JNK pa hway ac i a ion and AOX induc ion we e b ough abou simul aneously by exp ession o he exogenous ansc ip ion ac o Gal4, bu his ime using a con ol plasmid ha bo ing a ca aly ically inac i e, mu a ed AOX, we again ound no e ec o AOX (Fig. 7C: see also he esul s o a pa allel expe imen in Table S4). AOX also p oduced no significan di e ence in AP-1-dependen ansc ip ion in cells whe e hep had been knocked down (Fig. 7D; Table S4). We conduc ed a simila exe cise in mammalian cells, using an HEK293 cell-de i ed epo e cell line (he e designa ed HEK-AP1), s ably ansduced wi h len i i al con- s uc s exp essing AOX o , as a con ol, he mu a ed, ca aly ically inac i e a ian (mu AOX). Success ul ansduc ion and cell cloning a limi ing dilu ion we e e ified ia he fluo escence con e ed by he co ansduced ma ke GFP, and AOX unc ionali y was e ified by espi ome y (Table S5). Al hough indi idual HEK-AP1 cell-de i ed clones showed a a iable deg ee o AP-1-dependen ansc ip ional ac i i y, AOX-exp essing and con ol cell clones showed a simila suscep ibili y o he e ec s o he JNK an agonis s SP600125 and inhibi o V (Fig. 7E). Su p isingly, SP600125 inc eased a he han dec eased he ansc ip ional eadou , despi e he ac ha i inhibi ed c-Jun phospho yla ion (Fig. 5B), al hough i did modes ly supp ess PMA-ac i a ed ansc ip- ion in he epo e line (Fig. 7E). An imycin A di e en ially s imula es he mig a ion o AOX-exp essing cells. To gain insigh in o he in acellula p ocess(es) unde lying he enhanced mig a o y beha io o AOX-exp essing cells, we es ed he e ec s o suble hal doses o a ious me abolic e ec o s on he ela i e a es o mig a ion o AOX-exp essing e sus con ol iMEFs. In an ini ial expe imen (Fig. 8A), we es ed a ious oxida i e phospho yla ion inhibi o s, an ioxidan s, and p o ease inhibi o s in he wound-healing assay o a di e en ial e ec on AOX-exp essing cells. Fo u he s udy, we selec ed h ee ea - men s ha appea ed o gi e a di e en ial e ec (an imycin A, oligomycin, and mi o- quinone mesyla e [Mi oQ]), oge he wi h wo ha did no ( o enone and ca bonyl cyanide p- ifluo ome hoxyphenylhyd azone [FCCP]), and measu ed wound closu e in ou independen expe imen s. An imycin A had a significan ly di e en e ec on he mig a ion o AOX-exp essing MEFs e sus wild- ype MEFs (Fig. 8B), s imula ing he mig a ion o he o me bu supp essing ha o he la e , whe eas Mi oQ, o enone, oligomycin, and FCCP had no significan e ec s. To unde s and he implica ions o hese findings o he mechanism by which AOX p omo es cell mig a ion, we checked he e ec s o AOX exp ession on espi a ion in he cell lines es ed (Fig. 8C). AOX had no significan e ec on whole-cell espi a ion o on pe meabilized cell espi a ion on cI-, cII-, and cIV-linked subs a es. Howe e , in he p esence o an imycin A, i enabled AOX and Cell Mig a ion Molecula and Cellula Biology Decembe 2018 Volume 38 Issue 24 e00110-18 mcb.asm.o g 9 on Janua y 3, 2019 by gues h p://mcb.asm.o g/Downloaded om wo- ound PCR-based p ocedu e essen ially as desc ibed p e iously (112), bu using hep-specific p ime s (shown 5= o 3=) TGGAGGCAAAGCTCCAGGC and CGCGAACGAAGCAGCCAAGG o he fi s ound and GAATTAATACGACTCACTATAGGGGAGACATCCGCCACCCACGCACCTTC and GAATTAATACGACTCACTATA GGGGAGATCCCATTGCCCAGGTCGCCCAG o he second ound, ollowed by ansc ip ion using a MEGAsc ip T7 ansc ip ion ki (Li e Technologies). Knockdown a he RNA le el ( o ⬃85%) was e ified in ans ec ed cells (112) by qRT-PCR. Fo combined dsRNA/ epo e plasmid ans ec ions, 1 ⫻ 10 5 cells we e pla ed pe well in 24-well pla es. A e 30 min, cells we e ans ec