novo hnd .Nou oetKat ailC cnr,CrlC rgro n alA Krieg A. Paul and Gregorio* C. Carol Schnurr, C. Daniel Kraft, Robert Nworu, U. Chinedu during functions myofibrillogenesis overlapping skeletal have 4 and 3 Leiomodin ARTICLE RESEARCH ß eevd7Mrh21;Acpe oebr2014 November 4 Accepted 2014; March 7 Received ([email protected]) correspondence for 85724, *Author AZ Tucson, St, in Mabel E. muscle, 1656 Arizona, USA. of Cardiovascular striated Molecular University Sarver Program, Medicine, In Research Molecular and Cellular 2012). of Department recent al., a in et (for neurons Yamashiro distinct prominently striated and see cells with including expressed fiber review, lens cells, is erythrocytes, differentiated family muscle, Tmod1 terminally Tmod postmitotic, related patterns. four expression most are vertebrates, the In the (Tmod1). is there of tropomodulin1 length One filament capping investigation. thin filament of active lengths regulators of contractile studied thin area uniform generating intensively an and actin precisely remains turnover force which maintain dynamic al., undergoing by and et while Gokhin mechanisms assemble 2009; al., the filaments al., et et Ottenheijm Understanding Witt 2006; 1995; 2009). al., al., et et cellular Gregorio Bang and 1991; 2006; al., generation et force muscle (Granzier contractile striated function efficient in for length essential filament thin is actin of regulation Precise INTRODUCTION Skeletal Sarcomere, Tropomodulin Leiomodin, muscle, filament, thin Actin WORDS: KEY of value the demonstrate studies Xenopus these Furthermore, assembly. structurally ends, are pointed filament (Lmod1–3) actin to but localize leiomodins also and The Tmod1–4 to related ends. at depolymerization filament filament filament and thin elongation thin thin regulating to The by contributes uniformity optimal (Tmod) contraction. length for tropomodulin muscle essential protein is during capping length generation filament thin force of regulation Precise ABSTRACT prpit eeso ohTo4adLo3aerqie for that required time are Lmod3 first and the Tmod4 for both locomotion of demonstrate embryonic levels Tmod4, results and appropriate additional rescued These with was improved. supplemented assembly Lmod3- were sarcomere and Lmod3, embryos additional deficient with supplemented were and embryos Tmod4-deficient assembly when and Remarkably, sarcomere movement. of embryonic Tmod4 disruption impaired severe of in resulted roles Lmod3 the or examined during we Lmod3 Here, nucleators. robust ucinrdnatyduring can redundantly proteins function both unexpectedly, and, myofibrillogenesis embryonic 05 ulse yTeCmayo ilgssLd|Junlo elSine(05 2,2920doi:10.1242/jcs.152702 239–250 128, (2015) Science Cell of Journal | Ltd Biologists of Company The by Published 2015. nvitro in yfbi assembly. myofibril o h nlsso otatl rti ucinduring function protein contractile of analysis the for iceia tde niaeta md c nta as instead act Lmods that indicate studies biochemical Xenopus kltlmoirloeei.Ls fTmod4 of Loss myofibrillogenesis. skeletal nvivo in kltlmsl hnfilament thin muscle skeletal de ur ta. 03.Mueebysnl o md i at die Tmod1 for null embryos 1998; Mouse al., 2003). et abnormally Sussman al., to 1995; leads et al., et Tmod1 Mudry (Gregorio of 2011). Littlefield filaments depletion 1999; thin al., whereas al., long et et 2001), (Sussman al., Tsukada filaments in et actin 2006; results of cultures Greenfield Kedes, myocyte shortening primary a 2006; and in al., Tmod1 Kong of et 2003; Overexpression 2005; al., Kostyukova et al., 2005; (Fowler et functions al., these in et affect mutations Kostyukova regions that capping these given of actin-binding and domains, any two localization on -binding dependent two The be thin and to 1994). actin appear the Tmod1 al., of of the functions end et limits pointed (Weber and the at to filaments monomers binds actin of Tmod1 exchange tropomyosin, with cooperation speeta h -iergo ftesroeei cardiac development, in embryo sarcomere chick Skwarek- the During 2010; 2010). of al., al., et region Tsukada et 2008; Maruszewska Miano, M-line al., et and the (Chereau Nanda myofibrils at Lmod2 2001; that present indicate al., studies is overexpression et and cardiac (Conley Antibody both 2012). in muscle present skeletal are adult smooth-muscle- and Lmod3 and be and distinct to Lmod2 Lmod2 exhibit reported whereas specific, Lmod1 Lmod1, proteins with vertebrate proteins, distributions, tissue three of These family Lmod3. (Lmod) and leiomodin (Gokhin 2010). matures al., muscle et skeletal Gokhin filament as 2011; Tmod1 Fowler, thin ends of for correct absence compensating pointed structurally the maintaining filament organization, translocates sarcomere thus thin and In length Tmod4, Tmod3 cap 2011). with to muscles, together Fowler, sites Tmod1-null extrasarcomeric and adult from (Gokhin in reticulum addition, sarcoplasmic is is the Tmod3 cytoplasmic Tmod3 muscle, stabilize whereas skeletal to murine 2001), thought In al., ubiquitous. et be to Conley considered 1996; al., et (Watakabe (Gokhin T- 2011). Z-line or Fowler, the reticulum and at sarcomeric also compartment the is membrane either with tubule-associated Tmod4 with correlated 1999b). associate fast-twitch al., to be in et reported predominating to (Almenar-Queralt Tmod4 muscle appear with skeletal but speed, directly have twitch muscle muscle measured skeletal mammalian been in relative Tmod4 The not 2010). and al., Tmod1 1999a; et of to al., Gokhin ratios equivalent et 1999b; (Almenar-Queralt role al., muscle a et cardiac play Almenar-Queralt in to Tmod1 believed of is that it and where muscle, assembly skeletal 2010). the al., normal et be muscle, (Gokhin to embryos appears skeletal Tmod1-null myofibrils Although in 2008). muscle in al., skeletal et of expressed McKeown maintenance 2003; is (Fritz-Six al., defects et Tmod1 Chu cardiac 2003; to al., owing et (E)9.5–10.5 day embryonic ahrls skonaotteepeso n ucino the of function and expression the about known is less Rather tissue neuronal adult in primarily expressed is Tmod2 mammalian in isoform Tmod predominant the is Tmod4 c atn(omsl)flmnsin filaments (nonmuscle) -actin 239

Journal of Cell Science r losbtnildfeecs md aktesecond C- the extended the an lack within possess domain but Lmods actin-binding Tmods (third) differences. in additional is present substantial there domain Tmods, major which tropomyosin-binding the also to the contractions, similarities Tmod1, structural are Although some cardiac 2010). of share al., Lmods et expression (Tsukada the of heart in the expressed heart start isoform than Tmod the in later the expressed significantly be after to Lmod2 shortly revealed studies transcript ARTICLE RESEARCH 240 actin in the of Lmod2 elongation of 2010). in overexpression results al., that cardiomyocytes et discovered rat (Tsukada was cultured Lmods it the Finally, of properties biochemical protein capping a as deleted, function vitro is can Tsukada Lmod2 Lmod 1994; of truncated extension al., the C-terminal however, et the assays, (Weber When 2010). biochemical property al., this in et exhibit filaments to the fail limit actin Lmods to (cap) ability to of the to have function polymerization Tmods contrast whereas Lmods In Furthermore, that 2008). 2001). formation filament indicate al., actin nucleate et studies robustly Conley biochemical 1A; Tmods, (Fig. region terminal ugsigta h -emnletninmdltsthe modulates extension C-terminal the that suggesting , nvitro in Ceeue al., et (Chereau in pcfccnrciecmoet during components of properties assembly. functional contractile the study sarcomere the to specific system that during powerful demonstrate a is functions studies for embryo our redundant required Moreover, are largely assembly. Lmod3 share and proteins in other Tmod4 development muscle both skeletal each can that normal of show for Tmod4 results levels our sufficient and together, compensate Taken Lmod3 myofibrillogenesis. physiologically during that and assembly. determined sarcomere structurally compromises we severely Unexpectedly, Lmod3 either or of levels Tmod4 protein the Reducing myofibrillogenesis. precedes in muscle skeletal the developing in expressed a isoforms predominant that and regulation. Tmod1, length for required of filament is thin activities appears antagonist protein correct it Lmod an and observations, Tmod as filament between these balance the functions on of Lmod2 Based end 2010). that the al., at et Tmod1 (Tsukada of displacement and filament h eut rsne eeso htTo4adLo3aethe are Lmod3 and Tmod4 that show here presented results The Xenopus eooi Lo)adtoooui To)gn families. (Tmod) the tropomodulin of and expression (Lmod) developmental leiomodin and Structure 1. Fig. xrse thg eesi eeoigseea muscle. skeletal developing in levels high at skeletal Only expressed developing tissues. indicate heart Arrows and embryo. muscle the in marker tissue muscle muscle striated definitive the actin, supplementary of cardiac in Expression presented S1. is Fig. (st15) material expression stage Earlier neurula (st30), myofibrillogenesis. the stage of during tailbud stages early early left. the the the at during to shown heads are with patterns positioned Expression are embryos All hybridization. expression gene during family analyzed (F–I) domain Tmod was C- binding/WH2 and a (C–E) actin Lmod have third (B–I) proteins a (WH2/A3). Lmod contains Tmods. that the extension to terminal unique is tropomyosin-binding (TM2) second domain A and domains. (TM1) (LRR) tropomyosin-binding leucine-rich-repeat A2), Tmods and (A1, Lmods actin-binding domains. share unique are possess proteins yet (Tmod1–4) related, tropomodulin structurally and (Lmod1–3) leiomodin (A) ora fCl cec 21)18 3–5 doi:10.1242/jcs.152702 239–250 128, (2015) Science Cell of Journal mroadta xrsino ohtranscripts both of expression that and embryo actna1 Xenopus B,wsue oidct ada n skeletal and cardiac indicate to used was (B), mroi eeomn using development embryonic lmod3 Xenopus E and (E) enovo de tmod4 n httetwo the that and , I are (I) myofibril Xenopus nsitu in

