The Journal of

JCB

and Joshua R.Sanes and itsneuromuscular andmyotendinous junctions isoforms of Tyrosine-phosphorylated andnonphosphorylated the ofmusclecells.Micelacking ( produces two main nous junctions(MTJs).Innormalmuscle, junctions (NMJs)and,asshown here,abnormalmyotendi- muscular dystrophy, defectsinmaturation ofneuromuscular , ,( include (Ervasti andCampbell,1993).ConstituentsoftheDGC intracellular cytoskeletonandtheextracellularbasallamina forms amultimolecular,transmembranelinkbetweenthe found throughoutthesarcolemmaofskeletalmusclecells, whichis The dystrophin–glycoproteincomplex(DGC),* common NH 3 1 dominant isoforminextrajunctional regions,andisalso phosphorylated, isconcentrated attheNMJsandMTJs. Introduction R. Mark Grady, finding thatmutationsingenesencodingnumerousDGC al., 2002).InterestintheDGCstemmedoriginallyfrom al., 1990;YoshidaandOzawa,forreviewseeBlake et  rBTX, rhodamine junction; NMJ,neuromuscular nNOS,nitricoxidesynthase; dystrobrevin; DGC,dystrophin– complex;MTJ,myotendinous *Abbreviations usedinthispaper: AChR,acetylcholinereceptor;DB, [email protected] Louis, MO63110.Tel.:(314)286-2796.Fax:286-2892.E-mail: ington UniversitySchoolofMedicine,PediatricResearchBldg.,St. Address correspondencetoR.MarkGrady,Dept.ofPediatrics,Wash- http://www.jcb.org/cgi/doi/10.1083/jcb.200209045 The Journal ofCellBiology junction; junction; neuromuscular Key words: myotendinous dystrobrevin; dystrophin;musculardystrophy;

Department ofCellandDevelopmentalDepartment Biology, State University ofNew York Upstate Medical University, Syracuse, NY13210 ofPediatricsDepartment and St. Louis,MO63110

DB2, which isnottyrosinephosphorylated,thepre- DB1, whose COOH-terminalextensioncanbetyrosine 2), dystrobrevins(DBs; TheRockefeller University Press, 0021-9525

Article

dystrophin–glycoprotein complex,isfoundthroughout -Dystrobrevin (DB),acytoplasmic componentofthe

2

-terminal butdistinctCOOH-terminaldomains.

,

-bungarotoxin.

1 Mohammed Akaaboune, ,

, , Volume 160, Number 5,March 3,2003 741–752

DB isoforms,

2

,

and

and

2

-dystrobrevin: roles inskeletalmuscle

Department ofAnatomyandNeurobiology,Department Washington University SchoolofMedicine,

), (

), andsarcospan(Ervastiet

/2003/03/741/12 $8.00

DB1 and

2

AlexanderL.Cohen,

DB exhibit

1, DB2, with

and

1, and

), Roles oftheDGChavebeen lessextensivelystudiedatthe et al.,2000;Jacobson2001; Akaabouneetal.,2002). etal.,1997a;Grady1997a, 2000;Adams (Deconinck (AChRs) embeddedwithin thepostsynapticmembrane ations inthedensityandpatterning ofacetylcholinereceptors ness of ness of folds). Site-directedmutagenesisrevealed thattheeffective- turnover, andlow density)andMTJs(shortenedjunctional NMJs (abnormalacetylcholine receptorpatterning,rapid dystrophies; andmutationsinlaminin of thesarcoglycanshavebeenlinkedtolimb-girdlemuscular results inDuchennemusculardystrophy;deficienciesfour components causemusculardystrophy:lossofdystrophin with distinctsignalingorstructural properties. in muscleby meansofdifferentialdistributionisoforms , utrophin, of thepostsynapticmembrane.Forexample,myotubeslacking but contributesimportantlytothematurationandstabilization for initialstepsinpostsynapticdifferentiation, dispensable form load-bearingattachmentstotendons.TheDGC is myotendinous junctions(MTJs),atwhichmusclefibers (NMJs), atwhichmotoraxonsinnervatemusclefibers,and domains onthemusclefibersurface:neuromuscularjunctions importantinformationormaintenance of twospecialized also dystrophy (CohnandCampbell,2000;Blakeetal.,2002). cellular ligandfortheDGC,leadtocongenitalmuscular however, only isoform in present atNMJsandMTJs. Transgenic expressionofeither ability toserve asatyrosinekinasesubstrate. Thus, remodeling. Moregenerally, phosphorylation may beakeyregulatorypointforsynaptic In additiontoitsroleinmyofiberintegrity,theDGCis 2 Margaret M.Maimone, DB1 instabilizingtheNMJdependspartonits DB / DB1 completelycorrecteddefectsatthe miceprevented musclefiber degeneration; 1-, or DB may play multipleroles 3 Jeff W. Lichtman, 2,themainextra- DB havealter- DB1 741 2

The Journal of Cell Biology 742 junction; furthermore,only membrane, theirdetailedlocalizationdifferswithinthe sequences andtheshort(16aa)COOHterminusof al., 1993;Balasubramanianet1998),whereascommon able tomaintain musclestabilitybutthat COOH termini(Fig.1A).The188aaterminusof identical overmostoftheirlength(551aa)buthavedistinct Enigk andMaimone,1999;Neweyetal.,2001a)Theyare muscle (Blakeetal.,1996;Sadoulet-Puccio ing, ofwhichthree, MTJs. Thus, how isoformdiversitycontributestofunctionaldiversity. both other isoformsandhasnotbeenstudiedindetail.Although syntrophin- anddystrophin-bindingsitespresentinthe do notappeartoundergophosphorylation. on anotherDGCcomponent, distribution (KramarcyandSealock,2000).Here,wefocus wise, eachofthethreesyntrophinshasadistinctsarcolemmal MTJ (Khuranaetal.,1991;Ohlendieck1991).Like- autosomal homologue,utrophin,isconfinedtotheNMJand dystrophin ispresentthroughoutthesarcolemma,whereasits least, fromsitetowithinasinglecell.Inmusclefibers, Moukhles andCarbonetto,2001)and,formusclecellsat among celltypes(Straubetal.,1999;Loh2000; roles, butoneimportantfactoristhatitscompositionvaries coninck etal.,1997b). or bothdystrophinandutrophin(Ridgeetal.,1994;De- MTJs arestructurallyabnormalinmicelackingdystrophin et al.,1991;Chang1996;Crosbie1999]),and and nNOS[Chenetal.,1990;Byers1991;Khurana region (e.g.,dystrophin,utrophin,syntrophin,, MTJ, butseveralDGCcomponentsareconcentratedinthis Previously, weshowedthat in skeletalmuscle(Petersetal.,1997a;Blake1998). 1997b, 1998;Sadoulet-Puccioetal.,1997).Ahomologue, phin (Carretal.,1989;Wagner1993;Peters etal muscle,whereitbindsdystrophin,utrophin,andsyntro- nous phenotypes.Weshow that either form to“rescue”thedystrophic, synaptic,andmyotendi- 2001a). Basedonthesedifferences,wehypothesizedthat in extrasynapticregions(Petersetal.,1998;Newey addition, weshowherethat NMJs (Gradyetal.,1999,2000;Akaaboune2002).In both musculardefects(musculardystrophy)andabnormal phosphorylation of Salter, 2001),wewantedtotestthetheorythattyrosine lates theplasticityofneuron–neuronsynapses(Aliand tion, basedonevidencethattyrosinephosphorylationregu- locations withinthemusclecell. or structure. DB, hasbeendescribedbutisexpressedatlowlevelsifall DB isfoundthroughoutthesarcolemmainvertebrateskel- DB2 in DB1 and DB1 isasubstratefortyrosinekinasesinvivo(Wagneret Little isknownabouthowtheDGCplaysthesedisparate Several isoformsof To directlyassesstheseideas, weexpressed The JournalofCellBiology DB1 and DB DB2 mighthavedistinctfunctions.Inaddi- DB influencesDGCfunctionatthreedistinct / miceandanalyzedtheability ofeachiso- DB2 areconcentratedatthepostsynaptic DB1 affectsneuromuscularmaturation DB1–3, havebeendetectedinskeletal DB aregeneratedbyalternativesplic-

