Dystrobrevin: Roles in Skeletal Muscle and Its Neuromuscular and Myotendinous Junctions R

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2-25-2003 Tyrosine-phosphorylated and nonphosphorylated isoforms of α-dystrobrevin: roles in skeletal muscle and its neuromuscular and myotendinous junctions R. Mark Grady Washington University School of Medicine in St. Louis

Mohammed Akaaboune Washington University School of Medicine in St. Louis

Alexander L. Cohen Washington University School of Medicine in St. Louis

Margaret M. Maimone State University of New York Upstate Medical University

Jeff .W Lichtman Washington University School of Medicine in St. Louis

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Recommended Citation Grady, R. Mark; Akaaboune, Mohammed; Cohen, Alexander L.; Maimone, Margaret M.; Lichtman, Jeff .;W and Sanes, Joshua R., ,"Tyrosine-phosphorylated and nonphosphorylated isoforms of α-dystrobrevin: roles in skeletal muscle and its neuromuscular and myotendinous junctions." The ourJ nal of Cell Biology.160,5. 741-752. (2003). http://digitalcommons.wustl.edu/open_access_pubs/625

This Open Access Publication is brought to you for free and open access by Digital Commons@Becker. It has been accepted for inclusion in Open Access Publications by an authorized administrator of Digital Commons@Becker. For more information, please contact [email protected]. Authors R. Mark Grady, Mohammed Akaaboune, Alexander L. Cohen, Margaret M. Maimone, Jeff .W Lichtman, and Joshua R. Sanes

This open access publication is available at Digital Commons@Becker: http://digitalcommons.wustl.edu/open_access_pubs/625

The Journal of Cell Biology

JCB

Published February25,2003

and Joshua R.Sanes and itsneuromuscular andmyotendinous junctions isoforms of Tyrosine-phosphorylated andnonphosphorylated the sarcolemma ofmusclecells.Micelacking dystrophin, utrophin,dystroglycans( 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. R. Mark Grady, sarcoglycans ( produces two main nous junctions(MTJs).Innormalmuscle,alternative splicing junctions (NMJs)and,asshown here,abnormalmyotendi- muscular dystrophy, defectsinmaturation ofneuromuscular Introduction finding thatmutationsingenesencodingnumerousDGC al., 2002).InterestintheDGCstemmedoriginallyfrom al., 1990;YoshidaandOzawa,forreviewseeBlake et © http://www.jcb.org/cgi/doi/10.1083/jcb.200209045 The Journal ofCellBiology rBTX, rhodamine junction; NMJ,neuromuscular nNOS,nitricoxidesynthase; dystrobrevin; DGC,dystrophin–glycoprotein 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- junction; neuromuscular 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

-terminal butdistinctCOOH-terminaldomains.

-bungarotoxin.

1 Mohammed Akaaboune, ,

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

DB isoforms,

and

-dystrobrevin: roles inskeletalmuscle

Department ofAnatomyandNeurobiology,Department Washington University SchoolofMedicine,

), syntrophins(

), andsarcospan(Ervastiet

/2003/03/741/12 $8.00

DB1 and

AlexanderL.Cohen,

DB exhibit

1, DB2, with

and

1, and

), dystrophies; andmutationsinlaminin of thesarcoglycanshavebeenlinkedtolimb-girdlemuscular results inDuchennemusculardystrophy;deficienciesfour components causemusculardystrophy:lossofdystrophin ness of ness of folds). Site-directedmutagenesisrevealed thattheeffective- turnover, andlow density)andMTJs(shortenedjunctional NMJs (abnormalacetylcholine receptorpatterning,rapid however, only isoform in present atNMJsandMTJs. Transgenic expressionofeither with distinctsignalingorstructural properties. in muscleby meansofdifferentialdistributionisoforms ability toserve asatyrosinekinasesubstrate. Thus, dystroglycan, 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 Roles oftheDGChavebeen lessextensivelystudiedatthe et al.,2000;Jacobson2001; Akaabouneetal.,2002). etal.,1997a;Grady1997a, 2000;Adams (Deconinck (AChRs) embeddedwithin thepostsynapticmembrane ations inthedensityandpatterning ofacetylcholinereceptors remodeling. Moregenerally, phosphorylation may beakeyregulatorypointforsynaptic In additiontoitsroleinmyofiberintegrity,theDGCis 2 Margaret M.Maimone, DB1 instabilizingtheNMJdependspartonits DB / DB1 completelycorrecteddefectsatthe miceprevented muscleﬁber degeneration; 1-syntrophin, or DB may play multipleroles 3 Jeff W. Lichtman, 2,themainextra- DB havealter- DB1

