The Journal of Cell Biology

JCB

Ryan E.Mudry, expressing greenfluorescent – volution microscopy. Real-timeimagingoflive myocytes thin filaments,asrevealed by immunofluorescence decon- into chick cardiacmyocytes causedadramatic lossofthe invitro.MicroinjectionofmAb17ormAb8 against Tmod1 thatspecifically disrupteditsinteraction with generated monoclonalantibodies(mAb17andmAb8) protein, tropomodulin1(Tmod1),withtropomyosin, we stabilizes thinfilamentsincardiac myocytes The interaction oftropomodulin withtropomyosin Along thelengthofthinfilaments,tropomyosin,an from elongatingorshorteningtheirbarbedends. capped byCapZ;thereby,thethinfilamentsareprevented barbed (plusorfast-growing)endsofthethinfilamentsare other sarcomericproteins. involve severalmechanisms,includinginteractionswith are criticalforpropercontractilefunctionandappearto filaments withstrikinglyuniformlengths.Theseproperties monomersarepreciselyassembledandmaintainedin (thin)filamentdynamicsbecausewithinmyofibrils, actin proven tobeapowerfulmodelsystemforinvestigating trusion, cytokinesis,andcontraction.Striatedmusclehas for diverseprocessesincludingmigration,membranepro- The regulationofactindynamicsineukaryoticcellsisessential A 3 1 in ahead-to-tailfashion.Onewell-establishedroleoftro- Introduction  filament Key words: sarcomere;myofibrillogenesis;cardiacmuscle;actin;thin LLP, SanDiego,CA92101. Meredith Richards’presentaddressis Luce,Forward,Hamilton&Scripps, email: [email protected] 85724. Tel.:(520)626-8113.Fax.: (520) 626-2097. Anatomy, UniversityofArizona,1501N.CampbellAve.,Tucson,AZ Address correspondencetoCarolC.Gregorio,Dept.ofCellBiologyand http://www.jcb.org/cgi/doi/10.1083/jcb.200305031 The Journal ofCellBiology

Department ofCellBiology,Department The ScrippsResearch Institute, LaJolla, CA92037 ofCellBiologyDepartment andAnatomy Tucson, AZ85724

TheRockefeller University Press, 0021-9525 -helical rodlikemolecule,formshetero-andhomodimers -helical

Article

At theZ-lines,bordersofindividualsarcomeres,

the roleofactinfilament pointedendcapping essential forstriatedmusclefunction. To determine ctin (thin)filament lengthregulationandstabilityare

1 Cynthia N.Perry, , Volume 162, Number 6,September 15,20031057–1068 /2003/09/1057/12 $8.00 1 Meredith Richards, -tropomyosin and 2 Department ofMolecularDepartment andCellularBiology, University ofArizona, 3 Velia M.Fowler, in regulating theCa in pomyosin istocooperatewiththetroponincomplex example, homozygous critical rolefortropomyosininpropermusclefunction.For seeCooper, 2002).Invivostudieshaverevealeda review (Ono andOno,2002;Nyakern-Meazzaetal.,for from thedepolymerizingeffectsofADF/cofilinorgelsolin filament assemblyandphysicallyprotectsthinfilam actin 1990; Adamietal.,2002).Tropomyosinalsoenhances ends (Wegner,1982;Broschatetal.,1989;Weigt monomers fromtheirpointed(minusorslow-growing) tion andbending,preventsdepolymerizationofactin Tropomyosin increasesfilamentstiffness,preventsfragm that tropomyosinalsofunctionstostabilizethethinfilam mounting evidencefromanumberofinvitrostudiesindi (Huxley, 1969;forreviewseeCooke,1997).However, However, theprecisemechanisms bywhichtropomyosin actin filamentsandmuscleparalysis (OnoandOno,2002). tropomyosin expressionleads todisorganizedsarcomeric with tropomyosin prevents thinfilament depolymerization. prevents thinfilamentelongation,whereas itsinteraction protein:itsactinfilament cappingactivity multifunctional together withpreviousstudies,indicatethat Tmod1 isa iscriticalforthinfilament stability.tropomyosin These data, These studiesindicatethattheinteraction of Tmod1 with addition ofmAb17prevented thelossofthinfilaments. assay,reconstitution stabilizationofthefilaments beforethe depolymerized fromtheirpointedends.Inathinfilament withmAb17revealed thatthethinfilaments microinjected sin isoform( al., 1998).In obvious phenotype(Blanchardetal.,1997;Rethinasamy et onic lethal,whereasheterozygousknockoutmiceshowno in in force-generatingproperties (Kreuzetal.,1996).Studies not central,myofibrillarorganization,aswellalterations Caenorhabditis elegans Drosophila Tm1 3 ) resultindisruptionofperipheral,but andCarol C.Gregorio 2 , mutationsofonemuscletrop -dependent actomyosininteraction -tropomyosin nullmiceareembry- demonstratethatsuppression of 1,2 omyo- enta- ents. 1057 ents cate The Journal of Cell Biology 1058 Tmod1’s actin cappingactivity(butnotits interaction with jection ofamonoclonal antibodythatspecifically blocked using primaryculturesofchick cardiacmyocytes.Microin- end cappingactivityhasbeen investigateddirectlyinstudies chitecture invivo. maintaining thelengthsofthin filamentsandmyofibrilar- Therefore, thelevelsofTmod1 expressionareimportantin assembly(Chuetal.,2003;unpublisheddata). mately dayE10,suggestinganessentialroleforTmod1in logue) in and chickcardiacmyocytes,orSanpodo(aTmodhomo- et al.,1998a).Incontrast,overexpressionofTmod1inrat cytes resultedinabnormallylongthinfilaments(Sussman creasing levelsofendogenousTmod1inratcardiacmyo- have beeninvestigatedinseveralstudies.Forexample,de- filament lengthregulation. muscle suggeststhatTmod1mayplaymultiplerolesinthin ence ofseveralTmod1-interactingmoleculesinstriated their uniformlengths(McElhinnyetal.,2001).Theexist- ulin’s proposedfunctionasathinfilamentrulerthatdefines is intriguingtospeculatethatitsroleconsistentwithneb- Although thesignificanceofthisinteractionisunknown,it minal modulesofthegiantstriatedmuscleprotein,nebulin. 2003). Incontrast,Tmod1’sNH primary actinfilamentcappingactivity(Fowleretal., the et al.,2000).Itisthishalfofthemoleculethatpossesses terminal halfiscompactandtightlyfolded(Kostyukova filaments arereflectedbyitsdistinctstructure.ItsCOOH- Fowler etal.,1993). pomyosin cappingmolecule(Wegner,1979;Fowler,1990; pointed endsoftheactinfilaments,thusactingasatro- ing contribute tothinfilamentlengthregulationbyprevent- chemical analyses,itwasproposedthatTmod1mayalso fold (Weberetal.,1994,1999).Basedonadditionalbio- al., 1998b),andTmod1 myofibril disarrayanddilatedcardiomyopathy(Sussman et pressing Tmod1(TOT)intheirmyocardiumexhibited Dumesnil andFowler,2001).Transgenicmiceoverex- (Sussman etal.,1998a;Littlefield2001;Mardahl- al., 2000).Tmod1alsointeractswiththeextremeNH muscle tropomyosins(BabcockandFowler,1994;Veraet tains overlappingbindingsitesforbothmuscleandnon- flexible andelongated(Kostyukovaetal.,2000),con- ( actin filamentsinthepresenceoftropomyosinvitro elongation anddepolymerizationfromthepointedendsof Tmod1 alsobindstropomyosin.completelyblocks see Weber,1999).Unlikeotheractincappingproteins, cardiac musclecellsaswellinothercelltypes(forreview Tmod), whichcapsthepointedendsofthinfilamentsin filament lengthregulationistropomodulin1(Tmod1/E- ularly invertebratemuscle. contributes tothinfilamentstabilityremainunclear,partic- 0.2 Tmod1 hasaloweraffinityforthepointedends( K The functionalroleofTmod1’s actinfilamentpointed The physiologicalrolesofTmod1incardiacmusclecells Tmod1’s dualinteractionswithtropomyosinandactin Another sarcomericproteinknowntobecriticalforthin d

