4170 Erratum

The function of the M-line obscurin in controlling the symmetry of the in the flight muscle of Drosophila Anja Katzemich, Nina Kreisko¨ther, Alexander Alexandrovich, Christopher Elliott, Frieder Scho¨ck, Kevin Leonard, John Sparrow and Belinda Bullard

Journal of Cell Science 125, 4170 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.119552

There was an error published in J. Cell Sci. 125, 3367-3379.

In the section ‘Effect of reduced obscurin expression on other in IFM’, the second sentence was published omitting the term ‘M-line’. The correct sentence should read as follows: In wild-type , zormin was in both Z-disc and M-line (supplementary material Fig. S3A).

On p.3373, left column, second paragraph, second line, the word ‘line’ was published twice.

We apologise for these mistakes. Journal of Cell Science otoln h ymtyo h acmr nteflight the in of sarcomere muscle in the of obscurin symmetry protein the M-line controlling the of function The Article Research po muolbln I)adfboetnlk F3 domains. (Fn3) fibronectin-like and The small (Ig) for immunoglobulin- sense responsible of to proteins up stiff The al., of be frequency. stiffness must et (AL- high the fibres Tregear at filaments The 2011; changes 2010). thin al., length al., et on et Perz-Edwards Wu sites 2003; 1998; on target al., crossbridges actin et Khayat and between and of filaments match the rapid contraction stretches thick a on to alternating periodic IFM depends of the response stretch by The 1978). of by (Pringle, activated muscles (IFM) maintained opposing is muscle are flight muscle oscillations indirect The Gautel, the 2005; the insects. of for Perriard, essential contraction is and sarcomere oscillatory well-ordered (Agarkova A 2008). Z-disc Linke, under 2011; distortion the them to prone than prevent vertebrate more In is stress and region stretched. a M-line are contract, the the fibres fibres, within fibres hold when muscle alignment links the filaments of These as out thick coming M-line. register and the in in Z-disc, filaments crosslinked the are in sarcomere crosslinked neighbouring from thick are filaments parallel Thin of filaments. composed thin are and muscle striated of sarcomeres The Introduction in sarcomere symmetrical a of and development thin length the short the for or words: sarcomere, long necessary abnormally Key the is in In resulted IFM filaments. zones the bare the in shifted of misaligned. filaments; obscurin thick middle thick was that Isolated adjacent the absent. H-zone conclude of from or symmetry the We misplaced away the filaments. H-zones normal, on shifted and depends was missing were filaments RNAi were Z-disc that thin M-lines and of the irregular: zones polarity was Although bare flies IFM with and affected. mutant the larvae asymmetrical, of was P-element Embryos, structure were sarcomere. IFM the the Using filaments the and from the pupa. fly of predicted however, to structure and were unable pupa, the that were larva on the domains Adults expression embryo, the In obscurin all the decreased normally. has of and of developed isoform effect (IFM) development pupae muscle IFM the investigated flight larger throughout indirect have The M-line the we larva. in flies, the the two knockdown in in flies: found adult is is of isoform Obscurin muscles fifth the sequence. in A found muscles. are other isoforms in Four two UNC-89. protein nematode the to in similar Unc-89 as known (also Obscurin Summary Canada 1B1, H3A Quebec Montreal, University, § McGill Biology, of ` Department address: *Current 5 4 3 2 1 Katzemich Anja nw sTtn.Teeaetoioom nIM etn(4 kDa) (540 kettin IFM: in isoforms two are There ). as known o:10.1242/jcs.097345 doi: 3367–3379 125, Science Cell of Journal 2012 February 27 Accepted ei Leonard Kevin urn drs:MrkKa,FakutrSr 5,623Drsat Germany Darmstadt, 64293 250, Str. Frankfurter KGaA, Merck address: Current uhrfrcrepnec ( correspondence for Author igsCleeLno,TeRnalDvso o eladMlclrBohsc,NwHn’ os,Lno E U,UK 1UL, UK SE1 1SD, London CB10 House, Cambridgeshire Hunt’s Hinxton, Canada New Campus, 1B1, Biophysics, Genome Quebec Molecular Trust Montreal, and Wellcome University, Cell EMBL-EBI, McGill for Biology, Division of Randall Department Germany The Marburg, UK London, 35043 5DD, College 8, YO10 King’s Karl-von-Frisch-Straße York Philipps-Universita¨t Marburg, York, Biologie, of Fachbereich University Biology, of Department 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. Drosophila -ie bcrn n-9 hc filament, Thick Unc-89, Obscurin, M-line, Drosophila gene 5 onSparrow John , 1, ,Nn Kreisko¨ ther Nina *, [email protected] sallimus Drosophila F aeamdlrsrcuemade structure modular a have IFM noe o sfrso l (also Sls of isoforms for encodes Drosophila 1 n eid Bullard Belinda and ) 1,2, salremdlrpoeni h -ieof M-line the in protein modular large a is ) ` lxne Alexandrovich Alexander , Drosophila Muscle , 1,§ rdmntl nteMln ein(uuaae l,2008; myofibrils, al., of et periphery (Fukuzawa the region mature M-line at In the and is C-terminus. in ankyrin-binding the Obscurin-A obscurin predominately an near 2001). fibres, domains either al., skeletal have two et that or 2008; Young region isoforms al., 2002; et are Fukuzawa al., obscurin-B 2001; are al., et domains et DH-PH Russell (Bang and C-terminus SH3 the the near that except 2007). structure, al., domain et (Burkart IFM the of a M-line with the was in protein UNC-89 identified of A region the affected. C-terminal from the hardly predicted of is that to worms similar the sequence muscle: of of motility body-wall absent, integration often but the are the M-lines and in for myofibrils disorganised needed A-bands have mutants is regular protein into The 2008; myosin 2004). al., et al., (Qadota et C-terminus N-terminus, the Small near the domains near kinase domains two and signalling (RhoGEF) DH-PH and elegans Caenorhabditis Z-disc the in al., both M-line. et found Burkart the is 2005; in zormin, al., and isoform, et to Sls (Bullard Z-disc the filament Another near thick 2007). actin the link of which end of the both kDa), (700 Sls700 and h etbaehmlgeo N-9 bcrn a similar a has obscurin, UNC-89, of homologue vertebrate The in identified originally was UNC-89 protein M-line The 3 hitpe Elliott Christopher , Drosophila Drosophila Bna ta. 96.UC8 a SH3 has UNC-89 1996). al., et (Benian eoe(ml ta. 04 and 2004) al., et (Small genome muscles. 1 ree Scho Frieder , Drosophila Drosophila bcrnis obscurin ¨ck IFM. unc-89 3367 4 , Journal of Cell Science rjci Blade l,20) ftee nyteSsisoform The Sls M-line. the the only in these, found In of been 2005); myomesin. has al., or zormin et titin (Bullard contain projectin not does Drosophila M-line The different. 