anchorage andmotorneuroninnervation anchoring bothsynapticandnon-synapticmyonucleithatareimportantforpropermotorneuroninnervationrespiration. within 20minutesofbirth.TheseresultssuggestthattheKASH-domain-containingproteinsSyne-1andSyne-2playcrucialroles disruption ofneither branches.Althoug nuclei inSyne-1KASH-knockoutmicesignificantlyaffected theinnervationsitesandcausedlongermotornerve nuclei anddisruptedtheorganizationofnon-synapticinskeletalmuscle.Furtheranalysisindicatedthatlosssy (also knownas *Author forcorrespondence (e-mail:[email protected]) University ofColorado,Boulder, CO80309,USA. nuclear envelope duringcelldivision, respectively (Hedgecockand anchorage ofsyncytial cellsandassociationofcentrosomeswiththe nuclear envelope andtoplayrolesinnuclearmigration, 83, ANC-1andZYG-12have beenshown tobeassociatedwiththe Zhen etal.,2002).In 2002; Wilhelmsen etal.,2005;Yu etal.,2006;Zhang2001; al., 2004;Gradyet2005;Malone2003;StarrandHan, association ofthenuclearenvelope withKASHproteins(Fischeret to bindnuclearenvelope andarelikely toberesponsibleforthe (StarrandFischer, 2005).KASHdomainshave beenshown 60 aminoacidsthatislocatedattheC-terminusofKASH-family al., 1999;StarrandHan,2002;et2001;Yu etal.,2006). processes (Gradyetal.,2005;Malone2003;Mosley-Bishop et roles innuclearpositioningduringvarious cellularanddevelopmental proteins thatareassociatedwiththenuclearenvelope playimportant Klarsicht/ANC-1/Syne homologue(KASH)-domain-containing analyses inseveral modelorganisms have shown thatthe processes (Morris,2003;StarrandHan,2003).Inrecentyears,genetic systemshave beenfoundtoplayimportantrolesinthese extensively studied,andboththe microtubule andtheactin other cellfunctions.Nuclearmigrationandanchoragehave been processes, includingfertilization,celldivision, cellmigrationand incellsiscrucialformany biological The properpositioningofnuclei INTRODUCTION Academy ofSciences,Shanghai,200031, China. Laboratory ofNeurobiology, ShanghaiInstitutesforBiologicalSciences,Chinese Accepted 14December 2006 1 MSP-300, Mouse ANC-1, KASH,SUNdomain,Nesprin, KEY WORDS:Synapticnuclei,Neuromuscular junction,Neonatallethality, Nuclearenvelope, generating micewithsingleanddouble-disruptionoftheKASH-domain-containinggenes nuclear anchorageandmigration.We investigatedthemechanismandfunctionofnuclearanchorageinskeletalmusclecellsby the KASH-domain-containingproteinshavebeenshowntobeassociatedwithnuclearenvelope,andinvolvedinboth Proper nuclearpositioningisimportanttocellfunctioninmanybiologicalprocessesduringanimaldevelopment.Incertaincel Xiaochang Zhang Syne-1 (2007)doi:10.1242/dev.02783 Development 134,901-908 06520, USA. and DepartmentofGenetics,Yale UniversitySchoolofMedicine,New Haven,CT Fudan University, Shanghai,200433,China. NC 27706,USA. unZhuang Yuan Institute ofDevelopmentalBiologyandMolecularMedicine,SchoolLifeScience, The KASHdomainisaconserved proteinmotifofapproximately 4 Department ofImmunology, DukeUniversityMedicalCenter, Durham, 5 Howard HughesMedicalInstituteandDepartmentofMCDB, Syne-2 and 1,4 and MinHan C. elegans 1 Syne-1 ). We showedthatthedeletionofKASHdomainSyne-1abolishedformationclusterssynaptic , RenerXu Syne-2 nor , theKASH-domainproteinsUNC- Syne-2 2 1, Institute ofNeuroscience andKey 1,5 *, BinggenZhu 3 Howard HughesMedicalInstitute affected viabilityorfertility, play crucialrolesinmyonuclear 1 ija Yang , Xiujuan Starr andHan,2002;etal.,2001).In Thomson, 1982;HorvitzandSulston,1980;Maloneetal.,2003; (synaptic nuclei) clusterundertheneuromuscular junction(NMJ), (Bruusgaard etal.,2003).Noticeably, except for a3-8nuclei nuclei arestablyanchoredatthe peripheryofeachindividual cell each myotubematuresintoa large syncytial muscle fiber and nuclei undergo migration(EnglanderandRubin,1987).Later on, myoblasts fusetogethertoform multinucleatedmyotubes,and During earlydevelopment oftheskeletal muscle,hundredsof in nuclearpositioningmultinucleatedskeletal muscle cells. developmental processes. involved innuclearanchorageduringimportantcellularand proteins toANC-1andMSP-300suggestthatthey mayalsobe et al.,2001;Zhen2002).ThestructuralsimilaritiesofSyne to thenuclearenvelope (Apeletal.,2000;Grady2005;Zhang KASH domainsofSyneproteinshave beenshown totarget proteins a KASHdomainattheirC-terminus(StarrandFischer, 2005).The -binding domainsattheirN-terminus,alarge middlepartand all fourproteinsarevery large (>6000aminoacids)andcontain proteins. Syne-1andSyne-2areorthologsofANC-1MSP-300: Nesprin-3 isamuchsmallerproteincomparedtotheothertwo Syne Wilhelmsen etal.,2005;Zhang2001;Zhen2002). Gough etal.,2003;Mislow etal.,2002;Padmakumar etal.,2004; 2; andasNUANCE inhumans)andNesprin-3(Apeletal.,2000; Nesprin-1, Enaptin165),Syne-2(alsoknown asSyne2andNesprin- in ,namelySyne-1(alsoknown asSyne1,Myne1, oogenesis (Yu etal.,2006). and toplayacrucialroleinanchoringnurse-cellnucleiduring 2002), was alsoshown tobeassociatedwith thenuclearenvelope overall structure(StarrandHan,2002;Volk, 1992;Zhangetal., Drosophila droplets (Mosley-Bishop etal.,1999;Welte etal.,1998).The migration duringeye development andforthemovement oflipid domain proteinKlarsichthasbeenshown tobeimportantfornuclear ye1 Syne-2 Syne-1; Syne proteinshave beenimplicatedinplayingimportantroles Three KASH-domain-containingproteinshave beendiscovered 2 , XuDing MSP-300, ahomologueoftheworm ANC-1proteinin double-knockout micediedofrespiratoryfailure 1 , ShuminDuan Syne1 (also knownas RESEARCH ARTICLE 2 , TianXu Drosophila Syne-1 1,3 , ) and , theKASH- naptic Syne2 ls, in 901 h