Journal of Cell Science n mdpoen,sc steasneo h second the of 2001). the C-terminal patterns al., expression the et specific of (Conley in proteins (A3) Lmod absence presence in domain only the the actin-binding found and extension additional as Lmod an from such of (TM2) site proteins, A2). tropomyosin-binding 1A), Lmod Tmod and Fig. the between (A1 2001; differences and significant al., domains also are et actin-binding there However, (Conley conserved proteins has two of including others families Tmod by the Lmod between and work similarity structural Previous significant unambiguous the recognized . allow mammalian organization orthologous the of syntenic identification and conservation sn nomto rmDAsqec aaae,w have of (Lmod1– we Lmod families and databases, (Tmod1–4) Tmod sequence the 3) of DNA genes all from identified information Using all assembly during myofibril expressed of are stages transcripts Lmod3 and Tmod4 RESULTS ARTICLE RESEARCH oetal rc mut ntehatadwal ndeveloping in weakly whereas and muscle), heart (skeletal the that somites in amounts showed muscle trace striated potentially 1C–E) of of (Fig. marker Examination specific patterns 1984). highly al., a actin, et cardiac (Mohun is of which pattern the 1B), to (Fig. compared were These S1). the nteeryhat ncontrast, In heart. early the in nseea ucebtwsasn rmtehat(i.1) o the For 1E). (Fig. heart the from absent tmod was but muscle skeletal in yusing By Xenopus eefml,epeso of expression family, gene nsitu in mro Fg CI upeetr aeilFig. material supplementary 1C–I; (Fig. embryos eou laevis Xenopus Xenopus yrdzto,w eemndtetissue- the determined we hybridization, lmod3 mdadLo ee ihthe with genes Lmod and Tmod tmod1 tmod ohpiaysequence primary Both . lmod2 a xrse eystrongly very expressed was lmod1 lmod1–3 and a iil nteearly the in visible was a xrse weakly expressed was lmod a xrse at expressed was eeexpression gene eefmle in families gene actna1 hnflmnso qa rdfeetlntsta r o aligned), marker not Z-disc are prominent that the lengths (i.e. for different staining actin or and equal filamentous of visible. nonstriated time-point filaments not thin earliest detected were the sarcomeres staining first organized At Phalloidin (st)20], 2A–L). myofibrillogenesis [stage (Fig. which analyzed Tmod4 at of time localization which within the stages, developmental on occurs 2). three (Fig. centered at development were muscle performed skeletal was early Analysis during Tmod4 Lmod3 skeletal of localization and the subcellular and of expression model, the experimental assembly examined the we characterize initial better the To sarcomere. during muscle proteins Tmod4 of Lmod3 roles the and understanding towards directed are studies region Our M-line the in assemble during and localize Lmod3 and Tmod4 sntognzd(opr i.2,) fetvl identical Effectively 2C,K). Fig. (compare organized localization its Scho stage, not this and at is expressed Sparrow to is well-characterized Tmod4 see Although identical other 2010). reviews, are in (for observations myofibrillogenesis organisms These of puncta. descriptions dot-like isolated er n ekyvsbei kltlmsl,weestestaining the whereas muscle, skeletal for in pattern visible weakly and heart ihlvl ndvlpn kltlmsl ise,weesthe subsequent levels. whereas undetectable or tissues, low the at muscle expressed are skeletal members for family developing other in Importantly levels report, high 1I). this (Fig. in developing experiments the muscle in not for but muscle cardiac Transcripts skeletal in staining. abundant highly were background non-specific with enovo de r rsn tteMln eino h acmr rmteearliest 5 the bar: from Scale sarcomere assembly. the myofibril of of region proteins stages M-line Lmod3 the and regions, at M-line Tmod4 present and both are Z-line that the conclude at We respectively. detected was arrows) (W, Lmod3 the for throughout Staining broad visible somites. st34, were developing At filaments (U–X) actin visible. striated were sharply arrows) (S, staining In of Lmod3 (Q). bands filaments narrow developing actin regions, of striated these regions diffusely some narrow st24, showed At muscle (Q–T) of observed. localization was no (O) but filament visible, actin was continuous staining st20, muscle At skeletal (M–P) (M–X) during myofibrillogenesis. respectively. protein regions, Lmod3 M-line of and Localization Z-disc the at Sharp visible tissue. muscle skeletal of filaments the regions actin throughout organized) observed highly were At (i.e. (I) (I–L) striated visible. sharply were broad arrows) st34, (G, of staining filaments bands Tmod4 actin narrow and of regions, arrowheads) number striated small these a In in (E). visible were periodic striations no diffuse with (A), visible of was localization st20, staining At filament (A–D) actin myofibrillogenesis. continuous muscle skeletal during protein antibodies and against phalloidin Texas-Red-conjugated using examined during M-line Xenopus the to localize Lmod3 Xenopus and Tmod4 2. Fig. ora fCl cec 21)18 3–5 doi:10.1242/jcs.152702 239–250 128, (2015) Science Cell of Journal tmod2 a atnn md rLo3 AL oaiaino Tmod4 of Localization (A–L) Lmod3. or Tmod4 -, mro eescindadpoendsrbto was distribution protein and sectioned were embryos myofibrillogenesis kltlmyofibrillogenesis. skeletal Xenopus a and atnn(,arwed)adTo4sann K were (K) staining Tmod4 and arrowheads) (J, -actinin a atnn()o md tiig() EH tst24, At (E–H) (C). staining Tmod4 or (B) -actinin tmod3 oie.Frt eaaye the analyzed we First, somites. tmod4 a eeal ifs n consistent and diffuse generally was a and a atnn(,arwed)and arrowheads) (R, -actinin atnn(,arwed)and arrowheads) (V, -actinin lmod3 h oiergosof regions somite The a m atnn()o Lmod3 or (N) -actinin m. a r xrse at expressed are atnndetected -actinin a c 09 Ono 2009; ¨ck atnn(F, -actinin tmod4 241

Journal of Cell Science ta. 97 eevsbewt hlodnand phalloidin with visible Holtzer 1994; were al., to 1997) et referred al., Rhee (often et structures’; sarcomeres ‘I-Z-I-like or nascent ‘Z-bodies’ some as stage, this At 1988). eino idtp mro yti tg Fg 2I–L,U–X). (Fig. stage trunk by this marked the Z-discs by well-organized throughout Tmod4-knockdown exhibited embryos because embryos present wild-type st34 Control and are at of out sarcomeres region 3C–F) carried (Fig. ordered somite were of Assays highly control sections embryos. filament, frozen 3G–J) thin (Fig. on from Z-disc, proteins of tissue localization and the filament examine thick to used microscopy Deconvolution remarkably was 3A). showed (Fig. ( embryos appearance treated external dramatically the normal 3B), reduced or demonstrated (Fig. were treatment blots microscopy MO levels western Although immunofluorescence Tmod4 analysis. that for blot western prepared when stage, for tailbud were the until develop they to allowed were MO control during expression Tmod4 early reduce specifically to (MO) morpholino acmr omto in somite examined formation with next sarcomere stage, We aggregates 2O). same (Fig. distributed tissue organization the randomly clear at as no with Lmod3 appearing for staining Lmod3 made were observations ARTICLE RESEARCH sebe nTo4dpee mro,ee tteearliest 242 the at shown). not even (data embryos, development never of sarcomeres Tmod4-depleted stages that in revealed were assembled experiments studies knockdown timecourse These maintenance), might in performed. Tmod4 myofibril that for the normally (i.e. required address disrupted be formed subsequently To were but sarcomeres 3L,P,M,Q). embryos Fig. that staining (compare and phalloidin titin possibility perturbed of whereas distribution also The striations, was pattern. regular irregular no in showed or embryos myofibrils Tmod4-knockdown of puncta staining Specifically, from disruption somites 3G). Tmod4 complete the (Fig. in and near observed contrast, was filaments In thin 3C). (Fig. well-organized actinin, sarcomere during Tmod4 of role the about known assembly is assembly sarcomere Little prevents Tmod4 of Depletion organized