3368 nS3dmi erteNtriu,floe yDH-PH by followed domains, of N-terminus, Fn3 two mass domains, the Ig has 21 molecular gene near downstream, entire Further domain the predicted domains. of by SH3 structure a domain obtained an predicted have isoform The possible kDa. would The 475 largest splicing Flybase. The in 1A). (Fig. CG30171 or 36 CG33519 as Drosophila annotated ( and gene 2R obscurin The The is that Results can obscurin. IFM muscles of the amount other reduced of whereas a structure with contraction, function precise oscillatory for the needed of development the are maintenance for filament the essential thin of and is the symmetry Obscurin of obscurin. the polarity on that and and dependent length here the lost show and We was filament fly. of sarcomeres thick to expression IFM unable reduced of were with flies structure flies regular In the pupa. obscurin, and of development larva during embryo, followed was the sarcomere the in of obscurin IFM of in M-line the stabilises obscurin myomesin of and Institute 2000). Federal actin Zurich, Swiss thesis, Technology, PhD of muscle, striated Expression in isoforms isoproteins: diversity contractile Functional Agarkova, of (Irina myomesin encode not does in (Unc-89) homologue obscurin function Gautel, crosslinked The the 2008; 2009). of al., Obsl1, al., et et and (Fukuzawa Kontrogianni-Konstantopoulos 2011; domains obscurin Ig interacting myomesin, through titin, at including than rather 2008). myofibrils, al., contrast of et in interior (Fukuzawa and, the periphery muscle in the skeletal ankyrin-binding is of Obsl1 the M-line al., obscurin, et the to as Geisler in 2008; is well al., Obsl1 et 2007). as (Fukuzawa C-terminus SH3, the domains, the near lacks region kinase but to obscurin, and similar of is region that DH-PH N-terminal pattern the a al., in in seen et domains vertebrate Fn3 that Porter in and obscurin, Ig 2009; to Kontrogianni- has similar Obsl1 al., protein, fibres: second et a 2003; 2006; Lange is al., There 2003; 2005). et al., al., (Bagnato et Konstantopoulos the et and SR create may Kontrogianni-Konstantopoulos the this between and link (SR), a reticulum sarcoplasmic the to in binds ankyrins Obscurin-A 2003). al., et Kontrogianni-Konstantopoulos h i ftesuyw eciehr a ofn u how out find to was here describe we study the of aim The proteins, of network a of composed is M-line vertebrate The Drosophila ora fCl cec 2 (14) 125 Science Cell of Journal ii srpae ytesalrpoen l and Sls proteins smaller the by replaced is titin , eoesqec rdcsagn faot1 bwith kb 18 about of gene a predicts sequence genome bcrngn n t xrsinproducts expression its and gene obscurin Unc-89 slctda hoooearm at located is ) Drosophila Drosophila Drosophila h position The . srather is genome 3kai isn rmioomC hc scnitn ihgene with consistent is which C, isoform splicing. from than missing more Therefore, is the A. kDa isoform N-terminus, than 53 about smaller is the kDa 105 C or isoform from kDa, Obscurin 370 kDa. cleaved 422 N- were be would the protein kDa remaining of 53 N-terminal the from downstream If residues most 12 kDa C-terminus. were gene peptides The 53 C-terminal of most about The product cleavage. terminus. a were protein be identified to than peptides likely is rather protein of splicing, eclosion the of after that spectroscopy appeared showed that Mass pupae isoform C. the (6-day- isoform from Mature the IFM derived C. contain of peptides isoform more flies IFM contain Young the flies D. of old) and traces B and isoforms isoforms pupal non-IFM S1A). two amounts small Fig. the and A of material isoform IFM (supplementary larger the contain compared pupae wild-type Pharate the mature were and in flies flies eclosed Isoforms newly adult In otherwise. pupae, late isoforms. indicated the five of when thorax all except in anti-Ig14- antibody, used are we 16 following, shows the domains one in blot described 1 than are western that kinase experiments The more and blot. is the Ig14-16 there on that that bands individual possibility fly. in adult the the isoform in exclude found isoforms cannot the (isoform from We isoform We size single in a 1C). Ig16 contained differed (Fig. larva which The E), and Ig15, 1 IFM. A the (isoforms kinase in which Ig14, of were two and C) thorax, against the Ig14-16) in isoforms antibodies as four detected to using referred by (hereafter in identified were blots IFM, isolated and western thorax fly the of muscles the of in S1. isoform Table material largest catalytic supplementary in the the shown in are of IFM domains downstream in obscurin for exons coding by Exons encoded of sequences. domains regulatory encoded are the is exon; one 1 by the kinase 2 kinase or of and two, sequence exons, by two core by encoded The often exons. individual are three, domains by sometimes Ig encoded single domains derived that is the shows IFM of exons in Prediction transcript exons. largest 35 the Therefore, from 15. exon the bp except Flybase, 75 in predicted exons all contains IFM in transcript IQ the protein. lacks largest vertebrate and the domains The of Ig 1B). fewer domain has (Fig. it predicted homologues: vertebrate are domains Drosophila kinase two and tg 7 bcrnwsse costemsl nastriped a in muscle the across late during seen muscles was somatic of Obscurin at M-line embryo the 17. the in stage in and detected 16 first stage was early obscurin experiments, our In pupal and larval, embryonic, muscles in obscurin of Position h sfrso bcrnta r rsn ntelra n those and larva, the in present are that obscurin of isoforms The largest the to corresponds that cDNA the of sequence The bcrnioomi mle hntenmtd and nematode the than smaller is isoform obscurin ny ohatbde ece ihalfv bcrnisoforms. E obscurin isoform five has all larva with the reacted IFM; antibodies the in Both are in only. C isoforms and four A least Isoforms anti- at thorax. or are anti-Ig14-16 There with antibodies. incubated kinase1 were Blots (F). IFM antibodies. raise to used ( were that regions show H n h w iaedmis(i1adKin2). and (Kin1 domains kinase ( two (DH- RhoGEF the the and encoding insertion. PH) exons P-element show gene the the of above position Lines the marks and Epgy2 protein 1–36. the in muscles. domains the in gene, isoforms obscurin The 1. Fig. etr oso sfrsi av L,auttoa T and (T) thorax adult (L), larva in isoforms of bots Western C) B oan rdce ntepoen ie bv domains above Lines protein. the in predicted Domains ) ( xn ntegn r numbered are gene the in Exons A) Journal of Cell Science ic yfbisbcm ie nteaut n bcrnadkti are kettin Z- and 5 and obscurin bars: M-line and Scale the adult, striations. at the