DEVELOPMENT bar, 25 panels). Syne-1andSyne-2signals are visibleonthenuclearenvelopeofsamplesfrom thecontrol, butnotthehomozygous-knoc ( Bam samples were digestedby (+/+), heterozygous (+/–)and homozygous-knockout(–/–)mice.Probes usedare indicatedinFig.1A,B.(C)For for negativeselection.Restriction enzyme sites:A, exons of Syne-1 Fig. 1 generated transgenicmicethatoverexpress theSyne-2KASH domain-containing isoformsofbothgenes.Inaddition,wealso of involved inmyonuclearpositioning duringmuscledevelopment. the functionofSyneproteinsandwhichproteinisdirectly determined towhatdegree thisdominant-negative effect reflects spacing ofnon-synapticnuclei.However, itremainstobe positioning ofsynapticnucleibut hadnoeffect ontheeven expression ofthedominant-negative formofSyne-1disruptedthe (Grady etal.,2005).Thisstudyshowed that theectopic transgenic miceexpressing adominant-negative formofSyne-1 proteins innuclearpositioninghasbeenindicatedthestudyof Zhang etal.,2001).Morerecently, adirectinvolvement ofSyne levels intheskeletal muscle(Apeletal.,2000;Zhang2005; Both Syne-1andSyne-2have beenfoundtobeexpressed athigh anchoring mechanismforthosenucleihasalsobeenobscure. (Sanes andLichtman,2001;Schaeffer etal., 2001), but the essential inmaintainingthepostsynapticcomponentsofNMJ long beenproposedtobetranscriptionallyspecializedand anchorage remainsunknown. Inaddition,synaptic nucleihave However, theunderlyingmechanismresponsiblefornuclear other tominimizethetransportdistance(Bruusgaardetal.,2003). synaptic nucleiisspeculatedtoresultfromrepellingeach Lichtman, 1999).Theevenly spacedlocalizationpatternofnon- myonuclei distribute evenly inmusclefibers (Sanesand 902 E-H To thoroughlyunderstandthecellularandphysiologicalfunctions Syne-1 ) Frozen sectionsofskeletal musclewere stainedwithanti-Syne-1(green inEand F)oranti-Syne-2(green inGandH),DA Idgsini iil inalllanes).(D) For HI digestionisvisible . The generationofSyne-1andSyne-2KASH-domain-deletion mice. and ␮ RESEARCH ARTICLE Syne-2 m inFforE,F;10 and Syne-2 Syne-2 ) wasreplaced byaneomycin-resistance expression cassette(neo).A . Exonsare labeledandcodingregions are indicatedbyblackboxes.Inthetargetingvector, thelastexonof , wegeneratedmicedeficient intheKASH- Bam ␮ m inHforG,H. I hc ile . b(idtp lee n . kb(mutantallele)bands(noticethata4.1bandcaused by HI, whichyielded3.8kb(wild-type allele)and5.2 Syne-2 Avr , Spe II; B, I-digested genomicDNAyielded5.5 kb(wild-typeallele)and2.6(mutantbands. Bam HI; E, Eco RI; H, prcmy021. Primersfor request). (sequence detailsofallprimersmentionedinthispaperareavailable upon Primers for progenies werebackcrossedwithC57/B6Jmice. followed byinjectiontoobtain chimericmice.Chimerasandtheirpositive positive cloneswerescreenedbyPCRandSouthernblot,whichwas electroporated intoembryonicstem(ES)cells.Afterdoubleselections, cloned intopPNTat into the respectively. For fragments werethenclonedintopPNTatthe mice h eetreigvcosfrthe The -targeting vectors for Generation of MATERIALS ANDMETHODS activities ofeitherorbothproteins? and (4)Whatarethephysiologicalconsequencesofeliminating redundantly? nuclei? (3)Dothesetwo homologousproteinsfunction cells? (2)Dotheseproteinsplayaroleinanchoringnon-synaptic essential fortheanchorageofsynapticnucleiinskeletal muscle KASH-domain-containing isoformsofSyne-1and/orSyne-2 inskeletal musclecells.We aimtounderstand:(1)Are 1A was first clonedintopBluescript.The library (Invitrogen). For the constructed from129genomicDNA fragmentsscreenedoutofaBAC Genomic DNA was extracted andgenotypedusingathree-primerPCR. Hin ( A II ( dIII. , B Xho HSV-TK ) Schematicrepresentation oftheknockoutstrategies for C Syne-1 I siteofpPNT, whilethe , D ) Southern-blot analysesofgenomicDNA from) Southern-blot wild-type Syne-1 Syne-2 cassette waslinkedtothe5 genotyping wereprcmy016,prcmy017andprcmy018 Xba Syne-2 , the I- and Eco Hin Syne-1 Syne-2 genotyping wereprcmy019,prcmy020and RI. Eachvector was linearizedwith dIII- Syne-1 Syne-1 construct, the Bam single anddouble-knockout Spe HI fragmentwas blunt-endligated Avr and I- nlss genomicDNA analysis, Eco II- Not Ј Syne-2 Hin end (3 RI genomicfragmentwas I- Hin Development 134(5) dIII and Xho Syne-1 dIII fragmentinFig. KASH domainwere Ј kout, mice.Scale I and end for PI (blueinall Hin (the lasttwo Bam dIII- Syne-2 Not HI sites, Bam I and ) HI

