Journal of Cell Science o:10.1242/jcs.108688 doi: 4158–4169 125, ß Science Cell of Journal 2012 April 24 Accepted el omdb h uino udeso post-mitotic of hundreds of many fusion extend can fibers the muscle Adult by myocytes. mononucleated formed multinucleated large are cells fibers muscle skeletal surface Mammalian nuclear cells. the on translocation. microtubule nuclear present of the motors direction of overall of the polarity type determine the the both and examples, network these Starr, dynein polarized and In (Fridolfsson with a network 2010). this microtubules, of along backsteps plus-end of small driving the bundle toward non-centrosomal nucleus parallel the In 2007). moving al., motor in et cells Tsai precursor 2004; developing hypodermal al., the through migrate et nuclei Shu as 2003; contrast, nucleus al., et the the minus-end-directed (Xie surrounds that walks nucleus cage microtubule Dynein the a along as centrosome dynein. the toward requires protein, such this motor migration defects In microtubule disrupt 2011). nuclear developmental Hoogenraad, that and neurons, Kuijpers severe migration mutations by neuronal (reviewed in lissencephaly and to result development example, coupled movement they For tightly brain as development. is during cells during movement for differentiate nuclear important and particularly migrate is Microtubule-dependent movement 2010). Fridolfsson, nuclear & Starr cytoskeleton the by and (reviewed envelope controlled nuclear is the between systems connections many by in positioning and movement Nuclear Introduction crowded the and complex from the result positioning. through myonuclear to motility proper likely their for words: facilitate are allowing Key may myotube cell, nuclei muscle remodeled single myotube continuously developing a of also the is rotation within of that three-dimensional extent, cytoskeleton nuclei environment The microtubule lesser nuclear cellular individual bipolar move. drive a complex nucleus nuclei to for a the the but the with of observed interacting dynamics, as surface in motors dynamics the nuclear competing resulting from inhibits variable of acting translocation, also activity motors The stochastic depletion directed nuclear myotubes. oppositely Dynein inhibits homology Thus, the myotube. in nuclei. klarsicht/ANC-1/Syne significantly the to adjacent motility with of between and localize interaction spacing midline dynamic rotation altered dynein, by the a to nuclear part and at leading forms in nuclei abolishes kinesin-1 which least of kinesin-1 proteins, cytoskeleton, aggregation at Nuclear of motor microtubule abnormal mediated microtubule Depletion intact is cell. localization an minus-end-directed proteins. the Kinesin-1 as require and (KASH) of myotubes. that rotation the plus- in cell find within and length The chromocenters envelope We muscle translocation and nuclei. the nuclear elucidated. nucleoli Both During envelope, around been along unit. nuclear types. network not The a regularly cell dimensions. have bipolar as three positioned many involved in together rotate mechanisms in nuclei rotate they the cell, nucleus nuclei with the but of through myofibers microtubules, translocate positioning actively requires syncytial nuclei and myotubes form developing movement in to the translocation fuse drive cytoskeleton myoblasts the development, with interactions Dynamic Summary 19104-6085, PA Philadelphia, Building, Richards D400 Pennsylvania, of ( correspondence University for the *Author at Medicine of Holzbaur* School USA F. Perelman Physiology, L. of Erika Department and Wilson H. cells Meredith muscle developing nuclear in robust dynamics drive motors microtubule Opposing 4158 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. rprncerpstoigi lociia nseea muscle skeletal in critical also is positioning nuclear Proper yen iei,Mcouue uce Nuclei Muscle, Microtubule, Kinesin, Dynein, .elegans C. [email protected] mro iei- stepredominant the is kinesin-1 embryo, ) rqetyi irtn el n sdie ydni motors dynein migrating by in more rotation driven of occurs is function The rotation and 2008). Holzbaur, This cells al., and two (Levy et 2008). migrating in (Ji in Holzbaur, attachment rotate frequently and substrate nuclei Levy of plane where 2007; the fibroblasts, within been cultured dimensions, has rotation in Nuclear 1961). explored Capers, Konigsberg, 1958; and Konigsberg, Cooper and 1960; (Cooper translocate they as rotate yet not Rubin, have and translocation and this explored. (Cooper (Englander drive been Konigsberg, dependent that cytoskeleton cell mechanisms be microtubule the and to 1987), the intact shown Cooper an was of translocation on 1960; this axis Although Capers, 1961). long 1958; the Konigsberg, along translocate function. muscle proper nuclear for correct muscular required that be suggesting in Emery-Dreifuss may 2011), found positioning al., dominant been et (Mattioli have autosomal (Bruusgaard dystrophy nuclei cells with clustered long Abnormally patients extraordinarily 2003). This al., these and 2004). nuclei et in the between al., cytoplasm distances transport et the minimize to Kummer capacity the as transcriptional well of sufficient 2003; as ensure long-axis to al., the thought is along et positioning specialized the spaced (Bruusgaard at of evenly found cell, fiber cluster are the nuclei a the of for junction, periphery neuromuscular except the and, at nuclei length in centimeters al tde npiaymoue ugse htnce may nuclei that suggested myotubes primary in studies Early to observed been have myotubes chick developing in Nuclei eerhArticle Research Journal of Cell Science rce nteX n lnsoe ie h ih aesi n hwrpeettv rcso niiulcrmcnesdrn oaino h nucl the of rotation during chromocenters individual of tracks representative show D and C in