|

Volume 160,Number5,2003 DB DB2 ispresentathighlevels DB DB, toaddresstheissueof / / knockoutmiceexhibit micehavemalformed DB1 or DB1 issignifi- DB3 lacksthe

DB1 or

DB2 is

DB2

Results cantly betterthan to showthattheenhancedefficacyof tendinous defects.Wealsousedsited-directedmutagenesis on itstyrosinephosphorylation. from themuscle creatinekinasegenewerelinked tocDNAs We generatedtransgenicmice inwhichregulatoryelements Generation oftransgenicmice Figure 1. in bothtransgeniclines. with apan-DBantibody. in kD.(C)Immunostainingofskeletalmusclefromthefourgenotypes each genotype(wt;wild-type).Molecularweightmarkersareshown pan-DB antibody.Equalamounts(50 vivo (P).(B)Immunoblotofwholemuscleextractsstainedwitha area), andsitesin dystrophin (DYS;blackarea),sequencesuniqueto shown arebindingregionsforsyntrophin(SYN;hatchedarea)and recognized byanti- showing thetwomainisoformsexpressedinmuscleandregions

Generation oftransgenicmice.

DB1 thatundergotyrosinephosphorylationin

DB antibodies(pan-DB,DB1,andDB2).Also

DB2 inpreventingsynapticandmyo-

DB isexpressedin

g) ofproteinwereloadedfor (A)Structureof DB1 dependsinpart 95% ofmusclefibers DB2 (grayhatched DB, The Journal of Cell Biology recombinant similar inthetgDB1andtgDB2lines(Fig.1B).Levelsof isoforms showedthatlevelsoftransgeneexpressionwere firmed withtheotherlines(seeMaterialsandmethods). and 28,respectively.However,themainresultswerecon- figures, thedesignationstgDB1andtgDB2refertolines12 present in levels oftotal anterior musclesof the diaphragm,quadriceps,soleus, sternomastoid,andtibialis fer intheirabilitytomaintain muscular integrity,weexamined (Grady etal.,1999).Totest whether and centrallynucleatedregenerating myotubes(Fig.2) ized bydegeneratingmyofibers, infiltratingmonocytes, cantly higherthenlevelsofendogenous or regenerationwasseenineither degenerated. Nohistologicalevidenceformusclefiberdegeneration nucleated, indicatingthatsomeoftheinitialcohortfibershad mice. The diaphragm providedaparticularly stringenttest encoding Figure 2. Muscular dystrophy skeletal musclefromwild-type, show highpowerimages. combinant tochemical analysisshowedthat that expressed lated withfibertype(unpublisheddata). there werenodetectabledifferencesamongfibersthatcorre- cluding tibialisanterior,sternomastoid,anddiaphragm, the transgenewasexpressedinallskeletalmusclestested,in- transgenic linesthatexpressed of endogenous in DB DB DB DB Immunoblotting withantibodiesthatrecognizeall DB / / / / ,tgDB2 mice.Regeneratedmusclefibersarecentrally / ,tgDB2 mice,respectively(Fig.1C).Inbothlines, mice(Gradyetal.,1999,2000).Westudiedtwo miceexhibitamildmusculardystrophycharacter- mice. DB1 and DB1 or 95% ofallmusclefibersin DB1 in Hematoxylinandeosin–stainedsectionsof DB2 in DB2 (11A,11B,and28).Inthetext DB2 incontrolmice,whereaslevelsofre- DB incontrols(Fig.1B).Immunohis- DB2 bothpreventmusclefiberdegeneration DB2. Transgenicmicewerebredto DB DB DB / / / ,tgDB1 and DB ,tgDB1 muscleweresignifi- ,tgDB2 weresimilartolevels DB DB1 (12and14)three / , / DB1 and transgenicline.Insets DB DB1 and DB1 butsimilarto DB / ,tgDB1, and DB / ,tgDB1 and DB2 were / DB2 ,tgDB2 DB dif- predominant extrasynapticisoform.Bothisoformsareabsentfrom which areindicatedbyarrowheads.(A)Inwild-typemuscle,both Sections werecounterstainedwithrBTXtoidentifysynapticsites, with antibodiesthatrecognize than incontrols.In but theextrasynapticabundanceoftransgenic expressing (DB Figure 3. sites. For In molecular identityisseenusingtheDB2antibody(Gradyetal.,2000). extrasynaptic expression ofbothisoformsisreduced. Likewise,onlylowlevelsof decreases. (B)Inmicelackingdystrophin (mdx),sarcolemmal DB DB1 and2areenrichedatthesynapse,whereas DB ispresentthroughoutthesarcolemmaandenrichedatsynaptic DB1 (mdx,DB DB / / ,tgDB1/DB2), extrasynaptic muscle,althoughweaksynapticreactivityofunknown / ,tgDB1 and DB2, thispatternissimilartothatseenincontrolmuscle, Localization of DB1 and/or DB arepresentinmdxmiceexpressing recombinant / ,tgDB1). DB DB / DB2. muscleexpressingboth / DB isoformsin

Dystrobrevin isoforms| ,tgDB2 muscle,transgene-encoded Sectionsofskeletalmusclestained DB1, DB1 isretainedand DB2, orboth(pan-DB). DB

DB1 isgreater

/

andmdxmice DB2 isthe Gradyetal.