DEVELOPMENT primers XP154andXP155. obtained fromthePCRamplification ofC57/B6JgenomicDNA withthe genomic DNA samplesweredigestedby C57/B6J genomicDNA withtheprimersXP141andXP142.For Bam and Russell,2001).For Zmd n utrophin(Novocastra). and (Zymed) (Sigma),rapsynsynaptophysin antibodies wereused:MuSK (Harlow andLane,1999).Thefollowing commercial standard protocols (Leica). higher-resolution pictures light microscope(forlow magnification) oraconfocalmicroscope for extensive washing, diaphragmsweremountedandviewed undereithera incubated withgoatanti-rabbitIgG-FITC (Sigma),BTXandDAPI. After and anti-synaptophysin(Zymed).Themuscleswerethenwashed and fixed andstainedwithamixtureofrabbitanti-neurofilament (Chemicon) al., 2002). (Vectashield) andviewed underalightmicroscope (Misgeldet medium fibers wereteasedout,mountedinmounting washing, individual containing2%goatserumand0.4%Triton). Afterthorough (PBS (BTX) (Molecularprobes)andDAPI dilutedinblockingsolution thenstainedwithtetramethylrhodamine-conjugated were thoraciandthendissectingouttheirtriangularissterni,which their (Sigma). (Gradyetal.,2005).Themyctagwas labeledwith9E10-FITC previously was carriedoutfollowing theprotocoldescribed (leg muscle) anterior tibialis the To analyzemyonucleiofadultmice,whole-mountstaining Immunofluorescence stainingandmicroscopy inliquid-nitrogen-cooledisopentane.Sections(6-8 in OCTandfrozen For frozentissuesections,tissuesofinterestweredissectedout,embedded Histological analysis Bioscience). were affinity-purified withHiTrap NHS-activated HPcolumns(Amersham rabbits withthepurified 6 agarose column.Polyclonalantibodieswereproducedbyimmunizing induced usingIPTGin 123 aminoacidpeptideofSUN2.Productionrecombinantproteinswas peptide attheC-terminusofSyne-2;andprDX057prDX058for at theC-terminusofSyne-1;prDX061andprDX062for66aminoacid the115aminoacidpeptide following primers:prDX059 andprDX060for were clonedintopET28orpET32afterPCRamplification withthe DNA fragmentsfromcDNA clones(ATCC) of Preparation ofantibodiesagainstSyne-1,Syne-2andSUN2 the pTWM1totheC-terminusbetween insertion ofanhGHpolyAfrom Spe pBluescriptKS(–).A6xMyctagfrompCS2+MTwitha5 into was cutfrom The 549bpKASH-domain-containingfragmentbetween Generation of genesare crucialfornuclearanchorage Syne analyzed. Ultimately, five viableandfertilepositive founderswereobtainedand wereidentified byPCRwithprimersprDX032andprDX033. mice oftheFVBinbredstraintoobtaintransgenicmice.Positive embryos was linearizedwith pBMGH topositionthecassettebehindMCKpromoter. Thisvector with hematoxylinandEosin. embedded inparaffin. Sections(5 fixed in4%formaldehyde,dehydratedethanol,clearedwithxyleneand were thencollected. KASH-hGH polyAwas thenclonedintothe Southern blotwas carriedoutaccordingtostandardprotocols(Sambrook muoitlg of frozen tissuesectionswas carriedoutfollowing Immunohistology To analyzebranchesofthephrenicnerve, diaphragmsweredissectedout, Skeletal musclefibers ofE18.5embryoswere analyzedbyfixing For paraffin sections,embryosortissuesofinterestweredissectedout, sitewas thenaddedinframe attheN-terminus,followed bythe I Eco HI andhybridizedwithaprobeobtainedfromthePCRamplification of RV and Syne-2 Syne-2 Xho I sites.TheresultingcassetteofMyc6-Syne-2 Xho .cl BL21 E. coli Syne-1 cDNA (ATCC, Cat.No.7492527)andligated transgenic mice ϫ I and His fusionproteins,andtherabbitanti-sera , genomicDNA samplesweredigestedwith ␮ Sac m) werethencollectedandstained and proteinswerepurified usingaNi- II beforebeinginjectedintothe Spe I andhybridizedwithaprobe Syne-1 Spe I and , Syne-2 ␣ -bungarotoxin Xba Xho and I and I sitesof Syne-2 SUN2 Ј -end ␮ Pst m) I , shows thatmore than99% Axotape software (AxonInstruments). All dataweredigitizedat10kHzandcollectedonmagneticdiskswith microelectrodes thatmeasured20-70megaohms whenfilled with3MKCl. near themainintramuscularnerve. Intracellularrecordingsweremadewith stimulated throughasuctionelectrode,andthediaphragmwas penetrated mMglucose(pH7.3)atroomtemperature.Thephrenicnerve was 10 methods). BothSyne-1andSyne-2werefoundtolocalizethe against theC-terminalpartofeachprotein(seeMaterialsand to examine nuclearenvelope-localized Syneproteinswithantibodies (data notshown). Secondly, weusedimmunofluorescencestaining amplified, whereasamiddlesegment ofeach genewas transcribed that thecodingsequenceforeachKASHdomaincouldnotbe with totalmRNA frombothheartandskeletal muscle,andfound these mutants,wefirst carriedoutreverse transcriptase(RT)-PCR Syne-2 Generation of RESULTS mMCaCl KCl, 2 mM mMNaCl,2.5 and balancedinanoxygenatedsolutioncontaining125 Diaphragms withintactphrenicnerves wereisolatedfromE18.5embryos Electrophysiology Fig. 2 25 more nucle Myonuclei were foundtobe distributedevenlyin simultaneously stainedwithDAPI (blue) andanti-SUN2(green). ( formed clusters(B)andarrays(C) in shown), andthehomozygous-knockout mice ofeither 1C,Danddatanot was confirmed byPCRandSouthernblot(Fig. crossed withC57/B6Jmice.Homologousrecombinationinmice targeting was carriedoutin129EScellsandthechimeraswere codonandformatruncatedprotein.Gene translation-termination recombination, eachgenewas predictedtohave apremature 1A,B; seeMaterialsandmethodsfordetails).Afterhomologous substituting thelastexon withaneomycin-resistancecassette(Fig. Syne-2 proteinsinmice,wedeletedeachKASHdomainby To investigate thefunctionofKASH-domain-containingSyne-1and knockout mice A-C To furtherconfirm thattheKASHdomainswereeliminatedin ␮ igemsl ie a teasedfrom thetibialisanteriorand ) Asinglemusclefiberwas m. . Non-synaptic nucleiare disorganized in were viableandfertile. ar clusters outsideNMJ 2 2mMMgCl , 12 Syne-1 of fibers in and 2 . mMNaH , 1.3 s( Syne-1 n Syne-2 >110 foreachgroup RESEARCH ARTICLE Syne-1 –/– mice. ( 2 –/– PO KASH-domain- mice formedthree or Syne-1 4 5mMNaHCO , 25 Syne-1 D ) Statisticaldata +/– ) –/– . Scalebar (A), but Syne-1 mice. 3 903 and or :