741 2

on January 4, 2012 4, January on jcb.rupress.org from Downloaded The Journal of Cell Biology Published February25,2003 742 sequences andtheshort(16aa)COOHterminusof al., 1993;Balasubramanianet1998),whereascommon junction; furthermore,only membrane, theirdetailedlocalizationdifferswithinthe COOH termini(Fig.1A).The188aaterminusof identical overmostoftheirlength(551aa)buthavedistinct Enigk andMaimone,1999;Neweyetal.,2001a)Theyare muscle (Blakeetal.,1996;Sadoulet-Puccio ing, ofwhichthree, able tomaintain musclestabilitybutthat 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,sarcospan, MTJ, butseveralDGCcomponentsareconcentratedinthis MTJs. Thus, how isoformdiversitycontributestofunctionaldiversity. Previously, weshowedthat in skeletalmuscle(Petersetal.,1997a;Blake1998). 1997b, 1998;Sadoulet-Puccioetal.,1997).Ahomologue, phin (Carretal.,1989;Wagner1993;Peters etal muscle,whereitbindsdystrophin,utrophin,andsyntro- both other isoformsandhasnotbeenstudiedindetail.Although syntrophin- anddystrophin-bindingsitespresentinthe do notappeartoundergophosphorylation. nous phenotypes.Weshow that either form to“rescue”thedystrophic, synaptic,andmyotendi- phosphorylation of Salter, 2001),wewantedtotestthetheorythattyrosine lates theplasticityofneuron–neuronsynapses(Aliand tion, basedonevidencethattyrosinephosphorylationregu- 2001a). Basedonthesedifferences,wehypothesizedthat in extrasynapticregions(Petersetal.,1998;Newey locations withinthemusclecell. addition, weshowherethat NMJs (Gradyetal.,1999,2000;Akaaboune2002).In both musculardefects(musculardystrophy)andabnormal 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

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

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,

on January 4, 2012 4, January on jcb.rupress.org from Downloaded The Journal of Cell Biology Published February25,2003 firmed withtheotherlines(seeMaterialsandmethods). and 28,respectively.However,themainresultswerecon- figures, thedesignationstgDB1andtgDB2refertolines12 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, or regenerationwasseenineither degenerated. Nohistologicalevidenceformusclefiberdegeneration nucleated, indicatingthatsomeoftheinitialcohortfibershad encoding Figure 2. recombinant similar inthetgDB1andtgDB2lines(Fig.1B).Levelsof isoforms showedthatlevelsoftransgeneexpressionwere levels oftotal present in cantly higherthenlevelsofendogenous skeletal musclefromwild-type, mice. The diaphragm providedaparticularly stringenttest combinant lated withfibertype(unpublisheddata). there werenodetectabledifferencesamongfibersthatcorre- cluding tibialisanterior,sternomastoid,anddiaphragm, the transgenewasexpressedinallskeletalmusclestested,in- transgenic linesthatexpressed tochemical analysisshowedthat show highpowerimages. that expressed of endogenous Muscular dystrophy 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- which areindicatedbyarrowheads.(A)Inwild-typemuscle,both Sections werecounterstainedwithrBTXtoidentifysynapticsites, predominant extrasynapticisoform.Bothisoformsareabsentfrom with antibodiesthatrecognize than incontrols.In but theextrasynapticabundanceoftransgenic Figure 3. (DB expressing sites. For In molecular identityisseenusingtheDB2antibody(Gradyetal.,2000). extrasynaptic expression ofbothisoformsisreduced. Likewise,onlylowlevelsof decreases. (B)Inmicelackingdystrophin (mdx),sarcolemmal DB1 and2areenrichedatthesynapse,whereas DB 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