Chrom panels arrows. right and The arrowheads time. with over demarcated planes Z are and structures envelope-associated Y nuclear X, the Color of 3). in marks Movie tracked fiduciary material the (supplementary nucleus; of shown each long-axis are (GPP130) the in ( Golgi indicates chromocenters or rotation. (centrin-2) arrow bright membrane of Bidirectional nuclear visualization time. outer (emerin), over aid membrane shown to nuclear are inner chromocenters (LaminA), z-stacks bright confocal label of projections dots maximum colored and and dye Hoechst with labeled was DNA rcscrepn otecrmcneslbldwt ooe osi h iesre.Saebr:2 bars: Scale series. time the in dots colored with labeled chromocenters the to correspond tracks le h ro niae h one-lcws oaino ulu,vsbewt hs-otatotc ywthn rmnn uloi h arro 10 the bar: nucleoli; Scale prominent nucleus. watching adjacent an by p passes optics been and phase-contrast have deforms with myotube it visible the as nucleus, in nucleus nuclei a a visible of of all rotation direction; path counter-clockwise horizontal the the in indicates runs arrow myotube The the blue. of long-axis The 1). Movie material (supplementary myotube (mean shown are myotube dye; per Hoechst with stained were Nuclei (D3–D7). r seta o rprdsrbto fnce ntemature the in examined. been nuclei yet of not and has distribution this however, proper length fibers; rotation, muscle for and the dynamics translocation essential nuclear both that are Given possible including is development, centrality. it during myotube, the nuclear of important be complexity to maintaining suggested been has in but unclear, still is fibroblasts i.1 uliatvl oaeadtasoaedrn ytb differentiation. myotube during translocate and rotate actively Nuclei 1. Fig. surface, nuclear the to localize within both translocate rotation. dynein nuclei three-dimensional that and robust display find Kinesin-1 also We by and 2010). (reviewed myotubes al., vivo in et myofibril myotubes Sanger developing C2C12 and of closely faithfully myogenesis that dynamics developing and resembling early that organization cytoskeletal of in with system assembly, features model most movement well-established replicates nuclear a myotubes, of dynamics nti td,w s iecl irsoyt xmn the examine to microscopy cell live use we study, this In 6 s.e.m.; n . 0moue) cl a:50 bar: Scale myotubes). 30 a atnni ngen mgsaemxmmpoetoso ofclzsre.Mouelnt n ubro nuclei of number and length Myotube z-series. confocal of projections maximum are Images green. in is -actinin m .( m. m .( m. D B ulii ytbsepesn loecn osrcst ae h ula lamina nuclear the label to constructs fluorescent expressing myotubes in Nuclei ) hs-otatiae rmatm-as euneo ula irto naD7 a in migration nuclear of sequence time-lapse a from images Phase-contrast ) C ( ersnaieeape fncerrtto splmnaymtra oi 2). Movie material (supplementary rotation nuclear of examples Representative ) A rprdsrbto fnce ndvlpn ucecells. Results the muscle developing for in essential abnormal nuclei are of causes dynamics distribution nuclear proper dynein myotubes, normal in or that nuclei of indicating kinesin dispersal inappropriate either and this aggregation of in motor Loss dominant more system. the is kinesin-1 dynamics, Whi nuclear proteins. klarsicht/ANC-1/Syne the (KASH) with homology interactions by mediated likely ytb Fg A.Ms ytbsdslyarltvl even relatively a display myotubes Most per 1A). nuclei (Fig. of number the myotube cell in the increase of an length with concomitant the increases fuse, myoblasts additional As multinucleated cells. generate syncytial to fuse myoblasts as formed are in Myotubes dimensions three in myotubes rotate developing and translocate both Nuclei ersnaieiae fmoue ifrnitdfr37dy nvitro in days 3–7 for differentiated myotubes of images Representative ) ula yaisi eeoigmoue 4159 myotubes developing in dynamics Nuclear m m. ebt ooscnrbt to contribute motors both le ha olw the follows whead seudo-colored cneswere ocenters ddt label dots ed i Shaded ei. myotube Journal of Cell Science aeilMve1.Teetn n aeo oainvaries rotation of rate 2 than and more rotating extent nuclei of Occasionally The 50–60% myotube. with 1). considerably, the Movie of (supplementary rotation material axis of direction their their long change nuclei individual the of to of tracking perpendicular course accurate time the space. allowing XYZ over in another chromocenters min), trajectories (15 one of 2). to positions Movie observation relative the material fixed nucleus, supplementary remain individual 1C; an (Fig. focal to in Within time chromocenters microscopy stained over confocal brightly used the stacks of and DNA positions dye, the the labeled Hoechst track we live-cell precisely, a more phase- rotation with time-lapse Movie this material monitor in supplementary To arrow; nucleoli 1). 1B, of (Fig. imaging movement contrast the monitoring by on one dependent, not pass but influenced, nearby. that can is those suggesting nucleus nuclei arrowhead), each of 1B, and dynamics (Fig. the groups, translocate in they myotube as will or another either same nuclei individually, the individual in move within cell; can Nuclei migrate the direction. of change Nuclei long-axis occasionally the 1). along of Movie direction course material the continuous, supplementary vary than over can nuclei rather greatly individual of episodic, velocities Translocation by movement. characterized dynamics, analysis. form accurate for to strongly substantial begin too undergone contract myotubes and had networks point branched that time cells this in in past myofibrillogenesis; dynamics days study remained 4–7 in to days 7 for velocities order differentiated myotubes differentiated on primarily Average focused myotubes We culture. 