additional tropomyosinmoleculesfrombindingtothe The JournalofCellBiology M) anditscappingactivityisdown-regulated 50pM).However,intheabsenceoftropomyosin, Drosophila,

resultedinshorterthinfilaments

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mouseembryosdieatapproxi-

Volume 162,Number6,2003

2

-terminal halfishighly

K d

1,000- 0.1– 2 -ter- proper musclefunction. indicate thattheregulatedactivityofTmod1isessentialfor ing depolymerizationfromtheirpointedends.Thesestudies of Tmod1appearstostabilizethethinfilamentsbyprevent- rio etal.,1995),whereasthetropomyosin-bindingdomain tion andmaintainsthelengthsofthinfilaments(Grego- The actincappingactivityofTmod1inhibitselonga- of Tmod1haveuniqueandcomplementaryfunctionalroles. indicate thattheactin-andtropomyosin-bindingactivities curred fromtheirpointedendstowardtheZ-line.Ourdata ance ofthinfilamentswasvisualizedinrealtimeandoc- ments andsubsequentcontractileactivity.Thedisappear- live cardiacmyocytesresultedinadramaticlossofthinfila- that blockingtheinteractionofTmod1withtropomyosinin ment lengthandstabilityincardiacmuscle.Wedetermined the interactionofTmod1withtropomyosinonthinfila- tions inthinfilamentlengthregulation. what exactlyisthefunctionofTmod1–tropomyosininterac- with thethinfilaments.Therefore,ithasremainedunclear these studies,Tmod1andtropomyosinremainedassociated the maturethinfilamentsincardiacmuscle.Interestingly, ament cappingactivityisrequiredtomaintainthelengthsof tile activity(Gregorioetal.,1995).Thus,Tmod1’sactinfil- tin filamentsfromtheirpointedendsandabolishedcontrac- tropomyosin), resultedinanabnormalelongationoftheac- dots. Thedots wereincubatedwithincreasing concentra- chick Tmod1 (25 nM)wasabsorbedonto nitrocellulose pomyosin bindingtoTmod1, full-lengthrecombinant Tmod1 (Fig.1c). kD polypeptide,corresponding tothemolecularmassof that mAb17andmAb8specifically recognizedasingle40- chick cardiacmyocyteextracts wereperformed,revealing of theseantibodies,Westernblotanalysesembryonic tropomyosin bindingsite.Todemonstratethespecificity dues 6–34ofTmod1,whichcontaintheskeletalmuscle mAb8 recognizeepitopeswithintheNH mAb17). TheseresultsdemonstratedthatmAb17and including residues35–359and95–359(datashownfor 6–184, 6–94and6–57,butfailedtorecognizefragments 1), andrecombinantTmod1fragmentsincludingresidues antibodies recognizedfull-lengthTmod1(Fig.1b,lane end ofTmod1byWesternblotanalysis(Fig.1b).Both that theextremeNH teraction ofthemoleculesinvitro.Previousworkshowed monoclonal anti-Tmod1antibodiesthatdisruptedthein- Tmod1 withtropomyosin,wefirstsoughttoidentify To studythefunctionalsignificanceofinteraction and perturbitsinteractionwithtropomyosin recognize theNH Two anti-Tmod1monoclonalantibodiesspecifically mAb17) thatspecificallyrecognizedtheNH monoclonal antibodiestochickenTmod1(mAb8and pomyosin (BabcockandFowler,1994).Wegeneratedtwo ids 6–34)isessentialforbindingtoskeletalmuscletro- Results Here, wesoughttoinvestigatethefunctionalpropertiesof To determinewhethermAb17 andmAb8inhibitedtro- 2 -terminal regionofTmod1 2 -terminal endofTmod1(aminoac- 2 -terminal resi- 2 -terminal The Journal of Cell Biology on ment cappingactivity(Gregorioetal.,1995),hadnoeffect Tmod1’s COOH-terminalendandblocksitsactinfila- mAb9 (closedtriangle),anantibodythatrecognizes of to Tmod1(Fig.1d).Incontrast,a28-foldmolarexcess incubated with incubated tropomyosin thatboundtoTmod1,ascomparedwithmAb9whichdidnotdisrupttheinteraction(closedtriangle).(e)Tmod1dots squares) completelyabolished Tmod1 ofeithermAb17(opencircles)ormAb8(closed periment demonstratedthata10-foldmolarexcessover dots ofTmod1werepreincubatedwith ciation of whether mAb17andmAb8wouldalsopromotethedisso- of time.AdditionmAb8resultedinasignificantdissociationtropomyosinfromTmod1. with tropomyosininvitro. and mAb8specificallydisrupted theinteractionofTmod1 lished data).TheseresultsdemonstratedthatbothmAb17 cross-linking Tmod1bythebivalentantibody(unpub- pomyosin indicatingthatthiseffectwasnotcausedby mAb8 alsoinhibitedtheinteractionofTmod1withtro- of tropomyosin fromTmod1(opencircles).Fabfragments MOPC-21 didnotresultinasignificantdissociationof squares). Incontrast,additionofequivalentmolarratios within 60min(Fig.1e,datashownformAb8,closed mAb8 promoteddissociationoftropomyosinfromTmod1 those antibodieswithrespecttoTmod1.BothmAb17and followed byincubationwitha13-foldmolarexcessof designed toinvestigate theinteractionofTmod1 withtro- We nextused mAb17 andmAb8inmicroinjection studies disassembly ofthinfilaments andcessationofbeating in livecardiacmyocytesresults inareversible Disrupting theinteractionof Tmod1withtropomyosin tion of tions (0–700nM)ofmAb8ormAb17beforetheaddi- 125 I-tropomyosin bindingtoTmod1(Fig.1d).Totest 125 I-labeled skeletalmuscletropomyosin.Thisex- 125 125 I-tropomyosin fromTmod1,nitrocellulose I-tropomyosin ( 125 I-TM), followedbyincubationwithmAb8(closedsquares),orMOPC21(opencircles)forvaryingperiods 125 I-tropomyosin binding

