hni Li Zhenlin skeletal during molecules hypertrophy signalling of muscle regulator a as acts Synemin ARTICLE RESEARCH ß eevd2 etme 03 cetd1 uut2014 August 13 Accepted 2013; September 24 Received cells 1 stellate hepatic and muscle cells nervous retinal later the in including cells, was types, endothelial cell expression filaments and system, protein tissues synemin vimentin different 1980), in high-molecular-mass detected and Lazarides, a protein and desmin the (Granger as is with exception identified associated An First markers. of identity most synemin. 1980). makes cell connect IFs (Lazarides, useful of mechanically space expression them cell-type-specific cytoplasmic or that tissue- the The diameter of structures the 10-nm of various components of filamentous are cytoskeleton (IFs) filaments Intermediate INTRODUCTION filament, muscle Intermediate Skeletal hypertrophy, Muscle Desmin, WORDS: KEY tissue, ( muscle synemin-deficient in functions generated its in protein we roles understand functional of better its To about targeting process. known spatial either is little and However, A-kinase-anchoring (PKA). temporal A an provides kinase As also membranes. it cellular protein, bridge a and forms IFs protein, (IF) between filament intermediate IV type a Synemin, ABSTRACT Atosfrcrepnec zelnl@pcf;[email protected]) ([email protected]; correspondence for *Authors Paris-France. Myologie, de Institut UMR7215, CNRS olsai xrsin utemr,teatvt fmuscle-mass of activity RII increased PKA the (the and molecules control expression, Furthermore, FBXO32) expression. as GDF8) follistatin known as (also known atrogin (also myostatin and decreased capacity by remodelling accompanied increased level, was molecular with the compared At resistance mice. fatigue control and force maximal increased with 16,Booia dpainadAen,Isiu eBooi ai-en,Paris, Paris-Seine, Biologie de Institut France. Ageing, F-75005 and Adaptation Biological U1164, nsroem ebae.Floigmcaia overload, mechanical Following mild membranes. a Synm showed sarcolemma defects but and myofibres in in fertility phenotype regeneration and and degeneration development normal displayed ae oehr u eut hwta yei sncsayto molecules necessary signalling is regulates hypertrophy. synemin muscle and during that cells. integrity show these membrane results of maintain our differentiation together, the and affect Taken self-renewal could cell synemin between satellite of absence balance muscle the of that analysis suggested Finally, behaviour mice. mutant in increased aqeieGao-Li Jacqueline obneUniversite Sorbonne 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,48–61doi:10.1242/jcs.143164 4589–4601 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 2 / 2 iemslssoe ihrhprrpi capacity hypertrophic higher a showed muscles mice 1, ,AaParlakian Ara *, 2 obneUniversite Sorbonne ´ ,UM nvPrs0,URCR 26ISR ERL 8256/INSERM CNRS UMR 06, Paris Univ UPMC s, 1 oeyeBlanc Jocelyne , a ´ ,UM nvPrs0,ISR U974, INSERM 06, Univ-Paris UPMC s, uui,p06 n RB)was CREB1) and p70S6K subunit, 1 Synm ai Coletti Dario , 2 / 2 mice. ) 1 radFerry Arnaud , Synm 1 oi Alonso-Martin Sonia , 2 / 2 mice 2 eiePaulin Denise , npuioetebyncse el,weesboth whereas cells, development 2004). stem al., embryonic embryonic et pluripotent Xue and during 2001; in splicing al., alternative regulated et by (Titeux produced differentially are – are kDa) (41 L synemin in al., differing kDa), – isoforms et 180 synemin tails Tawk Three 2006; C-terminal 2006). al., their al., 2009; et et al., Schmitt-Graeff et Uyama Izmiryan 2010; 2003; 2006; al., al., et et Izmiryan Izmiryan 2003; al., et (Hirako 01.Ipraty yei xrsini peuae in upregulated is al., expression et Martins the synemin Souza in (de cells Importantly, brain stem adult 2011). the neural of multipotent zone in subventricular expressed are synemin ta. 08;Sne l,20b u ta. 00) Indeed, 2010a). al., et Sun 2008b; (Sun al., roles et different Sun have 2008a; might al., isoforms et these that suggesting 2010a). al., et Sun 2001; not al., et Mizuno 2008; al., also et Synemin utrophin 2004). dystrophin, al., complex, and et protein Xue dystrophin-associated 2007; the Izmiryan al., 2003; 2006; et or with al., al., Jing interacts desmin et 2009; lacking Izmiryan et al., mice 2000; et Hirako al., of et cells 2011; (Carlsson the vimentin are in al., filaments delocalised synemin and et Consequently, unstable 2008). Chourbagi al., keratin, et 1999; (Bellin Khanamiryan or structures filamentous al., vimentin co- form to et desmin, appropriate type, cell the as an on depending requires such therefore partner, its polymerisation and about networks, known filament is of significance; little contributions because clinical the disease functions. about human potential to unknown 2014). a synemin al., remains et much highlight Pekny 2010; however, al., al., features et et (Jing Noetzel 2007; cancer These Alexandre al., breast et of expression Jing or forms its 2005; aggressive of with neurotrauma loss associated and is as disorder, neurodegenerative a such disease, conditions pathological yfbilrZlnsi entlcrimoye,whereas cardiomyocytes, neonatal with in IFs desmin Z-lines of association the myofibrillar mediate to believed is synemin agtn fpoenkns PA nautadneonatal and adult spatial in and (PKA) temporal A modulate kinase can protein it focal of and and targeting Z-lines (AKAP) as protein such structures, cellular and profiles other adhesions. interaction between protein and various bridging Sun a as these IFs 2007; act to al., on proposed et is Based synemin Jing activities, 2010a). 2005; cell al., al., for et essential et (Jing is migration and synemin and dynamics, that adhesion adhesion demonstrates focal evidence in Further participates 2012). al., et cardiomyocytes (Lund between complexes junctional stabilises synemin yei suiu nta tcno oooyeiet form to homopolymerise cannot it that in unique is Synemin yei a nadtoa oea nA-kinase-anchoring an as role additional an has Synemin a b dsrbei Bli ta. 99 hsee l,20;Hijikata 2006; al., et Bhosle 1999; al., et (Bellin -dystrobrevin snmn neat ihvnui,mtvnui n talin, and metavinculin vinculin, with interacts -synemin, 2 hitpeHourde Christophe , b 1 snmn(lokona yei ,10ka and kDa) 150 M, synemin as known (also -synemin hgn Xue Zhigang , a atnn lci ,zxn n he opnnsof components three and zyxin, 1, plectin -actinin, a snmn(lokona yei H, synemin as known (also -synemin 1 n ni Agbulut Onnik and ´ 1 ireJoanne Pierre , b snmni expressed is -synemin a snmn but -synemin, 1 a , 1, and - * 4589 b a b - - -

Journal of Cell Science xrsino aZi eecya el.N ifrne were differences No and specific cells. skeletal drives mesenchymal between in in observed promoter desmin LacZ LacZ vimentin The of expression of the system. expression muscle; of cardiovascular specific cardiovascular development the drives embryonic and promoter during muscle synemin skeletal for role potential uo ta. 94 into 1994) (Colucci- al., LacZ et vimentin-promoter-driven Guyon or 1997) in al., et levels (Li LacZ protein 1B,C). and (Fig. mRNA brain and the heart at muscle, confirmed skeletal was synemin of hc xn1 noig8%o h yei o oan was domain, rod synemin- in Heterozygous synemin 1A). ( the mice (Fig. knockout sequences of synemin loxP 85% two loxP-floxed by encoding flanked the 1, exon generated which first We method. oni h xetdMneinrtot idtp ( wild-type to ratio Mendelian expected the in born bandb nebedn eeoyosmc ( mice heterozygous interbreeding by obtained nte sargltro inltasuto ntePAadAkt and PKA during the Thus, in signalling transduction signal pathways. 