DB1 and2

DB2

743

The Journal of Cell Biology 744 levels, whereasin density in of AChRsineachthefourgenotypesasassessedbytheirmeanfluorescence intensityafterincubationwithasaturatingdose synapses, AChRsarepatchilydistributed,butspiculesofatbranch bordersareshorterthanthoseofmutants.(B)Synapt branches fromsynapsessuchasthoseshowninAtohighlightdifferences inAChRpatterning.Spiculesareindicatedbyarrowhea models ofmusculardystrophy, including because itisthemostseverely affectedmuscleinseveral Figure 4. fragmented branchbordersandradiatingspicules.In smooth borders.Withinbranches,AChRsareevenlydistributedor faintlystriated.In wild-type, bers, orinfiltration bymonocytesweredetected inany degenerating fibers, regenerating(centrally nucleated)fi- et al.,1998).Rescuebyboth transgeneswasdramatic.No (Stedman etal.,1991;Grady etal.,1997b,1999;Duclos The JournalofCellBiology DB DB DB1 isbetterablethan / / synapses( , DB DB / / ,tgDB2 synapses( ,tgDB1, and n

|

Volume 160,Number5,2003 49) is DB2 torestorenormalAChRdensityanddistribution 30% ofwild-type( DB n

/ ,tgDB2 musclestainedwithrBTX.Inwildtypesynapses,AChRsform multiplebrancheswith 19) densityremainsintermediatebetweenwild-typeand DB DB / ,tgDB1 synapses,AChRpatternisessentiallynormal.Inthemajorityof / n mice

18). At dominantly slow (typeIandIIA)fibers(soleus). Thus,ei- diaphragm, and sternomastoid),andinmuscles withpre- fast (typeIIBandII)fibers (tibialisanterior,quadriceps, ing inagefrom1–7mo, muscleswithpredominantly (Fig. 2andunpublisheddata). Thiswastrueinmicerang- muscles ofeither DB / ,tgDB1 synapses( DB / synapses,AChRsarepatchilydistributedwith DB n DB

/ 49), AChRdensityrecoverstocontrol / mice. ,tgDB1 or DB (A)Longitudinalsectionsof / . (C)Highpowerimagesof DB / ic density( ofrBTX.AChR ,tgDB2 mice DB ds. / ,tgDB2 SE) The Journal of Cell Biology be explainedsolelybyitsprimarysequence. calization of was abundantextrasynaptically(Fig.3A).Thus,selectivelo- was barelydetectableextrasynaptically,recombinant tributed similarly.However,whereasendogenous trasynaptic levelsof present extrasynaptically,butitspresencefailedtoreduceex- synaptic sitesrequiresthepresenceof 1 B).Analternative,however,isthatrestrictionof muscle mightresultfromitshigherthannormallevels(Fig. We immunostainedmuscleswithantibodiesspecificfor Synaptic localizationof cle fiberintegrity. ther and levels. In creased when membrane-associated sites. Totestthishypothesis,wegenerateddoublytransgenic aptic-binding sitesandtherebydisplace sites (markedwithrhodamine ports, bothisoformsarepresentathigherlevelssynaptic not bindselectivelytoextrasynapticsites. 1998; Gradyetal.,2000).Likewise,recombinant than inextrasynapticregionsofthesarcolemma(Petersetal., which bindsspecificallyandquasiirreversiblytoAChRs) test whether to thesarcolemma.Levelsofendogenous remained lowinmdx, the membrane. Wefoundthatlevelsofextrasynaptic and theseassociationsmightbe sufficienttotether tions of ever, thereisevidencefordystrophin-independent associa- trophin-deficient (mdx)mice (Gradyetal.,1997b).How- DB2 wereconcentratedatsynapticsitesin DB2, theendogenousandrecombinantproteinsweredis- DB2 mighthaveahigheraffinitythan DB1 and DB DB2, aregreatlyreducedin extrasynaptic regionsofdys- The abundanceofextrasynaptic We alsoexploitedthepresenceofextrasynaptic DB1 or / DB micethatexpressed DB withthesarcolemma(Crawford etal.,2000), / DB DB2 (Fig.1A).Consistentwithpreviousre- ,tgDB2 mice,respectively(Fig.3A).For DB1 dependsontheDGCforitslocalization DB1 totheNMJinwild-typemusclecannot DB1 waspresent,suggestingthat DB2 aloneiscapableofmaintainingmus- / ,tgDB1/DB2 mice, DB1 (Fig.3A).Indeed,levelsof DB2 immunoreactivitywerede- DB DB isoforms DB1 and ,tgDB1 mice despite the -bungarotoxin [rBTX], DB1 in DB2. Forexample, DB1 forextrasyn- DB2 wasclearly DB1 fromsuch DB2 atsimilar DB, primarily DB DB / DB1 and / DB2 did DB1 to DB1 to DB1 to ,tgDB1 ,tgDB1 DB1 DB1 DB1 trophic symptomsinmdx, withthisresult,therewasnoattenuationofdys- sistent overexpression ofrecombinant absence ofdystrophin. sociation withthemembranenorattenuatedystrophyin quences in Thus, but significantlygreaterthan inmutants(Fig.4,BandC). were eitherabsentorshortand AChRdensitywasslightly spicules radiatingfromtheedge oftheAChR-richregion distinguishable fromthosein micelackingall lesser degreethan mdx, In brane, leadingtoastriatedappearancewithineachbranch. the junctionalfoldsthatinvaginatepostsynapticmem- AChRs incontrolNMJsareenrichedalongthecrestsof thatradiatebeyondtheterminal’s edge.Second, spicules borders inmutantsarefragmentedwithlongfinger-like smoothly outlineeachbranchincontrols,whereas (Grady etal.,2000;Akaaboune2002).First,AChRs fect thearrangementofitsAChRsinthreeways(Fig.4) greatly affecttheoveralltopologyofNMJbutdoesaf- branch ofthenerveterminal. AChR-richpostsynapticmembraneunderlyingeach an Normal adultmouseNMJsarepretzelshapedwith AChR distributionanddensity In contrast,in 4). Thus, able fromnormalat defects: AChRdistributionanddensitywereindistinguish- pared withcontrols. other hand,most the linearstriationscharacteristicofnormalmuscle.On comparable tothoseseenat agethepostsynapticmembraneremainedabnormalat of AChRs isreducedby larly spacedaggregatesorclumps.Finally,thedensityof Grady etal.,2000)withreceptorsformingsmall,irregu- the crestsandfoldsisblurred(shownultrastructurallyin 90% ofsynapticsites:AChRsformedsmallaggregates Expression of DB DB DB2 supports normal postsynapticmaturation toa / DB1 alonecansupportpostsynapticmaturation. synapses,incontrast,thedistinctionbetween DB1 neithermediateaDGC-independentas- mice(unpublisheddata).Thusuniquese- DB DB1 inmicepreventedallthreeofthese DB1. DB / 95% ofsynapticsitesexamined(Fig. t similar tothatofcontrolmice,whereas in was intermediatebetweencontroland to Figure 5. synapses hadanaveraget synapses ( ( to calculatet a 3-dperiod.(A)Grayscaleimagesused intensity inrBTX-labeledsynapsesover assessed bythechangeinfluorescence 1/2 ,tgDB2 miceexaminedat4–6wk DB DB2 torestorenormalAChRturnover

of / Dystrobrevin isoforms| 70% at SE, DB DB ,tgDB2 postsynapticsiteswere / DB n DB .