125

I-tropomyosin

data). However,themajority( for Tmod1andunperturbedactinfilaments(unpublished a thinfilamentpointedendstriatedstainingpattern strated few cellsmicroinjectedwithmAb17ormAb8demon- were fixedandstainedforthinfilamentcomponents.A and incubatedfor1hafterinjection.Themyocytes mAb8 cardiac myocytesweremicroinjectedwithmAb17or pomyosin inthecontextoflivingcardiacmyocytes.Day3–5 ing anyanti-Tmod1 monoclonalantibodyinto cardiacmyo- mAb17 wasalso notduetononspecificeffects ofintroduc- tion inthecells(Fig.2,iand j).TheeffectofmAb8and staining wasnotduetoTmod1 cross-linkingorsequestra- type, indicatingthattheloss ofsarcomericactinfilament with FabfragmentsofmAb17 exhibitedanidenticalpheno- cells remnantsremained(Fig. 2,d,f,andj).Cellsinjected filaments weredetected(not depicted),whereasinother turbed bythemicroinjectionofmAb17ormAb8,noactin ends (Fig.2,cande).Inthemajorityofcells(70–75%)per- promoted Tmod1’sdissociationfromthinfilamentpointed that disruptingtheinteractionofTmod1withtropomyosin the cellsmicroinjectedwithmAb17ormAb8,suggesting detected (i.e.,Tmodappeareddiffusedinthecytoplasm) loidin staining(Fig.2j).Notably,noTmod1striationswere staining wasnotduetoanartifactfrominhibitionofphal- actin antibodiesdemonstratedthattheabsenceof (Fig. 2d,bottom).Additionally,stainingwithanti–cardiac MOPC-21 (Fig.2b)orinsurroundinguninjectedcells tin stainingincellsinjectedwiththecontrolantibody pared withthenormalstriatedappearanceofsarcomericac- fluorescently conjugatedphalloidin(Fig.2,dandf),com- matic lossofactinfilaments,asdeterminedbystainingwith with mAb17(Fig.2c)ormAb8e)exhibitedadra- Interaction ofTmod1andtropomyosin| with mAb17, ormAb9,followedbyincubat dots werepreincubatedwithmAb8, ing toTmod1.(d)Tmod1nitrocellulose detected abandat mAb17, ormAb8.mAb8andmAb17 full-length (lane1)andNH were probedwithmAb17thatrecogn (b) Westernblotsofsamplesinpanela 95–359, 6–94,and6–5,respectively). (lanes 2–6,residues6–184,35–359, (lane 1,1–359)andTmod1fragments blue–stained geloffull-lengthTmod1 action withtropomyosin. chick heartlysates,anddisruptitsinter- 6–57, recognizeTmod1inembryonic dramatic decreaseinthepercentageof dramatic or mAb8(opencircles)resultedina concentrations ofmAb17(closed bating theTmod1dotswithincreasing bind totheNH Figure 1. antibodies alone(2 antibodies extracts wereprobedwithsecondary (c) Westernblotsofembryonicheart COOH-terminal Tmod1(lanes3and4). Tmod1 (lanes2,5,and6),butnot 125 I-tropomyosin ( 80%) ofmyocytesinjected mAb17 andmAb8specifically 2 -terminal Tmod1resid

alone), MOPC-21, 40 kD,correspond- 125 Mudryetal. I-TM). Preincu- (a)Coomassie 2 -terminal squares) were ized 1059 ues ion The Journal of Cell Biology 1060 filaments (o).Bars,10 filaments binding siteresultedinalossof actin fragm 1 h.MicroinjectionoftheTmod1 into cardiacmyocytesandincubated for identify injectedcells)wasmicroinjected mixedwithMOPC-21(n,to 1–130, Tmod1 fragmentcontainingresidues this timepoint(m,arrows).Arecombin Actin filamentswereeasilyvisualizedat were incubatedfor48hbeforestaining. (l). Totestforrecovery,injectedcells ofactinfilamentswasseen perturbation injected withmAb17(kandl);no mAb95 (h).Fibroblastswerealsomicro- with MOPC-21(b,arrows)or filaments wereseeninmyocytesinjected bodies (j,arrowheads).Normalactin arrowheads) orusinganti– AlexaFluor 488phalloidin(dandf, a lossofactinfilamentsasdetectedby injection ofmAb17ormAb8resultedin injected antibody(a,c,ande).Micro- conj observed usinganAlexaFluor594– mAb95 (gandh).Injectedcellswere fragments (iandj),mAb8(ef),or and b),mAb17(cd),Fab (a were microinjectedwithMOPC-21 actin (thin)filaments. live cardiacmyocytesresultsinalossof between Tmod1andtropomyosinin Figure 2. actin disassemblywasaccompaniedbyof tin filamentswasreversible. (Fig. 2m).Thesedataindicatethatthelossofsarcomericac- mAb17 Fabs,buthadnodetectablestainingforthemAbs covery wasobservedinallcellsthathadbeeninjectedwith filament striationswasclearlyseenat24h.By48h,afullre- bly, attheselatertimepoints,apartialreappearanceofactin mAb17 Fabs,incubated24–48h,fixed,andviewed.Nota- numbered grids(toidentifyinjectedcells)werewith this experiment,cardiacmyocytesplatedoncoverslipswith antibodies bynewlysynthesizedTmod1beforefixation.In tial degradationofthemAb17Fabsand/orsequestration croinjected cellsforlongertimeintervalstoallowpoten- whether thephenotypewasreversiblebyculturingmi- antibodies hadtoxiceffectsonthemyocytes,wetested (Fig. 2,kandl). 1995) resultedinnoobservableeffectsonactinstaining fibers butnodetectableTmod1(GregorioandFowler, injection ofmAb17intofibroblaststhatcontainactinstress nonspecific effectsonactinfilaments,wefoundthatmicro- 1995). Inadditionalcontrolexperimentstoexamineforany tin filamentstriations(Fig.2,gandh;Gregorioetal., disrupts itscappingactivity,didnotresultinthelossofac- that recognizestheCOOH-terminalregionofTmod1and molecule (unpublisheddata),ormicroinjectionofmAb9, body thatrecognizesanepitopeclosetothemiddleof cytes. MicroinjectionofmAb95,anotheranti-Tmod1anti- Next, todeterminewhethermAb17-inducedTmod1and To determinewhetherinjectionofthefunction-blocking ugated anti–mouseIgGtodetectthe The JournalofCellBiology ent containingthetropomyosin Disruption oftheinteraction Cardiacmyocytes m. -actin anti-