ed wi h 300 ng o each ele an plasmid and 4 ␮ go hep-specific dsRNA pe well in a o al olume o 100 ␮ l. A u he 4 ␮ go he dsRNA was added 72 h la e , and luci e ase assays we e conduc ed 110 h a e he ini ial ans ec ion. Fo luci e ase assays, 75 ␮ l o suspended cells om each well was ans e ed in iplica e o he wells o a 96-well mic opla e (Lab Sys ems) and analyzed using a Dual-Glo luci e ase assay sys em (P omega), acco ding o he manu ac u e ’s p o ocol. Luminescence was measu ed using a The mo Labsys ems Luminoskan Ascen pla e eade . Luci e ase epo e assays in mammalian cells. Fi efly luci e ase epo e assays we e ca ied ou in HEK-AP1 cells and in AOX/mu AOX-exp essing clones de i ed om hem, as ollows: 30,000 cells we e pla ed in echnical duplica e (2 wells pe sample) in luminome e -compa ible Nunc Mic oWell 96-well pla es wi h lids (The mo Fishe Scien ific). A e 24 h, he medium was eplaced wi h medium con aining ei he 0.2% DMSO, 20 ␮ M SP600125 in 0.2% DMSO, 20 ␮ M JNK inhibi o V, o no added d ug. Cells we e incuba ed o 2ha 37°C. Fo PMA ea men , a second eplacemen medium con ained 8 nM PMA plus 20 ␮ M SP600125 in 0.2% DMSO, 20 ␮ M JNK inhibi o V, o no o he added d ug, as app op ia e, and he cells we e incuba ed o a u he 6 h. Luci e ase assays we e ca ied ou using he Dual-Glo luci e ase assay sys em (P omega), acco ding o he manu ac u e ’s p o ocol, and luminescence was measu ed using a Pe kinElme UV/ isible pla e eade . P o ein analysis by Wes e n blo ing. Ba ches o 300,000 MEFs o HEK-AP1 cells o 250,000 BJ-5 a cells we e pla ed on 6-well pla es (CellS a ; G eine Bio-One). A e 24 h, he medium was eplaced wi h medium con aining ei he 0.2% DMSO, 20 ␮ M SP600125 in 0.2% DMSO, 20 ␮ M JNK inhibi o V, 8 nM PMA, o no added d ug and he pla e was incuba ed o 2 h (o 40 min, in he case o PMA). Cells we e ca e ully insed in ice-cold phospha e-bu e ed saline (PBS) and hen sc aped ee on ice using a Cy One cell sc ape (220 mm long, 11-mm blade) in 75 ␮ l o esuspension bu e con aining 100 mM NaCl, 10 mM T is-HCl, and 1 mM EDTA, pH 7.8, supplemen ed wi h cOmple e, Mini, EDTA- ee p o ease inhibi o and phospha ase inhibi o cock ails (a he manu ac u e ’s ecommended amoun ; Roche) and 1 mM phen- ylme hylsul onyl fluo ide. P o ein concen a ions we e de e mined using he B ad o d assay. A e lysis by he addi ion o an equal olume o SDS sample bu e (Laemmli 2⫻concen a e; Sigma-Ald ich), samples we e hea ed o 5 min a 100°C and b iefly cen i uged o emo e pa icula es, and 20 ␮ g o each ex ac was loaded on o 18-well p ecas Any kD C i e ion TGX S ain-F ee p o ein gels (Bio-Rad), which we e un and blo ed as desc ibed p e iously (43). Blo s we e p ocessed as desc ibed p e iously (15), bu wi h blocking in 5% bo ine se um albumin (BSA) in PBS-Tween o 1honashake and using he p ima y an ibody phospho-c-Jun (Se 73) abbi monoclonal no. 3270 (1:1,000; Cell Signaling Technology) o phospho-c-Jun (Se 63) II abbi polyclonal 9261 (1:1,000; Cell Signaling Technology), wi h ep obing using an i- ␣ -ac inin abbi polyclonal C-20 (1:7,000; sc-7454-R; San a C uz Bio echnology). Seconda y an ibody was pe oxidase-labeled goa an i- abbi IgG (1:10,000; PI-1000; Vec o Labo a o ies). The chemiluminescence o all blo s was documen ed bo h wi h film and by using a Bio-Rad ChemiDoc image . Respi ome y. Whole-cell and pe meabilized cell espi a ion was measu ed essen ially as desc ibed p e iously (118). iMEFs we e seeded 24 h be o e he expe imen and g own in DMEM con aining 4.5 g/li e glucose, 10% e al bo ine se um (The mo Fishe Scien ific), 2 