containing of regions exhibited The tissue that arrowheads). regions 2F, myogenic same (Fig. staining the phalloidin in striated visible were striations spaced most Although 2E). (Fig. yfbilgnssadaelclzdi ls rxmt othe to visible. are proximity sarcomeres close as in early in as localized present M-line are are we and proteins observations, myofibrillogenesis Lmod3 Tmod4 M- these and the on Both Tmod4 Xenopus at Based both observed 2I–L). that sarcomeres. were (Fig. conclude mature 2W,X) (Fig. of somites were Lmod3 lines and myofibrils the 2K,L) organized of (Fig. throughout highly muscle embryos, skeletal visible these the doublet In a in detected. as assembly in (st32), than embryos sarcomere tailbud rather Finally, present sarcomere. examined band, the of single length we sides both a from sarcomere molecules as representing observed the was for ends At observed that nascent 2S,T). developing at the to (Fig. staining Lmod3 identical of of M-lines striations nearly region narrow pattern clear was M-line the with Tmod4, again, staining the Once Lmod3 at arrows). of 2G,H, Tmod4 (Fig. of sarcomere nascent presence the showed Xenopus , or ae,a h ieo h aletdetectable earliest the of time the at later, hours 5 nvivo in ygnctsu tteeris tgsof stages earliest the at tissue myogenic Xenopus eeomn.Ebysijce with injected Embryos development. oadesti su,w sda antisense an used we issue, this address To . uce tiiga hnflmn pointed filament thin at staining muscle, , Xenopus 5husatrtefrtsroee were sarcomeres first the after hours 15 a a atnnwsognzda isolated as organized was -actinin atnnapae ucae regularly punctate, appeared -actinin s2,HagadHockaday, and Huang (st24, a atnnadF-actin and -actinin a atnnstaining -actinin . tmod4 0)b the by 90%) Oor MO a - raiaino ii n ysnwsas disordered were also filaments was thin was ( regions staining organized some phalloidin myosin in The with areas, observed and most sarcomeres 4H,I). (Fig. in nonstriated, titin Although, filaments) 4P,Q). clear (Fig. actin of no sarcomeres (i.e. of nonstriated organization organization was organized staining observed 3H,I), repeating Lmod3 phalloidin (Fig. of previously and As depletion of punctate absence 4C–R). of Tmod4 (Fig. depleted for controls complete Staining with embryos on compared a 4A,B). tailbud-stage (Fig. effect revealed detectable from development little somites embryonic having while overall muscle embryonic is,w eosrtdta h diino sequence-specific a of addition the development. the examining muscle that lmod3 experiments skeletal demonstrated of embryonic we during First, series Lmod3 similar of a role out carried assembly sarcomere We prevents Lmod3 of Depletion o h bec fTo4 eused We all Tmod4. of levels compensate of the to examine we absence effort the 3B), an (Fig. in for upregulation embryos other transcriptional MO-treated that undergo possibility in the levels considered protein Tmod4 eetbyatrdi epneto studies response in for These altered expression detectably (st34). gene examined that at was stage showed developmental structure same sarcomere the at which performed was assay The S2). and Tbe1 upeetr aeilFg 3–) odrcl test was directly To myofibrils S3A–D). studies rescue the Fig. the No material for of supplementary used S3A–D). GFP–Tmod4 1; structure of (Table doses Fig. or the at material dimensions observed endogenous in supplementary of location alteration 3T; at the (Fig. position from appropriate Tmod4 the indistinguishable to we M-line, localized control, the it preliminary that embryos demonstrated a untreated and into As GFP–Tmod4 we encoding treatment, experiments. mRNA MO introduced of rescue effect out non-specific a carried not was be and Tmod4 of should Lmod experiments endogenous of subsequent proteins. Tmod expression and in compensatory of observed independent results the vrl yfbi sebya sesdb bevto of observation by assessed as assembly restored myofibril effectively the GFP–Tmod4 overall of addition with resulting that the showed together of somites Examination 3S–V). embryo Fig. material the (supplementary into structure myofibril GFP–Tmod4 rescue could Tmod4 in of addition the whether 25 ess59) ae oehr hs tde demonstrate studies muscle. for these essential is together, Tmod4 Taken that 3Aa, 5.9%). we (Fig. versus embryos phalloidin Tmod4-depleted 62.5% in MO-treated the gaps with M-line experiments, of of compared the sarcomere presence staining, loss the organized to by in assessed multiple the as increase GFP–Tmod4 structure, significant that for of a produced conclude From addition responsible embryos we that is studies, assembly. determined rescue Tmod4 In the of 1). sarcomere of (Table ablation success embryos specific the unmanipulated of of were 3S–Z). view embryos those lengths (Fig. Tmod4-rescued to in filament organization comparable lengths thin sarcomere myosin that and (TFLs) showed and quantification titin Additionally, F-actin, actinin, lhuhwsenbossoe eyefcierdcinof reduction effective very showed blots western Although ocnimta h ako acmr tutr a u oloss to due was structure sarcomere of lack the that confirm To tmod4 tmod4 Oefcieydpee md xrsin( expression Lmod3 depleted effectively MO ora fCl cec 21)18 3–5 doi:10.1242/jcs.152702 239–250 128, (2015) Science Cell of Journal Otetdebys eitoue RAencoding mRNA introduced we embryos, MO-treated Otetdebys(upeetr aeilFig. material (supplementary embryos MO-treated tmod enovo de , 4) o ecesuis efirst we studies, rescue For 24%). and tmod4 tmod lmod yfbi sebyi skeletal in assembly myofibril a tmod atnnlclzto was localization -actinin Otetet Therefore, treatment. 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Journal of Cell Science EERHARTICLE RESEARCH ora fCl cec 21)18 3–5 doi:10.1242/jcs.152702 239–250 128, (2015) Science Cell of Journal O eutdi eceo acmr tutr.Dt hwthe show Data structure. sarcomere of the rescue with in together resulted GFP–Tmod4, MO, controls). encoding in mRNA 62.5% of to Addition compared staining phalloidin following 5 same the bar: at Scale presented scale. are controls (C–Z) images unmanipulated fluorescent of All that (C–F). proteins to sarcomeric comparable other was and (X,Y) (S–V) of filament Organization thin GFP–Tmod4. both encoding mRNA with embryo In experiments. studies, Tmod4 rescue these the by of demonstrated specificity was that The knockdown with (S–Aa) compared (K–N). heavy disorganized controls myosin severely of and also (P) was titin (Q) of chain region components, N-terminal sarcomeric the other including of stage Localization developmental (O–R) same (C–F). the with at compared embryos sarcomeres control Tmod4 disrupted of severely depleted show embryos protein of through tissue in Sections muscle resulted (G–J) developing protein assembly. Tmod4 sarcomere of of Loss disruption (C–R) protein. Tmod4 of the that and Tmod4) specifically antibody anti-Tmod recognizes the that demonstrate blots myofibril and muscle skeletal for essential assembly. is Tmod4 3. Fig. mean eutdi rmtcrdcini h ecnaeo correctly of percentage ( the sarcomeres embryos in st34 structured reduction in dramatic protein a Tmod4 in of resulted depletion that showed (Aa) tmod4 6 ... *** s.e.m.; A mro rae ihcnrlM uprpanel) (upper MO control with treated Embryos (A) O(oe ae)dvlpnral.()Protein (B) normally. develop panel) (lower MO Xenopus tmod4 P , m tmod4 .Qatfcto feprmna results experimental of Quantification m. .0 ( 0.001 md rti ln aee GFP– labeled (lane protein Tmod4 Owsc-netdit the into co-injected was MO , .%o iessoigMln gaps M-line showing fibers of 5.4% Oefcieybokdtranslation blocked effectively MO x 2 analysis). Xenopus 243

Journal of Cell Science al .Ti iaetadsroeelnt analysis length sarcomere and filament Thin 1. Table ARTICLE RESEARCH atr ihdsic -iegp Fg E.A expected, As was 5E). protein 244 (Fig. Tmod4 gaps endogenous depletion, M-line Tmod4 staining distinct organized) following (i.e. with with when striated Tmod4- supplemented regular structure pattern embryos a Although 5A), filament showed (Fig. thin Myc–Lmod3 structure. phalloidin a little with sarcomere observed showed stained embryos we embryos of Tmod4-depleted depleted Myc–Lmod3, When rescue in additional formation. striking examined of with sarcomere amount was supplemented total was during the muscle protein present raise skeletal to Lmod3 protein was Myc–Lmod3 endogenous Lmod3 adding of of analysis result level by blot altered the Western Tmod4-depleted not Myc–Lmod3. that in of we formation showed expression idea, sarcomere this by during test rescue muscle, To properties to muscle. functional embryonic attempted share in are might assembly members myofibril Tmod4 proteins whether family determine Lmod3 to filament sought Tmod and we thin issue, To and this Introduction). the explore (see Lmod further distinct of biologically the cell and end that biochemically pointed suggest the the date within all positions near However, 2K,W). Furthermore, identical or (Fig. or 2012). at similar al., at sarcomere, et Tmod4 located and are Yamashiro Lmod3 the proteins 2001; see