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Volume 162,Number6,2003 ant

, wemicroinjectedarecombinantNH tibodies todisruptTmod1’sinteractionwithtropo- myocyte sarcomeres. sociation oftropomyosinwiththeactinfilamentsincardiac of Tmod1withtropomyosinisnecessarytostabilizetheas- had disassembled.Thisresultsuggestedthattheinteraction Fig. 4b),indicatingthatthe entire thinfilament aments (see clearly observed,whichisconsistentwiththelossofactinfil- stained fortropomyosin.Alossoftropomyosinstainingwas trop jected cellsthatwerebeatingattheonsetofexperiment cells injectedwithMOPC-21(TableI).100%oftheunin- to beat1hafterinjection,comparedwith cally diminishedincellsmicroinjectedwithmAb17.Only would beinhibited.Indeed,beatingactivitywasdramati- jected cellssuggestedthatcontractileactivityofthe is criticalforactinfilamentstability. pothesis thattheinteractionofTmod1withtropomyosin nous tropomyosin.Theseexperimentssupportthehy- prevented endogenousTmod1frombindingtoendoge- Tmod1 fragmentcontainingaminoacids1–130,likely dominant-negative mechanism;thatis,theinjected a of actinfilamentswasobserved(Fig.2o),likelydueto Fowler, 1994;Greenfieldand2002).Again,aloss taining thetropomyosinbindingsite(Babcockand fragment ofTmod1(1–130)intocardiacmyocytes,con- 5% ofthosemyocytesinjectedwithmAb17wereobserved As acomplementaryapproachtousinganti-Tmod1an- The dramaticdisappearanceofthinfilamentsinmicroin- omyosin, cellsmicroinjectedwithmAb17Fabswere 88% ofthose 2 -terminal The Journal of Cell Biology 1999). AfterinjectionwithmAb17,striatedGFP– in allmyocyteswhereitwasexpressed(Helfmanetal., sin assembledalongtheentirelengthofthinfilaments the pointed endsofthethinfilamentsinlivecells(i.e., pomyosin wasobservedtodissociatedirectlyfromthe fluorescence intensityofGFP– In contrast,injectionofMOPC-21hadnoeffectonthe was lostfromthesidesoffilamentsorZ-lines. time-lapse imagesofcellsexpressingGFP– 5.6 mechanism ofhowthisdisassemblyoccurred,wecollected of Tmod1withtropomyosinwasdisrupted.Toaddressthe stability wascompromisedincellswhichtheinteraction The datapresentedthusfarindicatedthatthinfilament 87.8 in livecardiacmyocytes Thin filamentsdepolymerizefromtheirpointedends 255 ity incardiacmyocytes. is criticalforthinfilamentstabilityandcontractileactiv- these datasuggestthattheTmod1–tropomyosininteraction 251 remained beatingafter1h(unpublisheddata).Insummary, No.ofinjected 17 MOPC-21 mAb Table I. (Fig. 3);i.e.,nodetectableGFP– gressive dissociationonlyfromthepointedendsupto60min the pointedendswasfirstnoticeableat30min,withpro- pointed endsgreatlydiminished).Thisdissociationfrom atsimilarintensities,whereastheintensityfrom mained We speculatethatthisslowerdepolymerizationeffectwas myocytes injectedwithmAb17,fixed,andthenstained. peared tooccurwithslowerkineticsthanthatobservedin ingly, thedepolymerizationeffectonthinfilamentsap- that weremicroinjectedwithmAb17.GFP– cardiac myocyte a P 0.001by GFP– mAb17 inhibitsthecontractileactivityofchick t test. -tropomyosin detectableattheZ-linesre- myocytes -tropomyosin. Interest- -tropomyosin intensity -tropomyosin Percentage of -tropomyo- cells beating 3.0 5.1 -tro- a pomyosin topreventactinfilamentdepolymerization. pointed ends(Fowleretal.,1993),functioningwithtro- bilize tropomyosin’sassociationwiththeactinfilament observation alsosuggeststhatTmod1actsasa“cap”tosta- ments fromtheirpointedendsincells.Furthermore,this thin filamentsisduetothedepolymerizationoffila- sults fromthisexperimentdirectlyshowthatthelossof that mayhavefurtherstabilizedthethinfilaments.There- in thestoichiometryoftropomyosinisoformexpression pomyosin (GregorioandFowler,1995)and/oralterations typically donothaveaconsiderablepoolofsolubletro- mAb17 Fabs, andM-line epitopesoftitin.Incellsinjectedwith I-band myosin, themajorthickfilamentcomponent;aswell due tooverexpressionof pattern for gions, closelyspacedremnantsofaperiodicstriatedstaining ments (Fig.4,d,f,h,andj,green).However,insomere- red), appeareddisruptedalongwiththelossofactinfila- red), andtitinN2AA168–170epitopes(Fig.4,hj, antibodies against usingAlexaFluor488phalloidin,togetherwith costained croinjected withMOPC-21FabsormAb17were disassembly hadonoverallsarcomereorganization,cellsmi- To determinetheeffectthatmAb17-inducedthinfilament and titinfilaments by perturbationofZ-lines,thickfilaments, Thin filamentdisassemblyisaccompanied Therefore, theperturbationofothersarcomericfilamentsys- thin filaments(vonArxetal.,1995;Linke1999). components, remainedrelativelyintactintheabsenceof organization ofthickfilaments,aswellothersarcomeric These datasupportpreviousstudiesthatrevealedthe in theabsenceofactinfilamentsidenticalmyofibrils. (corresponding toalternatingZ-andM-lines),wasobserved an alternatingstainingpatternof M-line (blue).InsomecellsinjectedwithmAb17Fabs, l) weretriplelabeledforactin(green), firm thisobservation,cellsinjectedwithmAb17Fabs(Fig.4 clearly intheabsenceofactin(Fig.4,arrowheads).Tocon-

Interaction ofTmod1andtropomyosin|

-, titin,andmyosinwereobserved

-actinin (Fig.4d,red),myosinf,

-actinin, themajorZ-linecomponent;

MOPC-21. Bar, was observedincellsmicroinjected with ends (a–f,arrows).Nodepolymerization filam injected withmAb17displayed thin 60 min).After30andmin,cells 1 h(datashownforpreinjection,30and Images wererecordedevery15minfor with MOPC-21(a–c)ormAb17(d–f). GFP– mAb17. visualized inlivecellsexpressingGFP– fromtheirpointedendswas filaments Figure 3.

-tropomyosin andmicroinjectedwith

-tropomyosin inmyocytesthat

ent shorteningfromtheirpointed

-tropomyosin weremicroinjected

Cardiacmyocytesexpressing Depolymerization ofthin -actinin andM-linetitin

5 m. -actinin (red),and Mudryetal. 1061 The Journal of Cell Biology 1062 detection. Actin filaments(Fig.5,dandg), aswellthe Fowler, 1995), asshownbyalackofimmunofluorescent b) andTmod1(Fig.5c), were removed(Gregorioand a) andthethinfilamentcomponents, tropomyosin(Fig.5 comeric componentsincluding thethickfilaments(Fig.5 ing themin0.5MKCl.Under theseconditions,manysar- permeabilizing cardiacmyocytes withsaponinandextract- Fowler, 1995).Inbrief,ghostmyofibrilswerepreparedby actin filamentsin“ghostmyofibrils”(Gregorio and the required thepriorreconstitutionoftropomyosinalong binding ofTmod1tothepointedendsactinfilaments tions. Usingthisassay,wepreviouslydemonstratedthat well asinterferewithspecificthinfilamentproteininterac- nents (intheabsenceofthickfilamentcomponents),as the assemblypropertiesofindividualthinfilamentcompo- constitution assay.Thisassayallowsustoselectivelystudy filament disruptionphenotype,weusedathinre- To gainadditionalinsightsintothemechanismofthin thin filamentdepolymerization interaction ofTmod1withtropomyosin,prevents reconstitution assay,beforethedisruptionof Stabilization ofactinfilamentsinathinfilament the thinfilamentsobserved. tems, aswellZ-lines,waslikelysecondarytothelossof (l, green).(inset)Enlargedviewofl.(arrowheads)Closelyspacedremnantsstriatedstainingpatterns.Bar,10 myocytes injectedwithmAb17FabsdisplayedalternatingM-linetitin(l,blue)and Myocytes injectedwithmAb17orFabsdisplayedaperturbedstainingpatternfor titin (iandj,red;kl,blue).MyocytesinjectedwithmAb17Fabsshowedalossoftropomyosinconsistentthe 1 h,fixed,andstainedfortropomyosin(ab,red), components. Figure 4. The JournalofCellBiology Disrupting theinteractionbetweenTmodandtropomyosininlivecardiacmyocytesresultsaperturbationofothersarcomeric Cardiac myocytesweremicroinjectedwithMOPC-21Fabs(a,c,e,g,i,andk)ormAb17(b,d,f,h,j,l),incubatedfo