DEVELOPMENT on anti-SUN2signals.Synapticnucleidisappeared in Syne-1 diameter) (Fig.2B,C).Statistical datashowed thatover 99%of with thedistancebetween adjacent nucleilessthantheir cluster was defined asthreeormorenucleigroupedtogether, for skeletal musclecellsin nuclear envelope. We thusanalyzednucleipositioningin syncytial sufficient toeliminatetheendogenousSyne-1proteinfrom 2005), but thedominant-negative effect ofthetransgenewas not nuclei organization insyncytial skeletal musclecells(Gradyetal., domain ofSyne-1didnotdetectobvious defectsinnon-synaptic- Previous work usingtransgenicmiceoverexpressing theKASH mice referred toas established Syne-1andSyne-2KASH-domain-knockout mice, 1E-H).We concludedthatwehad homozygous-mutant mice(Fig. nuclear envelope inheterozygous skeletal musclebut notin Fig. 3 904 Non-synaptic nucleiare disorganizedin nuclei) clustered undertheNMJ.( DAPI; Green, anti-SUN2-labeledmyonuclei;Red,BTX.Scalebar:25 nuclei clusteredtogetherabnormally in 2A).Bymarked contrast, along thewholemusclefiber (Fig. X.D.). muscle cells(Fig.2A-C,andseetext below; unpublisheddatafrom the antibodyspecifically recognizesthenuclearenvelope ofskeletal anti-SUN2 antibodyasanadditionalmarker tolabelmyonuclei, as outside musclecellswhenDAPI stainingwas used,weemployed an interference ofnucleifromconnective tissues andSchwann cells In wild-typemice,non-synaptic nucleidistribute uniformly Syne-1 . Anchorage ofsynapticnucleiisabolishedin –/– RESEARCH ARTICLE –/– ucefbr otie oeta he nuclear muscle fibers containedmorethan three , P Syne-1 <0.0001); thenumberofperisynapticnucleiwasslightlyincreased in –/– and Syne-1 Syne-2 B-C –/– –/– ٞ once eeosre ne theNMJof ) Nonucleiwere observedunder mice. Inordertoexclude the mice, respectively. Syne-1 –/– Syne-1 mice (anuclear Syne-1 Syne-1 –/– mice (4.7in –/– –/– mice. ␮ m. ( A-A Syne-1 the numberofsynaptic nucleiwas significantly reduced(data not of asite.Statisticaldatabasedon DAPI stainingdemonstratedthat BTX-positive sitebut was lessthanhalfitsdiameterfrom theedge was countediftheDAPI andSUN2signaldidnotoverlap withthe overlapped with theBTX-positive site,andaperisynaptic nucleus defined assynapticwhen atleast25%oftheDAPI andSUN2signal to define anucleusto besynapticorperisynaptic:anucleuswas study, wefollowed themethodsofGradyetal.(Gradyal.,2005) DAPI andanti-SUN2.Inthis labeled myonucleisimultaneouslywith Syne-1 it isessentialtoexamine dominant-negative effect ofthistransgene,asmentionedearlier, unchanged (Gradyetal.,2005).Given thecaveats ofthe essentially total numberofnucleiassociatedwithasynapsewas peripheral tosynapsewas almostequallyincreasedso thatthe nuclei was drasticallyreducedwhilethenumber of nuclei expressing theC-terminusofSyne-1,numbersynaptic (Apel etal.,2000).Inprevious analysisusingtransgenicmice Syne-1 hasbeenshown tobeconcentratedatsynapticnuclei abolished in Anchorage ofsynapse-associatednucleiis space betweenthem. essential toproperlyanchornon-synapticnucleiandcreatethe anchorage floatfreelyin (Fig.2D).Theseresultsindicatethatmyonucleilacking clusters ٞ esandmsl ieswt T (markingtheAChRs) and We stainedmusclefibers withBTX ) Representative +/+ Syne-1 Syne-1 in synaptic-nucleipositioning. and Syne-1 –/– –/– mice. ( ie(. ncnrlvs0.8inKO, mice (0.0incontrol Syne-1 Syne-1 +/– s0.0in vs D , E +/– ) Statisticalanalysesofsynapticnucleibased –/– Syne-1 muscle fiberwithfournuclei(synaptic Syne-1 mice Syne-1 –/– –/– –/– mice tounderstandtheroleof , n mice, andthatSyne-1is =140 for Development 134(5) P <0.0001). Blue, Syne-1 + /? and 210

DEVELOPMENT yass(’.Saebr:25 synapses (F’).Scalebars: ra (green) compared withwild-type(E,E’).Noticeably, myonucleiexpressingthe NMJtoperipheralregion thetransgene (F,F’), envelope inskeletalmusclecells(D).Blue,DAPI;green, anti-myc.( yncerpositioningin Myonuclear in skeletal muscle. that tibialis anteriorandtriangularissterni.Theseobservations indicate different piecesofskeletal muscle,includinginthediaphragm, 3A approximately 4-5nucleiclusteredundereachNMJ( myonuclei; red, BTX.( defects ofmyonucleiin in perisynaptic nuclei)was dramaticallyreducedin number ofsynapse-associatednuclei(synapticplus greater than2.5nuclei(Gradyetal.,2005).Therefore,thetotal Fig. 3).In mice, comparedwithzerointheirlittermatecontrols( mice ( based onSUN2signals. positioning nuclei stainedbyDAPI, wefurtheranalyzednuclear shown). However, consideringthepotentialnoiseofnon-musclecell genesare crucialfornuclearanchorage Syne there was anaverage ofonly0.8perisynapticnucleiin theNMJ(Gradyetal.,2005).Surprisingly, under still observed Syne-1 KASH significantly moresevere thantheprevious observation using Fig. 4 synaptic nucleiwereproperlypositioned in described above, wefoundthatbothsynapticnucleiandnon- 2002; Zhangetal.,2001;Zhen2002).Usingthesamemethods also expressed inskeletal muscle(Apeletal.,2000;StarrandHan, Syne-2 hasasimilarproteinstructuretoSyne-1and,like Syne-1, is effect transgenic protein displays adominant-negative normal, butanMCK-drivenSyne-2KASH dominant-negative effect. [0.8 in mice carryingthe KASHfragmentofSyne-2 driven bythe MCK of Syne-1intheanchoringmyonuclei, wegeneratedtransgenic positioning process. myonuclear and datanotshown). Thus,Syne-2aloneisnotessentialforthe In