jcb.rupress.org from Downloaded on January 4, 2012 4, January on

The Journal of Cell Biology Published February25,2003 744 levels, whereasin models ofmusculardystrophy, including because itisthemostseverely affectedmuscleinseveral Figure 4. smooth borders.Withinbranches,AChRsareevenlydistributedor faintlystriated.In density in of AChRsineachthefourgenotypesasassessedbytheirmeanfluorescence intensityafterincubationwithasaturatingdose synapses, AChRsarepatchilydistributed,butspiculesofatbranch bordersareshorterthanthoseofmutants.(B)Synapt fragmented branchbordersandradiatingspicules.In branches fromsynapsessuchasthoseshowninAtohighlightdifferences inAChRpatterning.Spiculesareindicatedbyarrowhea bers, orinfiltration bymonocytesweredetected inany degenerating fibers, regenerating(centrally nucleated)fi- et al.,1998).Rescuebyboth transgeneswasdramatic.No (Stedman etal.,1991;Grady etal.,1997b,1999;Duclos wild-type, The JournalofCellBiology DB DB DB1 isbetterablethan / / synapses( , DB DB / / ,tgDB2 synapses( ,tgDB1, and n

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

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

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

on January 4, 2012 4, January on jcb.rupress.org from Downloaded The Journal of Cell Biology Published February25,2003 tributed similarly.However,whereasendogenous trasynaptic levelsof present extrasynaptically,butitspresencefailedtoreduceex- We immunostainedmuscleswithantibodiesspecificfor Synaptic localizationof cle fiberintegrity. ther 1 B).Analternative,however,isthatrestrictionof muscle mightresultfromitshigherthannormallevels(Fig. synaptic sitesrequiresthepresenceof and levels. In was barelydetectableextrasynaptically,recombinant creased when membrane-associated sites (markedwithrhodamine ports, bothisoformsarepresentathigherlevelssynaptic sites. Totestthishypothesis,wegenerateddoublytransgenic aptic-binding sitesandtherebydisplace be explainedsolelybyitsprimarysequence. calization of was abundantextrasynaptically(Fig.3A).Thus,selectivelo- 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- DB1 and DB2 mighthaveahigheraffinitythan 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 absence ofdystrophin. sociation withthemembranenorattenuatedystrophyin trophic symptomsinmdx, withthisresult,therewasnoattenuationofdys- sistent overexpression ofrecombinant quences in 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 Thus, but significantlygreaterthan inmutants(Fig.4,BandC). were eitherabsentorshortand AChRdensitywasslightly spicules radiatingfromtheedge oftheAChR-richregion distinguishable fromthosein micelackingall lesser degreethan mdx, AChRs isreducedby larly spacedaggregatesorclumps.Finally,thedensityof Grady etal.,2000)withreceptorsformingsmall,irregu- the crestsandfoldsisblurred(shownultrastructurallyin comparable tothoseseenat agethepostsynapticmembraneremainedabnormalat of pared withcontrols. other hand,most the linearstriationscharacteristicofnormalmuscle.On 4). Thus, able fromnormalat defects: AChRdistributionanddensitywereindistinguish- In contrast,in 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 was intermediatebetweencontroland to Figure 5. in synapses hadanaveraget synapses ( ( to calculatet a 3-dperiod.(A)Grayscaleimagesused intensity inrBTX-labeledsynapsesover assessed bythechangeinfluorescence ف 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- ف 2.5 d.AChRt Gradyetal. 1/2 n DB, inthat of

n ف 45) was

10.5 d DB 33) 745 1/2

on January 4, 2012 4, January on jcb.rupress.org from Downloaded The Journal of Cell Biology Published February25,2003 with enrichmentatthesynapticsites(arrowheads).In 746 nNOS and antibodies tonNOSor and transgenicmice. AChRs migrateandturnover fasterat AChR turnover Figure 6. However, thepercentageofnormal-appearingsynapsesin tgDB2 synapseswasalsoevidentin3–6-mo-oldanimals. (Akaaboune et al.,2002).Becauselittleisknown aboutthe part by than incontrols,supporting theideathat membrane levelsofbothproteinswerereduced.Expression described below,provideindependentevidencethat (Fig. 1B).Moreover,studiesoftransfectedmusclefibers, specific sequencesplayadistinct role. even therelativelyineffective age-related increasesuggeststhatthecontinuedpresenceof lower thannormalatmanysites. were similarin it isimportanttonotethatlevelsoftransgeneexpression raises thepossibilitythatdifferencesbetween ize thestructureofinitiallyabnormalsynapses.Moreover, it DB1 restorednormallevelsofbothproteinsto DB DB DB2 arequantitativeratherthanqualitative.Inthisregard, The differencebetween The JournalofCellBiology / / ,tgDB2 muscle,levelsofnNOSand ,tgDB2 increasedto tethering AChRs tothepostsynapticcytoskeleton Localization ofDGC-associatedproteinsin 1-syntrophin arepresentthroughoutthesarcolemma DB Sectionsofskeletalmusclewerestainedwith 1-syntrophin ( / ,tgDB1 and