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Volume 162,Number6,2003 -actinin (c,d,k,andl,red),myosin(ef,I-bandtitin(gh,red)orM-line addition ofmAb17 ormAb8withTmod1 wouldaffect merization ofthethinfilaments (seeDiscussion). Tmod1 andtropomyosinmust beintacttopreventdepoly- ments arecappedbyTmod1, theinteractionbetween These dataindicatethatonce theactin–tropomyosinfila- (unpublished data)hadno effect ontheactinfilaments. with Tmod1intheabsenceofreconstitutedtropomyosin sence ofTmod1(Fig.5,t–v),ortheadditionmAb8 MOPC-21 (Fig.5,h–k),theadditionofmAb8inab- bytheadditionofamixtureTmod1with followed intact cells.Incontrast,reconstitutionoftropomyosin phenotype thatappearedidenticaltoobservedin loss ofactinfilaments(Fig.5,mandq,respectively): a Tmod1 withmAb8(Fig.5,p–s),resultedinadramatic of recombinantTmod1withmAb17(Fig.5,l–o),or addition ofexogenoustropomyosinfollowedbyamixture room temperature(unpublisheddata).Remarkably,the major Z-linecomponent pomyosin plusTmodalone,werestablefor butions. Infact,actinfilamentsalone,orthosewithtro- (Fig. 5g)andassembledintheirtypicalsarcomericdistri- (Fig. 5f)werereconstitutedontheexistingactinfilaments rified tropomyosin(Fig.5e)andrecombinantTmod1 mained intactintheextractedcells(unpublisheddata).Pu- To determine ifstabilizingtheactinfilaments before -actinin (l,red)stainingintheabsenceofdetectableactin -actinin (d),myosin(f),andtitin(hj).Strikingly, -actinin, andtitinfilamentsre-

m.

actinfilaments (b). 60 minat r The Journal of Cell Biology addition ofthe Tmod1–mAb17mixture (Fig.6e)or mained striated alongthestabilizedmyofibrils afterthe (Fig. 6,c,g,andk)Tmod1 (Fig.6,d,h,andl)re- for phalloidintreatment).Strikingly, bothtropomyosin stitution oftropomyosinand Tmod1(Fig.6,datashown lized withjasplakinolideor phalloidin, beforetherecon- the lossofthinfilaments,actin filamentswerefirststabi- addition ofmAb8(t)intheabsencerecombinantTmod1hadno effectontheactinfilaments(u)ortropomyosin(v).Bars, mAb8 (p–s)resultedinthelossofactinfilaments(mandq),tropomyosin (nandr),Tmod1(os)o;rs,bot tropomyosin (j),andTmod1(k)uponadditionofMOPC-21(h i;jandk,bothcostained).Incontrast,theadditionofmAb17 with biotinylatedtropomyosinfollowedbyamixtureofTmod1 MOPC-21(h–k)showednodisruptionofthestriatedactinfila tropomyosin (e)andrecombinantTmod1(f)werereconstitutedonto theghostmyofibrils(g).(B)Cardiacmyocytesextractedand or Tmod1(c):allthreesarcomericcomponentswereextracted,whereas theactinfilamentswereunaffected(candd,costained). filaments. actin Figure 5. The disruptionoftheTmod1–tropomyosininteractioninpermeabilized andextractedcardiacmyocytesresultsinthelossof (A) Cardiacmyocytespermeabilizedwithsaponinandextractedin 0.5MKClwerestainedformyosin(a),tropomyosin(b), rect effectof disrupting theinteractionof Tmod1with the thinfilament disassemblyphenotypeobserved isadi- These datafromthereconstitution assayalsosuggestthat was sufficienttopreventthin filamentdepolymerization. disruption oftheinteraction ofTmod1andtropomyosin, the actinfilamentsviaphalloidin orjasplakinolide,before Tmod1–mAb8 mixture(Fig. 6i).Thus,stabilizationof Interaction ofTmod1andtropomyosin| Mudryetal. h costained).The reconstituted 10 Biotinylated

(l–o) or

ments (i), m. 1063 The Journal of Cell Biology 1064 tropomyosin (gandk),Tmod1(hl),whichwerestabilizedbyphalloidin(e,f;i,j;g,h;k,l,allcostained).Bar,10 both costained).Notably,theadditionofmAb17(e–h)ormAb8(i–l)alongwithpurifiedTmod1hadnoeffectonactinfilame MOPC-21 wasaddedtothemyocytes(a–d)andhadnoapparenteffectonactinfilaments(b),tropomyosin(c),Tmod1(d)(aan wereextractedandactinfilamentsstabilizedwithAlexaFluor488phalloidinbeforetheaddition oftropomyosina myocytes cytes withGFP– mechanism ofthisobservedphenotype,wetransfectedmyo- cessation ofcontractileactivity.Tofurtherinvestigatethe sulted inastrikingphenotype:lossofthinfilamentsand taining thetropomyosinbindingsite.Theseapproachesre- tinct structural andligand-bindingregions, the NH vertebrate muscle. ForTmod1,wefoundthat itstwodis- proposed roleasathinfilament stabilizingcomponentin For tropomyosin,ourstudy provides directsupportforits exciting, novelfunctionsforboth tropomyosinandTmod1. with phalloidinorjasplakinolide. Theseobservationsreveal prevented bythepriorstabilization oftheactinfilaments bation oftheinteractionTmod1withtropomyosin, is further revealedthatthelossofthinfilaments,uponpertur- their pointedends.Resultsfromacellpermeabilizationassay that thethinfilamentsdepolymerizedprogressivelyfrom to visualizethelossofthinfilamentsinrealtime,revealing cells withmAb17.Thislivecellimagingstrategyallowedus lengths ofthethinfilaments,andthenmicroinjectedthese Discussion against theNH interaction bymicroinjectingfunction-blockingantibodies pomyosin, inlivechickcardiacmyocytes.Wedisruptedthis Tmod1 withoneofitsthreeknownbindingpartners,tro- In thiswork,wespecificallyblockedtheinteractionof regulatory, orsarcomericcomponents. tropomyosin andislikelynotinfluencedbyothersoluble, Figure 6. jecting arecombinantNH The JournalofCellBiology Stabilization ofthinfilamentsbyphalloidinbeforeadditionmAb17ormAb8preventsfilamentdisruption. 2 -terminal regionofTmod1andbymicroin- -tropomyosin, whichassembledalongthe