in dots in labelled apart as wider further appears become are myofibrils myofibrils Myofibrils hours, narrow disc. 72 the at band of Z-disc; broad labelling the hours, a at and 48 as kettin At and appears M-line labelling strands. the obscurin amorphous in and in packed is closely kettin are and myofibrils distribution punctate a has obe lsrt h pdra tahetst hnosui nteM-line. the in obscurin 25 a than in site is attachment and epidermal ( Z-discs the the to marks closer (green) doublet GFP-kettin (red); Anti-obscurin gene. larval and embryonic in kettin sites protein attachment Z-disc epidermal The the 2A). to muscles close which (Fig. is at epidermis the sites to the attached to are laterally positioned was and pattern a sfr fSs rd.Osui sete ieo h pdra attachment epidermal ( the embryo. of type side either is ( Obscurin site. (red). Sls) of attachment isoform epidermal (an the at ( gap a (arrows). with site muscles, the across flies. striations wild-type ( showing of anti-obscurin. pupa with and larva labelled embryo, were the in Obscurin 2. Fig. E F npp t3 or,4 or n 2husAFadatrelso (at eclosion after and APF hours 72 and hours 48 hours, 30 at pupa in IFM ) ˚ ) niosui gen n nikti mgna.A 0hus obscurin hours, 30 At (magenta). anti-kettin and (green) anti-obscurin C); mroo -lmn uat aeln ssmlrt hto h wild- the of that to similar is Labelling mutant. P-element of Embryo C) B D mrolble ihat-bcrn(re)adanti-kettin and (green) anti-obscurin with labelled Embryo ) avlbd almsl,fo l iewt GFP- a with line fly a from muscle, wall body Larval ) idtp mro(aesae17) stage (late embryo Wild-type A) m m. Muscles sls bcrnadkti perdi tie atr.Teearly The into 2E). assemble pattern. (Fig. to kettin striped begins before obscurin and a structures that hours discrete in shows 72 labelling appeared at of After than pattern kettin appearance. wider dot-like and were a myofibrils obscurin Z-disc pupae, and of M-line 72 eclosion at the myofibrils gave narrow kettin increased The hours the length separated. and sarcomere more were as obscurin Z-disc; myofibrils hours, and both 72 and at of defined kettin M-line better Striations became lined and side-by-side. the myofibrils in narrow closely in at obscurin was dots up labelled striations by kettin hours, formed were broad protein striations 48 Z-disc as At the appeared strands. while to showed APF obscurin periodicity, undifferentiated hours hours, 30 of 30 At from 2E). signs followed (Fig. adult of was emerged position IFM newly The the pupal shown). not the (data in (APF) hours formation obscurin 48 puparium at after strongly, hours more 32 and, at IFM from the amplified midway at embryo. kettin, doublet was the than in a site Obscurin seen attachment as was the 2D). from as resolved further (Fig. and was Z-discs site between and attachment 2007) GFP-Sls Z-disc epidermal al., expressing the marks et GFP-Sls line 2D). (Burkart fly (Fig. a antibody from anti-obscurin with larvae labelling by M-line, similar 2C). is (Fig. below) embryos (see wild-type gene of obscurin that the of to start the near in obscurin inserted of position The 2B). unc89[EY15484] (Fig. kettin than sites attachment (Kreisko muscle unc89-IR h F sfr n oermiiglra isoform larval remaining some of and traces isoforms, A non-IFM isoform two the IFM had the pupae the isoform; using siRNA the in by down pupae knocked and was obscurin larvae which with expression effectiveness obscurin The reduced of effects Developmental The article. unlikely. this in were described siRNA are lines, target two the lines 34 the of fly exon for the same effects for Table the off-target Results material was muscles (supplementary that the 7 showing on and that effect 12 6 exon The to 5, S1). other kinase domains the encodes domains, Ig Fn3 that and gene encodes Ig obscurin preceding the the of and 34 2 1996). exon al., to different targeted et two one Ranganayakulu with crossed 2007; were al., lines Driver et Dietzl 2006; (Cripps, P-element the in a and muscles IFM, as particular were target lines to to Driver used fly. refererred be is can the driver with and appropriate gene combined 1A) obscurin siRNA (Fig. the of mutant. intron here first the used of a start was the of to close inserted insertion mutant, is expression The reduced The gene. in the result obscurin. can of gene a targeting absent. into by transposon or siRNA P-element and reduced insertion of was P-element by IFM protein expression reduced the the was of which expression assembly in Obscurin the in flies obscurin in of sarcomere function the studied We expression obscurin of Reduction rvr a sesdi etr lt.Lk h idtp,larvae wild-type, the Like blots. western of in assessed was drivers uigdvlpeto h ua bcrntasrpscudbe could transcripts obscurin pupa, the of development During the in is muscles wall body larval of obscurin that found We UH3-GAL4 Mef2-GAL4 r eerdt sRA lines. RNAi as to referred are bcrni h lgtmsl -ie3369 M-line muscle flight the in Obscurin UH3-GAL4 ; te ta. 06 n bcrni ute rmthe from further is obscurin and 2006) al., et ¨ther UAS-unc89-IR uatebysi hc -lmn was P-element a which in embryos mutant ; hc rmtsepeso nalmuscles all in expression promotes which , UH3-GAL4 UAS-unc89-IR unc89[EY15484], le a h avlobscurin larval the had flies hc rmtsepeso in expression promotes which , UH3-GAL4 GAL4-UAS and nwihaP-element a which in UAS-unc89-IR MEF2-GAL4 and ytmwt an with system Mef2-GAL4 ; UAS- lines, Journal of Cell Science titosaei h -iergo;i h dl,moi aeln si o tteMln n nasnl ada h -ic yfbisaenroe hni the in than narrower are Myofibrils Z-disc. myosin the hours, at 72 band single at a myofibrils); in single and isolate M-line to the fragile at better too dot H-zone were a the fibres in wider, (the is become 1.5 myosin striations labelling myofibrils at the myosin broad adult, in myofibrils adult, the in doublet single to the appears a are APF in myosin is Inserts hours region; there and 48 wild-type. M-line and H-zones From the sharper no band. in are single are are striations a there striations myosin in hours, and Z-disc 48 obscurin the hours, at at 72 labelled strands; ( at is longer. myosin M-line; sarcomeres adult, the and the at defined in dots Z-disc; in the labelled near are label myosin and obscurin and H-zones 3370 i.3 bcrnadmoi nteIMo h pupa. the of IFM the 29 in at myosin grown and Obscurin 3. Fig. ora fCl cec 2 (14) 125 Science Cell of Journal ˚ .