2012). of signalling Akt bridging regulator and structures, al., a a in cellular as as other another and one et role IFs functions: between important protein (Pitre a cytoskeletal two have play proliferation might synemin to cell synemin shown recently, glioblastoma More been 2006). al., has et (Russell myocytes cardiac ARTICLE RESEARCH 4590 Synm of mice characterisation behavioural and Phenotypic synemin of ( knockout role the analyse To gene-targeted of Generation RESULTS knockout synemin generated we muscle, an as skeletal and/or ( protein in cytoskeletal a the AKAP as define synemin better of to roles Therefore, functional remain. questions important many nert,mxmlfrepouto n aiu eitneof resistance fatigue membrane in and involved production muscle. is skeletal force adult synemin maximal in that changes integrity, demonstrate phenotypic We and molecular mice. of investigation the eoeln,adta t bec fet h eaiu fmuscle of behaviour the affects muscle cells. absence satellite during its molecules signalling that of and regulator remodelling, a cytoskeletal as a synemin, acts that protein, show results our together, Taken satellite cells. muscle in between programmes balance differentiation the and self-renewal affect the might synemin of In absence control. the mass addition, muscle in implicated higher molecules of signalling forms expression, phosphorylated several of FBXO32) levels as higher and known expression, follistatin as (also known atrogin (also myostatin and lower GDF8) oxidative, more become to switching type fibres in muscle by characterised increased was This capacity muscle. remodelling skeletal in overload mechanical to response ute nlsso 2we-l aemc e ihstandard with fed parameters functional mice and biochemical male similar revealed 12-week-old diet chow of analysis Further xrsigCei h emie Homozygous germline. the in Cre expressing ytmadteptenn fseea uce(i.1D,E). cardiovascular (Fig. the muscle skeletal of of of development patterning absence the the and the affect system that not does indicating synemin stages, developmental different Synm Synm Synm enx nrdcdtasee o desmin-promoter-driven for transgenes introduced next We vdnefrteerlso yei otne oeeg,but emerge, to continues synemin of roles these for Evidence 2 + 2 2 / / 2 / 2 / 2 2 and mice. ) iedslyn vr opooia abnormalities. morphological overt no display mice iewr on ob ibeadfrie n were and fertile, and viable be to found were mice Synm Synm Synm 2 + Synm Synm / / Synm 2 2 2 iewr bandb sn os line mouse a using by obtained were mice ) ieuigteCelx-eitdexcision Cre-loxP-mediated the using mice ) / 2 2 2 ie nadult In mice. + Synm / / 2 / 2 + , iedslydahge hypertrophic higher a displayed mice iewr ibeadfrie enabling fertile, and viable were mice Synm 2 / 2 Synm + nvivo in ie ogi ute nih na on insight further gain to mice, / 2 and 2 Synm / 2 egnrtdsynemin- generated we , Synm mice 2 / 2 Synm 2 / ie h absence the mice, 2 mro during embryos 2 Synm / 2 iewere mice Synm Synm 2 / 2 +/ + / 2 + ), ). h xrsino te Fpoen in affect proteins not IF does other synemin of of expression absence the the that suggesting S1A), Fig. gd(18-month-old) to aged (3-month-old) adult young from muscle, weight-bearing a soleus, shown). not (data fibres muscle mouse perdwike n reua in irregular well-defined and and wrinkled sarcolemma smooth appeared the the 3A,E), to (Fig. muscles Contrary control bordering 3H). membranes Fig. in arrows littermates myofibres, control (see with the compared of when sarcomeres organisation abnormal the with in observed were irregularities 3% o l eoye,but genotypes, all for fseea uce yei n ee te F desmin, and – 18 IFs cytokeratin other 8, seven cytokeratin and syncoilin, Synemin nestin, vimentin, muscle. PCR real-time skeletal quantitative performed of we IFs, other of 2F). expression (Fig. occurring was regeneration that confirmed of myosin fibres muscles) muscle as regenerated presence known newly (8 also immature, the in (MHCnn, MYH8) chain Moreover, perinatal, muscle heavy focal myosin regeneration. of neonatal occurrence and the indicating degeneration 2D,E), Fig. 12-week-old (0.4% in control arrows between to compared fibres as muscles muscles the with anterior, the in differences stained of tibialis major size Synm muscles no of and revealed soleus shape eosin sections and and haematoxylin of plantaris gastrocnemius, analysis Morphological in Synm damage sarcolemmal increased with associated Myopathy xrsinlvlo hs ee F nautpatrsmuscles plantaris adult in IFs seven these between the of in level observed were stages expression various differences significant at muscle No skeletal differentiation. in of expressed are – 19 cytokeratin gs(i.3,) nodrt uniyti hnmnn we phenomenon, ages. this both 18-month- old in quantify frequent at more invaginations were invaginations to that membrane showed Results of order frequency the In assessed 3C,G). (Fig. ages ucefbe splmnaymtra i.SB.Similar synemin-binding S1B). in other vinculin, and Fig. for dystrophin as observed such material partners, were patterns (supplementary localisation fibres muscle lgmn n itiuino iohnra a bevdin observed was mitochondria, of Z-line Synm distribution organisation, sarcomere and of alignment terms in ultrastructure, normal ess03 invaginations/ 0.31 versus invaginations/ ciiyol taon m(o eal e supplementary see details (for am 6 S1). around spontaneous Table material and at consumption only oxygen activity increased an phenotype, atr iharglrproiiyi both striated in expected periodicity the regular gave a with proteins pattern (phalloidin) filament actin ( Z-line-associated for immunostaining irsin fibres ofre h bec fsnmnin synemin of synemin absence for fibres the muscle confirmed of Immunostaining partners. performed different synemin we against directed proteins, antibodies associated using immunohistochemistry its of localisation subcellular month-old 6 sn lcrnmcocp,w xmndteutatutr fthe of ultrastructure the examined we microscopy, electron Using ovrf hte h bec fsnmnifune the influences synemin of absence the whether verify To oeaietesroemlitgiyo h kltlmuscle skeletal the of integrity sarcolemmal the examine To oass hte h bec fsnmnafcsthe affects synemin of absence the whether assess To 6 Synm Hn-oiiefbe nwoeslu n plantaris and soleus whole in fibres MHCnn-positive 2 2 . fmsl irsa en etoulae in centronucleated being as fibres muscle of 0.6 2 2 / / / 2 2 ora fCl cec 21)17 5940 doi:10.1242/jcs.143164 4589–4601 127, (2014) Science Cell of Journal 2 2 Synm Synm mice / n oto ie(i.2.Hwvr equantified we However, 2). (Fig. mice control and uce(i.3–) oee,a 8mnh old, months 18 at However, 3A–D). (Fig. muscle Synm 2 iea oprdt 3-month-old to compared as mice m 2 2 fsroem nmsl rm18-month-old from muscle in sarcolemma of m / 2 2 / 2 / 2 n oto itrae splmnaymaterial (supplementary littermates control and ie eijce vn lede(B)in (EBD) dye Blue Evans injected we mice, mice, Synm Synm m P 2 fsroem nmsl rm3- from muscle in sarcolemma of m , / 2 2 0.05). ie(i.3.A otsod a old, months 3 At 3). (Fig. mice / 2 aemc a lgtlean slight a had mice male Synm Synm 6 a 2 atnnaddsi)and desmin) and -actinin .5 (mean 0.05) / Synm 2 2 Synm Synm / ucefbe tboth at fibres muscle 2 Synm muscle. 2 2 / 2 2 / 2 2 / 2 / uce and muscle, 6 2 n control and n control and ... see s.e.m., ie(0.51 mice Synm 2 / 2

Journal of Cell Science oee,acetn iaeatvt sa fmueserum mouse of assay activity kinase creatine a However, ieietfe rae hntrefl nraei h ubrof number the in in fibres increase three-fold EBD-positive than greater a identified mice EERHARTICLE RESEARCH B eutdi rae paeo h y ntemsl irsof fibres muscle the in dye the of of uptake Synm Injection greater damage. in mm. sarcolemmal resulted 0.5 mark bar: EBD diaphragm to Scale the systems. muscles into nervous plantaris uptake and and its vascular analysed the and of intraperitoneally development mice the affect not does synemin of absence that suggests embryos ( mice synemin-knockout of Generation 1. Fig. B of (B) fmcaial overloaded muscle mechanically plantaris the in observed of were results Similar 4F,M). (Fig. uniiaino ucescin from sections muscle of quantification otiigslto.Epeso fLc ndsi-aZebysi kltl n ada uceseii,weesvmni-aZi anyexpress mainly and is control vimentin-LacZ in whereas expression muscle-specific, desmin-LacZ cardiac of and pattern skeletal- similar is A embryos systems. desmin-LacZ nervous in and LacZ vascular of Expression solution. containing aZ epciey xrsinpten ncnrland control in patterns expression respectively) LacZ, h ieeoso yei r hw sprl oe.Hmlgu eobnto nrdcdlx eune notepooe n nrn1 sidctdw indicated as 1, intron and promoter the into sequences loxP introduced 5 recombination the Homologous of boxes. purple position as The shown arrows. are green synemin of exons five The E15 n atrig(1.)i o fetdb h bec fsnmn iial,teietclvmni-aZepeso atr ncnrland control in pattern expression vimentin-LacZ identical the Similarly, synemin. of absence the by affected not is (E13.5) patterning and (E11.5) ietnLc-xrsigmc o w eeain ooti obetasei mro (desmin-LacZ/ embryos double-transgenic obtain to generations two for mice vimentin-LacZ-expressing yei ( synemin Synm yei in synemin 2 2 Synm / 2 / 2 rvimentin-LacZ/ or Synm Synm + ieta ntoeo oto ie(i.4.A 4). (Fig. mice control of those in than mice / + 2 , / + Synm 2 .Atrecso,termiigfu xn r hw sprl oe.()Epeso fsnmnRAi er H,msl M n brain and (M) muscle (H), heart in RNA synemin of Expression (B) boxes. purple as shown are exons four remaining the excision, After ). / + , Synm + / 2 Synm and Synm + / 2 and , Synm + 2 / 9 2 / 2 n 3 and .Ebyswr olce tdfeettm ons(mroi a,E uigdvlpetadstained and development during E) day, (embryonic points time different at collected were Embryos ). Synm 2 uce oprdwt control with compared muscles / Synm 2 iea eeldb TPR oeteasneo yei N in RNA synemin of absence the Note RT-PCR. by revealed as mice 9 2 rmr sdfrPRaaye r niae yarwed.Tebto iga hw h r-xie leeof allele Cre-excised the shows diagram bottom The arrowheads. by indicated are analyses PCR for used primers / 2 2 os iilsatro uce.