/ / DB n ,tgDB1 synapses( mice. 20), whereasthoseof

/ DB deficiencydoesnot DB1 isbetterablethan 1/2 DB1 (Fig.3B).Con- ,tgDB2 synapses( 56) were ,tgDB1 comparedwith / . (B)AChRsinwild-type DB TurnoverofAChRsas synapsesratherthan / synapsescom- 2.5 d.AChRt Gradyetal. 1/2 n DB, inthat of

n 45) was

10.5 d DB 33) 745 1/2 /

The Journal of Cell Biology 746 with enrichmentatthesynapticsites(arrowheads).In nNOS and antibodies tonNOSor and transgenicmice. were similarin it isimportanttonotethatlevelsoftransgeneexpression However, thepercentageofnormal-appearingsynapsesin tgDB2 synapseswasalsoevidentin3–6-mo-oldanimals. (Akaaboune et al.,2002).Becauselittleisknown aboutthe part by than incontrols,supporting theideathat AChRs migrateandturnover fasterat AChR turnover Figure 6. membrane levelsofbothproteinswerereduced.Expression specific sequencesplayadistinct role. described below,provideindependentevidencethat (Fig. 1B).Moreover,studiesoftransfectedmusclefibers, lower thannormalatmanysites. even therelativelyineffective age-related increasesuggeststhatthecontinuedpresenceof raises thepossibilitythatdifferencesbetween ize thestructureofinitiallyabnormalsynapses.Moreover, it DB1 restorednormallevelsofbothproteinsto DB DB2 arequantitativeratherthanqualitative.Inthisregard, DB The differencebetween The JournalofCellBiology / / ,tgDB2 muscle,levelsofnNOSand ,tgDB2 increasedto tethering AChRs tothepostsynapticcytoskeleton Localization ofDGC-associatedproteinsin 1-syntrophin arepresentthroughoutthesarcolemma DB Sectionsofskeletalmusclewerestainedwith 1-syntrophin ( / ,tgDB1 and

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Volume 160,Number5,2003 DB DB2 caneventuallynormal- 50% at6moofage.This / 1-syn). Inwild-typemuscle, ,tgDB1 and 1-syntrophin remained DB DB DB / DB , DB2mice / DB mutant DB actsin / muscle.In DB1 and synapses / muscle, DB DB1- / , creased recruitingability. maintaining synapticarchitecturemayreflectinpartitsin- whereas thet methods) toaskwhether quantitated rBTXfluorescenceinvivo(seeMaterialsand molecular mechanismsthatregulateAChRturnover,we directed mutagenesis(GFP-DB1-P3 directed al., 1998])hadbeenchanged tophenylalaninebysite- rosine residues(aa698,706,and723[Balasubramanian et form of three tyrosine residues did.Thetwoconstructs wereintro- preclude itsphosphorylation, whereasthe ofits (Fig. 7A).Thus,theaddition ofGFPto DB1 fusionproteinunderwent tyrosinephosphorylation constructs inheterologouscells showedthatonlytheGFP- termediate betweencontrolsandmutants(t 60% ofAChRswerelostoverasimilartime,indicatingt whereas density, Thus, consistentwiththeresultsonAChRdistributionand modulator ofAChRclustering(JonesandWerle,2000). et al.,2000),andstudiesinvitrohaveimplicatednNOSasa synaptic defectssimilartothoseseenin (Grady etal.,2000).Moreover, greater abilityof To furtherinvestigatemechanismsthatmightaccountforthe Synaptic localizationof of the NH for itssynapticfunction.Totestthisidea,wefusedGFPto the possibilitythattyrosinephosphorylationisimportant phorylated invivo(Balasubramanianetal.,1998),raising The COOH-terminaldomainof Role oftyrosinephosphorylationin and nitricoxidesynthase (Akaaboune etal.,1999,2002).In est levelsof the NMJ,increasedefficacyof did notdiffersignificantlyfromthatofcontrols(t when viewedenface, indicates at were lostfromthecellsurfaceafter3d.Thisdegreeofloss intensity. Inwild-typemice, located andimagedagaintoassesschangesinfluorescence ual synapseswereimaged.3dlater,thesame beled withasinglenonsaturatingdoseofrBTX,andindivid- fects onthisprocess.Adultsternomastoidmuscleswerela- in manysynapsesof contrast, levelsof phin andnNOSweresimilartothoseincontrols(Fig.6).In duced synapticallyandextrasynapticallyin neuronal nitricoxidesynthase(nNOS).Levelsofbotharere- centration atsynapticsitesrequires ration, westudiedthedistributionoftwoproteinswhosecon- ther AChR distribution(unpublisheddata).Thus,althoughei- In 2.5 d(Fig.5).AChRstabilityin DB isoformcanrecruit DB 2 terminusof DB1 inwhichthethreemajorphosphorylatedty- DB2 isonlypartiallyeffective. DB1 alonecansupportnormalAChRturnover, / 1/2 1/2 ,tgDB1 muscle,synapticlevelsof 1-syntrophin alsohadthemostabnormal of ofAChRsin DB1 than 1-syntrophin andnNOSremainedlow 10.5 d,similartopreviousreports DB DB DB1 (GFP-DB1)ortoamutant DB1 and 1-syntrophin / / DB2 tosupportsynapticmatu- ,tgDB2 synapseswiththelow- 20% ofthelabeledreceptors ,tgDB2 mice.Interestingly, DB 1-syntrophin andnNOSto 1-syntrophin mutantshave DB: / DB1 istyrosinephos- DB2 haddifferentef- ). Expressionofthese ,tgDB2 micewasin- DB1 over DB DB DB DB1 function 1-syntrophin and / 1/2 / / DB1 didnot mice,nearly mice(Adams