Volume 160,Number5,2003 ف 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- / , 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 the NH for itssynapticfunction.Totestthisidea,wefusedGFPto the possibilitythattyrosinephosphorylationisimportant phorylated invivo(Balasubramanianetal.,1998),raising greater abilityof To furtherinvestigatemechanismsthatmightaccountforthe and nitricoxidesynthase Synaptic localizationof of The COOH-terminaldomainof Role oftyrosinephosphorylationin three tyrosine residues did.Thetwoconstructs wereintro- preclude itsphosphorylation, whereasthe mutation ofits (Fig. 7A).Thus,theaddition ofGFPto DB1 fusionproteinunderwent tyrosinephosphorylation constructs inheterologouscells showedthatonlytheGFP- (Akaaboune etal.,1999,2002).In termediate betweencontrolsandmutants(t 60% ofAChRswerelostoverasimilartime,indicatingt creased recruitingability. maintaining synapticarchitecturemayreflectinpartitsin- est levelsof density, Thus, consistentwiththeresultsonAChRdistributionand modulator ofAChRclustering(JonesandWerle,2000). et al.,2000),andstudiesinvitrohaveimplicatednNOSasa when viewedenface, (Grady etal.,2000).Moreover, did notdiffersignificantlyfromthatofcontrols(t in manysynapsesof contrast, levelsof phin andnNOSweresimilartothoseincontrols(Fig.6).In the NMJ,increasedefficacyof whereas indicates at were lostfromthecellsurfaceafter3d.Thisdegreeofloss intensity. Inwild-typemice, located andimagedagaintoassesschangesinfluorescence ual synapseswereimaged.3dlater,thesame beled withasinglenonsaturatingdoseofrBTX,andindivid- fects onthisprocess.Adultsternomastoidmuscleswerela- synaptic defectssimilartothoseseenin duced synapticallyandextrasynapticallyin neuronal nitricoxidesynthase(nNOS).Levelsofbotharere- centration atsynapticsitesrequires ration, westudiedthedistributionoftwoproteinswhosecon- ther AChR distribution(unpublisheddata).Thus,althoughei- In ف 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 ف 10.5 d,similartopreviousreports DB DB DB1 (GFP-DB1)ortoamutant DB1 and 1-syntrophin / ف / 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- ف DB2 in /

5.5 d). ف mice 9 d),

1/2

on January 4, 2012 4, January on jcb.rupress.org from Downloaded The Journal of Cell Biology Published February25,2003 toration ofpostsynaptic structureatmostsynaptic sites,and (Fig. 7,BandC). cence, andtheirpostsynapticmembraneswereanalyzed later, transfectedfiberswereidentifiedbyGFPfluores- mice byelectroporation.Muscleswereremoved10–14 d duced intotibialisanteriormusclesof2-wk-old GFP-DB1 in fected AChRdistributionin differentways.Expressionof at synapticsitestoasimilar extent (Fig.7B),yettheyaf- levels basedonfluorescenceintensity andwereconcentrated 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 to restorenormalAChRdistributionin GFP-DB1, orGFP-DB1-P3 HEK293 cellstransfectedwithnoDNA, phosphorylation sites. muscle by category 3or4withnonein1. Expression ofGFP-DB1in (D) MusclefibersstainedwithrBTX. highly concentratedatsynapticsites. 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 postsynaptic structurein Figure 7. were synapses). Categories1and4 both GFP-DB1andGFP-DB1-P3 transfected musclefibersshowingthat (C) Highpowerimagesofsynapsesfrom with concentrationsatsynapticsites. fluorescence alongtheentiresarcolemma either fusionproteindemonstratesimilar stained withrBTX.Myofibersexpressing earlier. Afterdissection,muscleswere synapses) orGFP-DB1-P3 electroporated withGFP-DB1(118 fibers (90synapses),andin 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 ) /