wild-typeand To examine apotentialoverlapping functionofSyne-2withthat Syne-1 KASH ٞ Syne-1 .B otat once eese ne theNMJin ). Bycontrast,nonucleiwereseenunder . Myonuclei positioningisnotaffected in Syne-1 n =210, MCK-Syne-1 KASH plays acrucialroleinthepositioningofsynapticnuclei –/– transgenic mice,where1.5nucleionaverage were vs 4.7inwildtype( P transgenic mice].Furthermore,theanchorage <0.0001; Fig.3B C , D Syne-1 ) AnMCK-promoter-driven C-terminalfragmentofSyne-2containingtheKASHdomainwaslocalizedtonuclear Syne-1 ␮ ( m inBforA,B;25 A +/– , B –/– ) Anchorageofsynapticnucleiin transgenic mice,thisaverage was mice wererepeatedlyobserved in mice, therewere,onaverage, Syne-2 P 000) oprdwt 5.0 <0.0001), comparedwith ٞ ,C ٞ Syne-2 ,D). Thisdefectis –/– ␮ m inCforC,D;25 mice is Syne-2 –/– Syne-1 mice (Fig.4B n –/– =140) (Fig. P Syne-1 <0.0001; Syne-1 mice, butanMCK-drivenSyne-2KASHtransgenicprotein displaysa –/– MCK- E-F mice Syne-2 ␮ Ј –/– –/– ) Overexpressed Syne-2KASHfragmentexpelledsynapticnucleifrom under m inFforE-F –/– which werethencrossedtoproduce mutants werecrossedtogeneratedouble-heterozygousmice, mice whengenotypingatorafter postnatalday7. Syne-1 1 deleting bothKASHdomains. redundant functions.Thus,weexamined theconsequencesof transgenic micesuggestthat overlapping expression patternsandtheabove resultsinSyne-2 The conserved proteinstructurebetweenSyne-1andSyne 2,their mice failtobreathe anddieshortlyafterbirth Syne-1 andSyne-2KASH-domaindouble-knockout anchoring ofmyonuclei. nuclear envelope andthatSyne-2could playaregulatory roleinthe that Syne-1andSyne-2mightsharethesamedockingsiteson effect tothatofSyne-1(Gradyetal.,2005).Thoseresultsindicate theKASHdomaindisplayed asimilardominant-negative contained peripheral region (Fig.4F theNMJ tothe NMJ, andsynapticnucleiwereexpelled fromunder the lines, nucleicarryingtransgenicSyne-2rarelystayedunder expressed (Fig.4Danddatanotshown). Additionally, inthosetwo significantly decreasedintwo lineswherethetransgenewas highly muscle, whereasendogenousSyne-1atthenuclearenvelope was transgenic proteinwas localizedtothenuclearenvelope inskeletal promoter (Jaynesetal.,1988).Inallfive linesthatweobtained,the (Fig. 5Banddata notshown). Their heartsbeatforafew minutes and unabletobreatheeven thoughthey wereabletoopenthemouth weight andanatomytotheirlittermates, they werecyanotic atbirth mothers. Whilethesedouble-homozygous pupshadasimilarbody but diedwithin20minutes andweresooncannibalizedbythe expected Mendelianratio,wecouldnotobtainviable 1 Although homozygous-knockout (referredtohereafteras then inter-crossed toobtain fertile. mice (B)wassimilartowild-type(A).Green, anti-SUN2labeled +/– +/– Careful analysisindicatedthat ; Syne-2 ; Syne-2 –/– Ј Syne-1 . ; Syne-2 Syne-1 –/– –/– +/– iewr ona ecnae ossetwt the consistentwith atpercentages mice wereborn and ; Syne-2 +/– +/– ; Syne-2 Syne-1 mice. TheSynedouble-heterozygous, –/– –/– Ј ). Thus,theSyne-2fragmentthat and +/– ; Syne-2 Syne-1 Syne-1- , Syne-1 Syne Syne-1 Syne-1 RESEARCH ARTICLE –/– DKO micewere bornalive +/– Syne-1 and –/– –/– and and Syne-2 mice wereviableand ; Syne-2 ; Syne-2 Syne-2- Syne-2 +/– rely stayedunder Syne ; Syne-2 +/– +/– homozygous DKO) mice. –/– Syne mice were may have and double- –/– Syne- Syne- DKO 905 and

DEVELOPMENT ipae rsl omlmsl ntm (D)compared to displayed grossly normalmuscle anatomy ( double-heterozygous littermates(A). compared totheir 1 birth. indicating thefailure ofbreathing. A thatthealveoliairsacsofDKO micewere notexpanded(F), showed iheeysnpewr essentiallyeliminatedin with every synapsewere (nearly onenucleiperNMJforbothstrains),theassociated Phrenic nervesdisplaylongerbranchesin for thelethality. synaptic-nuclear-anchorage defectaloneisnotsufficient toaccount Syne-2 Syne-2 Fig. 5 of phrenicnerves in nerves andplaysanessentialroleinbreathing.AtE18.5, branches branching processinthediaphragm,whichiscontrolledbyphrenic examined whetheralossofsynapticmyonucleiaffected thenerve- from thelossofsynapticnucleiinskeletal musclecells.We therefore may beduetothemalfunctionofmotorneurons,possiblyresulting The neonatal-lethalityorlack-of-breathingphenotypeofDKO mice and minimum ofonecopy ofeither normal architecture(Fig.5C,D). expanded (Fig.5F),althoughtheirskeletal muscle displayedgrossly demonstrated thatthealveoli airsacsofthesemicewerenot ribcages. Postmortemhistologicalanalysisofthelungs legs inresponsetoapainfulstimulus,but failed tomove their prior todeath.Inaddition,the 906 sda h oto (E). Scalebars:25 thecontrol used as E,F. nuclei numberof Syne-1 of 2 DKO miceexhibited significantly longerbranchesthanthedouble- examined theanchorageofsynapticnucleiin the potentialroleof perform anessentialfunctionimmediatelyafterbirth.Considering their littermatesatE18.5(Fig.6).Although C +/– –/– , D The robust lethalphenotypeof Syne-1 ; Syne-2 ogtdnlscin fE85diaphragms. LongitudinalsectionsofE18.5 ) ie(vrg f0.0perNMJ, of mice (average . Syne ( +/– Syne-1 A +/– +/– RESEARCH ARTICLE , B (C). ( +/– ; Syne-2 mice (0.0vs0.0, ) Newborn ) Newborn ; Syne-2 +/– DKO mutants E and , F mice, ) Postmortemhistologicalanalysis ofthelung Syne +/– Syne-2 +/– Syne Syne-1 mice hadsimilarnumbersofsynapticnuclei Syne-2 P mice, but both DKO micewas