( C. A idtp ua t3 or P,osui slble nbodsrain n ysni naopossrns t4 or,teeare there hours, 48 at strands; amorphous in is myosin and striations broad in labelled is obscurin APF, hours 30 At pupa. Wild-type ) B ) e2GL;UAS-unc89-IR Mef2-GAL4; 6 h anfcto ftemi aes cl as 5 bars: Scale panels. main the of magnification the F a aeldwt niosui gen,at-ysn(aet)adpalii rd.Ppewere Pupae (red). phalloidin and (magenta) anti-myosin (green), anti-obscurin with labelled was IFM ua hr sn bcrni h F taysae t3 or P,moi si amorphous in is myosin APF, hours 30 At stage. any at IFM the in obscurin no is There pupa. m m. Journal of Cell Science o-lgtmsls oosui ol edtce ntelarvae the in detected of be pupae could and obscurin No muscles. non-flight IFM-specific the Therefore, UH3-GAL4 S1B). Fig. material (supplementary o tteMln n loi adcoet h ic h dot-like The disc. Z the to close band a in a in as seen also detected those and was myosin M-line like fly, the adult sarcomeres at the dot In regular stage. no this at at than were flies separated wild-type there more but were M-line hours, the 48 within hours, myosin 72 of At dots hours. 48 fuzzy H-zones at APF no myofibrils although hours phalloidin-stained type, in 48 wild visible to from and were myofibrils hours of due 30 that to at similar be labelling was Myosin to 3B). (Fig. likely obscurin is stage. Z-disc adult the development. the and hours to 72 between close A-band the band double of the a lengthening from single in labelling hidden myosin a in are change to epitopes The end A-band. the the the anti-myosin at because of and rest monoclonal probably H-zone the A-band, The in a the IFM Z-disc. in of adult and labels the M-line here the to used at antibody close labelled Z- was the band to myosin close 72 fly, single doublet adult a At the in defined; myofibrils. and In better M-line disc. single was the M-line at in labelled the was best and antibody myosin seen wider of were were Narrow myofibrils dots M-line M-line. hours, in and the the together, in at actin closely were label and packed myosin 3A). were and APF, myosin (Fig. obscurin stage myofibrils hours same and hours, the 30 48 at striations, the kettin At at like to broad – pupae APF strands in undifferentiated hours wild-type 30 was In from and obscurin adult. stages myosin obscurin, pupal emerged of in newly position followed The was with pupae. compared actin was wild-type obscurin lacking of flies of that pupae the in IFM the ako bcrnafce h sebyo ysndrn pupal during myosin of assembly the affected obscurin of Lack rvrde o prcal eueosui eesin levels obscurin reduce appreciably not does driver e2GL;UAS-unc89-IR Mef2-GAL4; e2GL;UAS-unc89-IR Mef2-GAL4; ua a odetectable no had pupae le.Teasml of assembly The flies. o h idtp nautIM(upeetr aeilFg S3B). Fig. material (supplementary IFM adult in type wild the for unc89-IR ol l swl stewl-ye hwn httewild-type the that showing wild-type, flies the restored. rescued was as gene The well was obscurin P-element. mutant as the the fly of gene, could obscurin excision the precise on by effect flightless mutant rescued an P-element to the were that of due phenotype Flies solely insertion the was of IFM. that P-element confirm effect the To the 4A). the of (Fig. check for function to the homozygous tested on were was levels flies obscurin of reduced capability flight flies adult The in expression obscurin Reduced itiuino etn(i.2)wr nfetdin unaffected were 2E) that the (Fig. suggests in kettin changes Z-disc of Developmental distribution the delayed. was near A-band The labelling the of antibody. elongation of antibody the penetration in of H-zone better appearance better-defined allowing late a myofibrils, to the due of be core may M-line the at labelling h iae2dmi,aoiino h xrsino l ieobscurin to five targeted all of was expression siRNA the of Since abolition IFM. domain, 2 the kinase the in The or IFM. muscles the thoracic in the obscurin detectable no the UAS-unc89-IR was Mef2-GAL4; there of and P- muscles D, and the thoracic B by the unaffected expected, UAS-unc89-IR was UH3-GAL4; As isoform insertion. two larval the element The of traces isoforms. retained IFM IFM the whereas D, isoform obscurin idtp le Fg B.Tetoai uce fteP- the of muscles thoracic of The that 4B). with mutant, (Fig. compared element was flies flies wild-type knockdown obscurin of IFM affected. was UH3-GAL4 bcrnpeeti h oa hrccmslsadi isolated in and muscles thoracic total the in present Obscurin le ntson,adtepsto fkti a h aeas same the was kettin of position the and shown), (not flies bcrni h lgtmsl -ie3371 M-line muscle flight the in Obscurin or Mef2-GAL4 unc89[EY15484], imtro h yfbi.Saebr 5 bar: Scale myofibril. the the of across diameter obscurin show (top) the Z-stacks in lines. missing RNAi are (arrowheads) H-zones the lines. in reduced is M-line unc89[EY15484] wild-type the in (red). Obscurin phalloidin and antibodies, anti- (magenta) (green), anti-obscurin with labelled the the of of purity ( the lack sample. confirms the IFM isoform): in isoform kDa Sls 540 largest the is (kettin Sls the in UAS-unc89-IR obscurin no was there isoforms; isoform. UAS-unc89-IR larval GAL4; wild-type a IFM and reduced isoforms isoform, thoracic smallest had the mutant a P-element as The (bottom). anti-Sls control or loading were (top) larva anti-obscurin and with (F) IFM incubated (T), thorax of flies. blots knockdown Western RNAi obscurin and element GAL4 P- with the flies excising Control by element. restored was flight flightless; unc89[EY15484] ncdw le;tepooto ffisal ofly to able flies shown, of is proportion the flies; knockdown expression. obscurin reduced ( of Effect 4. Fig. lgttsso idtp,Peeet n RNAi and P-element, wild-type, of tests Flight A) rvr lw ( flew. drivers eefihls,soigta h IFM the that showing flightless, were UAS-unc89-IR iehdtetonnIMisoforms non-IFM two the had line bcrni h F.Moirl were Myofibrils IFM. the in Obscurin C) iehdn eetbeosui in obscurin detectable no had line n 5 0frec eoye The genotype. each for 20 le.Toae a he sfrsof isoforms three had Thoraces flies. uatadmsigi h RNAi the in missing and mutant uatadRA ie were lines RNAi and mutant akdalbttesmallest the but all lacked B bcrnioom nP- in isoforms Obscurin ) le a h w non-IFM two the had flies le rvnb either by driven flies UAS- Mef2-GAL4 Ni(sNi or (dsRNAi) RNAi Mef2-GAL4; m a m. UH3- -actinin ; UAS- Journal of Cell Science 3372 i.SB.I h -lmn uatand material mutant were (supplementary P-element the H-zone ends In wild-type the S3B). filament the in Fig. In was thin tropomodulin. tropomodulin of misaligned position IFM, the and affect to 2001) expected in Fowler, H-zone and the of borders flies knockdown abnormal. in appeared but the spaced, the of regularly with core were the Z-discs M-line. in predominately Mef2-GAL4 was whereas – In M-line wild-type the P-element myofibril. the in the as in – In other Z-disc zormin S3A). the affect in Z- Fig. was both to zormin material in mutant, likely (supplementary was zormin and was myofibrils, disc obscurin wild-type In of proteins. M-line amount the Reducing proteins other on IFM expression in obscurin reduced of Effect crawling jumping. larval fly for (Elliott neither for leg needed nor middle is the for obscurin both on power Therefore, TDT The 2007). in the al., flies. of et control Flies action as the well S2C). from as comes jump Fig. jumping to able material were (supplementary lines RNAi controls the as was were there but IFMs mutant, P-element was knockdown the obscurin the of or of three in M-line none labelling Ig14-16 the Faint all in wild-type. against detected the in in antibodies than Myofibrils narrower with 1. case lines the labelled was RNAi kinase this two flies; myofibrils the wild-type the in throughout for myofibril distributed of the was of that M-line section the with cross in IFM compared Obscurin the 4C). of was structure (Fig. flies the on adult mutation P-element in the flies of knockdown effect obscurin The in domain. myofibrils 1 of kinase Structure the as well as domain, 2 kinase the have the by isoforms h -oe hra etni h -icwsunaffected. was RNAi the Z-disc of myofibrils the in H-zone in clear no kettin was there whereas in Although myofibril the H-zone, of core the the to confined was tropomodulin drivers, control with compared of whereas reduced in slightly larvae, ability was affected obscurin crawling were lacking Fig. larvae The muscles material non-flight flies. that (supplementary these confirmed normal This appeared S2A,B). Z-discs whereas the of TDT) UAS-unc89-IR trochanter, the of depressor (tergal muscle due probably length. is filament flies the thin knockdown in in obscurin variability ends in to filament enter IFM of the they misalignment of where H-zone the well-aligned Therefore, were Z-disc. filaments the in not thin were showed that sarcomere lines half showed regular a RNAi contrast, in the filaments By of thin register. The the sarcomeres of type. the ends wild in the the that H-zone in an than of that longer lack suggests somewhat mutant, were the P-element been filaments and the thin has H-zone in obscurin narrower sarcomere The downregulating longer 2010). slightly of effect al., result similar et A a (Schnorrer lines. in described as RNAi resolved H-zone the was the in not on sarcomere but wild-type mutant the P-element the in H-zone The GAL4. rpmdlncp h one n fti iaet tthe at filaments thin of end pointed the caps Tropomodulin eddntepc bcrni h avlmsl rtejump the or muscle larval the in obscurin expect not did We Mef2-GAL4 ora fCl cec 2 (14) 125 Science Cell of Journal omnwsmr ifs nbt h -icadthe and Z-disc the both in diffuse more was zormin , UAS-unc89-IR UAS-unc89-IR le.Osui a isn rmtemuscles, the from missing was Obscurin flies. H-A4 UAS-unc89-IR UH3-GAL4; Mef2-GAL4 rvr h de fZdssoccasionally Z-discs of edges the driver, Drosophila ie rvnby driven lines rvr hw htalteeisoforms these all that shows driver, a le rvnby driven flies atnnlbligi l h IFMs the all in labelling -actinin e2GL;UAS-unc89-IR Mef2-GAL4; F (Mardahl-Dumesnil IFM UAS-unc89-IR avecalda well as crawled larvae UH3-GAL4 UH3-GAL4 MEF2-GAL4; ihboth with or Mef2- and xeddit h -oe oe o h feto akof lack of effect the for model A sometimes H-zone. filaments the thin lines, into both RNAi extended In and observed. mutant not P-element was a this of the zone; side bare either of filament same thick the the been displaced short that length, have normal or would the crossbridges followed long of retained polarity filaments abnormally filaments thin in Had thin filaments. resulting the thin of filaments, of middle thick the polarity adjacent from The far zones sarcomere. bare some with for asymmetrical, (shown The lines other. RNAi UAS-unc89-IR the the thin GAL4; than in pronounced and sarcomere more half zone, was one bare effect in the of longer polarity of rigor were The side filaments of H-zone. either opposite zones displaced reversed direction bare the the was in the crossbridges the were mutant, have filaments P-element by thick zone the of recognised In bare 6A). be half (Fig. the in crossbridges can filaments of which Z-disc Thin 1985). the side polarity, C., towards either M. end Reedy, IFM sarcomeres barbed and K. the normal M. with (Reedy, the arrowheads filaments thin In as and and thick zone, zone. appear between bare the crossbridges bare of conditions, edge rigor the the in to sarcomere, extend form filaments the thin anti- to sarcomere, of in assembled middle fashion are molecules by the myosin parallel formed of in regions is rod filaments H-zone the where The thick filaments. of thick alignment of asymmetry to due (supplementary IFMs sarcomeres knockdown the S4). in Fig. and disruption material wild-type of Z-disc. extent in the the myofibrils bypassed affected shows filaments of less was some overview normal myofibril where An the by periphery, of extreme core the accompanied more The than showed H-zone. was lines the RNAi fly in the shifts to in IFMs ability structure. The muscle the gene sarcomere. obscurin restored, wild-type the the was when of that of confirming excision middle by P-element, into reversed the the were up structure from sarcomere broken in abnormalities H-zones further was phenotype, shifted M-line severe more the mutant were the a and from In P-element wandered aggregates. sarcomere, electron-dense H-zone the the the the cases, of of marking mild the middle phenotype in M-line across severity; region denser The in clear varied the 5). a with (Fig. mutant. is sarcomere, mid-point P-element IFM the the wild-type of in the middle than which in lines M-line, H-zone RNAi and The in H-zone severe the more micrographs in were Electron abnormalities flies. lines showed wild-type RNAi of (EMs) and that mutant P-element with the compared flies in was IFM knockdown the obscurin of in structure IFM The of structure fine The eito ntelnt fflmnsi oeeteea the at extreme more is filaments thin of and of periphery, length ends the periphery. at the the than in aligned that better Thin suggests deviation specifically are core filaments. 