,tgDB1 mice DB 1/2

1-syntro- DB2 in /

5.5 d). mice 9 d), 1/2 The Journal of Cell Biology toration ofpostsynaptic structureatmostsynaptic sites,and GFP-DB1 in fected AChRdistributionin differentways.Expressionof at synapticsitestoasimilar extent (Fig.7B),yettheyaf- levels basedonfluorescenceintensity andwereconcentrated (Fig. 7,BandC). cence, andtheirpostsynapticmembraneswereanalyzed later, transfectedfiberswereidentifiedbyGFPfluores- mice byelectroporation.Muscleswereremoved10–14 d duced intotibialisanteriormusclesof2-wk-old GFP-DB1 andGFP-DB1-P3 DB / myofibersresulted insignificantres- wereexpressedatequal DB / NMJs (Fig.7,D–F).ExpressionofGFP-DB1-P3 AChR distributionwasqualitativelynormalatasubsetof with among transfectedfiberswas greaterthanthatseenin normal (Fig.7,D–F).The variabilityof“rescue”seen 0.0001 byChi-squaretest),and nosynapticsitesappeared was significantlylessthanthatseenwithGFP-DB1(p stored AChRdistributiontosomeextent,buttherecovery for tyrosinephosphorylation inthefunction of able proteinlevels. Nonetheless,theseresults indicatearole DB / DB functionorbecauseelectroporation ledtovari- transgenicmice,possiblybecause GFPinterfered GFP-DB1, orGFP-DB1-P3 HEK293 cellstransfectedwithnoDNA, phosphorylation sites. muscle by to restorenormalAChRdistributionin category 3or4withnonein1. Expression ofGFP-DB1in (D) MusclefibersstainedwithrBTX. highly concentratedatsynapticsites. were synapses). Categories1and4 both GFP-DB1andGFP-DB1-P3 transfected musclefibersshowingthat (C) Highpowerimagesofsynapsesfrom with concentrationsatsynapticsites. fluorescence alongtheentiresarcolemma either fusionproteindemonstratesimilar stained withrBTX.Myofibersexpressing earlier. Afterdissection,muscleswere fibers expressing 4. Incontrast, category1or2with into apses infibersexpressingGFP-DB1fell examined. Approximately70%ofsyn- represent percentagesoftotalsynapses 2 and3wereintermediate.Numbers wild-type fibers(87synapses), (F) ComparisonofAChRdistributionin highlight differencesinAChRpatterning. synapses suchasthoseshowninDto power imagesofbranchesfrom fragmented branchborders.(E)High having patchyclustersofAChRsand synapses) orGFP-DB1-P3 electroporated withGFP-DB1(118 fibers (90synapses),andin postsynaptic structurein Figure 7. borders. GFP-DB1-P3 linear striationsandrelativelysmooth pattern withsynapticbrancheshaving leads toaqualitativelynormalAChR vivo withGFP-DB1orGFP-DB1-P3 vivo muscles thathadbeenelectroporatedin (B) Fiberbundlesfrom treated cellswastyrosinephosphorylated. Only GFP-DB1frompervanadate- an antiphosphotyrosineantibody(P-tyr). with eitherananti- phosphatases. Proteinwasidentified beforelysistoinhibittyrosine pervanadate were treatedwith( with anti-GFPantibodies.Cells constructs, lysed,andimmun DB DB

Dystrobrevin isoforms| synapses typicalofwild-typeand / / muscle,respectively;categories musclewithmanysynapses Impaired restorationof DB1 thatlackstyrosine over 70%ofsynapsesin GFP-DB1-P3 DB antibody(DB)or ) orwithout( (A)Immunoblotof haslessability DB Gradyetal. 1% incategory DB DB (161 expression opurified DB1. / skeletal fell into / / alsore- DB muscle

are 2wk ) / 747

The Journal of Cell Biology 748 of similar tocontrols, whereasthose were muscle, bothformswereabsent.In the MTJ. transgene-encoded Folds in are halfthesizeofnormalfolds( retain manyfeaturesof of thetendon(T).In interdigitating withthecollagenfibrils long slenderprojectionsofmuscle(M) characterized bymultiplefoldswith intermediate between controland that foldsin (B) MeasurementsofMTJfoldsrevealed leled thedifferencesdescribedabovebetween ther of granularinteriors.Incontrast,althoughexpressionei- tic siteswithsharp,spicule-freebordersandstriatedinstead ther transgenicallyorbytransfectionresultedinpostsynap- in Figure 9. fore askedwhether enriched attheMTJinwild-typemice(Fig.8).Wethere- In initialstudies,wefoundthatboth The MTJ fibers transfectedwithGFP-DB1andGFP-DB1-P3 at theMTJ(arrowheads).(B)In muscle, both were stainedwithantibodiesto at theMTJ. Figure 8. projections. In are shallower withbluntedmuscle whereas in whereas in the tgDB1 and wild-type, (A) ElectronmicrographsofMTJsfrom completely by rosine phosphorylation. over These parallelssuggestthattheenhancedabilityof remained granular(Fig.4BcomparedwithFig.7D). led torestorationofsharp(spicule-poor)borders,interiors DB DB DB2, orboth(pan-DB).(A)Inwild-type Interestingly, thequalitativedifferencesbetween MTJs appearsimilartowild-type, DB DB The JournalofCellBiology / / ,tgDB1 andDB ,tgDB2 mice.NormalMTJsare DB2 tosupportsynapticstructuredependsonitsty- DB2 orGFP-DB1-P3 / / ,tgDB2 MTJs( DB miceandrestoredmore MTJ structureisabnormal Localization of Sectionsofskeletalmuscle DB DB DB / DB1 and2wereenriched DB ,tgDB1 MTJs( DB1 thanby DB / / / , ,tgDB2 micetheMTJs MTJs( DB / DB wasrestoredto / DB ,tgDB1 mice, / ,tgDB2 fibers.Expressionof n DB isrequiredfortheintegrityof DB ,tgDB2 muscle,

/ / n ,tgDB1, and

230)were MTJs,folds DB isoforms

/ | 437folds) n MTJs.

n DB2.