that al., suggests review, et staining (Conley immunofluorescent a and family Tmod for protein structural larger places and several single analysis a share phylogenetic within families and Lmod 1A) protein (Fig. Lmod domains and Tmod The during equivalence assembly functional sarcomere of exhibit Lmod3 stages and initial Tmod4 muscle. the skeletal during embryonic demonstrate role in results essential assembly These an myofibril 1). plays to (Table Lmod3 comparable embryos were that control that embryos of lengths Lmod3-rescued sarcomere those that and revealed TFLs lengths exhibited filament with thin MO- treated organized of embryos multiple in to of increase that from Myc–Lmod3 lmod3 with significant compared a of Data structure in sarcomere addition resulted depletion. embryos was that the Lmod3 treated structure showed of sarcomere to success experiments (sarcomere) of specifically The absence filament due that 4S–Z). (Fig. demonstrates thin Myc–Lmod3 rescue of of actin disruption expression that by and showed caused we organization assembly not Second, did 1). material myofibril Table alone (supplementary S3E–H; structure Myc–Lmod3 or Fig. dimensions of myofibril expression alter that demonstrated hnflmn n acmr iesoswr qiaett otosfraltetet xetfrteGPTo4rsu fLo3M ru.TL hnfil thin TFL, group. MO Lmod3 of Mean rescue GFP–Tmod4 the for except Student’s treatments A all length. for controls sarcomere to SL, equivalent length; were dimensions sarcomere and filament Thin rescue+ Myc–Lmod3 GFP–Tmod4+ GFP–Tmod4+ Experiment y–md 0.85 0.85 0.83 Myc–Lmod3+ Myc–Lmod3 GFP–Tmod4 Control Oaoe(i.4;7.%vru 36) Quantification 23.6%). versus 70.2% 4I; (Fig. alone MO lmod3 lmod3 tmod4 tmod4 O0.83 MO O0.74 tmod4 MO O0.84 MO Otetet(aantson,s the so shown), not (data treatment MO O0.86 MO lmod3 nvitro in Owsefcetyrsudby rescued efficiently was MO n elclueeprmnsto experiments culture cell and t ts a sdfrsaitclaayi (* analysis statistical for used was -test F SL TFL 6 6 6 6 6 6 6 6 .407–.62.05 1.99 2.08 2.01 0.73–0.96 0.65–0.86 0.74–0.94 0.72–0.89 0.04 0.04*** 0.05 0.04 .307–.82.05 2.07 2.04 0.75–0.88 0.77–0.91 0.80–0.88 0.03 0.03 0.01 ..( s.d. m )Mnmx( Min–max m) fLo3wr nbet ecemoirloeei nembryos in myofibrillogenesis rescue to unable were versions truncated Lmod3 observation, S3E–H), of M-line this with material the Consistent (supplementary into Fig. S4A–H). embryos Fig. normal assemble in to sarcomeres material failed of region Lmod3 Unlike of (supplementary versions S4). truncated muscle Lmod3 Fig. skeletal material regulate full-length (supplementary and assembly extension, myofibrils C-terminal sarcomere whether actin- into the tested incorporate lacking additional We Lmod3, could 1A). an of (Fig. with versions proteins truncated Lmod extension in C-terminal domain binding a of presence Lmod3. in deficient filament severely These embryos skeletal thin in 2001). of myofibrils assembly al., the muscle in for et sufficient is Littlefield results Tmod4 that 1999; indicate results Tmod1 al., GFP–Tmod4, et of excess (Sussman of overexpression shortening presence the that to due given likely most was TFL 2.04 length, egh 1.99 length, md-eltdebyswr prcal hre hntoeof Tmod4-rescued those for than ( shorter lengths controls appreciably sarcomere were embryos and compared structureLmod3-depleted TFLs 68%, sarcomere that to with 24% observed treated in embryos from of gaps) increase that M-line with an distinct by in (assayed resulted 5R,S). of (Fig. addition M-line that GFP–Tmod4 GFP– evident showed the samples was muscle. experimental of of multiple structure of region Analysis filament presence the thin skeletal at organized the Lmod3, when rescue endogenous Lmod3-depleted revealed not to but staining Tmod4, sufficient in triple was described Furthermore, GFP–Tmod4 structure knockdown Tmod4 of of sarcomere addition rescue the 5Q–X). Lmod3 (Fig. above, the GFP–Tmod4 of to addition Similar by 5M–P) (Fig. to depletion sufficient is Tmod4. the Lmod3 in of muscle, absence that skeletal controls near embryonic indicate of in assembly those results myofibril facilitate from These different 1). statistically (Table not Myc–Lmod3-rescued were in lengths embryos filament actin and sarcomere that diino y–md eut na nraei ci thin of actin that with in compared increase 27%, with to an treated 4% samples in from results organization filament Myc–Lmod3 staining, phalloidin of by overall of an visualized addition on structure analysis Based sarcomere 5Y. from of Fig. analysis Results in summarized 5I–L). are (Fig. experiments four Myc–Lmod3 following restored of also other Myc–Lmod3 was of addition Assembly myosin but 5F). including 5G), (Fig. proteins (Fig. location sarcomere this region at observed M-line was the protein in detectable not P m , h ao ifrnebtenTo n mdpoen sthe is proteins Lmod and Tmod between difference major The enx eemndwehri a osbet eceLmod3 rescue to possible was it whether determined next We )Mean m) .5 *** 0.05; ora fCl cec 21)18 3–5 doi:10.1242/jcs.152702 239–250 128, (2015) Science Cell of Journal lmod3 P 6 6 , .5 al ;mean 1; Table 0.05; 0.001). .8 esscnrl F,0.85 TFL, control: versus 0.08; OGPTo4 F,0.74 TFL, MO+GFP–Tmod4: 6 6 6 6 6 6 6 6 0 .721 56 44 37 26 1.87–2.19 1.77–2.24 1.83–2.47 1.85–2.20 .07 0 0.08* 0.13 0.09 .619–.623 62 31 1.94–2.16 1.84–2.21 1.87–2.13 0.06 0.08 0.05 ..( s.d. tmod4 m )Mnmx( Min–max m) Oaoe esrmnsshowed Measurements alone. MO 6 lmod3 ...Teosre eraein decrease observed The s.d.). Oaoe(i.5) We 5Z). (Fig. alone MO m m) 6 6 .1 sarcomere 0.01; .4 sarcomere 0.04; n ament

Journal of Cell Science EERHARTICLE RESEARCH httetuctdvrin fLo3wr xrse at expressed were Lmod3 of versions confirmed S4U–Zb). S4Ac) truncated Fig. Fig. the material material that (supplementary (supplementary blots Tmod4 Western of depleted qiaetlvl otefl-eghLo3poenthat protein Lmod3 (supplementary full-length structure S4Q–T). the Fig. sarcomere material rescued to successfully levels equivalent ora fCl cec 21)18 3–5 doi:10.1242/jcs.152702 239–250 128, (2015) Science Cell of Journal hwtemean the show the with together Myc–Lmod3 lmod3 encoding mRNA of addition The oto Otetdcmae o2.%in 23.6% to in compared (70.2% MO-treated the sarcomeres control in structured reduction correctly in dramatic of protein a percentage Lmod3 in of resulted depletion embryos that st34 showed (Aa) results 5 of bar: of Scale that (C–F). to controls comparable unmanipulated other was and (X,Y) (S–V) proteins filament sarcomeric thin of Organization Lmod3. the into was injected knockdown experiments. Lmod3 rescue the by of demonstrated controls specificity of The that (S–Aa) with (K–N). compared was disorganized (Q) severely myosin also and other (P) of titin including localization components, same The sarcomeric the (O–R) of (C–F). embryos stage control developmental of that structure with sarcomere compared Lmod3 disrupted of severely depleted show embryos protein in through tissue in Sections muscle resulted (G–J) developing protein assembly. Lmod3 sarcomere of of Loss disruption (C–R) protein. Lmod3 of the that and Lmod3) specifically antibody recognizes blots anti-Lmod Protein the (B) that normally. demonstrate and develop panel) panel) (upper (lower MO assembly.MO control myofibril with skeletal treated for Embryos essential (A) is Lmod3 4. Fig. Orsoe acmr tutr ocnrllvl.Data levels. control to structure sarcomere restored MO Xenopus 6 Xenopus ... *** s.e.m.; lmod3 md rti ln aee Myc– labeled (lane protein Lmod3 mrowt RAecdn Myc– encoding mRNA with embryo P Oefcieybokdtranslation blocked effectively MO , .0 ( 0.001 x lmod3 2 analysis). lmod3 m .Quantification m. Owsco- was MO MO-treated). lmod3 245

Journal of Cell Science EERHARTICLE RESEARCH mro rae ihM ln Fg ) wn othe the to of rescue of Owing that 246 distribution predicted 6). we and (Fig. embryo, of live levels the alone in the that protein MO controlling rescuing with of with compared and complexity treated locomotion that Lmod3, displayed embryos revealed improved Tmod4, mostly mostly assay significantly external expressing an expressing This embryos to Lmod3-depleted response forceps). embryos their for with embryos Tmod4-depleted 0–5 34 of (touching Stage scale stimulus a 2010). qualitative on al., a scored et were using (Sadikot matrix assessments index-scoring subcellular (motility) the locomotion conducted we at function, embryo recovery physiological improved structural to translated partial level whether assess To Tmod4 of rescue Lmod3 upon and improved is locomotion Embryo opnaesrcual o ahohrbtta hyapa to appear they that can functions. However, but redundant Lmod3 relevant other and physiologically achieve. each Tmod4 possess locomotion for that to only structurally embryo not compensate difficult in indicating detected, improvements be were significant would statistically locomotion full uceasml.Ntol osteeby omfunctional form embryo the does only Not assembly. muscle during powerful a proteins regulatory family Lmod filament and Tmod actin the of functions of the explore to stages sought early