2 | -terminal Tmod1fragmentcon-

Volume 162,Number6,2003 2 -termi- because binding oftheterminaltropomyosin moleculeat ments (Broschat, 1990;Weberetal.,1994, 1999).Thisis pomyosin overtheamountnecessary tocoattheactinfila- depends onthepresenceofa considerable excessoffreetro- vent actindepolymerizationfrom thepointedendsinvitro aments. However,theability oftropomyosinalonetopre- slowly fromthepointedends compared withnakedactinfil- with tropomyosin(butlackingTmod1)depolymerizemore trop polymerize intolong,stablefilamentsintheabsenceof are stableintheabsenceofTmod1.Forexample,actincan studies, thatshowtropomyosin–coatedactinfilaments zling basedonmanyobservations,mainlyfrominvitro interactions arecriticalforthinfilamentstabilitypuz- and stabilityofthethinfilaments. tein incardiacmuscle,involvedmaintainingthelengths gether, weconcludethatTmod1isamultifunctionalpro- pointed ends(Gregorioetal.,1995)(Fig.7,IV–VI).To- abnormal elongationofthethinfilamentsfromtheir vealed thatTmod1’scappingactivityisrequiredtoprevent hibiting theinteractionofTmod1withactin,whichre- is strikinglydifferentfromthephenotypeobtainedin- cardiac myocytes(Fig.7,I–III).Thephenotypeweobserved thin filaments,bypreventingtheirdepolymerizationinlive at thepointedendsiscriticalformaintainingintegrityof live myocytes.TheinteractionofTmod1withtropomyosin the thinfilamentsthatcanbedistinguishedincontextof tin cappingdomain,haveuniquerolesatthepointedendsof nal tropomyosin-bindingdomainandCOOH-terminalac- The exactmechanismsbywhichTmod1–tropomyosin omyosin andTmod1;actinfilamentssaturated m. d b;cand d, nts (fandj), nd Tmod1. Cardiac

The Journal of Cell Biology through itsinteractionwithtropomyosin,andinhibitingelongation/maintenanceofactinfilamentslengthsca pointed ends(VI).TheseresultssuggestatleasttwofunctionalrolesforTmod1:stabilizing/preventingdepolymerizationofth Tmod1; however,theaddition oftheanti–NH ghosts) intheabsence(orpresence) oftropomyosinand appear toberelativelystable inextractedcells(myocyte myocyte thinfilamentreconstitutionassays.Actinfilaments not fullyaccountforasurprisingresultfromourcardiac actin dynamics. affecting pointedendstability,aswelltropomyosinand both theterminaltropomyosinandactinmolecules,thereby or mAb17.Inthismodel,Tmod1functionsasacapfor Tmod1–tropomyosin interactionswereinhibitedbymAb8 actin filament depolymerization,asweobservedwhen by polymer fromthethinfilaments.Thiswouldbefollowed tive ecule atthinfilamentpointedendsfollowedbycoopera- lead toweakenedbindingoftheterminaltropomyosinmol- Tmod1–tropomyosin interactionsincardiacmyocytesmay ment pointedendsinvivo.Thus,specificperturbationof chor interactions withtropomyosinmaybeessentialtoan- sin concentrationsarelimiting),wespeculatethatTmod1 sembled ontothethinfilamentsandasaresult,tropomyo- and Fowler,1995)(i.e.,alltheavailabletropomyosinisas- cess freetropomyosininchickcardiacmyocytes(Gregorio ends (Weberetal.,1999).Becausethereisessentiallynoex- ability tobindbothactinandtropomyosinatthepointed tropomyosin moleculetothethinfilamentsbyvirtueofits may specificallypromotestrongerbindingoftheterminal depolymerization (Weberetal.,1994,1999).Thus,Tmod1 enhances theabilityoftropomyosintoreducepointedend pointed endsaresignificantlymorestablebecauseTmod1 coated withtropomyosinandcappedbyTmod1attheir ner, 1979;Weberetal.,1999).Incontrast,actinfilaments neighboring tropomyosinsalongthethinfilaments(Weg- the othertropomyosinmolecules,whichself-associatewith the pointedfilamentendissignificantlyweakerthanthatof In otherwords, intheseexperiments,Tmod1 plusmAb8or ity onwhatwould otherwisebearelatively stable structure. Tmod1 (Fig.7III)suchthat it catalyticallyimpartsinstabil- nal Although thismechanismisanattractivemodel,itdoes Tmod1 antibodiesmAb8ormAb17 appeartotransform dissociation or“unzipping”oftheentiretropomyosin the terminaltropomyosinmoleculestothinfila- 2 -termi- assembly ofI-Z-I bodies,precursorstructures thatmature of thishypothesis, nebulinparticipatesinthe initialstagesof al., 2003;forreviewseeMcElhinny etal.,2003).Insupport in cardiacmuscle(Fockand Hinssen, 2002;Kazmierskiet of thethinfilamentsinskeletal muscleand,morerecently, cule forfunctioningasa“ruler” tospecifythepreciselengths tropomyosin-binding protein, astheprimecandidatemole- ever, severalgroupshaveidentifiednebulin,theTmod1and that Tmod1isinvolvedinspecifyingtheirlengths.How- and stabilizingthinfilamentlengths,thereisnoevidence this occursremainstobedetermined. tion andoverallstabilityofthethinfilaments.Exactlyhow and Tmod1butarethecriticalsiteforbothlengthregula- with respecttotherapidexchangeofactin,tropomyosin, pointed endsofthethinfilamentsarenotonlydynamic These studiesandourresultsindicatethattheslow-growing, aments inadultratcardiacmyocytes(Micheleetal.,1999). preferentially assembleontothepointedendsofthinfil- molecules, butnotepitope-taggedtroponinI field etal.,2001).Additionally,epitope-taggedtropomyosin pointed endsinchickembryoniccardiacmyocytes(Little- actin monomersrapidlyassociateanddissociatefromthe nents aredynamic?GFP-Tmod1andfluorescentlylabeled thin filamentsgiventhefactthatpointedendcompo- does Tmod1functionwithtropomyosininstabilizingthe dressing theseintriguingissues. interactions areperturbed.Ourcurrentexperimentsad- the filamentstobecomeunstableifTmod1–tropomyosin once Tmod1hasassociatedwiththepointedends,causing ble thatastructuralalterationoccursinthethinfilament of thefilament,resultinginitsdisassembly.Itisalsoplausi- sion), causingapropagateddestabilizationdownthelength act differentlywiththegiantproteinnebulin(seeDiscus- teractions. Alternatively,suchanalteredTmod1mayinter- now activelydisruptsactin–actinoractin–tropomyosinin- tibodies leadstoastructuralalterationinTmod1sothatit pomyosin–actin filaments.Perhapsthebindingofthesean- mAb17 appearstobehavelikea“depolymerase”fortro- Although Tmod1isacriticalcomponentformaintaining An interesting,yetcomplex,questionthatarisesis,how Interaction ofTmod1andtropomyosin| elongation ofactinfilamentsatthe capping activitywithmAb9resultsinan (V) Incontrast,disruptingTmod1’sactin depolymerization fromthepointedends. inalossofthinfilamentsvia results is unknown.(III)Theconferredinstability new functionality,buttheexactalteration meant toindicatethatTmod1acquiresa filaments. Note,theshapechangeis atthepointedendsofthin instability of Tmod1 (bluesquare),whichimparts toalterthefunctionalproperties appears (red) withmAb17ormAb8(black) ofTmod1andtropomyosin interaction Tmod1 (bluecap).(II)Disruptingthe actin (gray),tropomyosin(red),and Stable thinfilamentsarecomprisedof functional domainsofTmod1. Figure 7. Model oftwoidentified Mudryetal. in filaments pping activity. (I andIV) 1065