Volume 160,Number5,2003

DB DB1, 245) DB 203). in

/ /

. DB DB1 and / muscleusually DB2 were DB1 ei- DB DB paral- DB1 / / , we couldnotidentifytransfectedfibersintheelectronmi- maintaining theintegrityofbothMTJsandNMJs.Because for maintainingnormalMTJarchitecture. Thus, those ofwild-typeor cantly differentfromwild-type(Fig.9B;p those incontrols(Fig.9,differencebetweencontroland present atmutantMTJsbutweresignificantlyshorterthan with thecollagenfibrilsoftendon.Foldswerealso are deeperthanthoseoftheNMJ,createaninterdigitation lel tothemyofiber’slongaxis(Fig.9A).Thesefolds,which brane attheMTJisinvaginatedtoformfoldsthatrunparal- MTJ. WeusedEMtoaddressthisissue.Themusclemem- folds in DB DB2 differedintheirabilitytomaintainMTJstructure.In DB Based ontheseresults,weaskedwhether / / DB1 wassignificantlymoreeffectivethan ,tgDB1 mice,thedepthoffoldswasnotsignifi- p DB 0.0001 byANOVA).Thus, / ,tgDB2 miceweresignificantlyshorterthan DB / ,tgDB1 animals(p DB isimportant 0.25), whereas DB1 and 0.0001). DB2 in The Journal of Cell Biology that mediatebindingof that sharedsequencessuffice.Shareddomainsincludesites not requiredforitseffectuponthemembraneandsuggest findings showthattheuniqueCOOHterminusof ing partnerstotheDGC. 2 canmaintainmyofiberintegritybyattractingsimilarbind- Newey etal.,2001b).Thus,itislikelythatboth screens(Bensonetal.,2001;Mizuno hybrid Ligands forthesesitesareunknownbutmayincludenovel teractions (forreviewseeEnigkandMaimone,2001). gion thatcanpotentiallymediateotherprotein–proteinin- Also includedaretwoEFhanddomainsandazincfingerre- muscle fiberdegeneration, requires anintact DGC,soweconsideredthe possibilitythat mice hasshown thattheextrasynapticlocalization of distinct distributionsofthetwo isoforms.Analysisofmdx therefore wonderedwhatfactors accountforthenormally capable ofassociationwith extrasynaptic membrane.We NMJ andMTJ,butwhenoverexpressed transgenicallyitwas In normalmuscle, Localization of In normalmuscle, Muscular dystrophyand utrophin or 2000; Akaabouneetal.,2002).Conversely,micelacking impact upontheNMJ(Hacketal.,1998;Grady of tion andnotothers.Forexample,absenceofdystrophinor shown thatlossofsomeDGCcomponentsaffectsonefunc- both theNMJandMTJ.Geneticstudiesinmicehave taining sarcolemmalintegrityandstabilizingthestructureof The DGCplaysatleastthreedistinctrolesinmuscle:main- Discussion lation playsaroleinMTJmaintenance. croscope, wewereunabletotestwhether able than MTJ maycontributetomusclepathology. that expressionof the DGCmaintainmuscleviability.Althoughwefound isoform, suggestingthatitplaystheprimaryroleinhelping per, weshowthat trast, 1997a; Kameyaetal.,1999;Adams2000).Incon- detectable dystrophy(Deconincketal.,1997a;Grady a mainroleoftheDGCatthissitemaybetorecruit 1994; Deconincketal.,1997b),andourresultssuggestthat plicated theDGCinmaintenanceofMTJ(Ridgeetal., et al.,1995;Miosge1999).Previousstudieshaveim- involved inthepathogenesisofsomemuscledisorders(Law bers totheskeleton,anddisruptionofthisstructuremaybe MTJ isthemajorsiteofforcetransmissionfrommusclefi- part throughalternativesplicingofthe selective localizationofthetwomainmuscleisoforms, DB1 and DB-binding proteinsidentifiedrecentlyinyeasttwo- On theotherhand,lossof - resultsinmusculardystrophywithlittle DB iscriticalforallthreeDGCfunctions.Inthispa- DB2 tomaintaintheintegrityofMTJ.The DB2. 1-syntrophin haveabnormalsynapseswithno DB isoforms DB2 alonein DB’s disparateeffectsareexplainedin DB2 isthepredominantextrasynaptic DB1 isselectivelyassociated with the DB todystrophinandsyntrophin. DB1 wasequallycapable.These DB DB1-specific functionsatthe DB DB transcriptand / DB1 phosphory- miceprevented DB1 isbetter DB1 and DB2 is DB1. DB2 phin havepostsynapticdefects similartothosein 2001a). Second, 1997a, Gradyetal.,1997a) andmicelacking creased intheabsenceof utrophin (Deconincketal., ences arelikelytoberelevant becauseAChRdensityisde- nian etal.,1998;Peters 1998)(Fig.6).Thesediffer- tightly withbothproteinsthan does phin, thereissomeevidencethat that (Grady etal.,2000;Akaaboune2002).Here,weshow between crestsandtroughsofjunctionalfoldsisblurred over isabnormallyhigh,densitylow,andthedistinction defects in muscle (Fig.1B).However,analysisofthe by thehigherlevelsof results fromthepreferentialbindingof the extrasynapticlocalizationof extrasynaptic predominanceof lodged fromitsextrasynapticpositionby expressed atsimilarlevels,showedthat DB2 doubletransgenics,inwhich cur in to extrasynaptic-bindingsites.Thisinteractionwouldnotoc- with theDGC(Yoshidaetal.,2000).Thus,innormalmice and 2(Nawrotzkietal.,1998)butisnonethelessassociated syntrophin- anddystrophin-bindingsitespresentin targeting informationencodedinthe3 mRNA orbysynapse-specifictransportoftheusing lows: First,theremaybeposttranscriptionallocalizationof al., 2001a).Tworemainingpotentialexplanationsareasfol- appear tobetranscribedfromthesamepromoter(Neweyet man, 2001).Thisisunlikely,however,becausebothisoforms expression ofAChRsubunits(forreviewseeSanesandLicht- transcribed fromsynapticnuclei,analogoustothe forms includethepossibilitythat not likelytobearesultofcompetitionbetweentheisoforms. COOH terminus of mice (Adamset al.,2000).Thus,sequences intheunique the mechanism couldexplain,atleastinpart,theappearanceof (Sanes andLichtman,2001;Akaabouneetal.,2002).This tional regions,whicharesitesofAChRinternalization result, thereisenhancedfluxfromthesynapsetoperijunc- mutants, themobilityofsynapticreceptorsisincreased;asa lizes AChRswithinthepostsynapticmembrane.In diated bycommonsequencesandone two distincteffectsonthepostsynapticmembrane,oneme- terminus isimportantfor common to enhance theirefficacy.Forexample,eventhoughregions all synapticeffects,with quences. Alternatively,commonsequencesmightmediate DB andtheNMJ DB3 mayconfine DB1 mRNAeitherbysynapse-specificstabilizationofthe DB1 totheDGC.Thiscompetitionmightbeaccentuated DB isacomponentofthemolecularmachinerythatstabi- Other reasonsfortheselectivedistributionoftwoiso- How might DB DB1 isbetterablethan DB / DB postsynapticmembraneinwhichAChRturn- / DB1 and2bindbothutrophin ,tgDB1 muscle,whichlacks DB act?Onepossibilityisthat / mice,indicatingthatitsuniqueCOOH DB3 mightplayarole. DB1 totheNMJbyblockingitsaccess DB1 couldenhance theabilityof