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on January 4, 2012 4, January on jcb.rupress.org from Downloaded The Journal of Cell Biology Published February25,2003 748 of similar tocontrols, whereasthose were the MTJ. at theMTJ(arrowheads).(B)In Folds in are halfthesizeofnormalfolds( retain manyfeaturesof of thetendon(T).In interdigitating withthecollagenfibrils long slenderprojectionsofmuscle(M) characterized bymultiplefoldswith transgene-encoded intermediate between controland in Figure 9. fore askedwhether enriched attheMTJinwild-typemice(Fig.8).Wethere- In initialstudies,wefoundthatboth The MTJ fibers transfectedwithGFP-DB1andGFP-DB1-P3 muscle, both were stainedwithantibodiesto at theMTJ. Figure 8. muscle, bothformswereabsent.In that foldsin (B) MeasurementsofMTJfoldsrevealed ther of granularinteriors.Incontrast,althoughexpressionei- tic siteswithsharp,spicule-freebordersandstriatedinstead ther transgenicallyorbytransfectionresultedinpostsynap- leled thedifferencesdescribedabovebetween projections. In are shallower withbluntedmuscle whereas in whereas in the wild-type, (A) ElectronmicrographsofMTJsfrom completely by rosine phosphorylation. over These parallelssuggestthattheenhancedabilityof remained granular(Fig.4BcomparedwithFig.7D). led torestorationofsharp(spicule-poor)borders,interiors tgDB1 and 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 DB DB2 differedintheirabilitytomaintainMTJstructure.In 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

on January 4, 2012 4, January on jcb.rupress.org from Downloaded The Journal of Cell Biology Published February25,2003 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- that mediatebindingof that sharedsequencessuffice.Shareddomainsincludesites not requiredforitseffectuponthemembraneandsuggest 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 that expressionof the DGCmaintainmuscleviability.Althoughwefound isoform, suggestingthatitplaystheprimaryroleinhelping per, weshowthat trast, 1997a; Kameyaetal.,1999;Adams2000).Incon- detectable dystrophy(Deconincketal.,1997a;Grady able than MTJ maycontributetomusclepathology. part throughalternativesplicingofthe a mainroleoftheDGCatthissitemaybetorecruit 1994; Deconincketal.,1997b),andourresultssuggestthat plicated theDGCinmaintenanceofMTJ(Ridgeetal., et al.,1995;Miosge1999).Previousstudieshaveim- involved inthepathogenesisofsomemuscledisorders(Law bers totheskeleton,anddisruptionofthisstructuremaybe MTJ isthemajorsiteofforcetransmissionfrommusclefi- selective localizationofthetwomainmuscleisoforms, DB1 and DB-binding proteinsidentifiedrecentlyinyeasttwo- On theotherhand,lossof -sarcoglycan 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 1997a, Gradyetal.,1997a) andmicelacking creased intheabsenceof utrophin (Deconincketal., ences arelikelytoberelevant becauseAChRdensityisde- nian etal.,1998;Peters 1998)(Fig.6).Thesediffer- tightly withbothproteinsthan does phin, thereissomeevidencethat 2001a). Second, muscle (Fig.1B).However,analysisofthe by thehigherlevelsof results fromthepreferentialbindingof the extrasynapticlocalizationof that (Grady etal.,2000;Akaaboune2002).Here,weshow between crestsandtroughsofjunctionalfoldsisblurred over isabnormallyhigh,densitylow,andthedistinction 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 common to enhance theirefficacy.Forexample,eventhoughregions all synapticeffects,with quences. Alternatively,commonsequencesmightmediate diated bycommonsequencesandone two distincteffectsonthepostsynapticmembrane,oneme- terminus isimportantfor extrasynaptic predominanceof lodged fromitsextrasynapticpositionby expressed atsimilarlevels,showedthat DB2 doubletransgenics,inwhich 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. cur in to extrasynaptic-bindingsites.Thisinteractionwouldnotoc- with theDGC(Yoshidaetal.,2000).Thus,innormalmice and 2(Nawrotzkietal.,1998)butisnonethelessassociated syntrophin- anddystrophin-bindingsitespresentin defects in DB andtheNMJ DB1 mRNAeitherbysynapse-specificstabilizationofthe DB3 mayconfine DB1 totheDGC.Thiscompetitionmightbeaccentuated DB isacomponentofthemolecularmachinerythatstabi- How might Other reasonsfortheselectivedistributionoftwoiso- 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