We from M-line the myofibrillogenesis at of coexist Lmod3 and Tmod4 DISCUSSION yfbilgnss u eut niaethat indicate results Our myofibrillogenesis. ora fCl cec 21)18 3–5 doi:10.1242/jcs.152702 239–250 128, (2015) Science Cell of Journal nvivo in acmr tutr oprdwt htosre nLmod3- in of observed rescue that dramatic with a compared in structure resulted sarcomere Lmod3 to of GFP–Tmod4 depleted of embryos addition embryos Similarly, Tmod4-depleted increase). of (5.6-fold normal that displaying with sarcomeres compared significant of structure a percentage in the resulted in Tmod4 increase Myc–Lmod3 of of depleted addition embryos the to that showed results 5 bar: experimental myosin Scale restored. of also localization was other the (W) of by structure assayed The proteins, (S). sarcomeric whereas detected (R,V), not M-line was the Lmod3 organization, at striated abundant mature was (M– showing alone GFP–Tmod4 regions Lmod3 in of that depleted Note P). embryos of that rescued with significantly compared was of assembly depleted sarcomere embryos to Lmod3, added was GFP–Tmod4 When (Q–X). restored. Xenopus also studies, was these (K) In myosin (M–X) proteins, of sarcomeric localization other by of not assayed structure was The Tmod4 (G). whereas (F,J), detected M-line in was the that Myc–Lmod3 at Note organization, abundant (A–D). striated alone clear Tmod4 showing of regions depleted embryos with of compared that Tmod4, rescued significantly of was depleted assembly embryos sarcomere to When added (E–L) was Myc–Lmod3. Myc–Lmod3 encoding mRNA with along embryo studies, assembly. these equivalent myofibril largely skeletal display during Tmod4 functions and Lmod3 5. Fig. eltdmsl 29fl) aaso h mean the show *** Data (2.9-fold). muscle depleted P , .0 ( 0.001 eeomna oe o h td fskeletal of study the for model developmental mroaogwt RAecdn GFP–Tmod4 encoding mRNA with along embryo x 2 tmod4 analysis). Owsc-netdit the into co-injected was MO lmod3 Owsc-netdit the into co-injected was MO m .(,)Qatfcto of Quantification (Y,Z) m. Xenopus enovo de mro are embryos 6 AL In (A–L) s.e.m.; Xenopus skeletal

Journal of Cell Science rvossuysoigta md s-md soitdwith associated with (sk-Tmod) consistent Tmod4 is that observation showing This detected study 2). previous (Fig. a were pattern distributed were punctate Lmod3 myofibrils a and in Tmod4 assembly, immature sarcomere to Prior and 2A– (Fig. embryos thin , st20 were in but we structure studies, D,M–P), sarcomere previous detect somites these to st24 with unable the Consistent of tissue 1975). muscle (Muntz skeletal developing in detected 2010). (Tsukada al., et expression Skwarek-Maruszewska 2010; Tmod1 Lmod2 al., where et precedes myocytes, cardiac substantially and in expression Tmod4 observed relevant that that most is from myofibrillogenesis different skeletal observation the early during are The coexpressed are isoforms Lmod3 assembly. these myofibril that during suggesting muscle skeletal 1), in levels (Fig. high at expressed were Lmod3 and Tmod4 elongation an developing and protein). nucleator capping a filament as functions actin Tmod1 robust whereas factor, a (e.g. molecules is the of Lmod2 properties functional localizations, and different patterns demonstrated assembly have members family Tmod EERHARTICLE RESEARCH h rsneo H-otiigCtria xeso nLmods. addition, in extension and In C-terminal Tmods WH2-containing embryonic a in of the domain presence tropomyosin-binding including the the second significant proteins, a two the of the of on presence of structure based assembly and domain unexpected in structural was differences initial similar result share This during Lmod3 sarcomere. roles and regulatory Tmod4 functional and that components time first protein myofibril a crucial building in involved pathways the of facilitates elucidation This development. its during manipulations level protein in muscle skeletal embryo *** in means; increase the significant show a Data GFP–Tmod4 in motility. or resulted rescue) rescue) (homologous (heterologous However, protein protein Lmod3 embryos. Myc–Lmod3 of control either depleted with of Embryos compared addition (B) movement motility. little embryo very in showed increase resulted significant rescue) GFP– a (heterologous either in Myc–Lmod3 little of or very addition rescue) showed However, (homologous 0–5 Tmod4 embryos. Tmod4 of of control scale depleted with index Embryos compared qualitative (A) motility a 2010). using al., locomotion et for function. (Sadikot st34 muscle at embryonic assayed for were assays Locomotion 6. Fig. md,1;and 13; Tmod4, 40; 12; control, MO+Myc–Lmod3, follows: as are assayed or ae ns2 mro HagadHcaa,1988). Hockaday, and (Huang embryos st24 in later hours 5 During the in analyzed was genes Lmod and Tmod all of Expression Xenopus Xenopus nvitro in tmod4 , tmod4 OMcLo3 32. MO+Myc–Lmod3, mro hs xeiet hwdta only that showed experiments These embryo. as u loi seteeycmlatto compliant extremely is it also but days, 3 eeomn,sroeesrcuei first is structure sarcomere development, n elcluesuiso te mdand Lmod other of studies culture cell and OGPTo4 24; MO+GFP–Tmod4, P , lmod3 .0 ( 0.001 O 30; MO, x 2 nlss.Eby numbers Embryo analysis). nvivo in tmod4 lmod3 efudfrthe for found We . O 35; MO, MO+GFP– Embryos lmod3 md n md oaiesmlaeul tteatnthin actin the at that simultaneously suggest localize observations ends Lmod3 our pointed Although filament and 2010). thin Tmod4 from al., Tmod1 et of (Tsukada displacement primary resulted the Lmod2 chick of in in expression experiments high-level where on did myocytes, based cardiac unexpected nor was Lmod3, This endogenous S3A– H). Fig. displace material (supplementary Tmod4 to Tmod4 displace Lmod3 epitope-tagged appear tagged studies, not At our Tmod4. did in and or protein Lmod3 used patterns for levels localization assembly expression of the differential more timing observe the be in not differences to did appeared We al., which 2010). et (Skwarek-Maruszewska bands, Tmod1 than M-lines broader from mature separated with two associated as be to myofibrils myocytes. found only cardiac was chick Lmod2 cultured Specifically, in myofibrils and within assembly differently the during roles striated studies myofibrils. have embryonic of localization proteins maintenance both during These that unknown. Lmod3 suggest was of development this Until Tmod4 localization muscle region. both M-line and the the embryo, in study, the later persisted of localization trunk At Lmod3 the and of 1999a). muscle throughout skeletal al., present the were chick myofibrils et mature cultured stages, (Almenar-Queralt developmental in myotubes myofibrils skeletal immature nonstriated developing the 2007; in Kostyukova, development muscle 2006; skeletal Xenopus early are al., During that et 2008). interactions Kostyukova, reported (Kostyukova been specific Tmod–tropomyosin has isoform it with and Lmod although of ends, Lmod–tropomyosin association pointed the on filament published actin date, been To model have 2006). its al., reports through a et no (Kostyukova molecules sites to tropomyosin binding two tropomyosin According two with filament interacting filaments. thin by caps thin Tmod ends colleagues, to and different localize Kostyukova they by or proposed whether clear same not still the is it ends, pointed filament rtisepesdwr o ufcett rmt hne nTFL in Tmod changes and promote vivo to Lmod in not sufficient tagged not additional do were myofibril of expressed Lmod3 S3A–H; levels skeletal proteins the early and that during Fig. Tmod4 or specification assembly that length material in either roles suggests in (supplementary have This alterations observed 1). no Table embryos, were normal TFL otherwise in overexpressed ends. of pointed filament association thin preferential specific influence with Lmods could or filaments Tmods thin composition tropomyosin the the that of possible is it Hence, 1999). al., et bevdi kltlmsl scnitn ihtepreviously the Tmod1- with of hearts consistent the in is structure we sarcomere that of muscle disruption knockdown reported skeletal Tmod4 in in of compromised effect observed resulted severely The is Tmod4 with formation. It of myofibrillogenesis sarcomere in Knockdown 3B). defects (Fig. TFLs. global more major controls altering much is simply of depletion Tmod4 than those of result the to that intriguing relative embryos treated inhibit the that to indicated blots tmod4 MOs Protein proteins. translation-blocking respective the this specific of synthesis address used To Tmod4 development. we of muscle question, roles skeletal precise during the Lmod3 concerning and known is little Relatively development muscle locomotion skeletal and for essential is Tmod4 ro tde aerpre htLo2adTo1localize Tmod1 and Lmod2 that reported have studies Prior hnadtoa F–md rMcLo3was Myc–Lmod3 or GFP–Tmod4 additional When a 2, Owsvr fetv nrdcn md rti eesin levels protein Tmod4 reducing in effective very was MO . a eea sfrso rpmoi r xrse,including expressed, are tropomyosin of isoforms several , 7, ora fCl cec 21)18 3–5 doi:10.1242/jcs.152702 239–250 128, (2015) Science Cell of Journal b 4and b sfrs(ad n Thie and (Hardy isoforms 5 ´ ad 92 Hardy 1992; baud, 247