(,)Dsi-advmni-rmtrdie aZ(emnLc n vimentin- and (desmin-LacZ LacZ vimentin-promoter-driven and Desmin- (D,E) muscles. anterior tibialis mouse / 2 Synm Synm ie(i.4J–M). (Fig. mice Synm 2 2 / 2 / 2 n nlsso mroi development. embryonic of analysis and ) 2 n control and / 2 mro.Cnrland Control embryos. nt ncnrlvru 111.2 (105.9 damage versus membrane control localised in this units detect to failed Synm 172.8 baseline; lnai uce nrsos onresiuain(i.5). (Fig. stimulation nerve to from response muscles in Plantaris by muscles production generation force force isometric plantaris maximal on the ablation measured synemin we of capacity, effect the investigate To of muscle plantaris the of properties Mechanical Synm 2 / 2 ora fCl cec 21)17 5940 doi:10.1242/jcs.143164 4589–4601 127, (2014) Science Cell of Journal Synm 2 t1mnhatroverload). after month 1 at / 2 iewr nebe ihete emnLc-xrsigor desmin-LacZ-expressing either with interbred were mice 2 / 6 2 mro niae htmsl n ada formation cardiac and muscle that indicates embryos 13uisi oto ess158.7 versus control in units 21.3 Synm A h o iga hw h N agtn vector. targeting DNA the shows diagram top The (A) 2 Synm / Synm 2 desmin-LacZ/ , 2 2 6 / 2 / 2 AC and (A–C) control 12-week-old soleus of and (SOL) (TA) anterior tibialis from muscles. mouse of Morphology 2. Fig. nioy(,wiearw) cl bar: 30 Scale arrows). white (F, MHC antibody anti-neonatal the by are stained that fibres newly muscle small, regenerated and arrows) black (D,E, fibres muscle of centronucleated presence the by confirmed is regeneration muscle of spontaneous presence The (C,F). antibody anti-MHCnn and A,B,D,E) (HE; haematoxylin-eosin with stained 26uisin units 32.6 ie C etr ltaayi of analysis blot Western (C) mice. ieehbtdadces in decrease a exhibited mice m m. Synm Synm + / 2 2 vimentin-LacZ/ , / rznsections Frozen Synm 2 ntoto in DF mice (D–F) 6 Synm 55uisin units 35.5 Synm 2 Synm / nX-gal- in 2 2 di the in ed / 2 2 6 tthe at / mice 4591 2 2 23.8 / 2 ith

Journal of Cell Science EERHARTICLE RESEARCH on htpatrsmsl asswr akdyicesdbt in 4592 both increased markedly were masses muscle plantaris that muscle found of soleus ablation surgical and by gastrocnemius overloaded fibre response were in muscles muscle resistance Plantaris mass, overload. fatigue to muscle and capacity in generation gains force transduction diameter, signal the mechanical examined in we synemin pathways, of role the analyse To mechanical to in response overload in remodelling muscle plantaris Increased of resistance fatigue Our the force. from initial that in muscles 20% of demonstrated decrease measuring a results and to muscle corresponding also time the was the stimulating muscle continuously plantaris of by resistance analysed fatigue The mice. control ( forces maximal oto ie( mice control Synm 2 Synm 20%, 2 2 / 19%, 2 2 mice P / 2 , .5 Fg 5C). (Fig. 0.05) iewsdcesda oprdto compared as decreased was mice P , .5 Fg B scmae with compared as 5B) (Fig. 0.05) .A ot fe vrod we overload, after month 1 At s. fices nbt aia oc n aiu eitnei epneto response in in resistance fatigue and overload force maximal both in increase of P (32.8 Synm epnet vrodi ohgntps(i.5C, (Fig. genotypes both in overload to of response resistance fatigue The 5B). (Fig. (+271.7% ie(+176.7% mice (+238.0% iia a,teices nmxmlfrei epnet overload to response in in force higher maximal in increase was the way, similar a anwshge in higher was gain nraewsas ihrin higher also was increase mean imtr(i-ert in (min-Ferret) diameter 5 .0) ae oehr u eut,dmntaigahge level higher a demonstrating results, our together, Taken 0.004). 6 6 2 / 1.6 2 ...,adwsacmaidb agrma ucefibre muscle mean larger a by accompanied was and s.e.m.), ora fCl cec 21)17 5940 doi:10.1242/jcs.143164 4589–4601 127, (2014) Science Cell of Journal 6 6 n oto ie(i.5)( 5A) (Fig. mice control and m 84mtn ess+197.4% versus mutant 18.4 12.4 m Synm Synm 6 Synm Synm 2 5.7 / 2 2 Synm / 2 2 2 iea oprdt oto,sgetthat suggest control, to compared as mice / Synm / 2 ess29.1 versus 2 eie ebaebreigcnrlmuscles; in well- control and bordering smooth membrane the defined to compared as of mice, sarcolemma the in the irregularity Note microscopy. a electron by transmission examined were mice (C,D,G,H) and (A,B,E,F) control (E–H) 18-month-old and (A–D) month-old muscles. of mouse analysis Ultrastructural 3. Fig. bomlsroee arw nH.Scale 0.5 H). bars: in (arrows with sarcomeres myofibres abnormal the of in organisation irregularities the minor certain Note also are C,G). there in that (asterisks irregular and wrinkled 2 ess+189.1% versus Synm / iea oprdt oto mice control to compared as mice 2 Synm Synm 2 / iea oprdt oto mice control to compared as mice 2 2 2 m / 2 2 lnai uceas nrae in increased also muscle plantaris ess+144.2% versus ACEG;1 (A,C,E,G); m / 6 2 / 2 ucefbe aclmaappear sarcolemma fibres muscle 1.7 iea oprdt control to compared as mice P ieta oto mice control than mice , m oesmslsfo 3- from muscles Soleus 6 .5.Ti ucemass muscle This 0.05). 6 control, m 78control, 17.8 . control, 9.5 m (B,D,F,H). m Synm 6 P , P . control, 8.8 Synm 5 2 .5.This 0.05). / .4) In 0.045). P 2 Synm P 5 2 5 0.006, / 2 0.01) 2 / 2

Journal of Cell Science fMC2-xrsigfbe nrsos ooela a higher was decrease overload of to level response in The in genotypes. fibres both MHC-2b-expressing the in of overload and decreased to significantly response increased in fibres MHC-2b-expressing significantly of proportion fibres proportion the overload, MHC-2a-expressing after month of 1 At overload. without mice ir) a xmnduigacso ar ocutMHC- plantaris count in between composition to differ MHC significantly macro not The custom did 5D–F). glycolytic muscle a (Fig. of cells using indicative MYH4; positive examined MHC- IIB, was and myosin fibre) as fibre), oxidative known of known fibres indicative (also (also MYH2; 2b of MHC-2a IIA, myosin isoforms, proportion MHC The as major overload. two the after expressing and overload without hs fmsl eoeln.mN xrsino the of expression between similar mRNA early was the remodelling. during molecules muscle occurring blotting days investigated changes of 7 western at analyse samples and phase to muscle on overload 6) performed (Fig. after were PCR Analyses 7). real-time (Fig. maintenance intracellular quantitative and of growth by muscle expression in protein involved and molecules signalling mRNA the examined we oto ape ihu vrod(aantson;after shown); not (data overload without samples control Synm EERHARTICLE RESEARCH oeuiaetemlclrmcaim mlctdi increased of in response implicated adaptive mechanisms muscle molecular the elucidate To in overload following mechanical molecules muscle-mass-regulating of Alteration MHC the muscle compare of muscles of to plantaris composition immunohistochemistry of MHC composition used to we related fibres, are resistance fatigue fatigue and hypertrophy. force muscle (maximal during gain resistance) performance in involved is synemin ie httecagsi ucefregnrto aaiyand capacity generation force muscle in changes the that Given Synm 2 / 2 2 / versus 2 iea oprdt oto ie( mice control to compared as mice 2 21.9% 6 Synm 04i control, in 10.4 Synm 2 / 2 2 mice / 2 Synm iefloigoverload, following mice 2 P Synm / 5 2 0.002). n oto mice control and 2 2 Synm 45.6% / 2 n control and 2 6 / 2 . in 7.4 and hni oto iebt ttetasrpinladpost- and transcriptional the overload. after at days 7 both as early mice as levels control translational in than overload, osqety h ee fpopoyae om a ihrin higher was forms phosphorylated of Synm level the Consequently, oe htmreso nlmainwr iia between similar were inflammation of be should markers It Synm 6D). that (Fig. insulin increased of noted was isoform (mIGF-1), muscle-specific factor-1 the growth mass, muscle of regulator eeipiae nngtv oto fmsl as a oe in E3-ligase-encoding lower was an mass, muscle atrogin, of Synm control of negative in Expression implicated gene 6B). (Fig. mice bevto,flittn natgns fmottn exhibited myostatin, of in overload antagonist after expression an higher follistatin, observation, iooa rti 6(P6 olwn vrodi ohcontrol both in overload following and (RPS6) S6 protein ribosomal II subunit regulatory kltlmsl htaeivle nmsl rwhand growth muscle in involved postnatal are in progenitors that myogenic muscle resident skeletal are cells in Satellite overloading response mechanical in muscle cells to satellite Pax7-positive of behaviour Altered GF) nmmaintre frpmcn(mTOR) rapamycin myostatin of for target genes. detected four mammalian of in was deactivator, expression an expression the (GDF8), mRNA in controls Reduced from differences eut eosrt htsgaln oeue mlctdin in implicated expression molecules higher have signalling hypertrophy that muscle demonstrate results nrae ncnrlmc olwn vrod u was but overload, following also mice was control in (Thr202/Tyr204) unchanged in ERK1/2 phosphorylated increased The 7). of (Fig. S6 protein level ribosomal and CREB1 (Thr389), p70S6K etr ltaayi eeldicesdepeso fPKA of expression increased revealed analysis blot Western Synm 2 2 2 / / / 2 2 2 ora fCl cec 21)17 5940 doi:10.1242/jcs.143164 4589–4601 127, (2014) Science Cell of Journal n oto t7dy fe vrod(aantshown). not (data overload after days 7 at control and uce fe vrod(i.6) hra positive a whereas 6C), (Fig. overload after muscles esscnrlatroela o PKARII for overload after control versus 2 Synm / 2 ie(i.7 upeetr aeilFg S2). 