similartothatof >0.1 byStudent’s K ie()apae cyanoticatbirth DKO mice(B)appeared double-knockout micediesoonafter n +/– =165 for in thepositioningofmyonuclei,we ; Syne-2 Syne Syne-1 Syne Syne-1 ␮ n DKO babiescouldmove their m inDforC,D;100 Syne =85). However, thesynaptic- Syne-1 –/– DKO miceindicatesthata +/– mice weresimilartothat or DKO and ; Syne-2 Syne-1 t –/– -test). Therefore,the Syne-2 Syne Syne Syne-2 ; +/– +/– Syne-1 DKO embryo DKO miceand n ; Syne-2 is requiredto littermate was =85 for Syne-1 +/– Syne-1 ␮ Syne-1 m inFfor –/– and ; Syne- –/– +/– Syne- Syne ; and –/– –/– ; Syne-1 ulu bu)sae ne h M rd nAadC u o nBo D. ( inAandC,butnotBor (red) stayedundertheNMJ (blue) nucleus Syne-2 disrupted. mro fteohrtogntps Scalebar:10 embryos oftheothertwogenotypes. embryos displayedasignificantlossofsynapticnucleicompared with different ( Fig. 6 in allfourgenotypes(Fig.7A hand, theAChR bandsco-localizedwithsynapsesofphrenicnerves heterozygous control(Fig.7A-D, Syne-1 In anchor themwhenthemuscle underwent violentcontractions. myonuclei atthepostsynapticregion, but was notsufficient tostably was sufficient totrapthemigrating in thetransgenicmice,which by thelow level ofnuclearenvelope-associated endogenousSyne-1 transgenic study(Gradyetal.,2005). Thedifference maybecaused nuclei, whichwas far moresevere thanthatdescribedintheprevious domain ofSyne-1isspecific tothe whether thedominant-negative effect ofoverexpressing theKASH proteins tomediatelocalizationthenuclearenvelope, itisnotclear skeletal musclecellsandbothcouldinteractwiththesame SUN Additionally, becausebothSyne-1andSyne-2areexpressed in Syne-1 proteinisrequiredfornuclearanchorageinmusclecells. from thenuclearenvelope, itwas thusnotcleartowhatextent the effect ofthetransgenereducedbut didnoteliminateSyne-1proteins normal inthesetransgenicanimals.Becausethedominant-negative cellular disorder, andtheanchorageofnon-synapticnucleiappeared severe physiologicaldefectswereobserved toaccompany the the NMJtoperipheralregion (Gradyetal.,2005). However, no synapticnucleifromunder 1 tothenuclearenvelope andexpelled thelocalizationofmajorityendogenousSyne- muscle blocked terminus, whichincludestheKASHdomain,inmouseskeletal A previous studyshowed thatoverexpressing theSyne-1C- positioning inskeletalmuscle Syne-1, Syne-2,SUNproteins andnuclear DISCUSSION bands thantheirlittermates. loss-of-function alleles. concern andindicatestheimportanceofcarryingoutanalysisusing 4)furtherraisedthis dominant-negative effect tothatofSyne-1(Fig. expression oftheKASHfragmentSyne-2displayedasimilar E A-D ttsia aasoe thatthe ) Statisticaldatashowed Syne-1 ) Representative NMJsfromofthefour E18.5triangularissterni . , respectively. Blue,DAPI;red, BTX.Noticethatonesynaptic Nuclear-anchorage defectsinmuscleof –/– –/– Syne ; Syne-2 –/– mice, boththetrappingand anchorage processeswere mice displayedanalmostcompletelossofsynaptic -knockout genotypes.Sy1andSy2represent +/– mice displayedsignificantly broaderendplate Ј -D Ј Syne-1 n ). Therefore, >10 foreachgroup).Ontheother Syne-1 –/– ; Syne-2 gene. Ourresultthatthe Development 134(5) Syne Syne ␮ +/– m. and DKO miceand DKO mice. Syne-1 Syne DKO and

DEVELOPMENT endplate bands(red) in showing theelongatedphrenic nervebranches(green) andthebroader yegenesare crucialfornuclearanchorage Syne branches are obviousin (green). Therighthemi-diaphragmofeachgenotypeisshown.Longer anti-synaptophysin and embryos were stainedwithanti-neurofilament approximately 2%ofthemusclecells not onlysynapticnuclei,but alsoofnon-synapticnuclei.Inaddition, Syne diaphragms (C’,D’).Scalebars:500 samples. ( Fig. 7 ta. 03 tr ta. 01.I isecluecells,Syne-1and et al.,2003;Starr etal.,2001).Intissue-culture recruiting KASH-domainproteins tothenuclearenvelope (Malone 1) have beenshown toplayimportantrolesinnuclearpositioning by prominent roleinmyonucleianchorage thandoes single-knockout miceindicate that below). However, thedifferences betweenthephenotypesoftwo with acommonfactor fortheirnuclearenvelope localization(see functions withSyne-1andthatbothproteinsarelikely to interact supports thehypothesisthatSyne-2couldhave overlapping andtheexpression ofSyne-2inmusclecells 4) transgene (Fig. that was sufficient toanchorthemyonucleiintransgenicmice. there was stillaconsiderablelevel ofSyne-1 onthenuclearenvelope KASH fragmentofSyne-1(Gradyetal.,2005),againindicating that was notobserved intheprevious transgenic miceexpressing the the normalfunctionsofskeletal muscle.Thisimportantphenotype large syncytial musclecells.Thiscouldthencausetheweakening of nuclear-cytoplasm transportation,aswellotherinteractions,inthe disrupted organization ofnon-synapticnucleicouldimpairnormal S1inthesupplementarymaterial).Theseverely group (seeFig. centralized nuclei,comparedwithlessthan0.5%inthecontrol A Ј -D Strikingly, wefoundthat .elegans C. The dominant-negative effect causedbytheSyne-2KASH Ј . . DKO mutants. Phrenic nervesdisplaylongerbranchesin A’-D’ SUN-domain-containing proteins (e.g.UNC-84,SUN- ) Enlargedviewsofasterisk-indicatedregions inA-D, ( A-D Syne-1 Syne-1 ) Whole-mountdiaphragmsofE18.5 Syne-1 –/– –/– ; Syne-2 ; Syne-2 ␮ nDfrAD 50 m inDforA-D; is crucialforthepositioningof +/– Syne-1 +/– and and Syne-1 Syne-1 Syne-1 plays amuchmore –/– Syne-1 Syne-2 –/– –/– mice exhibited ␮ ; Syne-2 ; Syne-2 m inD’for –/– . and –/– –/– showed nosignificant difference fromthatofthe obviously affected in of theNMJ(DeChiaraetal.,1996;Gautam1995),isnot Agrin-MuSK-Rapsn pathway, whichiscrucialforthedevelopment Syne anti-neurofilament, co-localizedwellwithAChR patchesinE18.5 nerves, whichwerelabeledwithamixtureofanti-synaptophysinand S2 inthesupplementarymaterial).Inaddition,terminiofphrenic 2 and thedecayofevoked EPPin E18.5 was blocked in potential (EPP)tofind outwhethertheneuromusculartransmission electrophysiological experiment andexamined theendplate the intracellular-Ca die ofdownstream defectsinsidetheskeletal musclecells,suchas its correspondingmusclecell.Theneonatal terminalto potentialsfromamotorneuron in transmittingsynaptic suggests thattheNMJin obviously depletedfromtheNMJsof andutrophin)componentswerefoundtobe (AChR, rapsyn,MuSK Surprisingly, neitherpresynaptic (synaptophysin)norpostsynaptic mice provide aneffective modelsystemtostudythisissue. nuclear anchorageisthefundamentalcauseoffatality. knocking outbothgeneshasondisrupting the additive effects that centralnervous system,itisstillconceivable that development ofthe associated withthetwo Syneproteins,suchasdefectsduring the neonatal lethalitymaybeduetoanunknown cellularfunction with themuscle. coupling of studied inmutantmicethatlacked them. Introduction), but theimportanceofthosenucleihasnever been specialized tomaintainthepostsynapticcomponents(see Synaptic nucleihave longbeenproposedtobetranscriptionally Synaptic nucleiandNMJdevelopment proteins andprobablyplayrolesinmyonuclearanchorage. mouse SUN1andSUN2arelikely tobethepartnersofSyne andX.Z.,unpublisheddata).Thus, (X.D. Padmakumar etal.,2005) a SUN-domain-protein-dependentmanner(Crispetal.,2006; Syne-2 have alsobeenshown tolocalizethenuclearenvelope in suggestions; Yuliang Shi,XiongliYang, Hai Huang,JianLiu,Yin Shen,Lihao Sanes, ZhenggeLuoandLinMeifor antibodiesandhelpful R. Grady, Joshua Lin SunandWufan Tao forhelpinmultiple aspectsofthiswork;R.Mark Chen forhelpingeneratingantibodies; BeibeiYing, XiaohuiWu, KejingDeng, setting uptheelectrophysiological experimentandforanalyzing data;Muyun forhelpin Wu chimeras andtransgenicmice;Wanhua ShenandXiumei We thankXiaohuiWu, Yanling Yang andYanfeng Tan forhelpinmaking displayed longerbranchesinboth observed thatthephenic nerves we Consistent withthisnotion, level ofthosegenesandweaken thenormalfunctionsofNMJ. depleted, thelossofsynapticnucleimightreduceexpression Syne-1 branchingbetweenthe morphology, NMJstructureandmotor-nerve Because wefailed toidentifymajordifferences inthemuscle The causeofneonatallethality muscle-nerve contacts. communication betweennerve andmuscleatthenewly formed maintaining theinnervation sitesbystrengtheningthe synapticnucleimay playimportantrolesinselectingor Therefore, +/– Thus, thecauseofrespirationfailure andlethalityassociated However, given thatNMJ-specific geneswerenotcompletely mro seFg S3inthesupplementary material).Thisresult embryos (seeFig. DKO embryos(datanotshown). Theseresultssuggestthatthe Syne –/– ; Syne-2 DKO miceremainsobscure.Althoughtheobserved Syne +/– 2+ and the DKO mutants.However, thepaired-pulseratio Syne-1 mobilization andtheexcitation-contraction Syne Syne –/– DKO mutantsmayremainfunctional or DKO embryos,wecarriedoutan Syne Syne-1 RESEARCH ARTICLE Syne DKO mice. Syne-1 –/– Syne Syne DKO mutants(seeFig. and Syne DKO mutantsmay –/– DKO diaphragms Syne Syne-1 and DKO mice DKO mice. Syne –/– ; Syne- DKO 907

DEVELOPMENT Mislow, J.M.,Kim,M.S.,Davis,D.B.and McNally, E.M. DeChiara, T. M.,Bowen,D.C.,Valenzuela, D.M.,Simmons,M.V., Crisp, M.,Liu,Q.,Roux,K.,Rattner, J.B.,Shanahan,C.,Burke,Stahl,P. D. Misgeld, T., Burgess,R.W., Lewis,R.M.,Cunningham,J.Lichtman,W. Bruusgaard, J.C.,Liestol,K.,Ekmark,M.,Kollstad,K.andGundersen, Apel, E.D.,Lewis,R.M.,Grady, R.M.andSanes,J. References http://dev.biologists.org/cgi/content/full/134/5/901/DC1 Supplementary materialforthisarticleisavailableat Supplementary material Life Science(Fudan). Morgan-Tan Centerof and (DivisionofScienceandTechnology) Government National NaturalScienceFoundationofChina,ShanghaiMunicipal comments onthemanuscript.Thisworkwassupportedbygrantsfrom the help andvaluablediscussions;DianaRonaiAileenSewellfor Ge, GuoyuanLiu,MinWan, MingyaoYing andmembersofIDMfortechnical 908 Malone, C.J.,Misner, L.,LeBot,N.,Tsai, M.C.,Campbell,J.M.,Ahringer, J. Jaynes, J.B.,Johnson,E.,Buskin,N.,Gartside,C.L.andHauschka,S.D. Hedgecock, E.M.andThomson,J.N. Harlow, E.andLane,D. Grady, R.M.,Starr, D.A.,Ackerman,G.L.,Sanes,J.R.andHan,M. Fischer, J.A.,Acosta,S.,Kenny, A.,Cater, C.,Robinson,C.andHook,J. Englander, L.andRubin, Horvitz, H.R.andSulston,J.E. Gough, L.L.,Fan,J.,Chu,S.,Winnick,S.andBeck,K.A. Gautam, M.,Noakes,P. G.,Mudd,J.,Nichol,M.,Chu,G.C.,Sanes, J.R.and interacts withlaminA/C. spectrin repeat transmembrane protein ofthemyocyteinnernuclearmembrane, neuromuscular junction. dystrophin- andKlarsicht-related protein associatedwithsynapticnucleiatthe neuromuscular junctionformationinvivo. J. S.etal. Poueymirou, W. T., Thomas,S.,Kinetz,E.,Compton,D.L.,Rojas,Park, complex. and Hodzic,D. Neuron development ofneuromuscular junctionslackingcholineacetyltransferase. and Sanes,J.R. mice studiedinvivo. (2003). Numberandspatialdistributionofnucleiinthemusclefibres ofnormal essential attachmentbetweenthecentrosome andnucleus. and White,J.G. elements, includingamuscle-specificenhancer. (1988). Themusclecreatine kinasegeneisregulated by multipleupstream 454. cell-lineage mutantsofthenematodeCaenorhabditiselegans. mitochondrial attachmentinthenematodeCaenorhabditiselegans. Harbor: ColdSpringHarborLaboratoryPress. Acad. Sci.USA Syne proteins anchormusclenucleiattheneuromuscular junction. localization ofSyne-1. envelope andmicrotubule localizationintheeye. Drosophila klarsichthasdistinctsubcellularlocalizationdomainsfornuclear nuclear movementinmusclefibersculture. 