Journal of Cell Science hntp.Orrslsso,frtefrttm,ta md is Tmod4 skeletal that embryonic time, in loss-of-function first assembly muscle. sarcomere the the successful for for of show, crucial specificity results swim Our the to phenotype. embryo demonstrating the of ability 6), the (Fig. partially, and, myofibril 3) and (Fig. organization assembly filament thin restored embryos treated ein,sroeesrcuewsasn.W r nbeto incomplete unable to due are assembly sarcomere was some We whether structure or protein Tmod4 partial absent. of knockdown of the majority was whether the structure determine in but sarcomere recognized, regions, be could Chu structure sarcomere 2003; al., 2005). al., et et (Fritz-Six derived Ono 2003; cells cardiomyocytes al., stem Tmod1-null et embryonic in murine and from embryos E9.5 null ARTICLE RESEARCH 248 the with two [approximately domains functional consistent family same larger the a of is many of most share members model that (Lmod are Lmods total second the and Tmods of This that observation case, levels protein. appropriate Tmod) this the plus be In might factor myofibrillogenesis. important similar broadly during perform might Tmod4 functions the and two In Lmod3 myofibrillogenesis. model, the with second consistent of parameters ratio within regulate TFL defined to required be during might a functions overlapping roles no example, unique with proteins play For proteins formation. Tmod4 and sarcomere the Lmod3 first, raise the during models. In different Tmod4 studies broadly two and propose These Lmod3 can We of assembly. sarcomere versa. functions the respective the the vice that of rescue observe question partially and to to embryos able surprising was MO-treated was Lmod3 excess it of factor) addition elongation nucleator a as an acts Lmod2 and and protein capping a as properties acts biochemical Tmod1 different (e.g. possess Lmod and Tmod Tmod4 Because and Lmod3 between functional redundancies reveal experiments rescue Alternate of those with compared Homologous-rescue thin organization 6). organization, (Fig. and lmod3 myofibril structure tadpole 2008). restored filament the al., effectively of et compromisedexperiments severely locomotion (Chereau also the levels cardiomyocytes Lmod3 knockdown reduced cultured Lmod2 Interestingly, the in developing to the similar studies are throughout results disorganization These musculature. myofibril severe with of treated sarcomeres, Embryos of TFL. formation simply Again, overall not myofibrillogenesis. the skeletal affected knockdown during Lmod3 function has essential Lmod3 whether determine an to studies equivalent out carried We and development muscle motility skeletal tadpole for essential is Lmod3 and Tmod total of isoforms levels assay. of low all time the very recognize at showed protein Lmod that 4B), Fig. antibodies 3B; polyclonal (Fig. using no (supplementary western blots and treatment staining First, immunofluorescent MO Second, S2). to reasons. Fig. response material two in observed family for was Lmod genes and unlikely Tmod of levels expression is in alteration detectable members family other by Compensation protein. Tmod Tmod4 of absence the in possible is eosrt,frtefrttm,ta h ucino nLmod studies an These of function 6). the myofibrillogenesis. (Fig. muscle that skeletal for time, essential activity is first member family the swimming for demonstrate, their the rescued to mRNA otipraty xrsino F–md in GFP–Tmod4 of expression importantly, Most partial embryo, the of regions muscle developing some In Otetdebys(al ) h diinof addition The 1). (Table embryos MO-treated lmod3 Otetdebysas significantly also embryos MO-treated lmod3 Osoe regions showed MO tmod4 lmod3 tmod4 MO- h eaieaonso md n md rtisduring proteins Tmod4 experimental and of different the Lmod3 during levels and of myofibrillogenesis the amounts normal relative at the protein studies. compete full-length these successfully in endogenous to used overexpression unable the is Lmod3 with of version of truncated levels assembly sarcomere significant of expression time that the in and/or at Given present protein. are myofibril Lmod3 truncated endogenous the the skeletal protein into of Lmod levels assembly endogenous between Lmod of for levels differences different the competing muscle, cardiac for the structural and considered including muscle examined, be results, Fig. might normal observed isoforms material reasons in of (supplementary of difference Tmod4 number context or of A the S4A–H), Lmod3 S4U–Ab). depleted Fig. of in embryos material in versions either (supplementary assembly to truncated sarcomeres, unable sarcomere of were into we incorporation proteins actin However, C-terminal any shorten 2010). the al., and observe et lacking myofibrils (Tsukada into protein, filaments incorporate Lmod2 could of extension, version 1A; (Fig. truncated Tmod of that resembles 2001)]. al., sequence et Lmod Conley the of thirds h er.Teadto fLo2poendrn ae tgsof stages later during protein in Lmod2 assembly of myofibril addition for The sufficient beating the heart. is that the is alone implies Tmod1 strongly heart observation of until the This presence tissue 2010). and al., cardiac occurred et chick (Tsukada has developing myofibrillogenesis for in transcripts present after that not This shown and are have differences. in Lmod2 Lmod studies formation the Previous myofibril the of muscle. than studies cardiac between important with consistent more is structure interpretation are case, protein families this In in Tmod by present. are similarities model protein this either the of extend provided amounts occur studies sufficient can GFP–Tmod4. that formation our tune sarcomere Tmod filaments, fine efficient additional that to thin and suggesting competition of of Lmod in length work that form the and shown properties the have distinct possess studies in the Tmod4. previous increasing Tmod4, plus by Although Lmod3 rescued of be of could levels total depletion crucial Lmod3 Similarly, the Lmod3 to provided situation, sufficient this severely In effectively was structure. and was Myc–Lmod3 assembly sarcomere assembly of rescue addition sarcomere However, models but disrupted. two unchanged, Lmod3 reduced, the were specifically between was levels distinguish Tmod4 When to above. us presented allow studies unique rescue a inadequate for is formation. Tmod4 required plus sarcomere Lmod3 efficient is of for amount Lmod3 total the Either structure plus or applies sarcomere function, disrupted. Lmod3 and situation reduced severely of equivalent are is An levels amount protein sufficient. total Lmod3 longer when the no a because is has Tmod4 or is Tmod4 because function that either protein unique compatible formation, not concentration sarcomere normal are levels efficient Tmod4 at a these with However, present shown). at remains When not (data protein levels present Lmod3 formation. clearly reduced, specifically myofibril are present. at they were proteins facilitates two structures the time, of sarcomere stoichiometry the this any know not as do microscopy M- we early the Although at as fluorescent detected were region proteins using Tmod4 line and Lmod3 showed both 2), we (Fig. As manipulations. nodrt nepe u eut,i ih eueu oconsider to useful be might it results, our interpret to order In a that indicated culture in myocytes cardiac of Studies ebleeta h eut fhmlgu essheterologous versus homologous of results the that believe We Xenopus ora fCl cec 21)18 3–5 doi:10.1242/jcs.152702 239–250 128, (2015) Science Cell of Journal Fg ) h otlkl xlnto sta the that is explanation likely most the 2), (Fig.