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Fig. ucpil odmg than damage to susceptible Synm vroddpatrsmuscle. plantaris mechanical overloaded the in result similar the m(,) 60 (A,B); mm 3 AE n lnai GL rmEBD- injected from (G–L) plantaris and (A–E) diaphragm of sections Frozen to integrity. used sarcolemmal was evaluate (EBD) dye blue Evans Synm P ,At,p06,CE1and CREB1 p70S6K, Akt1, ), , Synm .5between 0.05 Synm + 2 2 2 / / / / 2 2 2 2 Synm Synm ; 2 uce r more are muscles and + os ucefibres. muscle mouse b / P 2 / 2 , ieta ncontrol in than mice + Synm oto uce.Note muscles. control .5between 0.05 Synm 2 m / + (C–E,G–L). m / 2 Synm (C,G,J), mice 2 Synm 2 / 2 / 2 cl bars: Scale . + a / + 2 Synm Synm / (Ser96), and 2 4593 mice + + / / + 2

Journal of Cell Science Synm a7wspromdo lnai ucecyscin from cryosections muscle plantaris on performed Synm was Pax7 nhne nnoela,3.50 Notably, (non-overload, unchanged EERHARTICLE RESEARCH 4594 both in hypertrophy cells) muscle satellite M-cadherin- of in marker presence and surrogate its (a by shown cells is as positive synemin cells S3A, satellite Fig. in material present al., supplementary et in (Mitchell shown Pax7 As cell 2010). factor satellite transcription and the require growth self-renewal muscle Postnatal capacity. regenerative of composition MHC and performance mass, Muscle 5. Fig. sepce,tenme fPx-oiieclssignificantly (non- cells muscles mouse Pax7-positive control 2.90 determined. The in of overload, overload was to S3B). number response fibres in 100 the increased Fig. per expected, material cells As Pax7-positive supplementary of number 8A; (Fig. overload a7pstv aelt el in cells satellite Pax7-positive oc,ad()ftgerssac eeeaie ncnrland control in examined were resistance fatigue (C) and force, akro aeetmmrn) niois D ersnaiescin r rsne o oto and control for presented are sections Representative (D) antibodies. membrane), basement of marker h infcnl erae ucemxmlfreadftgerssac nnon-overload in resistance fatigue and force maximal muscle decreased significantly the oto ie ncnrs,1mnhatroela,n ifrne eeosre nmxmlfreadftgerssac ewe h w eoye ( genotypes two the between resistance in fatigue and mass force muscle maximal higher in a observed also were is differences there no that overload, Note after genotype). month each 1 contrast, In mice. control uce.Foe etoso lnai uce fcnrland control of muscles plantaris of sections Frozen muscles. oeso oiaie hntp,wt nices nMCapstv n eraei H2-oiiefbe fe vrod aaaerpre as reported are Data overload. after fibres muscle MHC2b-positive of switch in a decrease is and there that MHC2a-positive Note in macro. custom increase a an and software with means ImageJ phenotype, using (oxidative) counted were slow fibres more muscle MHC-2b-positive and MHC-2a- (E,F) overload. vrodadcnrlnnoela.Saebr 60 bar: Scale non-overload. control and overload nvitro in 2 2 6 / / 2 2 s.e.m. aelt el aldt epn ooela:tenme of number the overload: to respond to failed cells satellite Synm n oto iewtotoela n asafter days 7 and overload without mice control and oass h eaiu fstlieclsduring cells satellite of behaviour the assess To . a P 6 2 , .8 vrod 5.16 overload, 0.18; / 2 .5btenoela n non-overload; and overload between 0.05 iehv infcnl oePax7-positive more significantly have mice Synm 2 / 2 ie muoitceityfor immunohistochemistry mice, 6 Synm .7 vrod 3.63 overload, 0.27; 6 2 0.41; / 2 m os uce was muscles mouse m. P , .5.However, 0.05). Synm Synm b P , Synm Synm 2 .5between 0.05 6 / 2 2 nvivo in / 0.14). 2 iewr tie ihat-H-a(e) niMC2 bu)adat-elcn(re,a (green, anti-perlecan and (blue) anti-MHC-2b (red), anti-MHC-2a with stained were mice iewtotoela nnOL n n ot fe ehncloela OL.Note (OVL). overload mechanical after month one and (non-OVL) overload without mice 2 2 / / 2 2 os lnai muscle. plantaris mouse ieatroela.(–)MCcmoiinof composition MHC (D–F) overload. after mice Synm oiieclsprcln in colony per cells positive we myoblasts from First, cells in 1997). muscle al., expressed et skeletal (Li typically isolated the differentiation of myogenic is control early which the during under promoter, sequence localisation desmin nuclear a to fused omtett ygncdifferentiation myogenic to commitment omntrtemoei ifrnito oeta fsatellite of potential differentiation interbred we myogenic cells, the monitor To Synm est.Dsieasmlrnme fclsprmoei colony myogenic clonal per to cells them of cultured number and between similar birth a after Despite days density. 10 at muscles limb hs eut ugs ht nteasneo yei,primary synemin, of absence the in that, (28.36% suggest results (mean control cultures These than cell higher muscle skeletal 16% average on aelt el hncnrlmc eoeoela (3.50 overload before mice control Synm than cells satellite 2 2 / 2 2 Synm / / 2 ora fCl cec 21)17 5940 doi:10.1242/jcs.143164 4589–4601 127, (2014) Science Cell of Journal 2 vrodadcnrloverload; control and overload rmr uceclsso ihrlvlof level higher a show cells muscle primary 2.90 versus Synm 2 / 2 2 os lnai ucei oprsnt non-overload to comparison in muscle plantaris mouse / 2 A ucems wih) B smti maximal isometric (B) (weight), mass Muscle (A) Synm n oto ie h ecnaeo LacZ- of percentage the mice, control and Synm 6 .8i control, in 0.18 2 / 2 2 Synm / iewtotoela n ot after month 1 and overload without mice 2 iewt ieepesn LacZ expressing mice with mice 2 / 2 c P 6 Synm utrs(44.53% cultures , Synm s.e.m.; P .5between 0.05 , 2 0.05). / 2 2 / os plantaris mouse 2 P , rcnrlmouse control or 6 .5 Fg 8B). (Fig. 0.05) .8 primary 4.38) Synm 6 .6 was 4.46) 6 2 / n 2 .7in 0.27 5 non- 0for 10 oa to

Journal of Cell Science control, xmnd7dyatroela ncnrland control were in levels overload mRNA after (mIGF-1) day factor-1 7 growth examined insulin muscle-specific (D) and scmae ocnrl(7.8 control to compared as fstlieclspstv o ygnnol in only percentage myogenin the for higher by positive shown a cells as satellite mice revealed mutant of co-immunostaining in rate myogenin differentiation and MyoD Synm EERHARTICLE RESEARCH ubro a7pstv Px+ aelt el e ir in fibre per higher cells 2.3 significantly satellite versus a (Pax7+) cryosections, Pax7-positive Synm on of fibres isolated observed number freshly as of Immunostaining rates showed, 2004). auto-renewal al., and et differentiation (Zammit detection activation, culture allows cell the single-fibre model satellite of cell of the this pattern factors, colocalisation satellite using and myogenic of h) expression above-mentioned the 72 course on and Based time model. 24 the (0, during differentiation myogenin Pax7, as and such factors MyoD transcription myogenic of profile expression earlier differentiation myogenic to cells. commit wild-type to than tend cells muscle of levels mRNA of PCR regulators. real-time mass by muscle quantification Relative 6. Fig. ecnaeo h elpplto htwsdul-oiiefor the double-positive by was in that a evidenced MyoD population showed as and cell Pax7 Pax7 capacity the and activation of MyoD percentage cell with post-isolation, satellite h similar 24 at fibres, (9.8% rirr units. arbitrary iecus faayi.A 2hps-slto,teaverage the post-isolation, h 72 in At cells satellite analysis. Pax7+ the of of throughout number higher course remained number time average This S3C). Fig. el t4 otioainin post-isolation satellite h 48 myogenin-positive of at detection cells addition, In 8D,F). (Fig. aaaerpre smeans genotype. as in reported decrease are and Data levels. mIGF-1 mRNA and atrogin follisatin and in myostatin increase the Note muscles. obte hrceieti hnmnn eaaye the analysed we phenomenon, this characterise better To 2 2 6 / / 2 2 .)we oprdt oto (1.3% control to compared when 2.5) a P ess87.0% versus P iea oprdt oto (4.7 control to compared as mice 6 , , . ncontrol, in 0.3 .0)(i.8,) oimnsann fsingle of Co-immunostaining 8C,E). (Fig. 0.001) .5between 0.05 Synm A ysai ( Myostatin (A) 6 6 Synm . ncnrl.A 2hpost-isolation, h 72 At control). in 2.9 ...Tnaiaswr sdfreach for used were animals Ten s.e.m. 2 / 6 2 P 2 . in 0.6 , n oto ie(87.0% mice control and Hprt / Synm 2 .0)(upeetr material (supplementary 0.001) vrodadcnrloela.AU, overload. control and overload Synm a sda nitra reference. internal an as used was Gdf 2 Synm / 2 ) B olsai,()atrogin (C) follistatin, (B) 8), Synm 2 / iesget htthis that suggests mice 2 2 / iewsas higher also was mice 2 2 / 2 6 ess3.6 versus 6 os plantaris mouse . in 0.5 .)