The Journal of Cell Biology 1066 for propermyofibrilassembly,structure,andfunction. these threethinfilamentcomponentsappeartobeessential and stability.Furthermore,theregulatedinteractionsamong mentary, criticalrolesincontrollingthinfilamentlengths ulate thatnebulin,tropomyosin,andTmod1playcomple- lengths ofthethinfilamentsattheirpointedends.Wespec- tropomyosin polymers,thusstabilizingandmaintainingthe Once Tmod1assembles,itfunctionstocaptheactinand target Tmod1tothepointedendsofthinfilaments. axolotl; thatis,whenmouse portant formyofibrillogenesisintheamphibian,Mexican ray (Sehnertetal.,2002).Furthermore,tropomyosinisim- pomyosin levels,causingsarcomerelossandmyocytedisar- heart (sih)mutantsresultedinasignificantreductiontro- (TNNT2) mutants;thelossofTnnt2expressioninsilent myofibrillogenesis basedonanalysesofcardiactroponinT been implicatedinplayinganimportantrolezebrafish sembly mediatedbycofilin/ADF.Tropomyosinhasalso between actinstabilizationbytropomyosinanddisas- dicated thatactinfilamentassemblydependsonabalance strips wereincubated witheither0.5 buffer, runona10% gel,andtransferredtonitrocellulose. Nitrocellulose ground inliquidnitrogen.Thepowder wassolubilizedin2 embryonic chickhearts.Inbrief,hearts weredissected,snap-frozen,and probed asdescribedpreviously(Gregorio etal.,1995). massie bluestainingandWesternblot analysis,respectively.Blotswere Tmod1 andequivalentmolaramounts offragments,wereloadedforCoo- 0.5 Western blotanalysis erated in Recombinant full-lengthchickenTmod1andfragmentsweregen- Purified proteinsandanti-Tmod1antibodies Materials andmethods cofilin/ADF) mutantin Specifically, geneticanalysisoftheUnc-60B(homologue gested acriticalrolefortropomyosininmyofibrilassembly. myofibril assembly.Importantly,recentreportshavesug- act withtropomyosinasthinfilamentstabilizersduring et al.,1996;MoncmanandWang,1996).Nebulinmayalso filament architectureandrestrictslengths(Shimada which isconsistentwiththeideathatnebulindictatesthin pattern beforethethinfilamentsattaintheirmaturelengths, ditionally, inmanystudiesnebulinisobservedastriated into definitiveZ-linesandI-bands(Ojimaetal.,1999).Ad- tain theirmaturelengths,nebulin’sNH ment assembly:whentheactin–tropomyosinfilamentsat- is temptingtospeculatethefollowingmodelofthinfila- tivity wererestored(Zajdeletal.,1998).Giventhesedata,it pomyosin expression),myofibrillogenesisandcontractileac- into mutanthearts(thatshowedareductionofoveralltro- described previously(Fowler,1990). Q anionexchangecolumn.Rabbitskeletaltropomyosinwaspurified as pain-conjugated beads(Sigma-Aldrich),followedbyisolationonaMono MOPC-21 weregeneratedbyincubatingthepurifiedantibodieswithpa- Aldrich), wasusedasacontrol(Gregorioetal.,1995).FabsofmAb17and An irrelevantmonoclonalantibodyofthesameisotype,MOPC-21(Sigma- lyzed to1.0mg/mlininjectionbuffer(1mMTrisand25KCl,pH7.4). column (AmershamPharmaciaBiotech).Thepurifiedantibodiesweredia- from hybridomasupernatantsusingaproteinG–Sepharose4FastFlow ated byC.Grant(CustomMonoclonals,Sacramento,CA)andpurified Monoclonal antibodiesagainstrecombinantchickenTmod1weregener- The specificityoftheanti-Tmod1antibodies wasdeterminedfromday6 g offull-lengthorfragmentsTmod1, and20ngoffull-length The JournalofCellBiology Escherichia coli as describedinBabcockandFowler(1994). C. elegans

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Volume 162,Number6,2003 -tropomyosin wasintroduced g/ml ofMOPC-21, mAb8,mAb17 (OnoandOno,2002)in- 2 -terminal modules SDSsample mAb/Tmod dots wereincubatedwith330nMmAb8orMOPC-21(molarratioof MgCl muscle tropomyosininbindingbuffer(20mMHepes,80KCl,2 identification ofmicroinjectedcells. was performedintriplicate.Numberedgridsoncoverslipsallowedforthe mined. Greaterthan60cells/coverslipwereanalyzedandtheexperiment 37 cultured for4dwithmAb17orMOPC-21.Cellswereincubated1hat pendorf). Injectedcellswereincubatedfor1–48hbeforefixation. jector (model5246;Eppendorf)andmicromanipulator5171;Ep- mAbs, Fabs,orpurifiedTmod1fragmentsininjectionbufferusingaTrans- Cells culturedfor3–5dwereinjectedwitha0.3–1.0-mg/mlsolutionof mm culturedishescontainingCELLocategriddedcoverslips(Eppendorf). gorio andFowler,1995).Isolatedcellswereplatedat10 Cardiac myocyteswereisolatedfromday6embryonicchickhearts(Gre- Cell cultureandmicroinjectionprocedures 0–700 nMofmAb8,9,or17for3hatRTinafinalvolume200 nitrocellulose asspotsusingadotblotapparatusandpreincubatedwith 5 pmoloffull-lengthrecombinantchickenTmod1wereabsorbedonto Antibody competitionanddissociationassays with 7 pEGFP–rat– For livecellimaging,transfection was performedbyincubating1 Transfection andlivecellimaging convolved usingDeltaVisionsoftware(AppliedPrecision). sections) usingaCCDcamera(modelSeries300;Photometrics)andde- graphs wererecordedasdigitalimages(withZ-seriescontaining0.15- Cells wereanalyzedonamicroscope(modelIX70;Olympus).Micro- antibodies werepurchasedfromJacksonImmunoResearchLaboratories. phalloidin werepurchasedfromMolecularProbes.Allotherfluorescent bated inAlexaFluor594phalloidin.AlexaFluor-conjugatedantibodiesand conjugated donkeyanti–rabbitIgGantibodies(1:600).Cellswereincu- antibodies (10 mouse Fc-specificIgG(1:100).Anti–titinA168-A170(1:100)ortitinN2A Stanford University,Stanford,CA)followedbyFITC-conjugatedanti– 1984), oranti–myosinF59antibodies(1:10;providedbyF.Stockdale, and E.Ehler,InstituteforCellBiology,Zurich,Switzerland;Groveetal., Aldrich), anti–myomesinB4antibodies(1:50;providedbyJ-.C.Perriard monoclonal sarcomericanti– Fabs andincubatedfor1hbeforefixation.Cellswerewith bered gridsonthecoverslipwereinjectedwithmAb17FabsorMOPC-21 IgG (Fcspecific)antibody(1:100).Fortriplelabeling,cellswithinnum- search Products)followedbyadonkeyTexasred–conjugatedanti–mouse monoclonal anti–cardiacactinantibody(Ac1-20.4.2;1:10;AmericanRe- was visualizedusinganAlexaFluor488–,594–or647phalloidin,a anti–mouse IgG(Fabspecific;1:100)todetecttheinjectedantibody.Actin gated goatanti–mouseIgG(1:1,000)oradonkeyTexasred–conjugated 30 min.MicroinjectedcellswerestainedwithanAlexaFluor594–conju- for 15min.Coverslipswereblockedin2%BSA/1%donkeyserum/PBS for 10min,washedinPBS,andpermeabilized0.2%TritonX-100/PBS Cardiac myocyteswerefixed1hafterinjectionin5%formaldehyde/PBS Indirect immunofluorescenceanddeconvolutionmicroscopy overnight at4 centration used(700nM).Afterseveralwashes,dotswereincubated lowest antibodyconcentrationused(33nM)to28:1atthehighestcon- nM Tmod1).ThemolarratioofmAbtoTmod1variedfrom1.3:1atthe fied ina strate (PierceChemicalCo.)andexposedtofilm(BiomaxMR;Kodak). BlotswereincubatedinSuperSignalchemiluminescentsub- (1:20,000). or 1.0 nM Tmod1 wereadsorbedontonitrocellulosedotsandincubatedwith33 to removeunboundtropomyosin.Forthedissociationassay,5pmol the directionofdepolymerization. 15 min.Stainingwith anti– ber apparatus(Bioptechs)andZ-series ofinjectedcellswereimagedevery mAb17 orMOPC-21.Coverslipswere placedintoaFochtLive-cellCham- cytes culturedfor24h3–4dafter transfection, cellswereinjectedwith Technologies) for15minatRTand addingthemixturetocardiacmyo- Beating assayswereperformedbyinjectingbeatingcardiacmyocytes C andthepercentageofinjectedcellsthatwerebeatingwasdeter- 125 2 ,1 mMDTT,0.2%TritonX-100,and20mg/mlBSA),washed I-tropomyosin overnightat4 g/ml mAb17FabsfollowedbyHRP-conjugatedanti–mouseIgG l Cytofectene(Bio-RadLaboratories) and100 counter. -tropomyosin (giftfromJ.-C.Perriard; Helfmanetal.,1999) 13:1).Theamountofbound C with33nM g/ml) (Centneretal.,2000)wereadded,followedbyCy5- -actinin antibodies allowedustodetermine -actinin antibodies(1:1,500;EA-53;Sigma- 125 I-Bolton Hunter–labeledrabbitskeletal C. Afterseveralwashes,theTmod1 125 I-tropomyosin wasquanti- 6 l Opti-MEM(Life cells/dishin35- l (25 m g

