The Journal of Cell Biology
JCB
Ryan E.Mudry, expressing greenfluorescent protein– volution microscopy. Real-timeimagingoflive myocytes thin filaments,asrevealed by immunofluorescence decon- into chick cardiacmyocytes causedadramatic lossofthe tropomyosin 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 actin 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 myofibril 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