( 0.8) Synm P 2 Synm , 6 / 6 2 0.001) . in 3.0 . in 0.4 mice 2 / 2 naiainta sntfudi emnkoku ie We mice. desmin-knockout in found not is that irregular invagination have and wrinkled appear which membranes, sarcolemma g-ace oto itrae,w bevdml ucedefects muscle mild in observed we littermates, control age-matched aZrpre eeudrtecnrlo emno vimentin or desmin of control the that under show promoters gene reporter LacZ hs emnkoku iewudas oesnmnatvt in of activity feature synemin particular Indeed, lose One also 2004); 2001). muscle. al., would et mice Xue al., 2001; desmin-knockout al., et et thus, Titeux 2000; of Titeux al., presence et the (Carlsson on 2011; desmin depends tissue muscle al., in synemin of localisation et muscle (Chourbagi skeletal forms mature which fibres synemin, in of desmin This nature with 1996). heteropolymers the al., obligatory et to Milner linked 1997; al., be (Agbulut et might Li more 1996; difference disorganisation al., et Li in sarcomere 2001; al., et results mitochondria extensive of desmin and perturbations of localisation severe of that fibres, than absence muscle severe degenerated where less mice, is however, desmin-null phenotype, myopathic performance.The muscle reduced with associated aging. also with are increases defects These that disorganisation sarcomeric limited integrity a membrane and compromised regeneration, and degeneration uo ta. 94 ie l,1997). al., the et Li (Colucci- and 1994; of development desmin al., embryonic like et for proteins, Guyon development required IF not other are finding fibres), that vimentin, reports This with development. muscle consistent embryonic is subsequent myogenesis or the and system for cardiovascular myoblasts essential of of not organisation fusion is migration, synemin (proliferation, that indicate results hncnrlmc olwn vrod hshge epneto response higher This overload. in stimulation following hypertrophic resistance mice fatigue control higher by a accompanied than and force capacity, maximal hypertrophic isometric increased higher a exhibited mice IFs between protein Our bridging membranes. a be cellular to and proposed been has Synemin DISCUSSION n t bec eut namoahcphenotype myopathic a in Although results integrity absence membrane its sarcolemmal and for necessary is Synemin development embryonic for Synm required not is Synemin el hwahge ee fcmimn omyogenic to commitment of level higher a differentiation. show cells epne u eut teghntehptei htsynemin hypertrophic that this hypothesis in the participates strengthen results also Our post-translational cells of response. behaviour or altered satellite an mass expression that muscle muscle demonstrated in we gene Moreover, implicated control. molecules in signalling of changes modification to linked n euao fsga rndcinptwy,sc sPAand PKA as such protein pathways, signalling. cytoskeletal transduction Akt bridging signal of a regulator both a as and acting functions, dual has nmtnsadfrhrehnetento that notion the enhance further and a mutants showing cells observation in LacZ-positive 8D). previous desmin-promoter-driven the (Fig. of with percentage mice line higher mutant in are in results earlier These occurs process differentiation ebaeitgiyascae iha curneo focal of Surprisingly, occurrence regeneration. an of and with alteration degeneration and associated muscle resistance a to integrity fatigue leads force, inactivation membrane maximal synemin that functions in showed new decrease We demonstrated protein. have this muscle for skeletal in synemin of Synm 2 / 2 ora fCl cec 21)17 5940 doi:10.1242/jcs.143164 4589–4601 127, (2014) Science Cell of Journal 2 / ieaeval n etl,adepeso atrso a of patterns expression and fertile, and viable are mice 2 Synm ie hrceie yteocrec ffclmuscle focal of occurrence the by characterised mice, 2 / 2 iehv iia oywih oprdto compared weight body similar have mice Synm 2 Synm / 2 nvivo in mro eeo omly These normally. develop embryos 2 Synm / 2 netgtosit h roles the into investigations ieapast edirectly be to appears mice 2 / 2 iei h eet in defects the is mice Synm 2 / 2 Synm satellite 4595 2 / 2

Journal of Cell Science u eut niaeta h nraei aia oc n in and force in maximal higher was in overload increase to response the in resistance that and fatigue integrity indicate membrane Surprisingly, results synemin. decreased of our absence as the in such degeneration fibre defects muscle could to findings linked These resistance. be fatigue decreased show and force hsooia conditions, physiological ulfralfrso synemin. of forms all for null EERHARTICLE RESEARCH lct ihrmsl dpiersos nteasneof absence the 4596 in response in response resistance fatigue adaptive in overload increase muscle the thus, Moreover, higher muscle; synemin. control a fibre to and elicits compared mass muscle increase, larger diameter a by accompanied muscle, plantaris and mass muscle between in detected type is fibre difference obvious no Although following overload response mechanical hypertrophic the modulates Synemin more our the is because (Izmiryan isoform integrity synemin with cord which membrane yet for associated spinal know important cannot neurofilaments mouse We 2006). adult al., to et in binds compartment L membrane synemin been 2012), al., has and et It (Lund cardiomyocytes 2010a). between complexes whereas al., junctional cardiomyocytes, et neonate Sun in 2008b; Z-lines al., Sun 2001; et al., that Sun reported et Mizuno 2008a; 2008; al., al., et et Hijikata 2006; al., 1999; al., the et et Bhosle utrophin, (Bellin in dystrophin, talin and as vinculin involved zyxin, such 1, directly its defect. plectin dystrobrevin, structure, of be membrane perturbation might membrane of of that result integrity the related proteins observed be damage, with indirectly and synemin interaction be the of such could absence more the defect develop to to membrane fibres vulnerable to synemin this muscle Interestingly, less whereas time renders muscle contraction, leaving during desmin makes damage of deficiency to lack susceptible that propose b snmnmdae h soito fdsi F with IFs desmin of association the mediates -synemin Synm Synm 2 / 2 2 n oto ieudrnormal under mice control and / 2 iedvlplwrmaximal lower develop mice a snmnstabilises -synemin Synm 2 / 2 Synm oe is model 2 a / 2 - ta. 01,in (Musaro al., mass 2001), et muscle al., of (Sun regulator myostatin et positive a of mIGF1, higher antagonist and and 2010b), an 2011), follistatin, al., et of (Goodman expression genes synthesis myostatin, protein more and in atrogin a MHC-2b- involved of to in expression muscle decrease Lower of a fibres. switch by positive demonstrated a as by phenotype, explained oxidative be could overload to h oeipratices nmsl asin mass muscle in increase important part, more in of least the at expression explain, Increased could molecules overload. signalling to phosphorylated response in muscle plantaris evu ytm ecno xld htsm etrso the of features some the that in expressed exclude also cannot is Synm we synemin system, As nervous mice. control to compared yfbe Dsae,20) KP raiePAadits neonatal and adult and in AKAP an PKA as subcellular identified was specific organise Synemin at locales. complexes AKAPs macromolecular into 2009). substrates skeletal (Dessauer, in AKAPs myofibres muscle by restricted promotes spatially mouse is and signalling during cAMP-PKA CREB, 2005) proteins al., through et Wnt regeneration PKA, (Chen by development CREBs. embryo induced as myogenesis such controls factors proteins, of target and transcription release channels, ion numerous proteins, permits structural enzymes, phosphorylate metabolic subunits including that regulatory subunits PKA PKA. catalytic is to muscle skeletal binding in signalling cAMP cAMP of 2012). effector Stewart, major and The (Berdeaux muscle skeletal of regeneration and inligmlcls(k1 RB,RS n PKARII and RPS6 of CREB1, forms phosphorylated (Akt1, increased and Interestingly, molecules protein mass. total signalling muscle of levels in expression increase the supports ilhl oeaut h pcfccnrbto fec iset the to tissue each of contribution specific gene observed the synemin evaluate the to of help knockout will muscle) or (neural Tissue-specific mlctdi ucems oto eeosre in observed were control mass muscle in implicated APsgaln ly