DB2 than Dystrobrevin isoforms| DB1-specific sequencesservingto DB’s synapticfunction. DB2 torescuethesesynaptic DB2 innormalmuscleis DB2 seeninnormalmice DB1 mightbeselectively DB1 and DB1 associatesmore DB1 seeninnormal DB2 (Balasubrama- UTR (Neweyetal., DB1 wasnotdis- DB2 overthatof DB1-specific se- DB1–3. DB2. Thusthe DB DB3 lacksthe Gradyetal. DB2 were / DB exerts 1-syntro- ,tgDB1/ -syntro- DB DB1 DB 749 / The Journal of Cell Biology 750 similarly affectedin (Akaaboune etal.,1999).AChRturnoveranddensityare to decreasewithin1hafterimpositionofcompleteparalysis To generatethe Generation oftransgenicmice Materials andmethods NMJ, thet in responsetoalteredinput(ShengandLee,2001).Atthe postsynaptic receptors,canchangerapidlyanddramatically features, mostnotablythedistributionanddensityoftheir markably stable,theyarenotinert.Instead,severaloftheir growing realizationthateventhoughmaturesynapsesarere- occurs innumerousotherphosphoproteins. 1998) ormayservetorecruitadaptorsignalingproteinsas suggested bymodelingstudiesofBalasubramanianetal., boring sequencestoaffecttheiraffinityforotherproteins(as tion. Phosphorylationmightaltertheconformationofneigh- evidence that et al.,1998)(Fig.7A).Ourresults,therefore,providestrong sites oftyrosinephosphorylationin aptic efficacy.Theseresiduesarethemajor,ifnotonly COOH-terminal domainbyphenylalaninedecreaseditssyn- Replacement ofthreetyrosineresiduesin aptic membrane. thereby enhancing common sequencestobindutrophinandsyntrophin, ing tolearnwhether and Lichtman,2001;Smithetal.,2001).Itwillbeinterest- and Fischbach,2001;Laietal.,forreviewseeSanes (DeChiara etal.,1996;Gonzalez1999;Buonanno stability invitro;andtrkBaffectsAChRdistributionvivo postsynaptic differentiation;srcandfynmodulateAChR the postsynapticmembrane;MuSKplaysacriticalrolein aptic structure:erbBandephAkinasesareconcentratedin an activity-dependentfashion. state couldaffectAChRmobilityquickly,reversibly,andin tered efficacyof rapid activity-dependentalterations.Ontheotherhand,al- part oftheregulatorymechanism.However,actualloss scriptional startsite (Jaynesetal.,1988)(agiftfromS. Hauschka,Univer- atine kinasegene, extendingfrom which contains3.3 kbofregulatorysequencesfromthe mousemusclecre- the SalI/NotIsitesofpBK-RSV(Stratagene) andthenintoapEtCATvector, mouse muscleRNA.Theentirecoding regionwasthensubclonedfirstinto stop codonand muscle cDNAlibraryandligated.The 3 14.1.2 [EnigkandMaimone,1999]) wereisolatedfromaBC3H1mouse including synaptic plasticityandthepresenceofDGCcomponents, numerous reportsimplicatingtyrosinekinasesincentral trkB) affect erbBs, ephrinAforephA,agrinMuSK,andBDNF nases andwhetheractivatorsofthekinases(neuregulinfor the possibilitythatsimilarmechanismsactthere. Kroger, 2000;Levietal.,2002)itisintriguingtoconsider DB phosphorylationandsynapticplasticity DB isunlikelytobeaphysiologicalmechanismforsuch Our interestin Several tyrosinekinaseshavebeenimplicatedinpostsyn- The JournalofCellBiology 1/2 DB and ofAChRsincreases,andtheirdensitybegins DB1 phosphorylation.Inaddition,inviewof DB1 expressionvector,twocDNA clones(16.1Aand 125 bpofthe3 DB1 functionismodulatedbyphosphoryla- DB1 bychangesinitsphosphorylation DB1 phosphorylationstemsfromthe DB1’s abilitytostabilizethepostsyn- DB1 isasubstrateforanyoftheseki- DB DB, atcentralsynapses(Blakeand