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Fig. in Mef2- Journal of Cell Science h yfbi,weeteflmnsbpse h Z-disc. is the of It bypassed periphery length. filaments the normal the from where the originated myofibril, filaments twice the thick than and long more lines, that RNAi were likely the filaments in filaments few was of there a length although that the filaments, length in wild-type by variability a the more had of driven that flies to knockdown line similar in was the filaments shifted. thick than of was majority zones zone zone bare bare bare central central the 8B). a (Fig. majority, had zone UAS-unc89-IR lines the bare RNAi in the the in but, from shift variable filaments a Some with filaments P-element also the from the knockdown filaments of of of end mutant majority one IFM The towards 8A). the shifted (Fig. frequently in filament was with whereas in zone zone bare filament, flies the bare the flies, clear filaments. of a of isolated had middle IFM IFM of the wild-type images the the EM from in in filaments Thick seen filaments was thick obscurin reduced of asymmetry The obscurin in filaments flies thick knockdown IFM of structure The in distributed P-element UAS-unc89-IR the sparsely Mef2-GAL4; of was H-zone UAS-unc89-IR the Obscurin in remained mutant. 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These to regularity al., IFM the overall muscle. et of the (Small advantage wall for muscle taken needed body the is of UNC-89 the organisation that to shown have directed experiments siRNA by ahohr(ag ta. 09.I erfs,teeaetwo The are 2010). al., et there Raeker out in 2006; zebrafish, gene al., et single knocked In Raeker 2007; been al., 2009). et smaller (Geisler al., – two et and the far for obscurin (Lange compensate so and they other that – each possibility obscurin not the leaving encode have simultaneously, Obsl1, that in homologue either two vertebrates The downregulated RNAi. be using higher can by which or transformation gene, P-element single by a the by expression encoded of using the of is integrity advantage reducing The by and protein. the studied development of was in sarcomere muscle obscurin flight of function The Discussion 9B). (Fig. M-line the at mid-region filament the the to discrete corresponding of a probably in filaments, filaments was the obscurin thick of and region sections, filaments the thin from of separated parts were In images EM cryosections. that from labelled came confirmation filaments of the Further thick 9A). with and (Fig. associated is is IFM obscurin 2004), the for protein al., found in was skeletal shown same et vertebrate the been in filaments complex (Kontrogianni-Konstantopoulos previously a in that muscle thick had exist myosin It and of suggest obscurin filaments. that thick assembly obscurin with associated the lacking in sarcomeres abnormalities filaments thick The with obscurin of Association h ags sfr of isoform largest The bcrni h lgtmsl -ie3373 M-line muscle flight the in Obscurin .elegans C. obsl1 Drosophila ee,wihcmonsteproblem the compounds which genes, htecdsUC8 a entargeted been has UNC-89 encodes that yfbi eihr atrss.Saebar: 0.6 Scale the (asterisks). at periphery Z-disc myofibril the bypass zones. filaments bare Some shifted more and have H-zones lines irregular RNAi the in phenotype. IFMs the of rescues Excision P-element sarcomere. the the of from middle shifted the thick are of arrows) zones (black bare filaments case, severe In more arrow). a (white H-zone the are in there aggregates and wild-type compared the wavy with is mutant P-element the UAS-unc89-IR GAL4; UAS-unc89-IR GAL4; (D) (B,C) wild-type, (A) i.5 tutr fteIMi le with flies in obscurin. IFM reduced the of Structure 5. Fig. .elegans C. m unc89[EY15484 m. bcrni iteoe half over little a is obscurin Drosophila N-9o vertebrate or UNC-89 ( ece,(E) rescued, ] A–F unc89[EY15484 le.TeHzn of H-zone The flies. n (F) and sta obscurin that is Miae of images EM ) Drosophila Mef2- UH3- ], Journal of Cell Science 3374 i.6. Fig. e etpg o legend. for page next See ora fCl cec 2 (14) 125 Science Cell of Journal Journal of Cell Science ol eaot80n rmteMln,i sulkl there unlikely is SR. it the M-line, and obscurin the between from 80– vesicles interaction nm SR any extend the be 800 might as would M-line; about domains the be Ig from sarcomere would 21 Razzaq the no with along 1964; nm is Obscurin (Shafiq, 100 muscle like 2001). there skeletal al., myofibrils, and vertebrate along et M-line in longitudinally found the extending that and SR of Z-disc of SR network the reduced much between The midway SR. the the to Drosophila linked of directly that C-terminus unlikely is is the it obscurin at present, al., not region inside is et obscurin-A ankyrin-binding is vertebrate Kontrogianni-Konstantopoulos the Obsl1 and 2008; As larger myofibril, 2003). al., are the the -B et of of and periphery (Fukuzawa arrangement obscurin-A the the where to to vertebrates, confined in contrast isoforms in obscurin is sarcomere 2008). the al., et (Miller stiffness fibre been affect have may proteins isoforms However, these Sls in unexpected. and of obscurin lifespan was the the of throughout pupa observed amounts all IFM the the has smaller in of a IFM changes eclosion of the appearance after The in 1B. isoform isoform Fig. main in illustrated The domains in domains. identified kinase have the we isoforms five Drosophila The ankyrin-binding obscurin-A. the having of than rather domain in C-terminus, The the obscurin-B C-terminus. at domains vertebrate the kinase at resemble domains proteins kinase invertebrate two near and domains N-terminus, DH–PH and the SH3 with UNC-89, nematode in the 21 of the that domains: of architecture Ig domain of The number B. the to due Drosophila largely is this obscurin; iwdolqey cl a,20n.( nm. best 200 are the bar, arrowheads) of Scale and mid-point obliquely. asterisks or viewed by M-line (indicated M, changes chevrons zone. of filaments bare Rows filament of sarcomere. thick polarity shifted a the of that side show either in filaments shifted thin more on are (crossbridges) arrows) (black zones UAS-unc89-IR Bare Mef2-GAL4; In arrow). filament. 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Fig. vertebrate o rvn h le rmwlig hrfr,tels well less the the Therefore, in muscles non-flight walking. other and did from TDT, this the these flies muscles, like muscles of leg ordered the the thorax of the prevent case in the not in muscles obscurin; Other the lacked flies. also jump M-line, flies to wild-type the able as in were well flies obscurin as and detectable striations the regular no from this showed flies had sarcomeres in adult line in striations RNAi TDT irregular the same although be rather Similarly, not type. the might muscle obscurin maintaining and of 1985), muscle for wild-type al., are body-wall et essential of There the (Ball flies. in larva that adult wild-type M-lines than into the or develop H-zones less to well-defined slightly able no were only they was and the larvae, of crawl ability the to obscurin, larvae detectable no had flies UAS-unc89-IR were sites promoter P- larva internal the are the There of in type. the wild embryo expressed the in of the amount