eta oei yetoh,metabolism hypertrophy, in role central a plays signalling cAMP 2 / 2 ora fCl cec 21)17 5940 doi:10.1242/jcs.143164 4589–4601 127, (2014) Science Cell of Journal Synm hntp ol edet hne ntenua input. neural the in changes to due be could phenotype nvivo in 2 / 2 Synm phenotype. fe ct uceijr Seate l,2011). al., et (Stewart injury muscle acute after 2 / 2 lnai ucefloigoverload following muscle plantaris vrodi ohcnrland control both in following overload (S6) S6 protein and ribosomal (CREB) CREB1 p70S6K, (AKT), overload; II subunit regulatory PKA of expression increased the Note (OVL). overload mechanical after and (non-OVL) overload overload. non- control and non-overload as mean reported are loading Data a control. as used was GAPDH protein housekeeping The ERK, ERK1/2. overload. after control versus in S6 protein ribosomal and CREB1 (Thr389), p70S6K PKARII of forms (p-) phosphorylated higher Note mice. Synm in activity protein intracellular signalling in Changes 7. Fig. xrsini lnai ucefrom Synm muscle plantaris protein in detect expression to used was blot Synm between difference significant vrodadnon-overload; and overload 6 2 2 2 / / s.e.m. / 2 2 2 c vrodadcontrol and overload without mice control and os muscles. mouse P , .5between 0.05 a P , .5between 0.05 a Synm a PAI) Akt1 (PKARII), (Ser96), Synm 2 Synm Synm Synm b / Western 2 P 2 , / 2 mice 0.05 2 2 2 a / / / 2 2 2 ` )

Journal of Cell Science rvnLc-oiiemolssprmoei ooyotie rmcnrland control from obtained colony myogenic per myoblasts LacZ-positive driven fPAtre rti RBadRS r lohge in higher also are RPS6 and forms CREB phosphorylated protein the target overload; PKA of to response in muscle control EERHARTICLE RESEARCH ada yctsi hc titrcswt euaoysbnt of PKARII subunits regulatory with interacts PKARII it which in myocytes cardiac in cells satellite muscle of Behaviour 8. Fig. Synm orsodn htmcorps(ih ae) CD htmcorpso sltdfbe bandfo oto and control from obtained fibres isolated of Photomicrographs (C,D) panel). (right photomicrographs corresponding slto D.()Gah hwn h rprino a7ol,Px/yDadMo nystliecl ouain t0 4 n 2h(0,T4 n T72h) and post- T24h myog (T72) (T0h, and h h (MyoD/Myog), 72 72 myogenin and and and (T48) 24, MyoD 48 0, at both at only, (Myog) populations MyoD- myogenin cell experssing and satellite populations MyoD only cell for post-isolation. h MyoD satellite and 72 and (C), of and Pax7/MyoD post-isolation proportion 48 only, (T72) the at Pax7 h showing only of 72 Graphs proportion and (F) the (T24) isolation. showing 24 post Graphs at MyoD (E) and (D). Pax7 isolation for stained and muscles between 2 / 2 Synm a a lnai uce ne aeiecniin nnoela,nnOL n asatroela OL.()Aeaenme % fdesmin-promoter- of (%) number Average (B) (OVL). overload after days 7 and non-OVL) (non-overload, conditions baseline under muscles plantaris in Rsele l,20) h hshrltdfr of form phosphorylated The 2006). al., et (Russell 2 Synm / 2 vrodadcnrloverload; control and overload 2 / 2 ucei xrse oehgl hnin than highly more expressed is muscle a P , .5btenoela n non-overload; and overload between 0.05 Synm d P , 2 .0,frmoei-nystlieclsbtencnrland control between cells satellite myogenin-only for 0.001, / 2 mice. A vrg ubro a7pstv aelt el e 0 ucefbe ncnrland control in fibres muscle 100 per cells satellite Pax7-positive of number Average (A) Synm 2 / 2 Synm b RS/* hr h seikidctstephosphorylated muscle the evidenced in substrates as the PKA phosphorylated activity of PKA level indicates higher (against of the increase by asterisk an PKA confirmed we the by residue), phosphorylated where an Using substrates RRxS*/T*, activity. PKA against in increase antibody possible a indicating muscle, P , .5between 0.05 2 / 2 ent idibmsls(0dy otaa)(etgah n the and graph) (left postnatal) days (10 muscles hindlimb neonate ora fCl cec 21)17 5940 doi:10.1242/jcs.143164 4589–4601 127, (2014) Science Cell of Journal Synm 2 / 2 o-vrodadcnrlnon-overload; control and non-overload Synm Synm 2 2 / 2 / 2 cl a:53 bar: Scale . xesrdgtrmlongus digitorum extensor m m. c P , 4597 0.05 enin

Journal of Cell Science lorsl rmicesdatvto fAtsgaln oeue in molecules signalling muscle Akt Synm of activation and increased could from hypertrophy RPS6 result also and phospho-CREB1 muscle of expression synthesis, Increased an growth. protein to leading ultimately in RPS6, and We increase CREB1 in as muscle. increase such PKA of proteins an phosphorylation skeletal target the in that increases turn, results in in synemin which, speculate activity, of activity PKA absence We the PKA that S2B). restrict hypothesise Fig. could material synemin (supplementary mice esnfrti ifrnei o e la n arnsfurther warrants and clear The yet not cells. is between satellite difference balance muscle this the of for affect reason differentiation might synemin and of is self-renewal hypothesis absence One the culture. cell that primary in markers differentiation uha ucedsrpy ahxa n aging-associated and cachexia, dystrophy, might studies muscle sarcopenia. Future conditions as physiopathological cells. during such synemin satellite of role muscle the explore of affects signalling absence behaviour of its regulator that the and a remodelling, as muscle acts during molecules protein, cytoskeletal a synemin, PKA influence of regulation might the in synemin role its 2011), activity. through al., behaviour cell in et satellite signalling PKA (Stewart of involvement myogenesis the on Based characterisation. bec fepeso hnein change expression of absence dl ri d oz atn ta. 01.W lofudthat Pax7- found of number also The We cells. the 2011). satellite in of al., present et zone is Martins protein subventricular synemin Souza the (de in brain We cells adult stem cells. neural satellite multipotent both of and cells, stem behaviour embryonic that the reported examined previously we overload, oiiestlieclsin cells satellite positive ubro a7pstv aelt el nrae ncnrlmice, control in in the increased not cells overload but satellite muscle Pax7-positive during synemin of However, of number behaviour. influence cell potential satellite the in suggesting muscle, control in study. further requires of ARTICLE RESEARCH 4598 to bred ( synemin 2008) were heterozygous al., of mice et 1 male (Gary-Bobo exon Synemin-floxed mice excise 1A). transgenic (Fig. Cre-expressing 1 female at exon with sites of loxP ends harboured two Clinical mouse the Mouse transgenic of This help France). the (Illkirch, with Institute obtained was mouse synemin-floxed The gene-targeted of Generation METHODS AND MATERIALS increased in role a play in cells observed satellite hypertrophy muscle whether determine To during cells overload satellite mechanical of behaviour affects Synemin rmthe from eeso hshrltdEK/ r nrae nmsl from from muscle not in but increased mice are ERK1/2 control phosphorylated the overload, in of to particularly response levels in transduction, Interestingly, signal signalling. Akt of that and regulator hypothesis PKA a thereby the as support Akt, acts results from synemin Our away activation. 2A Akt favouring type phosphatase helping protein by 2012) sequester al., et (Pitre proliferation cell glioblastoma during eoyig l nmlsuiswr prvdb u ntttoa Ethics institutional our by approved were studies animal All genotyping. eiidb C eoyig eeoyosmc eeitrrdto interbred were mice Heterozygous genotyping. homozygous PCR generate by verified ae oehr h eut bandi hssuyso that show study this in obtained results the together, Taken Synm 2 / 2 2 Synm / ie ned yei ly oei k signalling Akt in role a plays synemin Indeed, mice. 2 Synm iei epnet vrodcmae otecontrol the to compared overload to response in mice Synm 2 2 / 2 / 2 + iehda nacdcpct oepesearly express to capacity enhanced an had mice / 2 ie nadto,w on htstliecells satellite that found we addition, In mice. mice. ) Synm nvivo in Synm b 2 snmni xrse npluripotent in expressed is -synemin / In-vivo 2 Synm Synm 2 ie hc a eiidb PCR by verified was which mice, a n iers ogermline-transmitted to rise give and / and - 2 Synm Synm 2 mdae xiino xn1was 1 exon of excision -mediated 2 ucei lgtyhge than higher slightly is muscle / 2 / 2 b mice ie h esnfrthis for reason The mice. 2 2 snmnaeepesdin expressed are -synemin / / 2 2 ucei epneto response in muscle iei o la and clear not is mice ta. 94 iefrtognrtost bandouble-transgenic obtain to generations two for (desmin-LacZ/ mice 1994) embryos al., (Colucci-Guyon vimentin-LacZ-expressing or et 1997) al., et (Li expressing aia oc a banddrn h eao) oc rdcin in productions Force which tetanos). at frequency (pulse the recorded length successively were during stimulation (muscle tetanic obtained to L0 response was at force made maximal were measurements duration. bipolar isometric ms a 0.1 All of by pulses distally wave previously square stimulated supramaximal using and electrode described silver proximally crushed as was Hourde nerve surgery. 