mM Glu aMAX (Gibco), and 100 U/ml penicillin plus 100 ␮ g/ml s ep omycin (Lonza). To ac i a e cell espi a ion, he g ow h medium was eplaced 1 h be o e he assay. Cells we e de ached wi h 0.05% ypsin and coun ed by ypan blue exclusion. Mi ochond ial espi a ion in pe meabilized cells was assayed using an O obo os oxyg aph-2K oxyg aph (O obo os, Innsb uck, Aus ia), wi h 2 ⫻10 6 iMEFs being di ec ly suspended in he oxyg aph chambe con aining 2 ml o espi a ion bu e B (10 mM KH 2 PO 4 , 20 mM HEPES-KOH, 20 mM au ine, 0.5 mM EGTA, 3 mM MgCl 2 , 1 mg/ml essen ially a y acid- ee BSA, 60 mM po assium-lac obiona e, 110 mM manni ol, 0.3 mM di hio h ei ol, pH 7.1). A e measu ing endogenous whole-cell espi a ion, subs a es and inhibi o s we e added in he ollowing o de : (i) digi onin (30 ␮ g), o pe meabilize he cells; (ii) sodium py u a e ( o 5 mM), sodium glu ama e ( o 5 mM), and sodium mala e ( o 2 mM) as a cI-linked subs a e mix, ollowed by ADP ( o 2 mM); (iii) o enone ( o 150 nM) ollowed by succina e ( o 10 mM) as a cII-linked subs a e mix; (i ) an imycin A ( o 30 ng/ml), o e eal AOX-media ed espi a ion; ( ) n-p opyl galla e (nPG; o 200 ␮ M), o e eal any esidual non-AOX-media ed oxygen consump ion o be sub ac ed; ( i) N,N,N=,N=- e ame hyl-p-phenylenediamine (TMPD; o 1 mM) plus sodium L-asco ba e ( o 2 mM) as a cIV-linked subs a e mix; and ( ii) sodium azide ( o 40 mM), o e eal any non-cIV-media ed oxygen consump ion o be sub ac ed. O 2 consump ion (in picomoles · second ⫺1 · millili e ⫺1 ) was no malized o he amoun o o al p o eins ex ac ed om 1 ⫻10 6 cells and assayed by he B ad o d me hod (119). All chemicals we e pu chased om Sigma-Ald ich. SUPPLEMENTAL MATERIAL Supplemen al ma e ial o his a icle may be ound a h ps://doi.o g/10.1128/MCB .00110-18. SUPPLEMENTAL FILE 1, XLS file, 0.1 MB. SUPPLEMENTAL FILE 2, XLS file, 0.1 MB. Andjelko ic´ e al. Molecula and Cellula Biology Decembe 2018 Volume 38 Issue 24 e00110-18 mcb.asm.o g 16 on Janua y 3, 2019 by gues h p://mcb.asm.o g/Downloaded om SUPPLEMENTAL FILE 3, XLS file, 0.1 MB. SUPPLEMENTAL FILE 4, XLS file, 0.1 MB. SUPPLEMENTAL FILE 5, XLS file, 0.1 MB. ACKNOWLEDGMENTS This wo k was suppo ed by he Eu opean Resea ch Council (ad anced g an 232738 o H.T.J.), he Academy o Finland (Cen e o Excellence g an 272376 and Academy P o esso ship g an 283157 o H.T.J.), he Finnish Cul u al Founda ion (a g an om he Vilho Rossin Fund o A.A.), he Uni e si y o Tampe e, he Tampe e Uni e si y Hospi al Medical Resea ch Fund, and he Sig id Juselius Founda ion. We hank Tea Tuomela, Ou i Ku onen, Me ja Jokela, Sina Saa i, and Samuli Ha - ikainen o echnical assis ance; Ma cos Oli ei a o use ul discussions; Di k Bohmann o he supply o epo e plasmids; Filippo Scialó o p o iding cells and plasmids; T oy Fai h ull o c i ical eading o he manusc ip ; Ma ia Aa onen and Tiina Pessa-Mo ikawa and he Flow Cy ome y Co e Facili y in he Depa men o Biosciences, Uni e si y o Helsinki, o assis ance wi h flow cy ome y; and Ou i Paloheomo and Teemu Ihalainen (Tampe e Imaging Facili y, Uni e si y o Tampe e) and Mika Molin (Ligh Mic oscopy Uni , Ins i u e o Bio echnology, Uni e si y o Helsinki) o help wi h mic oscopy. REFERENCES 1. Pandya P, O gaz JL, Sanz-Mo eno V. 2017. Ac omyosin con ac ili y and collec i e mig a ion: may he o ce be wi h you. Cu Opin Cell Biol 48:87–96. h ps://doi.o g/10.1016/j.ceb.2017.06.006. 2. Hun e MV, Fe nandez-Gonzalez R. 2017. Coo dina ing cell mo emen s in i o: junc ional and cy oskele al dynamics lead he way. 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