that in the to eclosed similar and obscurin pupae mutant, of and element flies, position knockdown The RNAi normally. the and P-element etersl ftasrpinfo nitra promoter. internal an also from might transcription isoform of small result The the 3B). isoforms be (Fig. larger reduced the considerably of insertion P-element expression is P-element whereas the the normally, having in expressed flies by isoform is adult smallest of affected the muscles Interestingly, thoracic not intron. the was first the that in by insertion regulated promoter be might The internal isoform the isoforms. an larval if the smaller smallest; sites, the of promoter of multiple expression one is to isoform lead larval can which 2002), hr r bcrnrmat ntedsotdHzn fthe of H-zone distorted the in remnants obscurin are There C) mroi n avldvlpetwsuafce nbt the both in unaffected was development larval and Embryonic lhuhlra uce fpoeyfo the from progeny of muscles larval Although .eeasunc89 elegans C. bcrni h lgtmsl -ie3375 M-line muscle flight the in Obscurin obscurin acmr.( sarcomere. uat eann bcrni nteMln region. M-line the in is obscurin remaining mutant, A nwl-yeIM bcrni nteMln.( M-line. the in is obscurin IFM, wild-type In ) ee(uuaae l,20;Rsele al., et Russell 2005; al., et (Fukuzawa gene The D) roetoswr aeldwt anti-obscurin with labelled were Cryosections ee(ml ta. 04 n nthe in and 2004) al., et (Small gene e2GL;UAS-unc89-IR Mef2-GAL4; Drosophila Mef2-GAL4; acmr has sarcomere UH3-GAL4; eehas gene B nthe In ) Journal of Cell Science 3376 hc iaet omn acmr t4 or P a 22 (at first APF the hours show 40 Beall at and sarcomere a Reedy is forming sarcomere. filament thick filaments the growing thick of a middle of middle the the the in that with obscurin ensure Evidently, associate to 1993). needed they Beall, is as and thin (Reedy formed between sarcomere and filaments are existing thick interdigitating filaments of of new array addition Z-bodies: initial by an assembled to is filaments flight pupa, the sarcomere the of of in IFM development contraction regularity During the 1997). oscillatory the al., for because et (Dickinson expected essential muscle is is This lattice fly. filament flies to knockdown obscurin able cannot the was IFM of the None thorax, obscurin. the without in function muscles fly non-flight the adult to and contrast pupa In adult. of IFM and in pupa filaments Obscurin larva, thick the in the muscles of the misalignment of development some during tolerate can thorax, sebyo ysnit ymtia A-bands. symmetrical subsequent the into their for myosin needed for of is necessary assembly Obscurin not structures. is repeating into it is arrangement no periodic myosin, is becomes or myosin there obscurin kettin where of before although sarcomere, Therefore, distribution the H-zone. of the the regular mid-region the in although in structures obscurin, abnormal periodic of Z- into the absence assembles obscurin of in Myosin unaffected development before largely is The and knockdown. myofibril, obscurin hours. kettin of 48 independent developing is at Interestingly, disc Z-disc the the myosin. hours along to that 30 or migrating strands at found actin in IFM we the kettin, distributed in Here, before periodicity sarcomeres formed. rudimentary APF, recognisable a are time has Z-discs obscurin which by well-defined becoming hours, hours, with 42 60 at M-line at the narrower at appearing density broad and ora fCl cec 2 (14) 125 Science Cell of Journal ˚ C), rao h hc iaet.Saebr 0 nm. 200 bar: small Scale a filaments. to thick confined the is of Obscurin area Protein–A-gold. and myosin antibody and anti-obscurin fly (Obs) the obscurin from were ( proteins anti-obscurin (Myo). of with extract pelleted an Proteins (lane with without thorax. and incubated 3) were and 1 anti-obscurin, (lanes 2) with beads myosin. 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Anti-obscurin protease 1:1000. al., developed diluted and et urea were and (Burkart M Tissues (Millipore) 6 thoraces. immuno-blotted with glycerinated buffer were from sample dissected in were homogenized IFMs dissection. after etiue.PoenAbaswt on niosui g,i idn buffer, binding 4 in at IgG, extract muscle anti-obscurin the bound with incubated with were beads Protein-A centrifuged. n D eedsetdi eaigslto,fxdimdaeyi 4% in kettin, immediately (Ig14-16), obscurin against and fixed and Antibodies TDT) primary muscle). zormin and larval solution, myosin, with (IFM and solution (embryo labelled relaxing relaxing PBT in and or diluted in were solution, Antibodies IFMs antibodies. relaxing Pupal dissected secondary in antibody. secondary washed were (Sigma), with paraformaldehyde, rhodamine-phalloidin labelled TDT and then antibody solution, and relaxing primary in with washed 2007). labelled al., et (Burkart were myofibrils into These separated and inhibitors, protease with EGTA) or.Tehmgnt a iue nbnigbfe 02MNC,2 MNa- MgCl mM 20 mM NaCl, 2 M (0.2 buffer 7.2, binding pH in diluted was phosphate homogenate The hours. 2 xmndwt es S50o xokp5 ofclmcocp ih63 were with microscope Samples confocal (Vector USA). software. 50 LSM Axioskop with ImmunoResearch, double-labelled embryos or processed and LSM510 (Jackson for objective for Zeiss oil-immersion IFM antibodies a used with labelled and examined were fluorescently larvae and antibodies embryos, USA) secondary 10 Laboratories, about Biotinylated to 1:50), 1:200. zormin, of case atnn A 7 Lkye l,19) a oolnlat-ysn A 147 MAC anti-myosin, monoclonal rat 1990); al., et (Lakey 276 MAC -actinin, o roetoigadimn-M Fsi eaigslto ihprotease with solution relaxing in IFMs immuno-EM, and cryosectioning For 2 eodr iaini %omu erxd,boksann n2 uranyl 2% in staining block tetroxide, osmium 1% in fixation Secondary . ora fCl cec 2 (14) 125 Science Cell of Journal m /ladat-ysnIGws0.4 was IgG anti-myosin and g/ml 2 a MNaN mM 5 , atnnwr fiiyprfe g,dltd110(r nthe in (or, 1:100 diluted IgG, affinity-purified were -actinin 3 ,fxdi %guaadhd,02 ancacid, tannic 0.2% glutaraldehyde, 3% in fixed ), 2 .