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/ Volume 160,Number5,2003 UTR,wasgeneratedbyRT-PCRfrom mice,suggestingthat 3,300 to endofthecDNA,including DB1 (Balasubramanian 7 withrespecttothe tran- DB1’s unique DB is also bredtomdxmiceand in anyoftheother linestested.Insummary,allfivetransgenic linestested but rescueofthesynaptic phenotypewassignificantly lessinthislinethan transgene-positive fibers,indicatingrescueofthedystrophic phenotype, line 11Amiceweretransgenepositive.Nocentralnucleioccurred in these than observedin in transgene-positivefibersandled toamorenormalsynapticstructure lines weresimilarinthatexpressionofthetransgenepreventeddystrophy bers expressed (lines 12and14)expresseddetectable were lowerinline11A,andonly cle andsynapticstructureAChRturnover). Levelsoftransgeneexpression Rabbit polyclonalantibodiesto min, cryoprotectedinsucrose,frozen,andsectionedenfaceat40 en faceviews,sternomastoidmuscleswerefixedin1%PFAPBSfor20 in PBS,andmountedusing0.1%p-phenylenediamineglycerol/PBS.For bit IgG(Alexa488;MolecularProbes)andrBTX(MolecularProbes),rinsed then incubated1hwithamixtureoffluorescein-conjugatedgoatanti–rab- 2% normalgoatserumfor2handthenrinsedwithPBS.Sectionswere same muscleswerestainedwithprimaryantibodydilutedinPBS/1%BSA/ with hematoxylinandeosin.Forimmunohistochemistry,sectionsfromthe isopentane andcrosssectionedinacryostatat8 For brightfieldmicroscopy,muscleswerefrozeninliquidnitrogen–cooled Of thefour Comparison oftransgeniclines rently. TostudylossrateofAChRs,anonblockingdoserBTX(0.1 compared withthatofanonbleachingfluorescentstandardviewedconcur- with afluorescencemicroscope.Theintensityatsynapseswas rated withrBTX(5 mice wereanesthetized,andtheexposedsternomastoidmusclewassatu- imaging techniquedescribedbyAkaabouneetal.(1999,2002).Briefly, AChR densityatNMJswascalculatedusingthequantitativefluorescence Quantitative fluorescencemicroscopyandinvivoimaging peroxidase-conjugated secondaryantibody(Roche)usingECL(NEN). tion Laboratories). Thisantibodywasdetectedwithgoatanti–mouseIgG1 gels andincubatedwithmonoclonalantibodyto amounts ofprotein(50 was determinedbyaBCAproteinassay(PierceChemicalCo.).Equal [CompleteMini; Roche]).Proteinconcentrationofwholemuscleextract in extractionbuffer(PBS,5mMEDTA,1%SDS,andproteaseinhibitors For immunoblots,sternomastoidmusclewashomogenizedandsonicated Immunoblotting taraldehyde/4% PFAinPBS,washed,refixed1%OsO from twotofouranimalswereanalyzedpergenotype. and allMTJsencounteredweremeasuredfromthemicrographs.Muscles acetate. Sectionsweresystematicallyscannedintheelectronmicroscope, embedded inresin.Thinsectionswerestainedwithleadcitrateanduranyl from from amined, expressionwasdetectedin three (lines11A,11B,and28).Results dose ensuredthattheturnoverratewasnotaffectedbyparalysis. tions wasmeasured(Akaabouneetal.,1999).Theuseofanonblocking 3 dlater,andtotalfluorescenceintensityofthepreviouslyidentifiedjunc- tensity atthefirstviewwasexpressedas100%.Micewerethenreexamined toxin hadcleared,individualsynapseswereimaged.Totalfluorescencein- was appliedtothesternomastoidfor2–5minand1dlater,afterunbound of (Peters etal.,1997b).Rabbitpolyclonalantibodiesthatrecognizeallforms phin (SYN17)weregiftsfromStanleyFroehner,UniversityofWashington onto an the carrying the ington UniversityMouseGeneticsCore.Fourindependentlinesofmice EcoRI siteofpBluescriptIISK( length clonewasisolatedfromtheBC3H1library,subclonedinto polyadenylation sequence.Togeneratethe sity ofWashington,Seattle,WA).Inaddition,thevectorcontainsanSV40/ nostar Inc.(no.24287).IllustrationswerepreparedinAdobePhotoshop 1997a). RabbitpolyclonalantibodytonNOSwaspurchasedfromImmu- ment of For ultrastructuralstudies,tibialisanteriormuscleswerefixedin4%glu- Linearized constructswereinjectedintoC57BL6oocytesattheWash- DB (calledpan- DB2 construct(tgDB2)wereidentifiedbyPCR.Eachlinewasbred DB DB DB andaffinitypurifiedusingtheimmunogen(Gradyetal., / ,tgDB2 lines11Band28weresimilar inallrespectstested(mus- DB1 transgeniclinesexaminedona / DB1 construct(tgDB1)andsixindependentlinescarrying background(Gradyetal.,1999). DB inline12butonly50–70%14.However,both DB g/ml) for60min,andsuperficialNMJswereviewed DB here)weregeneratedusingarecombinantfrag- / ,tgDB2 transgeniclines.Ofthesix g) wereresolvedon7.5%SDS–polyacrylamide DB ) (Stratagene)andthenintopEtCAT. DB1 ( / 70% ofmusclefibersin ,tgDB2 mice. DB1. Morethan95%ofmusclefi- DB638), DB2 expressionvector,afull- m; sectionswerestained DB (D62320;Transduc- DB DB DB2, and / 4 / background,two , dehydrated,and ,tgDB1 linewas DB2 linesex- DB 1-syntro- / ,tgDB2 g/ml) ® m. . The Journal of Cell Biology Benson, M.A.,S.E. Newey, E.Martin-Rendon,R.Hawkes, andD.J.Blake.2001. Balasubramanian, S.,E.T.Fung,andR.L. Huganir.1998.Characterizationofthe Ali, D.W.,andM.W.Salter.2001.NMDA receptorregulationbySrckinasesig- Akaaboune, M.,R.M.Grady,S.G.Turney,J.R.Sanes,andJ.W.Lichtman.2002. Akaaboune, M.,S.M.Culican,S.G.Turney,andJ.W.Lichtman.1999.Rapid Aihara, H.,andJ.Miyazaki.1998.Genetransferintomusclebyelectroporation in 28 ingreatestdetail. Adams, M.E.,N.Kramarcy,S.P.Krall,S.G.Rossi,R.L.Rotundo,R.Sealock,and References Accepted: 17January2003 Revised: 17January2003 Submitted: 10September2002 stitute undergraduatefellowshiptoA.L.Cohen. Health (toR.M.GradyandJ.R.Sanes)aHowardHughesMedicalIn- transgenic miceproduction,andCherylRiversformanuscriptassistance. for EM,andtheWashingtonUniversityMouseGeneticsCorefacility We thankRenateLewisfortissuecultureassistance,JeanetteCunningham Inc.), generatingafusionproteinwithGFPattachedtotheNH described aboveintoapEGFP-C1plasmid(CLONTECHLaboratories, Expression vectorGFP-DB1wasconstructedbycloningthe In vitroandinvivotransfection on theseresults,westudied cued synapticdefectsmoreeffectivelythananyofthetgDB2lines.Based exhibited rescueofthedystrophicphenotype,andbothtgDB1linesres- cal (Olympus)microscopy. rBTX andviewedbybothlight(CarlZeissMicroImaging,Inc.)confo- were isolatedunderafluorescencedissectingmicroscope,stainedwith fixed for20minin1%PFA.FiberbundlesexhibitingGFPfluorescence (BTX electroporator).Musclesweredissected10–14dafterinjectionand were deliveredatafrequencyof1Hz,givingfieldstrength site paralleltothemusclefibers.Ten80Vpulses,each20msinduration, (Genetronics) held4mmapartandinsertedoneithersideoftheinjection formed withapairof0.2-mmdiameterstainlesssteelneedleelectrodes electroporated (AiharaandMiyazaki,1998).Electroporationwasper- tized, thetibialiswasexposed,injectedwith25 mended byMcMahonetal.(2001).2hlater,themicewerereanesthe- a 4U/ their tibialisanteriormuscleswereinjectedtranscutaneouslywith25 (0.9% NaCl)ataconcentrationof2 tected usingPY20antibody(610000,TransductionLabs). ing anmAb(D62320,TransductionLabs), andphosphotyrosinewasde- min, andsubjectedtoimmunoblotting(seeabove). were precipitated,washed,resuspendedin1 Gsepharosebeads(AmershamBiosciences)for3hmore.Beads cific antibodies(A-11120;MolecularProbes)for1hat4 fractions werecollectedandimmunopurifiedbyincubatingwithGFPspe- sine phosphatasesasdescribedbyBalasubramanianetal.(1998).Soluble collection, cellsweresubjectedtopervanadatestimulationinhibittyro- cm dish.Cellswereharvested48haftertransfection.Immediatelybefore fectamine Plustransfectionreagent(Invitrogen)and4 shown tobemajorifnotsolesitesoftyrosinephosphorylationinTorpedo alanine. Theseresiduescorrespondtoaa685,693and710,whichwere change threeterminaltyrosineresidues(aa698,706,and723)tophenyl- DB (Balasubramanianetal.,1998). DB1. TocreateGFP-DB1-P3 This workwassupportedbygrantsfromthe National Institutesof For invivoelectroporation,DNAwasdissolvedintonormalsaline The Lett. tyrosine phosphorylationanddistribution ofdystrobrevinisoforms. biol. nalling inexcitatorysynaptictransmission andplasticity. offluorescentligands. unbinding Neurotransmitter receptordynamicsstudiedinvivobyreversiblephoto- muscular junctioninvivo. reversible effectsofactivityonacetylcholinereceptordensityattheneuro- vivo. neuromuscular synapsesdeficientinutrophin. S.C. Froehner.2000.Absenceofalpha-syntrophinleadstostructurallyaberrant L bovinehyaluronidase/salinesolution(Sigma-Aldrich)asrecom- DB1 constructsweretransfectedintoHEK293cellsusingLipo- 11:336–342. 432:133–140. Nat. Biotechnol. 16:867–870. DB Science. , PCR-directedmutagenesiswasusedto / ,tgDB1 line12and Neuron. 286:503–507. g/ l. 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