1987). in constant Rubin, relatively myotubes and primary in translocation (Englander nuclear of observations rate average initial an at developing 1) 11.7 complex Movie the of material of and supplementary 1B; length robust (Fig. the cell display along translocate nuclei Nuclei that dynamics. found and microscopy 22.7 be post n days to 7 1A), found myotubes (Fig. in (D7) cell nuclei differentiation between the distance of average length the the with throughout nuclei of distribution 4160 oerahn eoiisa iha 14.4 as high as velocities reaching some aiAt ae h ula aia olbldteDAwith DNA the co-labeled lamina, nuclear the RFP– label with to myotubes nuclei transfected andlaminA for we Levy true dimensions, also three 2007; is in al., this rotating et whether determine (Ji To structure 2008). intact Holzbaur, an and lamina as nuclear the envelope with nuclear rotate nucleus the of interior the within nuclear 1). of Movie deformation material rotation supplementary by 1B; observed accompanied (Fig. The shape often rotated. is nuclei in passing both and during nucleus, events, one these least at of of 93% rotation involved all in that events, indicates pass myotubes 29 75 The from nuclear series with translocation. time correlates of closely Analysis another by rotation. one pass accompanied neighboring to nuclei of usually of dynamics ability is the of Rotation individual independent for nuclei. rotation be with of to concert direction appear in and nuclei moving rate the nuclei rotate; as can well others, as nuclei, Isolated panel). ocs y oosrecrmcnesadeaie nuclear examined and chromocenters observe to dye Hoechst 5 ulirtt lcws rcutrlcws bu h axes the about counterclockwise or clockwise rotate Nuclei assessed as dimensions, three in rotate also nuclei Remarkably, complex exhibit and mobile are myotubes in nuclei Most phase-contrast time-lapse with myotubes C2C12 imaged We hnnce oaei w iesosi irbat,nucleoli fibroblasts, in dimensions two in rotate nuclei When 3 ulii 6myotubes). 66 in nuclei 630 6 7.8 ora fCl cec 2 (17) 125 Science Cell of Journal m /r(0.2 m/hr 6 0.13 m . /i;mean m/min; or fiaig(i.1B; (Fig. imaging of hours 3 6 0.95 ˚ mn(i.1,bottom 1C, (Fig. /min m (mean m 6 ..,smlrto similar s.d.), 6 s.e.m.; ˚ /min, 09.Ms el hwdasaloealba nmicrotubule in (58.6 bias myotube the overall of al., small et end T. one a Zhang, toward showed 2005; growth al., cells et Most (Pizon by 2009). described also 4), Movie h ogai ftecl Fg 2B1,1 (Fig. cell the along of organization axis microtubule long the anti-parallel of the an center 2B2,2 observe the (Fig. in we generally contrast, cortex cell, In are the 4). Movie microtubules toward material Steinmetz, myotube, plus-ends supplementary the and their the of with (Akhmanova with ends oriented microtubule the tracks the At fluorescently- that 2008). of a (GFP–EB3) end with protein microtubules growing of end-binding plus-tips tagged the labeled we 2010). move al., and et cortex Sanger cell by the (reviewed at initially inward showing form studies myofibrils with nascent array consistent that microtubule interior, the the of with toward 2A), more surround long-axis located (Fig. myofibrils nuclei the the In located and centrally 2005). microtubules along the al., the mononucleated et oriented both section, Pizon in array cross 1974; found (Warren, linear myotubes array a multinucleated centrosomal to radial, myoblasts reorganized the is cytoskeleton from microtubule the myogenesis, During myotubes in translocation rotation nuclear both and for necessary are Microtubules ) oehr hs xeiet eelta h ula interior nuclear the that 1997), reveal al., experiments Movie material these et supplementary Together, 1D; (Linstedt 3). (Fig. mCherry- GPP130 nucleus with the labeled protein, with Golgi, rotates matrix the that Golgi material found supplementary tagged al., also 1D; We et (Fig. 3). Bugnard nucleus centrin-2 Movie 1985; the individual al., that with shows rotate et Tracking puncta (Tassin 2011). al., cells et these surface Zaal the 2005; in from nuclei occur the to known of identifies nucleation, likely microtubule DsRed–Centrin-2 as (Bugnard 2009). of al., nucleus, sites myotubes et the in Srsen of 2005; proteins surface al., et centrosomal outer the other puncta at for in and described We localizes cytoplasm nuclei. centrin-2 the labeled in protein mCherry-GPP130 both the centrosomal of the or dynamics that DsRed-Centrin-2 found the observed a envelope, and the either construct with conjunction expressed in we move in also membrane rotates nuclear also nucleus the 3). construct chromocenters. of Hoechst-labeled (GFP–emerin) membrane the Movie with inner emerin tandem material the GFP-tagged that supplementary a reveals 1D; RFP– of (Fig. Expression the chromatin with in along rotating is the marks lamina nuclear the fiduciary that of show labeling laminA Comparison with microscopy. movements confocal chromocenter time-lapse using rotation 08,adi itntfo h he-iesoa rotation three-dimensional the Holzbaur, and from myotubes. Levy developed distinct fully 2007; more in al., is rotation in observed et nuclear and nuclei the (Ji 2008), resembles fibroblasts flattened which in the substratum, The described in the rotation. rotation of two-dimensional plane nuclear exhibit spherical occasionally in more myoblasts correlates are changes shape nuclei in in change with the This S1). nuclei myotubes Fig. structures material in While (supplementary disc-shaped fibroblasts, S1). in the three seen Fig. resemble myotubes myoblasts in in material those mononucleated to supplementary unit compared myoblasts 2A; in a (Fig. nuclei of as shape the together Golgi. the including rotate organelles associated with envelope along dimensions, nuclear and oepoeteplrt n yaiso h irtbl array, microtubule the of dynamics and polarity the explore To odtriewehrsrcue soitdwt h outer the with associated