Journal of Cell Science fMcLo3o F–md RAwsijce,tgte ihthe with together above. injected, described was doses mRNA pg MO 900 GFP–Tmod4 studies, or rescue Myc–Lmod3 Tmod4 and and of Lmod3 Myc–Lmod3 For encoding injected. mRNA was of GFP–Tmod4 pg 300 studies, localization protein for ng 37.5 at injected were GTAAGACATGTTGG-3 eeooosrsu xeiet,telcmto fthe many be of both can regulated of presence there In locomotion increased an Although level. that predict the restored. we physiological interpretations, partially experiments, was the tadpoles at rescue activity heterologous functional and al., of et lengthening (Tsukada proceeds observed development the 2010). as filaments with thin consistent cardiac is cardiogenesis ARTICLE RESEARCH hlodn(:0;Ivtoe)wsue osanFatn h secondary The F-actin. stain to used was Invitrogen) Texas-Red-conjugated a (1:400; Proteintech). and phalloidin (1:300; (1:500) antibody anti-Tmod anti-Lmod3 (1:300; rabbit rabbit anti-Myc 9E10), clone rabbit Upstate (1:300; Biotechnology/Millipore; CA), anti-Myc monoclonal Carlsbad, primary Biotechnology/Millipore), monoclonal Upstate Invitrogen, The UK), (1:300; Cambridge, anti- 2013). Abcam, anti-GFP al., (1:300; monoclonal anti-GFP immunofluorescent et rabbit a Aldrich), and (Nworu included previously cryosectioning antibodies described for as prepared staining were Embryos microscopy Immunofluorescence clone anti- MA; monoclonal Billerica, IL), and Biotechnology/Millipore, antibodies. anti-Tmod MO) 9E10) Upstate monoclonal Chicago, Louis, rabbit (1:1000, MA), St Proteintech, Myc Dedham, Sigma, using Covance, (1:30,000; (1:1000; 2013) anti-GAPDH (1:3000; al., anti-Lmod3 anti-GFP as monoclonal et performed rabbit stains were (Bliss S 20 blots (1:1000), Ponceau Western previously from and loading. assays protein (Bio- described protein gels acid total equivalent polyacrylamide Bicinchoninic verified Briefly, 10% CA). pre-cast Hercules, 2011). using Rad, al., separated was et embryos (Vaughan previously Xenopus analysis blot Western mismatch a used we experiments, control MO For ng). 50 (total MO2 TTTATTGGCAGGTTCCGCA-3 CTTGGTCGGAACCCTGGGACATTAT-3 0.2 in recover to allowed one-cell-stage into microinjected were Xenopus mRNAs and (MOs) Morpholinos Microinjections (Ambion/Life CA). kit GFP–Tmod4 mMACHINE Carlsbad, and mMESSAGE or Technologies T7 Myc–Lmod3 Myc the using encoding N-terminal prepared an mRNAs were Lmod3 containing Full-length vectors of tag. using pT7TS GFP amplified into regions inserted were coding and (NM_001016737) PCR truncated Tmod4 or and (NM_001079212) al., full-length RNA et synthesis, generate (Bowes to mRNA template Xenbase for a as using suitable used probes confirmed and linearized was were Plasmids genes 2008). of 7850238. orthology Lmod3, 7733245; 7689916; The Scientific, Lmod2, Tmod3, 7866366; (Thermo 7606904; Lmod1, 3396511; Tmod2, Scientific Tmod4, 6874337; Thermo Tmod1, PA): from Pittsburgh, obtained were containing sequences plasmids following constructions 1–4 The tropomodulin and 1–3 Leiomodin METHODS AND MATERIALS to interactions acto-myosin swimming. sufficient efficient allow maintains filaments thin n rcse ts3 tibdsae.Frkokonsuis eused we studies, knockdown For stage). (tailbud st34 at processed and md n md rnlto-lcigatsneMs( MOs antisense translation-blocking Tmod4 and Lmod3 u tde lomk ikbtenmoirlorganization myofibril between link a make also studies Our O(5 MO mro nuae n0.2 in incubated embryos yaepeaainwsaatdfo ehddescribed method a from adapted was preparation lysate 9 -TCTCCGATCATCGCATGAGCCATTG-3 nsitu in 6 9 eeTos L,Pioah R.MOs OR). Philomath, LLC, Tools, Gene ; yrdzto nlss(aln,19) For 1991). (Harland, analysis hybridization M/%Fcl,taserdt 0.2 to transferred Ficoll, MMR/2% tmod4 9 ; 6 n 5n ahof each ng 25 and tmod4 Xenopus M/%Fcl.Ebyswere Embryos Ficoll. MMR/6% a 9 atnn(:000 Sigma- (1:10,000; -actinin ; O 5 MO, lmod3 md– n Tmod1–4 and Lmod1–3 9 O,5 MO2, -AGCTCCTTCTG- lmod3 lmod3 9 t5 g For ng. 50 at ) 9 -AGGTTT- O and MO1 O,5 MO1, 6 MMR 9 - rcso) h mgswr eovle ihSfWR n then and CA). SoftWoRx Jose, San with (Adobe, deconvolved CS (Applied Photoshop were software using 3.5.1 processed images SoftWoRx The (Applied using Precision). AZ) system Tucson, RT (Photometrics, Olympus), Deltavision (IX70; 100 microscope a inverted a an using with Cy5- WA) imaged Issaquah, PA). Precision, were and Warrington, mounted Inc., sections (Polysciences, were (1:500) Poly/Mount The Coverslips Aqua with (1:500). anti-mouse-IgG slides onto anti-rabbit-IgG anti- goat donkey anti-rabbit-IgG conjugated goat Cy5-conjugated goat ImmunoResearch Alexa-Fluor-488-conjugated Alexa-Fluor-488-conjugated (1:500), Jackson (1:500), included and mouse-IgG Invitrogen Inc. from Laboratories, obtained antibodies o l tutrlsuis he mg eso ea-e–hlodn and Texas-Red–phalloidin- of sets image three anti- studies, structural all measurements For length and analysis Structural lea-url,A,Geoi,C .adFwe,V M. V. Fowler, and C. C. Gregorio, A., Almenar-Queralt, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.152702/-/DC1 online available material Supplementary months. material 12 after Supplementary release PMC in Deposited Expand C.U.N. to More Alliance to Western and Opportunities and C.C.G.; Student Alliance; to States Mountain HL083146] at and Education funding HL108625 Graduate P.A.K., to numbers and HL093694] [grant Foundation; number NIH Gootter [grant from (NIH) M. Health Steven of the Institutes from National funding the by supported was work This Funding contributed P.A.K. and work. C.C.G. this manuscript. experiments. to the the equally wrote P.A.K. evaluated and and respectively. C.C.G. planned studies, C.U.N., conceived, truncation C.C.G. Lmod3 the and the P.A.K. performed and R.K. C.U.N., and analysis D.C.S. hybridization experiments. situ the of in majority the out carried C.U.N. contributions Author interests. financial or competing no declare authors The interests Competing measurements. length DDecon Deconvolution for Distributed Bennett their Rachel for with and Fowler help software Velia for (DDecon) and Henderson Gokhin thoughtful Christine David for and blots, laboratories Cover western Krieg Cathleen and thank Gregorio We the discussions. of members thank We Acknowledgements ag .L,L,X,Ltlfed . rme,S,To,A,Kolo,K U., K. Knowlton, A., Thor, S., Bremner, R., Littlefield, X., Li, L., M. Bang, Fowler, and L. Pouplana, de Ribas A., C. Conley, A., Lee, A., Almenar-Queralt, nttt,L ol,C)(iteil n olr 02 ohne al., Research et Scripps Gokhin (The 2002; Fowler Fowler, Student’s and Velia (Littlefield A Dr CA) and 2010). Jolla, Gokhin La provided David Institute, generously was Dr software this by DDecon software; were the determine 1.42q using experiments ImageJ to quantified for were independent plugin used lengths four sarcomere was and of analysis TFLs analyzed. square Results at staining Chi significance. phalloidin recorded. in statistical Gaps were TFLs. M-line measure to the used were was phalloidin fluorescence and GFP showing sarcomeric merged; regions of somite Images analyzed. Only treatment. per nasaeo – o hi blt otic rsi nrsos oan to response in swim or twitch to ability their for 0–5 scored stimulus. were external of (Sadikot embryos scale experimental previously and a control described on st34 Briefly, as 2010). performed al., et was analysis Locomotion hnflmnsrarnet omsrae myofibrils. striated that evidence form muscle: to skeletal rearrange chick in filaments myofibrillogenesis thin in early assembles iaetlntsadrdcdcnrciefnto nseea muscle. skeletal in function 173 contractile reduced J. and muscle. Chen, lengths filament and skeletal L. fast R. in Lieber, ends pointed filament Chem. actin Biol. J. caps that tropomodulin, M. V. 1123. a 905-916. , atnnsandscin eeaqie rmtreo orembryos four or three from acquired were sections -actinin-stained 6 a 19b.Ietfcto fanvltoooui sfr,skeletal isoform, tropomodulin novel a of Identification (1999b). A13ojcieadaCoSA Qdgtlcamera digital HQ CoolSNAP a and objective 1.3 NA atnnwsue oietf acmrsadsroeelengths sarcomere and sarcomeres identify to used was -actinin ora fCl cec 21)18 3–5 doi:10.1242/jcs.152702 239–250 128, (2015) Science Cell of Journal 274 28466-28475. , t ts a efre ots o significance. for test to performed was -test 20) eui-eiin ieehbtsotrthin shorter exhibit mice Nebulin-deficient (2006). a atnnadpalii tiigwere staining phalloidin and -actinin .Cl Sci. Cell J. 19a.Tropomodulin (1999a). .Cl Biol. Cell J. 112 1111- , 249

Journal of Cell Science iteil,R,AmnrQeat .adFwe,V M. V. Fowler, and A. Almenar-Queralt, R., Littlefield, A. B. Rapp, and Hitchcock- A. and Choy, S., J. A. N. Kostyukova, Greenfield, A., Choy, A., B. Rapp, S., A. Kostyukova, L. Kedes, and Y. K. Kong, otuoa .S. A. Kostyukova, S. A. Kostyukova, R. A. Hockaday, and L. C. Huang, Franzini- F., Protasi, S., Holtzer, Q., Z. Zhang, X., Z. Lin, T., Hijikata, H., Holtzer, M. R. Harland, h,X,Ce,J,Rey .C,Vr,C,Sn,K .P n ug .A. L. Sung, and P. L. K. Sung, C., Vera, C., M. Reedy, J., Chen, X., Chu, Hayes, I., Fujiwara, A., Skwarek-Maruszewska, M., E., Boczkowska, D., Noumen, Chereau, C., Jarabek, C. R., C. Gibb, E., Gregorio, Segerdell, A., and K. Snyder, M. B., J. C. Bowes, Jones-Weinert, M., Chu, T., K. Bliss, ARTICLE RESEARCH 250 Thie and T. Mohun, B., Cooper, S., Hamon, S., Hardy, rgro .C,Wbr . odd . ens,C .adFwe,V M. V. Fowler, and R. Thie and C. S. Hardy, Pennise, M., Bondad, A., Weber, C., C. E. Gregorio, S. Hitchcock-DeGregori, and S. A. Kostyukova, J., N. Greenfield, E., N. Kim, H. Keurs, Ter B., and A. R. H. Akster, Nowak, H., Granzier, R., C. McKeown, L. A., R. Lieber, R. and Lewis, J. S., Chen, D. J., Gokhin, Zhang, L., M. Bang, S., D. Gokhin, M. V. Fowler, and S. D. Gokhin, Y. H. Zoghbi, C., C. Gregorio, B., Xu, S., R. Fischer, R., P. Cox, L., K. Fritz-Six, J. Moyer, and J. N. Greenfield, M., M. V. V. Fowler, Fowler, and A. Almenar-Queralt, L., K. Fritz-Six, A., C. Conley, one nsrgltsti iaetlnt nsrae muscle. striated in length filament 544-551. thin regulates ends pointed capping. filament actin 12075. for model novel a tropomyosins: capping. filament actin and binding E. tropomyosin S. DeGregori, irn ogbplrMCflmnsadIZIbodies. I-Z-I and L. filaments 93. H. MHC bipolar Sweeney, long microns and C. Armstrong, Xenopus of embryos. Xenopus lineages muscle striated the