The Journal of Cell Biology Chu, X.,J.Chen,M.C.Reedy,C.Vera, K.L.Sung,andL.A.Sung.2003.E-Tmod nofluorescence anddeconvolutionmicroscopy. and Tmod1,fixed,stainedasdescribedinthesectionIndirectimmu- treated withactin-stabilizingagentswerereconstitutedtropomyosin Probes) inDMSOfor7minafterthecellswereextracted.Myocytes aFluor 594phalloidinfor20minor1 din. StabilizationofactinfilamentswasperformedbyaddingeitherAlex- IgG antibodies(1:500).ActinwasvisualizedusingAlexaFluor594phalloi- 1844 antibodies(1:100)followedbyCy5-conjugateddonkeyanti–rabbit din (1:200;ZymedLaboratories)andTmod1usingrabbitanti-Tmod1 fixed andstainedforbiotinylatedtropomyosinusingFITC-conjugatedavi- EGTA, 10mMTris-HCl,pH6.8,and0.2mg/mlsaponin)thecellswere min. Afterrinsingthecellsinrigorbuffer(60mMKCl,5MgCl 0.2 mg/mlofmAb17,mAb8,MOPC-21or0.05mAb17Fabsfor25 KCl, 5mMTris-HCl,pH6.8,0.1CaCl 10 minatRT.Extractedcellswerewashedinincubationbuffer(20mM Centner, T.,F.Fougerousse,A.Freiburg, C.Witt,J.S.Beckmann,H.Granzier,K. Broschat, K.O.,A.Weber,andD.R.Burgess.1989.Tropomyosinstabilizesthe Broschat, K.O.1990.Tropomyosinpreventsdepolymerizationofactinfilaments Blanchard, E.M.,K.Iizuka,M.Christe,D.A.Conner,A.Geisterfer-Lowrance,F.J. Babcock, G.,andV.M.Fowler.1994.Isoformspecificinteractionoftropomodulin Almenar-Queralt, A.,A.Lee,C.A.Conley,L.RibasdePouplana,andV.M. Adami, R.,O.Cintio,G.Trombetta,D.Choquet,andE.Grazi.2002.Effectsof References Accepted: 29July2003 Submitted: 7May2003 and NSFDBI9912036 (toC.N. Perry). National ScienceFoundation(NSF)predoctoralfellowship(toR.E.Mudry), HL57461, HL03985(toC.C.Gregorio),and GM34125 (toV.M.Fowler), thoughtful comments. Mary Wooforhelpwithantibodygeneration;andtheGregoriolab alt forassistanceinperformingtheexperimentsthatledtothisproject; cellent technicalassistance;KathleenKunkeandAngelsAlmenar-Quer- and AdamGeachforisolatingcardiacmyocytes;JeanetteMoyerex- Abigail McElhinnyandYasukoOnoforediting;YewenWu,JoeBahl, We wouldliketothankAbigailMcElhinnyforinsightfuldiscussions; MgCl cubated withrecombinantTmod1at70 mM KCl,and0.1MHepes,pH7.5for10min,rinsed,thenwerein- cells werefirstincubatedwith50 AlexaFluor 488phalloidin.ToreconstituteTmod1ontothemyofibrils, followed byAlexaFluor594–conjugatedgoatanti–mouseIgG(1:800)and Tmod1 monoclonal95antibodies(1:50;Almenar-Queraltetal.,1999) with anti–myosinF59,anti–tropomyosinCH1(Linetal.,1985),oranti- saponin) for15s.Toconfirmtheextraction,cellswerefixedandstained (0.12 MKCl,4mMMgCl 1995). Day6cardiacmyocyteswerepermeabilizedinrelaxingbuffer The assaywasperformedasdescribedpreviously(GregorioandFowler, Cell permeabilizationassay MgCl in ahighsaltbuffer(0.5MKCl,10mMsodiumpyrophosphate,5 ATP, and0.2mg/mlsaponin)for10minat0 This workwassupportedbyNationalInstitutesofHealthgrants 2 2 mouse embryoniccirculation. capping ofactinfilaments attheslow-growingendisrequired toestablish study oftitin’sdifferentialexpression. Trombitas, C.C.Gregorio,andS.Labeit. 2000.Moleculartoolsforthe 28:8501–8506. pointed endofactinfilamentsbyslowing depolymerization. from thepointedend. ablation ofthemurine Schoen, D.W.Maughan,C.E.Seidman,andJ.G.Seidman.1997.Targeted 27510–27518. with skeletalmuscleanderythrocytetropomyosins. Biol. Chem. pomodulin, thatcapsactinfilamentpointedendsinfastskeletalmuscle. 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