structures whether determine To smoue eeo nclue entdadaai hnein change dramatic a noted we culture, in develop myotubes As 9 C upeetr material supplementary ,C; 6 . s41.4 vs 2.4 6 2.4; 9 ,C; Journal of Cell Science ( rnlcto olwn ramn fmoue ihDS rncdzl (mean nocodazole or DMSO with myotubes of treatment following translocation ulio h et hddtak orsodt h hooetr aee ihclrddt ntetm eis cl as 10 bars: Scale series. time the in dots colored orientati rotatio with the during labeled depict tracks chromocenters lines chromocenter White representative the time. shows to over panel correspond shown Right tracks are visualization. Shaded z-stacks aid S confocal left. to nuclei. of chromocenters the representative projections bright on of maximum label nuclei and migration dots dye colored the Hoechst and follow with myotube Arrowheads labeled the blue. was DNA pseudo-colored 6). been Movie 10 have material or myotubes (left) in DMSO nuclei with visible 10 treated all myotubes and in direction migration horizontal nuclear of sequences time-lapse n uliwr aee ihHehtde(le.Snl ln nX.Saebr:10 bars: Scale XY. in plane Z (10 Single nocodazole (blue). or dye DMSO Hoechst with treated with labeled 10 th were bars: show nuclei to Scale and (1–4) XY. overlaid in and plane respectively, ( red, Z myotube. pla and Single Y purple single dye. green, a with Hoechst is pseudo-colored with Shown were 1 nuclei. frames sequential growth. located three centrally microtubule and the of seconds around 3 tube every a obtained forming were network, myofibril developing 5 the bars: to Scale parallel run myotubes in Microtubules o ro;splmnaymtra oi ) ewe two between 5), Movie material supplementary smaller 2B3,3 arrow; (Fig. surface nuclear in top Microtubules the of dynamic. curve in are the found along surface polymerize Microtubules can nuclear arrowhead). the are with panel, contact microtubules top close (not 2D, myofibrils (Fig. only and invaginations panel) whereas bottom shown), 2D, (Fig. contain grooves microtubules wider nuclear panel, deeper, the both The on top arrowheads). grooves, 2D, 2D, or (Fig. invaginations, (Fig. surface within influence as nuclei well adjacent as may arrow) between lie polarity Microtubules microtubule that that in possibility movement. myotube nuclear the microtubule-dependent a differences raising within growth, local areas polarized were highly exhibited there Often respectively). mean rotation. and translocation nuclear both for required is and myotubes in nuclei around network dynamic a forms cytoskeleton ( microtubule The 2. Fig. I A uniiaino h ereo ula oaini rae ytbs(*** myotubes treated in rotation nuclear of degree the of Quantification ) mg famouei rs eto tie ihatbde otbln(T e)and red) (MT, tubulin to antibodies with stained section cross in myotube a of Image ) irtblsaeotnfudi ls poiint h nucleus. the to apposition close in found often are Microtubules m .( m. 6 G D ersnaieeape fncerrtto nmoue rae ihDS tppnl r10 or panel) (top DMSO with treated myotubes in rotation nuclear of examples Representative ) ... o oiatvru o-oiatdirection, non-dominant versus dominant for s.e.m.) irtblslebtenajcn uli(ro)adwti naiain arwed)o h ula ufc.Clswr tie o uui (re tubulin for stained were Cells surface. nuclear the on (arrowheads) invaginations within and (arrow) nuclei adjacent between lie Microtubules ) m .( m. B ytbswr rnfce ihGPE3t ae h rwn lsed fmcouue splmnaymtra ois4 ) Images 5). 4, Movies material (supplementary microtubules of plus-ends growing the label to GFP-EB3 with transfected were Myotubes ) 9 –3 9 eittedrcino ersnaiemcouuegot.4 growth. microtubule representative of direction the depict m /l o 0mna 37 at min 30 for g/ml) m pnl1;5 1); (panel m ˚ .Clswr tie ihatbde otbln cl a:20 bar: Scale tubulin. to antibodies with stained were Cells C. m 9 pnl n ) 2 3); and 2 (panels m , P , 0.001, 6 ula oainwsaoihdi ytbstetdwt 10 with treated myotubes in abolished was rotation nuclear epciey mean respectively; uli(otmarw,adwti naiain Fg 2B4,4 (Fig. invaginations within and arrow), (bottom nuclei rnlcto hncmae oteDS-rae control nuclear DMSO-treated of the with rate (0.082 to myotubes the compared myotubes reduced of when significantly treatment translocation 2E) microtubule that (Fig. the In depolymerize network found that 1987). nocodazole Rubin, we of of and concentrations (Englander inhibition cells, effect the no C2C12 whereas had myotubes, dynamics constantly actin primary and in complex translocation a forming nucleus. each myotubes, around network with in changing association close nuclei in is observations the network these microtubule the Together, that 5). indicate Movie material supplementary ula yaisi eeoigmoue 4161 myotubes developing in dynamics Nuclear ... *** s.e.m.; irtbl eoyeiainefcieyihbt nuclear inhibits effectively depolymerization Microtubule x m m 2 .( m. 9 ts;sonaepoe aafor data pooled are shown -test; /lncdzl rgt.Teln-xso h ytbsrni the in run myotubes the of long-axis The (right). nocodazole g/ml hw h naiaini h ula ufc.TeDAwslabeled was DNA The surface. nuclear the in invagination the shows a atnn(re) uliaelbldwt ocs y (blue). dye Hoechst with labeled are Nuclei (green). -actinin E ersnaieiae fmoue n urudn myoblasts surrounding and myotubes of images Representative ) P , m pnl4 (panel m .0,ANOVA; 0.001, 6 6 0.009 m s.e.m., 9 /lncdzl bto ae:supplementary panel: (bottom nocodazole g/ml .( ). C m oe fmcouueoinainwti a within orientation microtubule of Model ) /i s0.181 vs m/min n P . , 0nce nfv osvnmyotubes). seven to five in nuclei 30 .0;Fg FH.Additionally, 2F,H). Fig. 0.001; n . m .( m. 0 ulii 02 myotubes). 20–22 in nuclei 100 m .