2009; sciatic performed during al., intact were et remained measurements (Agbulut supply nerve force and to Active blood taken was the was care muscle that isometric Great plantaris Canada). ensure muscle Ontario, measures the Scientific, that Aurora system of (300C; arm force tendon lever dual-mode distal a The to clamps. attached with fixed limbs the were and mg/kg), 60 sodium, (pentobarbital anesthetised were Animals measurements force Muscle and Control analysis development Embryonic rnvrefoe etos(10- sections frozen Transverse Immunohistochemistry animals liquid Ten in analysis. point. experimental precooled further each isopentane for for paraformaldehyde used in were in the frozen fixed experiments, or the then nitrogen of weighed, end and were the muscles the At dissected and pentobarbital, determined. of overdose an then with killed was were animals 20% force initial by for taken fall allowed time The to was tetanus). Hz continuous min (submaximal 50 min 1 at stimulated 2 least continuously for was at muscle plantaris and The 5- ms) period. a rest after 500 min determined Hz, then was 143 resistance Fatigue to contraction. each 100 between and 50 25, from ietv 669EC.Oraia aiiyi ul iesdb the by licensed Commission insurance. fully welfare (European animal is has care facility and authorities animal animal competent French Our laws, on European 86/609/EEC). and regulations Directive French the and to directives, according conducted and Committee otiigslto L ta. 1997). al., stained et and (Li development solution during containing points time different LacZ/ srainptenaayi)foe etoswr rprdfor 2001), al., prepared et (Titeux polyclonal, France), rabbit Trappes, were France), (1:300, Dako-Cytomation, synemin Molsheim, monoclonal, Millipore, antibodies mouse sections monoclonal, (1:100, primary desmin rat with (1:400, frozen incubated perlecan were against sections analysis) The immunostaining. pattern (striation eosin and 8- hematoxylin using morphology staining; general examine to muscles the n xrse steaeaenme fPx-oiiestliecells/100 were 2010). satellite al., Pax7-positive section of et genotype) number (Mitchell per per average animals fibres four the myofibres or as three and expressed and and mice; cells per the sections Pax7-positive quantification, (three cell counted of satellite plot number For the software. using total ImageJ sections pattern of muscle The longitudinal function measured. on profile was of analysed section (min-Ferret) was muscle diameter striations whole (Hourde smallest of the The previously of the 2012). fibres described al., muscle using as et all Joanne made macro a 2013a; custom were al., using Zeiss a et analyses and Carl captured Morphometric software 700, France). were ImageJ Pecq, (LSM Images finally Le microscope DAPI. were SAS, laser-scanning slides Saint- with confocal PBS, mowiol motorised (Sigma-Aldrich, in washing using phalloidin After mounted FITC-labelled France). 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Journal of Cell Science esur .W. C. Dessauer, S., B. and Paulsen, J.-C., S. Larcher, N., Pournin, Diguet, O., Agbulut, D., C., S. Paulin, Martins, Souza I., de Dunia, M., M. Portier, E., Colucci-Guyon, Funding and D.P. D.C., A.P., Z.L, experiments. paper. data. the the the performed analyzed wrote O.A. O.A O.A. and and Z.X. Z.X. D.P., A.F., A.F., D.C A.P., J.B., A.P., Z.L, Z.L, J.G., experiments. P.J., the C.H., designed S.A., and conceived D.C., O.A. and Z.X. D.P., A.P., Z.L., contributions Author interests. competing no declare authors The interests Competing ARTICLE RESEARCH 4600 E. Lazarides, and L. You, B. C., H. Granger, Lincoln, L., B. Jacobs, M., D. Mabrey, W., J. Cle Frey, A., C. A., Goodman, C. Franco, B., Escoubet, A., Parlakian, G., Gary-Bobo, and D. Paulin, P., Vicart, Z., Xue, M., Carinci, F., Bruston, O., C.-M. Fan, Chourbagi, and D. D. M., Ginty, R. E., Robson, A. Chen, J., Breckler, G., M. Price, D., Paulin, L., Z. Li, L., M. R. Carlsson, Robson, and Z. Li, P., K. Campbell, E., D. Michele, C., R. Bhosle, R. Stewart, and R. Berdeaux, M. R. Robson, and W. T. Huiatt, W., Ip, B., Becker, W., S. Sernett, Butler- M., F., R. Bellin, Fougerousse, E., Mouisel, C., Hourde, A., Vignaud, O., Agbulut, Pe Z., Li, O., Agbulut, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.143164/-/DC1 online available material Supplementary material Supplementary a by supported Lefoulon-Delalande. was P.J. Fondation EME119]. the number University from the [Emergence fellowship by Curie and Marie 16117]; and & 16605 Pierre 14848, Franc 13607, Association numbers the [contract by (AFM) supported was work This Hourde Usukura, M., Okumura, Y., Nishizawa, Y., Tsujimura, H., Yamakawa, Y., Hirako, R., Ishikawa, K., Yuasa, M., Imamura, K., Isokawa, A., Nakamura, T., Hijikata, xrsino yei sfrsi os mroi tmclsadneural and cells stem embryonic mouse Z. Xue, in and isoforms Z. derivatives. Li, synemin D., of Paulin, V., expression Moura-Neto, K., S. Rehen, phenotype. obvious C. Babinet, 897. egtpoenascae ihdsi n ietnflmnsi muscle. in filaments vimentin and desmin with associated protein weight growth. load-induced mechanical of A. regulation T. Hornberger, and J.-S. al. focal induces et myocardium Z. the failure. in Li, heart gene D., and factor Paulin, response lesions serum D., the Daegelen, of inactivation D., Tuil, P., Bruneval, signaling. cAMP in dimension 941. next the complexes: assembly. filament heteropolymer synemin-desmin O. Agbulut, proteins. Wnt by induced myogenesis controls knock-out CREB desmin and wild-type of muscles skeletal mice. in and plectin and E. dystrophin paranemin, L. Thornell, with and G. Wiche, synemin protein filament intermediate utrophin. of Interactions regeneration. and metabolism, hypertrophy, filament synemin-containing intermediate anchor may the heterofilaments. alpha-actinin of with Interactions interactions synemin. and protein characteristics Molecular (1999). activity. muscle of absence A. Ferry, and S. G. Browne, structure. synaptic in 49 modifications and regeneration G. Butler-Browne, outr hsclatvt rtcsfo ucpiiiyt kltlmuscle mice. mdx skeletal in to function al. susceptibility heart et worsens from O. Pathol. but protects Agbulut, injury G., activity contraction-induced co- Butler-Browne, and physical cells: S., proteins Voluntary neuroglial Hatem, filament some A., intermediate Pannerec, and III cells protein type muscle O. filament with of Ohara, interaction keratins. classes intermediate and and four an K. localization all Owaribe, synemin, in W., mammalian present K. of Jackson, Characterization H., Matsumoto, J., alpha-dystrobrevin beta-synemin, actin. through H. and sarcolemma Yorifuji, costameric and to S. filaments Takeda, K., Kohama, 727-738. 51-66. , ,C,Jan,P,Mda . ognt . aqe,A,Musl E., Mouisel, A., Jacquet, N., Mougenot, F., Medja, P., Joanne, C., ´, itce.Cl Biol. Cell Histochem. 182 elTsu Res. Tissue Cell ice.Bohs e.Commun. Res. Biophys. Biochem. .Cl Sci. Cell J. 1509-1518. , M elBiol. Cell BMC 21) emnmttosi h emnlcnessmtfprevent motif consensus terminal the in mutations Desmin (2011). 19) ielcigvmni eeo n erdc ihu an without reproduce and develop vimentin lacking Mice (1994). .Bo.Chem. Biol. J. 20) dnllcyclase—A- Adenylyl (2009). Cell ´rie 20) ako emnrslsi briemuscle abortive in results desmin of Lack (2001). 121 ,S,Ldsy .A,Pui,D,Crad .and J. Cartaud, D., Paulin, A., M. Ludosky, S., ´, 79 u.J er Fail. Heart J. Eur. 2062-2074. , 313 679-694. , 21) APsgaigi kltlmsl adaptation: muscle skeletal in signaling cAMP (2012). 12 m .Physiol. J. Am. 114 20) lwmoi ev hi xrsini the in expression chain heavy myosin Slow (2009). 21) h oeo kltlmsl TRi the in mTOR muscle skeletal of role The (2011). 51. , 195-207. , 20) ifrne ntedsrbto fsynemin, of distribution the in Differences (2000). 39-47. , 274 29493-29499. , 18) yei:anwhg molecular high new a Synemin: (1980). 20) rti iaeAsgaln via signalling A kinase Protein (2005). m .Physiol. J. Am. 20) lci ik intermediate links 1 Plectin (2008). 296 10 346 635-645. , .Physiol. J. C205-C214. , ¸ 768-777. , Myopathies les contre aise iaeacoigprotein anchoring kinase x.Cl Res. Cell Exp. o.Pharmacol. Mol. Nature elMtl Cytoskeleton Motil. Cell 303 589 ,E1-E17. 433 5485-5501. , 21) Dynamic (2011). 20) Mosaic (2008). 317-322. , mn,S., ´ment, 317 76 (2013a). Cell (2006). (2003). m J. Am. 886- , 935- , 22 , Musaro Y. Capetanaki, and A. Bradley, D., Tran, G., Weitzer, J., D. Milner, ote,E,Rs,M,Svn,E,Hles .D,Hrmn,A,Nai A., Naami, A., Hartmann, R.