%Tio -0,2m T)and DTT) mM 2 X-100, Triton 0.1% , m /l nitoooui eu a diluted was serum Anti-tropomodulin g/ml. 2 MDT n nuae niefor ice on incubated and DTT) mM 5 , ˚ .Baswr ahdwt binding with washed were Beads C. m g/ml. m /l niosui (kinase1) anti-obscurin g/ml, 2 MAP mM 5 ATP, mM 5 , 2 immediately 6 2 , otmai,J . yad .R,Muhn .W n ioeu,J O. J. M. Vigoreaux, R. Cripps, and W. D. Maughan, R., L. Nyland, L., J. Contompasis, E. L. Thornell, and G. J. Yu, L., Carlsson, ant,P,Brn,V,Gaoel,E,Rsi .adSretn,V. Sorrentino, and D. Rossi, E., Giacomello, V., Barone, P., Bagnato, M. J. Squire, and C. T. Irving, K., M. Reedy, L., Hudson, A., H. AL-Khayat, ukr,C,Qu . rne,S,Bns . Ha V., Benes, S., Brendel, F., Qiu, C., Burkart, J. Sparrow, and S. Ball, E., Ball, ulr,B,Brat . aet .adLoad K. Leonard, and S. Labeit, C., Burkart, B., Ding, Bullard, D., Labeit, M., Gautel, C., M. Leake, A., Minajeva, C., Ferguson, B., Bullard, Benian,G.M.,Tinley,T.L.,Tang,X.andBorodovsky,M. The (1996). nuae tro eprtr o 0mn eaigslto (100 solution Relaxing min. 30 for temperature room at incubated ietmst eaaeteflmns h upninwscnrfgda 1500 at centrifuged was needle suspension 20G The a filaments. through the passed separate was to suspension times the five and added, were inhibitor calpain n hsppri eiae ohrmemory. preparations her (York EM to on dedicated is suggestions for mass-spectroscopy paper Renkawitz-Pohl many this Renate made and with and Reedy and advice, Mary Harrison Dowle helpful support. Peter for Sweeney, Adam Gautel Sean Mathias thank We and Facility). assisted Technology Stark EM Meg serum. anti-tropomodulin with for Fowler for Velia Nongthomba to Upendra and to grateful are We Acknowledgements mM 5 DTT, mM mM 20 2 ATP, NaCl, mM 5 mM EGTA, obscurin (10 mM 1981). or solution 5 Mg-acetate, al., wild-type relaxing mM NaN et 5 10–20 in Reedy 6.8, suspended of pH 2010; MOPS were IFMs al., to flies, glycerinated et calpain knockdown (Contompasis from with Z-disc digestion prepared mild the Myofibrils, by from myofibrils them from separate released were filaments Thick filaments Thick groa .adPrir,J C. J. Perriard, and I. Agarkova, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.097345/-/DC1 online available material Supplementary F.S. to MOP-93727 M. number a in of grant, Institutes operating A.A. B.B.; Canadian of Research a to and to Network Health London; fellowship College, FP6 511978 King’s postdoctoral group, Union number Gautel’s Council European project Research a MYORES, Medical by grant, supported Excellence was work The Funding ag .L,Cnnr . onf,F,Gah .J,Gthrt . cab M., McNabb, M., Gotthardt, J., A. Geach, F., Fornoff, T., Centner, L., M. Bang, nlsdb AD-O assetocp na ple isses4700 Biosystems Applied an a from on MASCOT. identified were spectroscopy were SDS-gels Proteins mass on analyser. Bands Proteomics MALDI-TOF buffer. SDS-sample by with eluted analysed were proteins and buffer iue ormv niscae myofibrils. undissociated remove to minutes 3 MET,1m GA Mmratehnl 0 lcrlwith glycerol 30% mercaptoethanol, mM 5 EGTA, mM 1 15 EDTA, mM 1 lgtni eesr o eghdtriain tutrlitgiy n ag bending filaments. large thick and muscle integrity, flight structural insect determination, of length stiffness for necessary is Flightin muscles. striated ysnha ofgrto nrlxdisc lgtmsl:xrymdldresting modeled binding. actin x-ray for muscle: poised are flight state 1063-1079. insect pre-powerstroke relaxed a in in cross-bridges configuration head Myosin fa nyi- sfr oosui ugssamlclrln ewe the between link molecular a suggests obscurin muscles. to striated in isoform myofibrils and reticulum ankyrin-1 sarcoplasmic an of ulr,B. Bullard, muscle. flight insect of contraction al. oscillatory Motil. et the in R. proteins K. Leonard, muscle. J., in titin Horwitz, with alphaB-crystallin chaperone S., Labeit, L., domains. transduction signal and Ig of 132 composed protein modular giant a elegans Caenorhabditis in development ioeu) ereon adsBioscience. Landes Georgetown: Vigoreaux). In formation. extensibility. different with muscles in expression their hc iaet ntecne ftesarcomere. the of center the in filaments thick n t neato ihosui dniyanvlZln oIbn ikn system. linking I-band to Z-line isoform, novel titin a al. identify 700-kDa Res. obscurin approximately et with unusual H. interaction an Granzier, its of and C., expression titin, C. of Gregorio, sequence D., gene Labeit, C., C. Witt, m 3 M ,te etiue n eupne n30 in resuspended and centrifuged then ), 835-848. , 1065-1072. 89, m 479-485. 26, api Clice) CaCl (Calbiochem); calpain 20) h otiuino eeist h td fisc lgtmuscle flight insect of study the to genetics of contribution The (2006). 20) oua rtisfo the from proteins Modular (2007). auesVraieEgn-Isc lgtMsl nieAdOut And Inside Muscle Flight Insect Engine- Versatile Nature’s rspiamlngse.Dv Genet. Dev. melanogaster. Drosophila itce.Cl Biol. Cell Histochem. eeuc8,rqie p uceMln seby encodes assembly, M-line muscle fpr required unc-89, gene 20) h -ad neatcwbta crosslinks that web elastic an M-band: The (2005). 18) uainafciglra muscle larval affecting mutation A (1985). 2 130 a de o2m n myofibrils and mM 2 to added was 20) e set fosui nhuman in obscurin of aspects New (2008). 91-103. , ˚a rnsCl Biol. Cell Trends g . aet . enr,K and K. Leonard, S., Labeit, P., ˚g, .Ml Biol. Mol. J. 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Reedy, Krz uloieecag atrpoenivle nsroeeassembly. sarcomere in 123-136. involved protein factor exchange nucleotide al. novel et reveals K. muscle flight M. interactions. insect Reedy, contracting actin-myosin E., isometrically Y. cryofixed, of Goldman, tomography C., Lucaveche, H., Sasaki, deduced properties sequence. coding and complete mapping its pattern, from developmental paramyosin: melanogaster er,seea uceadbandevelopment. brain and muscle skeletal heart, W. M. W. zebrafish. M. in myofibrils Russell, striated and of E. alignment S. Lyons, A., raiaino h xiaincnrcinculn ahnr fmsls u o for not but in muscles, endocytosis of vesicle machinery J. coupling synaptic C. excitation-contraction O’Kane, the and of J. organization N. Gay, P., A. Jackson, the of mesoderm ventral the Biol. in expression nautilus induces B. Bullard, muscle. and R. K. in 2432. (Obscurin) UNC-89 of domains M. G. mechanism. and function Biol. Membr. Mol. J. R. Bloch, muscle. stretch flight during insect movement of tropomyosin and activation connections myosin-troponin for evidence nue eet nnuohsooyadlcmto r eeae ymetabolic by generated are J. C. locomotion stress. Elliott, and oxidative and not T. neurophysiology and S. dysfunction in Sweeney, J., defects G. induced Evans, A., C. Middleton, muscle. flight insect in zones target 1451. actin to bind K. cross-bridges M. Reedy, and E. Towns-Andrews, n eui yeetohrssi %plarlmd lbgl teghndwith strengthened gels slab polyacrylamide 2% in electrophoresis agarose. by and domains. kinase protein MLCK-like containing iaetlyr n lrdXfrain rmisc lgtmuscle. flight insect from formations flared-X and layers filament Drosophila iaetasml n acmr tblt in stability 151 sarcomere and assembly filament heads. myosin ytmtcgntcaayi fmsl opoeei n ucinin al. function et and A. morphogenesis Stark, muscle M., of Radolf, Nature analysis A., genetic Azaryan, M., Systematic Fellner, K., Schernhuber, ein .M. G. 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