( m. F hs-otatiae from images Phase-contrast ) H eoiyo nuclear of Velocity ) 6 0.032 fteboxed the of n aebar: cale direction e m ei XZ. in ne m/min, m g/ml nof on d) 9 ; Journal of Cell Science nue grgto fnce ln h ytb,wt a respectively, EGFP, with expressing myotubes mean control myotube, to compared the as (13.0 along nuclei construct 19.3 adjacent vs between nuclei negative distance mean dominant of decreased 3D). the (Fig. aggregation cells EGFP–nesprin-2 of control induced in that expression those found than Furthermore, less myotubes displacement we rotated nuclei in this complete, positive Although envelope not S2). nuclear was Fig. the material from (supplementary EGFP–nesprin-2 kinesin-1 of displaced 2005). expression nesprins al., that et endogenous Grady found 2002; the al., We et displacing (Zhen localizes membrane by nuclear the negative domain from dominant acts This and 3C) a rotation. (Fig. as myotubes in nuclear envelope nuclear on the to specifically effect its assessed nesprin-2 of myotubes. in envelope result nuclear of localization the This the to 3B). part, kinesin-1 in (Fig. least construct at KLC1, mediates, nesprin-2 nesprin-2 of that the suggests binding to specific KHC observed and KLC2 and nesprin- C2C12 6348–6552 of residues from domain binding 2, assays KLC minimal pulldown the with GST interaction lysate this performed myotube examine we To 2011). myotubes, al., in et (Schneider to KLC shown been to recently bind has nesprin-2 protein Fridolfsson, KASH and the Starr and by 2010), (reviewed systems many 3A). in (Fig. cytoskeleton envelope proteins, nucleus led partner 2009), the Setou, of KHC and decoration Konishi to uniform 1997; to al., binds KIF5C et (Bi GFP-tagged which KLC1/2 also a including construct, We with domain myotubes shown). of tail not transfection a mAb, with that (SUK4 stained found were antibody myotubes pan-KHC when 3A). found second Fig. was in pattern shown similar decorate are A images abundantly (representative 1614) surface mAb nuclear (KHC, the myotubes. (KLC1/2) chain 2 heavy determine and C2C12 kinesin 1 chains and To D7 light envelope, kinesin nuclear 2009). on against directed the Antibodies al., immunofluorescence to performed et localized motor we X. kinesin-1 and (Levy Zhang, nucleus the neurons the whether 2008; of migrating surface Holzbaur, the as from and act such motors the rotation fibroblasts, and/or nuclear motor-dependent translocation microtubule displaying systems other In in rotation and myotubes dynamic translocation nuclear drives that rotation. Kinesin-1 or not suggesting translocation nuclear does for network, but essential not panel), microtubule are microtubules central the 7, eliminate Movie material growth microtubule supplementary (supplementary inhibits concentration ng/ml; This (100 7). myotubes nocodazole Movie in of material rotate dose lower and a translocate with to treated able still are nuclei dynamics, and translocation nuclear both Thus, for rotation. 6). necessary Movie are material microtubules supplementary 2G,I; (Fig. nocodazole 4162 I5,wihi h nyknsn1ioomepesdi skeletal in expressed isoform on kinesin-1 effects only against resulting the siRNA the is with evaluated which transfected KIF5B, and were Myotubes siRNA dynamics. with nuclear motor expression the the reduced we of myotubes, in rotation and/or translocation 3E). Fig. different; ofrhrts hspsiiiy eepesdteKS domain KASH the expressed we possibility, this test further To the and nucleus the between links mediate proteins KASH nuclear for necessary is network microtubule intact an While nodrt oedrcl eemn hte iei- drives kinesin-1 whether determine directly more to order In 6 6 0.9 ora fCl cec 2 (17) 125 Science Cell of Journal s.e.m., m a nEGFP–nesprin-2 in m (EGFP–nesprin-2 P , .01 enmouelnt a not was length myotube mean 0.0001; a KS)i ytbsand myotubes in –KASH) a KS-oiiemyotubes –KASH-positive a KS partially –KASH a 6 –KASH- 0.9 m m it;ls hn8 ftedt i bv 50 above lie data 50 the of 8% than less width; ereo ula oaini ytbstetdwt GPo EGFP– or EGFP with treated myotubes in nesprin2 10 rotation bar: nuclear Scale of dye). degree Hoechst with labeled was EGFP–nesprin-2 daetnce nmoue rae ihEGFP–nesprin-2 with treated myotubes in nuclei adjacent ( myotubes). 19–21 from ihHehtde cl a:10 labeled was bar: DNA Scale KHC. KLC1/2. dye. and with Hoechst KLC1/2 interacts for with methanol that with KHC fixed of were transfected domain Myotubes myotube the a tail, is shown EGFP-KIF5C 1/2 Also with (KHC)]. chains chain light heavy [kinesin kinesin kinesin-1 (KLC1/2); motor microtubule plus-end-directed the 0mb n H SK A) ltsoni ersnaieo three of representative is shown (63– blot ( KLC1/2 mAb); replicates. to (SUK4 independent in antibodies KHC boiled with and then probed mAb) were were 90 beads Blots first E1,E2); (Boil). eluted rounds: buffer was (two denaturing protein glutathione Bound mM lysate. 80 myotube with C2C12 with incubated beads GST–nesprin-2 oan epciey.Pl-onasy ihGST–nesprin2 with KASH assays and Pull-down domain respectively). transmembrane domain, repeats, spectrin domain, actin-binding xrsinlvl nmc iN-rae ytbs(i.4A). (Fig. myotubes siRNA-treated mock in levels expression i.3 iei- oaie otencerevlp nmoue n binds and myotubes nesprin-2. in protein, envelope LINC nuclear the the to to localizes Kinesin-1 3. Fig. bet ossetyrdc h eeso I5 to KIF5B of levels were the We reduce myotubes. D7 consistently nuclear in and later to differentiation hours of able 72 4 assessed day on were 2000), dynamics al., et (Kanai muscle m o clarity, for m a KS ( –KASH 6348–6552 a KS mxmmpoeto facnoa -eis DNA z-series; confocal a of projection (maximum –KASH n x . 2 -test; 8 uliin nuclei 382 oag,gen upeadbu hpsrpeetthe represent shapes blue and purple green, (orange, C E ersnaieiaeo ytb expressing myotube a of image Representative ) rqec itiuin ftedsac between distance the of distributions Frequency ) P 5 .8 hw r olddt for data pooled are shown 0.18; m ( .