-D., Hilgers, E., Sevinc, M., Rose, E., Noetzel, Imamura, M., Brosius, G., H. Lidov, R., J. Guyon, G., T. Thompson, Y., Mizuno, Panne J., K. Mitchell, and J. R. Bloch, A., M. Russell, Y., Zhang, J., Lupinetti, P., J. Kerr, M., L. Lund, twr,R,Fehe,L,Mnmn,M n edax R. Berdeaux, and M. Montminy, L., Flechner, R., Stewart, cmt-ref . ig . ishe . Desmoulie R., Nitschke, R., Jing, A., Bond, Schmitt-Graeff, and N. Cianciola, K., McKeegan, R., Haber, M., L. Lund, A., M. Russell, O. Skalli, and W. A. and Orr, O. M., Paul, Skalli, N., Davis, R., A., Matej, Pitre, A., M. Curtis, U., Wilhelmsson, M., Faiz, T., Pekny, L., Carlsson, G., Butler-Browne, O., Agbulut, M., Mericskay, Z., Li, Pinc E., Colucci-Guyon, Z., Li, Scime V., Seale, E., A. Jones, F., Grand, Le Hourde hnmra,L,L,Z,Pui,D n u,Z. O. Agbulut, and Xue, G. Butler-Browne, and P., Noirez, T., Launay, D. Paulin, Z., Li, L., Khanamiryan, Hourde O., Chourbagi, P., Joanne, Hourde P., Joanne, O. Skalli, and M. Pekny, Y., Pan, L., Li, W., Goodwill, O. U., Wilhelmsson, Skalli, R., Jing, and G. Gabbiani, M., G. R. Z. Robson, Xue, G., and Pizzolato, L. Z. R., Li, Jing, D., Paulin, T., Z. Federici, Xue, E., and Peltekian, A., Z. Izmiryan, Li, D., Paulin, A., C. T., Franco, Federici, A., F., Izmiryan, J. Leterrier, L., Khanamiryan, Y., Cheraud, A., Izmiryan, aaie,E. Lazarides, irpino uceacietr n ycrildgnrto nmc lacking mice in degeneration myocardial and desmin. architecture muscle of Disruption rngn xrsinssan yetoh n eeeaini senescent in Knu regeneration and N. hypertrophy Rosenthal, muscle. and sustains skeletal L. H. expression Sweeney, transgene R., E. Barton, M. M., L. Kunkel, desmin. USA and Sci. and -dystrobrevin Acad. alpha C. Natl. with Proc. interacts S. that protein Watkins, filament intermediate E., Ozawa, M., development. postnatal during Biol. progenitor Cell resident muscle A. cell D. non-satellite Sassoon, and G. Marazzi, cardiomyocytes. neonatal in filaments desmin M. Bond, 129-144. 20) yei xrsini iepedi ie irssadi nue in induced is and fibrosis cells. liver epithelial in biliary malignant widespread and is proliferating expression Synemin (2006). Biophys. Biochem. Arch. M. PP2A. antagonizing by Cell proliferation cell glioblastoma AKT-dependent disease. Alexander in fibers M. Pekny, with associated is gene Synemin relapse. the tumor of early methylation promoter aberrant cancer: myogenic for not but myofibrils of muscle. integrity skeletal and of D. fusion strength Paulin, and differentiation, and tensile commitment, C. the Babinet, for E., L. Thornell, symmetric C. the desmin. lacking Babinet, drive mice in and to myopathy D. pathway Paulin, polarity cell cells. stem planar satellite of the expansion activates Wnt7a space. aaiyadfaiiyi h ytohcmxmouse. mdx dystrophic the in force-generating muscle fragility on and factors capacity gender-related female of effect Protective (2013b). ciae ymsl nuyadpooe uceregeneration. muscle promotes and injury e24714. muscle by activated noptneo yei srltdt h rpryo t edadrod and dependent always head not activity. is phosphatase its calcineurin regeneration and muscle innervation of muscle during on chain property heavy myosin the slow to related is synemin domains. of myopathy.incompetence myofibrillar of models mouse create to mutants O. Agbulut, muscle. and J. skeletal Dumonceaux, al. dystrophic et in L. overloading Garcia, mechanical ONE L., PLoS to Arandel, response W., and adaptive Hadj-Said, neurotrauma E., Mouisel, in networks. astrocytes filament intermediate reactive GFAP Sci. and Cell in J. vimentin expressed with and differentially is interacts reactive Synemin astrocytoma human (2007). in in membranes ruffled present with is associates cells. synemin and astrocytes protein malignant filament central human Intermediate from cells neuronal and astroglial system. in nervous and isoforms vascular Synemin (2010). in partners of multiplicity G. systems. development: neuronal Z. mouse nervous during Xue, during isoforms neurons and and Z. glia Li, in isoforms D., development. synemin system Paulin, of V., expression Different Moura-Neto, E., Peltekian, R1508-R1514. 20) h nemdaeflmn rti,snmn sa KPi h heart. the in AKAP an is synemin, protein, filament intermediate The (2006). hl .adDh,E. Dahl, and R. chel, ¨ ´,C.,Joanne,P.,Noirez,P.,Agbulut,O.,Butler-Browne,G.andFerry,A. 23 . culg,K,Pu,A,Huho,L,Dboon,G,Molinaro, G., Dobrowolny, L., Houghton, A., Paul, K., McCullagh, A., , ` Glia 1243-1253. , ora fCl cec 21)17 5940 doi:10.1242/jcs.143164 4589–4601 127, (2014) Science Cell of Journal Nature .Cl Biol. Cell J. 12 Biochemistry 50 21) yei sfrsdfeetal raiecl ucin and junctions cell organize differentially isoforms Synemin (2012). 21) yei sepesdi ecieatoye n Rosenthal and astrocytes reactive in expressed is Synemin (2014). 120 7 257-266. , 18) nemdaeflmnsa ehnclitgaoso cellular of integrators mechanical as filaments Intermediate (1980). 107-120. , e35346. , 283 1267-1277. , a.Genet. Nat. erce.Res. Neurochem. ,C,Ohl,J,Caude J., Ochala, C., ´, rc . ao,B,Prain . esn . oe,E R., E. Gomes, V., Besson, A., Parlakian, B., Cadot, A., ´rec, 249-255. , x.Cl Res. Cell Exp. Oncogene 134 Glia 47 1255-1270. , 456 9531-9539. , 54 21) nemdaeflmn yaisadbreast and dynamics filament Intermediate (2010). 98 APMIS 27 204-215. , 204-213. , o-amn,M,Mrcky . ori,S., Pournin, M., Mericskay, M., ¸on-Raymond, 6156-6161. , 195-200. , 29 19) adoaclrlsosadskeletal and lesions Cardiovascular (1996). ,C,Fry . ulrBon,G,Vcr,P., Vicart, G., Butler-Browne, A., Ferry, C., ´, 21) dniiainadcaatrzto fa of characterization and Identification (2010). elSe Cell Stem Cell 315 4814-4825. , e.Biol. Dev. 21) ia-eitdepeso fdesmin of expression Viral-mediated (2013). 35 122 769-783. , 881-887. , 76-80. , rn . ej,F,Vgad A., Vignaud, F., Medja, Y., ´ran, ,A n unci .A. M. Rudnicki, and A. `, 175 AE J. FASEB uceNerve Muscle 4 362-366. , 19) emni essential is Desmin (1997). u.Pathol. Hum. 535-547. , 21) yei promotes Synemin (2012). r,A n kli O. Skalli, and A. `re, 20) Self-assembly (2008). 20) emsi,an Desmuslin, (2001). 20) oaie Igf-1 Localized (2001). 26 20) xrsinof Expression (2006). klt Muscle Skelet. m .Physiol. J. Am. 137-148. , .Cl Biol. Cell J. 21) RBis CREB (2011). 20) Synemin (2009). 21) Impaired (2012). 48 37 68-75. , LSONE PLoS 1200-1210. , o.Biol. Mol. (1996). (2009). (2005). (2006). 139 3 290 Nat. ,4. 6 , , ,

Journal of Cell Science u,Y,G,Y,Dnvc,J,Za,Y,Bn,M n hn J. Chen, and M. Band, Y., Zhao, J., Drnevich, Y., Ge, M. R. Y., M. Robson, Sun, and R. Z. Li, Robson, D., and Paulin, W., Z. T. Huiatt, Li, N., D., Sun, Paulin, R., D. Critchley, N., M. R. Sun, Robson, and Z. Li, D., Paulin, R., D. Critchley, N., Sun, ARTICLE RESEARCH ak . iex . alt . i . amsDpr,C,Cvlat,L A., L. Cavalcante, C., Daumas-Duport, Z., Li, C., Fallet, M., Titeux, M., Tawk, omladptooia ua eiaadlens. and retina V. human pathological Moura-Neto, and normal and skeletal D. in Paulin, follistatin and miRNA-1 regulates rapamycin myogenesis. of target Mammalian and adhesion cell for essential is and that zyxin protein migration. alpha-synemin domain LIM the mammalian with interacts within domain talin. repeated with directly a interacts of Identification metavinculin. and 657-667. vinculin with directly interacts alpha-synemin x.Cl Res. Cell Exp. .Cl Biol. Cell J. 189 316 x.Cl Res. Cell Exp. 1157-1169. , 491-505. , 20) yei xrsini developing in expression Synemin (2003). 314 1839-1849. , x.Neurol. Exp. 21a.Synemin (2010a). 183 ice.J. Biochem. 20a.Human (2008a). 499-507. , (2010b). (2008b). 409 , amt .S,Glig .P,Ngt,Y,Hdn . atig,T .and A. T. Partridge, V., Hudon, Y., Nagata, P., J. Golding, S., P. Zammit, and D. Paulin, M., Titeux, A., Izmiryan, V., Brocheriou, Y., Cheraud, G., Z. Xue, H., D. Adams, H., Reynaert, Y., Hirako, E., Rossen, Van L., Zhao, N., Uyama, iex . rceiu . u,Z,Go . elsir .F,Gihny P., Guicheney, F., J. Pellissier, J., Gao, Z., Xue, V., Brocheriou, M., Titeux, ehns o self-renewal? for R. mechanism J. Beauchamp, frames. reading open Res. different Cell and usage Exp. exon alternative by generated proteins Z. Li, adhesions. al. focal et to M. filaments intermediate Pekny, bridging R., Gut protein Robson, a synemin, Z., express Li, Z., Xue, 6435-6449. noigtodsic nemdaeflmn proteins. filament intermediate distinct two Z. Li, encoding and D. Paulin, 55 20) h os yei eeecdstreitreit filament intermediate three encodes gene synemin mouse The (2004). 1276-1289. , ora fCl cec 21)17 5940 doi:10.1242/jcs.143164 4589–4601 127, (2014) Science Cell of Journal 298 431-444. , 20) ucestlieclsaotdvretfts a fates: divergent adopt cells satellite Muscle (2004). 20) ua yei eegnrtsslc variants splice generates gene synemin Human (2001). .Cl Biol. Cell J. 166 347-357. , 20) eai tlaecells stellate Hepatic (2006). u.J Biochem. J. Eur. 4601 268 ,

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