in expression of laevis. patterns spatial and soito ihtooysn t ellrlclzto,adteitgiyof interactions. integrity the and specific. localization, cellular its tropomyosin, sarcomeres. with association larvae. laevis Xenopus skeletal to required is end slow-growing the at filaments circulation. embryonic actin mouse establish of R. capping Dominguez, E-Tmod cells. and muscle D. in T. nucleator filament Pollard, actin P., 239-243. an Lappalainen, is Leiomodin G., (2008). Rebowski, B., D. D. P. resource. Vize, and N. motility. Pollet, in roles and partners binding Cell new Biol. adhesion: Mol. cell in lasp-2 Investigating noigteseea n mohmsl eou avsbt tropomyosin beta laevis Xenopus muscle smooth and isoforms. skeletal the encoding myocytes. actin cardiac maintain chick to embryonic tropomodulin in by length capping filament pointed-end of Requirement (1995). domain capping N-terminal the 1. of tropomodulin properties of binding tropomyosin and Structure muscle. skeletal of relation 1060-1070. physiology. length force-sarcomere muscle the on skeletal and capping Biol. pointed-end Cell J. filament M. alters V. thin Fowler, and muscle L. regulate R. Lieber, skeletal S., R. Littlefield, nebulin-knockout in length properties. contractile filament isometric thin Reduced fibers. muscle skeletal in reticulum 194 sarcoplasmic the to link isoforms lethality. embryonic and development heart 163 aborted to leads M. mice V. Fowler, and domains. end-capping pointed 40009. filament actin two family. gene (Tmod) tropomodulin 73 the of members larger Leiomodins: 127-139. , 105-120. , 1033-1044. , eh Dev. Mech. a rh ice.Biophys. Biochem. Arch. tooysntasrpswt itnt3 distinct with transcripts -tropomyosin ici.Biophys. Biochim. uli cd Res. Acids Nucleic 189 el o.Lf Sci. Life Mol. Cell. .Bo.Chem. Biol. J. 19) nst yrdzto:a mrvdwoemutmto for method whole-mount improved an hybridization: situ In (1991). 24 bu,P. ´baud, 95-109. , 995-1006. , ipy.J. Biophys. 87 ehd elBiol. Cell Methods 20) bratmoirlasml ntoooui1null tropomodulin1 in assembly myofibril Aberrant (2003). 20) eooi/rpmoi neatosaeisoform are interactions Leiomodin/tropomyosin (2007). 20) rpmdln n tropomodulin/tropomyosin and Tropomodulins (2008). 20) tutrlrqieet ftoooui for tropomodulin of requirements Structural (2005). 199-202. , .Anat. J. 19) slto n hrceiaino DAclones cDNA of characterization and Isolation (1992). 20) ecn 3 ftoooui- euae its regulates tropomodulin-1 of 135 Leucine (2006). 20) ebs:aXnpsbooyadgenomics and biology Xenopus a Xenbase: (2008). 281 1131 36 88 9589-9599. , m .Physiol. J. Am. 65 159 D761-D767. , 21) Cytoplasmic (2011). 372-383. , 239-242. , 465 563-569. , 18) eeomn fmooa el in cells myotomal of Development (1988). 129-136. , 227-230. , 36 m .Physiol. J. Am. 19) needn sebyo 1.6 of assembly Independent (1997). 685-695. , 19) feto hnflmn length filament thin of Effect (1991). 20) rpmdlnbnstwo binds Tropomodulin (2006). 20) rpmdlncontains Tropomodulin (2003). 296 21) rpmdlnisoforms Tropomodulin (2010). Biochemistry 9 T aedfeettemporal different have UTR .Bo.Chem. Biol. J. 1123-1132. , 20) ci yaisat dynamics Actin (2001). elSrc Funct. Struct Cell c Nature Biochemistry atnadtropomodulin and -actin 284 bu,P. ´baud, m .Physiol. J. Am. 1827-1838. , 377 a.Cl Biol. Cell Nat. 44 4905-4910. , 83-86. , Science 278 19) Two (1999). .Cl Biol. Cell J. .Cl Biol. Cell J. 45 Genomics 40000- , 12068- , (2003). (2013). (2009). (2001). (2005). 22 83- , 320 260 3 , , , ur,R . er,C . ihrs . olr .M n rgro .C. C. Gregorio, and M. V. Fowler, M., Richards, N., C. Perry, E., R. Mudry, ad,V n in,J M. J. Miano, and V. Nanda, L. Muntz, B. J. Gurdon, and S. Fairman, N., Dathan, M. S., V. Brennan, Fowler, J., and T. A. Mohun, M. Sussman, J., Moyer, B., R. Nowak, R., C. McKeown, M. V. Fowler, and R. Littlefield, aahr,S,Gki,D . iua . oa,R .adFwe,V M. V. Fowler, and B. R. Nowak, S., Kimura, S., D. Gokhin, S., Yamashiro, M. D. Helfman, and R. Kobayashi, A., Watakabe, wr,C . re,P .adGeoi,C C. C. Gregorio, and A. P. Krieg, U., C. Nworu, ee,A,Pnie .R,Bbok .G n olr .M. V. Labeit, and H. Fowler, Granzier, Y., Wu, and M., McNabb, D., G. Labeit, C., Burkart, G. C., C. Witt, Babcock, R., C. Pennise, A., Weber, n,S. Ono, aio,T,Hmod .R n err,M B. M. Ferrari, and R. C. Hammond, T., Sadikot, W. J. and Sanger, H. and M. A. J. Sanger, Beggs, D., S., Rhee, Labeit, J., G. Stienen, C., C. Witt, A., C. Ottenheijm, C. C. Gregorio, and B. P. Antin, C., Schwach, Y., Ono, aga,E . ilr .L,Y,H .adBmn,W M. W. Bement, and Y. H. Yu, L., A. Miller, M., E. Vaughan, Scho and C. J. E., Sparrow, Kremneva, L., A. Zajac, M., Boczkowska, A., Skwarek-Maruszewska, skd,T,Ktynky,L,Hyh . ea,B,Nvk .M,Kajava, M., S. Novak, B., Desai, R., Huynh, L., and Kotlyanskaya, S. T., A. Tsukada, Kostyukova, B., P. Antin, N., Moroz, T., C. Pappas, T., Tsukada, Kirkpatrick, R., S. Daniels, R., P. Khoury, N., Gude, S., Welch, A., M. Sussman, Baque A., M. Sussman, 20) h neato ftoooui ihtooysnsaiie thin stabilizes tropomyosin with tropomodulin myocytes. of cardiac in interaction filaments The (2003). apl fXnpslei Dui 1802). (Daudin 774. laevis Xenopus of tadpole embryo. amphibian early the in genes actin of mouse Nature activation for sac type-specific yolk Cell the not but heart the development. in embryonic required is Tropomodulin1 (2008). images. 2564. fluorescence of analysis deconvolution distributed rhtcuei ies eltypes. cell filament diverse actin in regulate that architecture proteins capping pointed-end Tropomodulins: (2012). eedn agtgn xrse rfrnilyi ifrnitdsot muscle smooth differentiated in preferentially cells. expressed gene target dependent tutr nvivo. in structure filaments. actin S. of ends pointed brain the 1627-1635. to caps protein Tropomodulin binding major the as identified tropomodulin tropomyosin. of isoform novel eou ucefrsroei rti oaiainb ihrslto imaging. high-resolution by localization protein Methods sarcomeric for muscle Xenopus t oei myofibrillogenesis. in role its deficiency. nebulin with patients myopathy Genet. nemaline in weakness H. Granzier, in maturation. myofibril development arresting by cardiomyocyte 336-348. cells compromises stem embryonic gene murine (Tmod1) tropomodulin1 muscle. h Taecosakb Abr. by crosstalk GTPase Rho cardiomyocyte mature in tropomodulin and leiomodin sarcomeres. of behaviors cellular P. Lappalainen, and and R. Dominguez, T., Svitkina, ihntoooui- epnil o t oaiaina h one nso the of ends pointed myocytes. the cardiac at in localization filaments its S. actin for A. responsible Kostyukova, tropomodulin-1 and within C. C. Gregorio, muscle. V., A. cardiac in filaments thin the 3145. of end D. pointed J. C. C. Molkentin, Gregorio, and W. H. population Lim, mice. and 2101-2113. transgenic L., structural, tropomodulin-overexpressing molecular, R. in cardiomyopathy: analyses Price, dilated A., of Pathogenesis R. Walsh, D., myofibrils. of structure sarcomeric 94-105. the L. disrupts Kedes, cardiomyocytes and L. Terracio, D., way. the pave integrins maintenance. and assembly sarcomere Dyn. in C-terminus N-versus telethonin 20) eui euae hnflmn egh otatlt,adZ-disk and contractility, length, filament thin regulates Nebulin (2006). 239 .Bo.Chem. Biol. J. 21) yai euaino acmrcatnflmnsi striated in filaments actin sarcomeric of regulation Dynamic (2010). 311 18 17) ygnssi h rn n e uigdvlpeto the of development during leg and trunk the in Myogenesis (1975). 1124-1135. , 66 ora fCl cec 21)18 3–5 doi:10.1242/jcs.152702 239–250 128, (2015) Science Cell of Journal 2359-2369. , 716-721. , 370-379. , o.Bo.Cell Biol. Mol. .Cl Sci. Cell J. 20) hnflmn eghdseuaincnrbtst muscle to contributes dysregulation length filament Thin (2009). 21) eooi- sa naoito rpmdln1a the at tropomodulin-1 of antagonist an is Leiomodin-2 (2010). MOJ. EMBO 287 ,S,Um .S,Dnes .P,Pie .L,Simpson, L., R. Price, P., M. Daniels, S., C. Uhm, S., ´, 67 k F. ck, ¨ a.Rv o.Cl Biol. Cell Mol. Rev. Nat. 2459-2467. , 677-692. , 109 ic Res. Circ. 21) eooi ,anwsrmrsos factor- response serum new a 1, Leiomodin (2012). 25 21 2299-2310. , elMtl Cytoskeleton Motil. Cell 3843-3855. , 3352-3361. , 20) esrmn fti iaetlntsby lengths filament thin of Measurement (2002). .Cl Biol. Cell J. urBiol. Curr 20) h nta tp fmoirlassembly: myofibril of steps initial The (2009). 19) lee xrsino rpmdlnin tropomodulin of expression Altered (1998). Cytoskeleton. 103 .Bo.Chem. Biol. J. 1241-1248. , 19) h rmoirl vdnefor evidence premyofibril: The (1994). 21 .Ebyl x.Morphol. Exp. Embryol. J. 162 270-277. , 21) rprto fdeveloping of Preparation (2013). 21) dniiaino residues of Identification (2011). 1057-1068. , 69 10 21) ifrn localizations Different (2010). 337-370. , 293-298. , 286 19) -rpmdln a N-tropomodulin: (1996). 21) itntrlsfor roles Distinct (2010). 28 20) irpini the in Disruption (2005). .Cl Sci. Cell J. 2194-2204. , 1-24. , 20) oto flocal of Control (2001). ipy J. Biophys m .Pathol. J. Am. .Cl Biol. Cell J. e.Biol. Dev. ic Res. Circ. 123 u.Mol. Hum. 82 33 3136- , (1994). (1984). (1999). 2548- , 757- , Dev. 127 282 155 82 , , , ,

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