( m. A . ytbswr tie o uuisof subunits for stained were Myotubes ) 9myotubes). 29 B ceai fnesprin-2 of Schematic ) m ,s itiuini rnae at truncated is distribution so m, m .( m. D uniiaino the of Quantification ) a KS (1 –KASH 6348–6552 n . giant , 4 nuclei 140 0 of 20% rGST or and m bin m Journal of Cell Science lseddrce irtbl oo iei- knsnhaycan(H) iei ih his12(L12] N a aee ihHehtde Sc dye. Hoechst with labeled was DNA (KLC1/2)]. 1/2 chains light kinesin (KHC); chain heavy 10 [kinesin kinesin-1 motor microtubule plus-end-directed hooetrtak uigrtto ftebxdnce.Sae rcscrepn otecrmcneslbldwt ooe osi h ieseries. time the in dots and colored dye with repre Hoechst labeled ( show chromocenters with (E) below the function labeled panels to and/or The was correspond visualization. localization DNA aid tracks KIF5B 8). to Shaded chromocenters Movie nuclei. label material boxed dots (supplementary the blue 10 and of siRNA Red rotation KIF5B time. during or over tracks shown siRNA chromocenter are SCR z-stacks KIF5B confocal of with projections treated maximum myotubes in rotation nuclear h grgto fnce rmtecne eino hsmoue hc st h eto h eino h ytb hw) cl as 10 bars: Scale shown). myotube the of region the of left the to is which myotube, this of region center the from nuclei shows of 1 aggregation panel siRNA the in (inset G in shown otatmcocp o 0mnadaslt eoiywsqatfe (mean quantified was velocity absolute and myotubes. min in (mean 60 rotation for control microscopy and loading contrast translocation a nuclear as drive served to ( GAPDH surface (SCR). nuclear oligos the control from scrambled acts KIF5B 4. Fig. I5-eiin ytbsrte hnehbtn persistent, the in exhibiting unit a than as together translocation. rather squeeze independent and myotubes another one KIF5B-deficient of to top tended Nuclei on myotubes. control pile SCR in KIF5B in or those mock movement than the either different nuclear distinctly of were dynamics myotubes KIF5B-deficient the myotubes, siRNA- control the (SCR) scrambled in KIF5B treated decrease the small in translocation a of note rate did mean we Although 4B). (Fig. myotubes 48.7 to reduced was cells * P , h enncertasoainvlct nKIF5B-deficient in velocity translocation nuclear mean The m m .( m. .( m. .5 ** 0.05, G D , , H E uniiaino h ereo ula oaini ytbstetdwt iN D rGPKFCti oiatngtv osrc odisrupt to construct dominant-negative tail GFP-KIF5C or (D) siRNA with treated myotubes in rotation nuclear of degree the of Quantification ) P rae ytbswr tie o uui;DAwslbldwt ocs y.Rpeettv mgsfo h eta eino h ytbsare myotubes the of region central the from images Representative dye. Hoechst with labeled was DNA tubulin; for stained were myotubes Treated ) , .1 *** 0.01, P , 6 .0 sMc rGFP. or Mock vs 0.001 .%(mean 3.5% A ersnaieimnbosadqatfcto fKFBi ytbstetdwt iN n C iN o KIF5B; for siRNA SCR and siRNA with treated myotubes in KIF5B of quantification and immunoblots Representative ) x 2 a ts npoe aafor data pooled on -test atnnsann) ersnaieiaefo h n faKFBsRAtetdmouei hw nH(ne shows (inset H in shown is myotube KIF5B-siRNA-treated a of end the from image Representative staining). -actinin 6 6 ... ANOVA, s.e.m.; ... fmock-treated of s.e.m.) n 5 n needn elcts.( replicates). independent 3 . 2 ulifo 02 ytbs.( myotubes). 20–27 from nuclei 120 6 ... ANOVA; s.e.m.; yaisb cigfo h ufc ftences Consistent nucleus. the of surface the from acting nuclear by in dynamics 1997; decrease al., a et induced (Bi also 4E). (Fig. 2009), cargos rotation Setou, from a and motors In Konishi endogenous 2I). a (Fig. using displace acts which kinesin-1 nocodazole to construct, inhibiting tail-domain of GFP-KIF5C that negative effect found dominant the we experiment, to parallel similar material supplementary 8), 4C,D; (Fig. Movie rotation nuclear in reduction ula yaisi eeoigmoue 4163 myotubes developing in dynamics Nuclear oehr hs bevtossgetta I5 rvsnuclear drives KIF5B that suggest observations these Together, striking a to led also KIF5B of depletion that found We n B . ok rsRAtetdmoue eeiae ihphase- with imaged were myotubes siRNA-treated or Mock- ) 0nce n1–2moue) ( myotubes). 10–12 in nuclei 50 F rae ytbswr tie o uuiso the of subunits for stained were myotubes Treated ) ytbswr rae ihKFBsRAo with or siRNA KIF5B with treated were Myotubes C ersnaieeape of examples Representative ) m .Fralpanels, all For m. cl bars: Scale sentative l bars: ale Journal of Cell Science ulisc smoueed,mcouueognzto and organization 4H; (Fig. microtubule myotubes control ends, from myotube indistinguishable were as polarity of devoid the such regions 2; to in panel nuclei However, the relative siRNA 9). 4G, Movie comets (Fig. around material variable supplementary GFP–EB3 more frequently myotube of the the myotubes, of direction more long-axis of these the wrap In center with nuclei. the to nuclei, of in vicinity appeared network the microtubules complex in only more of but a 58% myotube, 1; form panel to siRNA tended and SCR that 4G, from microtubule (Fig. the myotubes, different cells alters on not control was in impact motor seen network turn microtubule the the in In myotubes, of movement. may nuclear the which loss of dynamics myotubes, that it in However, possible 4F). organization although (Fig. also cells, persists KLC1/2 siRNA-treated is for with signal treated to residual localized cells some longer no in is nucleus KHC that the found we hypothesis, this with 4164 ora fCl cec 2 (17) 125 Science Cell of Journal n 5 0.I 2 fKFBdpee el,microtubules cells, KIF5B-depleted of 42% In 50). . 