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on January 4, 2012 4, January on jcb.rupress.org from Downloaded The Journal of Cell Biology Published February25,2003 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 stop codonand muscle cDNAlibraryandligated.The 3 14.1.2 [EnigkandMaimone,1999]) wereisolatedfromaBC3H1mouse 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 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 ف 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

/ Volume 160,Number5,2003 UTR,wasgeneratedbyRT-PCRfrom mice,suggestingthat 3,300 to endofthecDNA,including DB1 (Balasubramanian 7 withrespecttothe tran- DB1’s unique DB is were lowerinline11A,andonly cle andsynapticstructureAChRturnover). Levelsoftransgeneexpression isopentane andcrosssectionedinacryostatat8 For brightfieldmicroscopy,muscleswerefrozeninliquidnitrogen–cooled Histology 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 also bredtomdxmiceand than observedin in transgene-positivefibersandled toamorenormalsynapticstructure lines weresimilarinthatexpressionofthetransgenepreventeddystrophy bers expressed (lines 12and14)expresseddetectable in anyoftheother linestested.Insummary,allfivetransgenic linestested but rescueofthesynaptic phenotypewassignificantly lessinthislinethan transgene-positive fibers,indicatingrescueofthedystrophic phenotype, line 11Amiceweretransgenepositive.Nocentralnucleioccurred in these from from amined, expressionwasdetectedin three (lines11A,11B,and28).Results from twotofouranimalswereanalyzedpergenotype. and allMTJsencounteredweremeasuredfromthemicrographs.Muscles acetate. Sectionsweresystematicallyscannedintheelectronmicroscope, embedded inresin.Thinsectionswerestainedwithleadcitrateanduranyl taraldehyde/4% PFAinPBS,washed,refixed1%OsO dose ensuredthattheturnoverratewasnotaffectedbyparalysis. tions wasmeasured(Akaabouneetal.,1999).Theuseofanonblocking 3 dlater,andtotalfluorescenceintensityofthepreviouslyidentifiedjunc- tensity atthefirstviewwasexpressedas100%.Micewerethenreexamined toxin hadcleared,individualsynapseswereimaged.Totalfluorescencein- was appliedtothesternomastoidfor2–5minand1dlater,afterunbound 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 onto an the carrying the ington UniversityMouseGeneticsCore.Fourindependentlinesofmice EcoRI siteofpBluescriptIISK( length clonewasisolatedfromtheBC3H1library,subclonedinto polyadenylation sequence.Togeneratethe sity ofWashington,Seattle,WA).Inaddition,thevectorcontainsanSV40/ of (Peters etal.,1997b).Rabbitpolyclonalantibodiesthatrecognizeallforms phin (SYN17)weregiftsfromStanleyFroehner,UniversityofWashington 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. ف 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. .

on January 4, 2012 4, January on jcb.rupress.org from Downloaded The Journal of Cell Biology Published February25,2003 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 Akaaboune, M.,R.M.Grady,S.G.Turney,J.R.Sanes,andJ.W.Lichtman.2002. Ali, D.W.,andM.W.Salter.2001.NMDA receptorregulationbySrckinasesig- Akaaboune, M.,S.M.Culican,S.G.Turney,andJ.W.Lichtman.1999.Rapid Aihara, H.,andJ.Miyazaki.1998.Genetransferintomusclebyelectroporation in 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- their tibialisanteriormuscleswereinjectedtranscutaneouslywith25 (0.9% NaCl)ataconcentrationof2 tected usingPY20antibody(610000,TransductionLabs). ing anmAb(D62320,TransductionLabs), andphosphotyrosinewasde- min, andsubjectedtoimmunoblotting(seeabove). were precipitated,washed,resuspendedin1 protein 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- 28 ingreatestdetail. 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/ DB (Balasubramanianetal.,1998). DB1. TocreateGFP-DB1-P3 This workwassupportedbygrantsfromthe National Institutesof For invivoelectroporation,DNAwasdissolvedintonormalsaline The Lett. tyrosine phosphorylationanddistribution ofdystrobrevinisoforms. offluorescentligands. unbinding Neurotransmitter receptordynamicsstudiedinvivobyreversiblephoto- biol. nalling inexcitatorysynaptictransmission andplasticity. 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. Micewereanesthetized,and 34:865–876. J. CellBiol. samplebuffer,boiledfor4 l ofDNA(50 DB wasdetectedus- 150:1385–1398. g ofDNAper10- DB Curr. 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