321-330. neuromuscular junctionsofrapsyn-deficientmice. Merlie, J.P. RESEARCH ARTICLE 36 J. CellBiol. , 635-648. (1996). Thereceptor tyrosine kinaseMuSKisrequired for (1995). Failure ofpostsynapticspecializationtodevelopat 102 (2006). Couplingofthenucleusandcytoplasm:role oftheLINC (2002). Rolesofneurotransmitter insynapseformation: (2003). TheC.eleganshookprotein, ZYG-12,mediatesthe , 4359-4364. 172 J. Physiol. Mol. Biol.Cell (1999). J. Biol.Chem. , 41-53. J. CellSci. 551 Using Antibodies:ALabManual (1980). Isolationandgeneticcharacterizationof (1987). Acetylcholinereceptor clusteringand , 467-478. 115 14 (1982). Agenerequired fornuclearand 275 , 61-70. , 2410-2424. Cell , 31986-31995. J. CellBiol. 85 Mol. Cell.Biol. Genetics , 501-512. Nature (2000). Syne-1,a 168 104 377 (2003). Golgi (2002). Myne-1,a Cell , 1385-1393. , 87-95. , 232-236. Genetics 8 . ColdSpring , 62-70. 115 Proc. Natl. , 825-836. Cell (2005). 96 (2004). 30 , 435- , Padmakumar, V. C.,Abraham,S.,Braune,Noegel,A.A.,Tunggal, B., Padmakumar, V. C.,Libotte,T., Lu,W., Zaim,H.,Abraham,S.,Noegel,A.A., Mosley-Bishop, K.L.,Li,Q.,Patterson,L.andFischer, J.A. Morris, N.R. Zhen, Y. Y., Libotte,T., Munck,M.,Noegel,A.andKorenbaum, E. Zhang, Q.,Ragnauth,C.D.,Skepper, J.N.,Worth, N.F., Warren, D. T., Zhang, Q.,Ragnauth,C.,Greener, M.J.,Shanahan,C.andRoberts,R.G. Zhang, Q.,Skepper, J.N.,Yang, F., Davies,J.D.,Hegyi,L.,Roberts,R.G., Sanes, J.R.andLichtman,W. Sambrook, J.andRussell,D. Sanes, J.R.andLichtman,W. Starr, D.A.andHan,M. Schaeffer, L.,deKerchove d’Exaerde, A.andChangeux,J.P. Starr, D.A.andFischer, J.A. Starr, D.A.andHan,M. Welte, M.A.,Gross, S.P., Postner, M.,Block,S.M.andWieschaus,E.F. Yu, J.,Starr, D.A.,Wu, X.,Parkhurst,S.M.,Zhuang,Y., Xu,T., Xu,R.and Starr, D.A.,Hermann,G.J.,Malone,C.Fixsen,W., Priess,J.R.,Horvitz,H. Volk, T. Wilhelmsen, K.,Litjens,S.H.,Kuikman,I.,Tshimbalanga, N.,Janssen,H., Cell Res. protein, isanelementofthenuclearmembraneandactincytoskeleton. Karakesisoglou, I.andKorenbaum, E. differentiating cellsoftheDrosophila eye. analysis oftheklarsichtgeneanditsrole innuclearmigrationwithin Cell Biol. envelope. Sun1mediatestheanchorageofNesprin-2tonuclear Gotzmann, J.,Foisner, R.andKarakesisoglou,I. Sci. NUANCE, agiantprotein connecting thenucleusandactincytoskeleton. neuromuscular junction. 673-687. envelope andformsasubcellularnetworkinskeletalmuscle. Nesprin-2 isamulti-isomericprotein thatbindslaminandemerinatthenuclear Roberts, R.G.,Weissberg, P. L.,Ellis,J.A.andShanahan,C.M. melanogaster muscleprotein MSP-300. (2002). Thenesprinsare giantactin-bindingproteins, orthologous toDrosophila membrane inmultipletissues. family ofspectrin-repeat-containing proteins thatlocalize tothenuclear Weissberg, P. L., Ellis, J.A.andShanahan,C.M. (3rd edn).ColdSpringHarbor:HarborLaboratoryPress. maintenance ofapostsynapticapparatus. cytoskeleton. transcription totheneuromuscular synapse. cargo-specific cytoskeletaladaptorproteins. nuclear positioning. forces andkinetics. (1998). Developmentalregulation ofvesicletransportinDrosophila embryos: 721-730. nuclear anchoringduringDrosophila oogenesis. Han, M. formation ofembryonicmuscleattachmentsinDrosophila. envelope andisessentialforproper nuclearmigration. R. andHan,M. 5050. protein plectin. novel outernuclearmembraneprotein, associateswiththecytoskeletallinker van denBout,I.,Raymond,K.andSonnenberg,A. 115 (1992). Anewmemberofthespectrinsuperfamilymayparticipatein , 3207-3222. 295 15 (2006). TheKASHdomainprotein MSP-300playsanessentialrole in J. CellSci. (2003). Nuclearpositioning:themeansisatends. , 54-59. , 330-339. Science J. CellBiol. (2001). unc-83encodesanovelcomponentofthenuclear Cell J. CellSci. 118 298 (2002). RoleofANC-1intetheringnucleitotheactin (2003). ANChorsaway:anactinbasedmechanismof 92 , 3419-3430. Annu. Rev. Neurosci. , 406-409. , 547-557. 171 (2005). KASH‘nKarry:thedomainfamilyof (2001). J. CellSci. 116 , 799-810. (1999). Developmentofthevertebrate (2001). Induction,assembly, maturationand , 211-216. Molecular Cloning: A Laboratory Manual Molecular Cloning:ALaboratory Genomics 114 (2004). Enaptin,agiantactin-binding Curr. Biol. Nat. Rev. Neurosci. BioEssays Neuron , 4485-4498. 22 Dev. Biol. , 389-442. 80 (2001). Nesprins:anovel (2005). Theinnernuclear 31 9 , 473-481. Development 134(5) , 1211-1220. 27 Development , 15-22. (2005). Nesprin-3,a , 1136-1146. 289 Development (1999). Molecular J. CellSci. 2 , 336-345. , 791-805. (2001). Targeting Curr. Opin. (2005). 128 118 (2002). , 5039- J. Cell 116 , Exp. ,

DEVELOPMENT