0 fKIF5B-deficient of 50% iN ae ne) iei--eltdmoue were mis-organization nuclei. myotubes the these the despite of that sarcomeres Kinesin-1-depleted suggesting functional again develop inset). cells spontaneously, contract 1 KIF5B- to observed noted panel also judged siRNA as YFP– We myofibrils, organized by develop 9). can myotubes Movie deficient material supplementary L12(i.3) hsmyrfetalmtto fepitope of limitation a reflect may and This KHC for 3A). observed on (Fig. that dynactin than and KLC1/2 irregular dynein more was of envelope cellular localization the of The range cytosolic 5A). broad strong a (Fig. with a functions consistent to motor minus addition the the of in at the localization myotubes, enriched that of also reveal periphery is nuclear dynactin complex motor and microtubule dynein end-directed both to Antibodies and translocation nuclear rotation to contributes also Dynein ** o iN scmae otesm okdt si i.4 ( 4D Fig. in as data Mock same the Data to siRNA. compared DHC is with treated siRNA myotubes for in rotation nuclear of degree olddt for data pooled o 0mnadaslt eoiywsqatfe (mean quantified was velocity absolute ANOVA; and min microscopy 60 phase-contrast for with 10 imaged bars: were Scale myotubes dye. siRNA-treated Hoechst with labeled was chain intermediate dynein p150 (DHC); (DIC); chain dynein/dynactin heavy minus-end-directed [dynein the complex of components for stained H C iN eesandfrDC I n p150 and DIC DHC, for or stained siRNA were DHC siRNA with SCR treated DHC Myotubes (mean (C) DHC; control replicates). for loading independent siRNA a with as dynein treated served and myotubes GAPDH (DHC) in chain (DIC) heavy chain dynein intermediate of (right) (left) quantification immunoblots and Representative (B) bars: (SCR). oligos Scale control dye. scrambled Hoechst with DHC labeled for was 10 acetone:methanol DNA 1:1 analysis. with DIC and and analysis, p150 for methanol i.5 yenlclzst h ula neoei ytbsand myotubes in rotation. envelope and nuclear translocation the nuclear to drives localizes Dynein 5. Fig. P m , a .( m. 0.01. atnnlclzto nD 21 el Fg 4G, (Fig. cells C2C12 D7 in localization -actinin B–D n . Glued ytbswr rae ihsRAt H rwith or DHC to siRNA with treated were Myotubes ) 0nce n1–2moue) ( myotubes). 10–12 in nuclei 50 n . uui fdnci] ytbswr ie with fixed were Myotubes dynactin]. of subunit 2 ulifo 02 ytbs.Fralpanels, all For myotubes). 20–27 from nuclei 120 6 ( E A m ... ANOVA, s.e.m.; uniiaino the of Quantification ) ytbswere Myotubes ) .()Mc-or Mock- (D) m. 6 Glued s.e.m.; DNA . x 2 n -test; 5 3 Journal of Cell Science yais h lseddrce I5 stemr dominant more the is data nuclear to KIF5B These contribute motor. plus-end-directed KIF5B 4D). and the Fig. dynein dynamics, both 5E, while (Fig. that myotubes suggest KIF5B-deficient in rae ytbs(i.5) ih2%o h ulicniun to continuing nuclei the 30 of than 25% more mock- translocation with rotate as of 5C), 64.7% nuclear (Fig. striking to myotubes Mean reduced as treated was motor. cells not dynein-deficient kinesin-1 in of was velocity the effect dynamics for the nuclear observed However, on 5D,E). depletion (Fig. dynein rotation and translocation myotubes dynein-depleted 5C). in (Fig. envelope nuclear the from depleted oksRAtetdmoue fe 2huso knock-down reduce of consistently to hours 72 to after DHC able myotubes were of siRNA-treated We mock levels motor. the the deplete the to less than is surface complex nuclear dynein the motor. the kinesin-1 across that distributed suggest homogenously may or accessibility, nncerlclzto,w lte h oiino ulia a Nuclei as 6E). (Fig. nuclei length of myotube position the the along distance plotted of we function localization, nuclear on may rotation aggregation. reduced nuclear 5), induce one 4, or to (Figs pass KIF5B sufficient normally to to nuclear be siRNA rotate order with not in treated proper do rotate myotubes dynein Live to in that nuclei. need nuclei nuclei As of another. indicate that aggregation suggest prevent between data data to cell distance necessary These average are dynamics 6D). the (Fig. reduced nuclei GFP–KIF5C significantly of also (expression function approach kinesin tail) negative of dynein- adjacent dominant disruption or a and between using KIF5B-depleted 6D), (Fig. in distance myotubes smaller depleted mean significantly was The was function dynein nuclei 6C,D). or was (Fig. kinesin nuclei either disrupted between when distance the of reduced the number the noticeably However, of in myotube. difference a per length detect nuclei we did the nor myotubes, Instead, along treated disruption. observed 6A,B). (Fig. kinesin-1 nuclear cells were dynein-depleted with in some nuclei myotube seen observed of the toward as we clusters enriched not center DHC, were in myotube aggregates for observed these but also siRNA aggregation, was myotubes with In aggregation myotubes construct. treated tail This GFP–KIF5C in the 6A,B). of expressing nuclei midline myotubes (Fig. the the at cell aggregate that densely 1A, the to (Fig. noted tended cells KIF5B we for control deficient of However, length 6A,B). the Fig. along even relatively nuclei a of display myotubes distribution differentiation, of days 7 myotubes After in distribution for nuclear necessary proper are dynamics nuclear Motor-dependent shown), blot (not p150 siRNA western of DHC with p150 levels Although treated for cells immunostaining expression in 5C). reduced (Fig. overall not were and that cell DHC indicates of the and depletion analysis myotubes a throughout Levy demonstrate of 2007; siRNA DIC al., analysis DHC et with Immunofluorescence (Caviston treated been types 2008). has DIC, as cell Holzbaur, destabilized, of other is levels in complex reduces observed dynein the concomitantly that siRNA suggesting DHC 5B). (Fig. ncdw fDCrdcdteaeaert fnuclear of rate average the reduced DHC of Knockdown ots h oeo yeni ula yais eue siRNA used we dynamics, nuclear in dynein of role the test To ofrhrcmaeteefcso oo elto rinhibition or depletion motor of effects the compare further To and control between different not was length myotube Mean ˚ n1 i,a oprdt ny2 fnuclei of 2% only to compared as min, 15 in Glued , ugssta yatnmyas be also may dynactin that suggests 0 fepeso eesin levels expression of 20% Glued ieymdae tlati atb neatoswt h KASH network. the microtubule with the along interactions translocation nuclear rotation nuclear by both and drive and part complex, abnormal LINC in the of least envelope,proteins and nuclear at the aggregation mediated to likely localize nuclear proteins motor The to nuclear distribution. normal leads for these of motors required inhibition myofibers. or Depletion are myotubes. mature developing dynein in dynamics of and even length kinesin-1 motor microtubule an the proteins the that achieve find we to hypothesis, along this necessary with Consistent nuclei is of mobility this distribution 1960). that (Capers, propose mobile very We are cells muscle developing in Nuclei cells. elongated these Discussion preventing of and ends This of the translocation rotation 6E). toward proper (Fig. out driving for nuclei cells necessary the to also control is addition the KIF5B aggregation, in to in similar that nuclei length, suggests, myotube of full distribution the their dynein-deficient the along despite in found nuclei Conversely, were the myotubes for as tail. aggregate, seen to GFP–KIF5C midline was propensity increased pattern with the same treated This near cells myotubes. clustered control to myotubes compared KIF5B-deficient in ta. 07 alee l,20)adncerrttosin rotations nuclear and 2009) al., et in 2008), Holzbaur, Vallee and Tsai (Levy 2007; 2004; fibroblasts al., et al., dynein (Shu neurons driver et while migrating primary in the 4), movement is (Fig. nuclear dynein Though of extent. myotubes lesser a in to contributes dynamics nuclear driving in distribution network. organelle microtubule and indir the or dynamics directly, may either nuclear flow myotubes, to This 1). contribute Movie streaming also material cytoplasmic (supplementary 2007) and the Shimmen, phase- resemble plants myofibril our that in During cell fluid described Thus, role. bulk the observed minor within often 11). we a dynamics dynamics, and only myotube translocate of play Movie imaging to to contrast able likely material still is were contraction (supplementary nuclei al., the et that (Cheung sulphonamide rotate myosin found muscle N-benzyl-ptoluene and skeletal of 2002), movement with inhibitor specific nuclear a contraction (BTS), influence inhibited also the 10) we might Movie of nuclei material with contractions contractions (supplementary contact microtubule as close their in such often and by are involved, Myofibrils forces network. abolished myofibril additional nucle be residual was could nocodazole- some in cells, inhibited rotation treated was the translocation While Nuclear on depolymerization. inform 2). (Fig. likely development. myotube here vertebrate and been Thus, during described occur 2010). that arrangement has dynamics al., dynamics et microtubule nuclear what Sanger 1974; the including to (Warren, assembly vivo, patterns myofibril myotubes in C2C12 developmental and described primary and similar Both translocate dimensions another. all nuclei exhibit one in that rotate pass myotube, even find 1961; the can of We Konigsberg, long-axis 1987). the and along Rubin, Cooper myotubes and 1960; chick Englander primary Capers, in 1958; observations (Cooper, initial with consistent tr,21) iei a infcnl ihrsalfrethan force stall higher significantly a has and Kinesin (Fridolfsson 2010). predominates Starr, activity kinesin-1 cells, precursor ula yaisi eeoigmoue 4165 myotubes developing in dynamics Nuclear h ula yaisw bev nCC2moue are myotubes C2C12 in observe we dynamics nuclear The u bevtosidct htknsn1i h rmr motor primary the is kinesin-1 that indicate observations Our network microtubule intact an require dynamics nuclear These Drosophila cl yifunigteplrt of polarity the influencing by ectly rmvmn ugssta there that suggests movement ar oivsiaet investigate To . oye Sru ta. 2005; al., et (Serbus oocytes .elegans C. i possibility, his hypodermal Journal of Cell Science eitn h oiino ulia ecnaeo h itneaogtemouelnt bnwidth (bin length myotube the along distance the of percentage a as nuclei of position the depicting isaoe50 above lies iN ytbs 3moue F-I5 al.Teed ftemoueaemre ihadr qae aapit ersn niiulnuclei. individual represent points data square; dark a with marked are myotube the of ends The tail). GFP-KIF5C ( myotubes 23 myotubes; siRNA fteecntut otencerevlp a displace can envelope nuclear the to localization the constructs motors. since endogenous these activity, motor of nuclear-based or for tail argues GFP–KIF5C the either EGFP–nesprin-2 expressing rotation myotubes drive nuclear to in Reduced surface observed suggests rotation. nuclear and the nucleus translocation from the nuclear acts both to motors of dynein population and a ( that kinesin with large both packed this of driver cellular of localization densely effective more motility complex is a the be the of which may within kinesin-1 myotube, myofibrils, Thus, developing the 2010). of al., environment et (Hendricks ( dynein mammalian myotubes. in nuclei of ( distribution ( z-series. proper tail. confocal for GFP-KIF5C necessary or are GFP dynamics GFP, nuclear Motor-dependent 6. Fig. 4166 C rqec itiuin ftedsac ewe daetnce nmoue rae ihsRAo F-I5 al(1 tail GFP-KIF5C or siRNA with treated myotubes in nuclei adjacent between distance the of distributions Frequency ) ora fCl cec 2 (17) 125 Science Cell of Journal m odsrbtosaetuctda 50 at truncated distributions are so m B a itiuino ulii ytbs ahln nteyai ersnsa niiulmoue raie codn olnt ( length to according organized myotube, individual an represents y-axis the on line Each myotubes. in nuclei of Distribution ) KS oiatngtv osrcsalso constructs dominant-negative –KASH , – Nfrknsnv . Nfrdynein) for pN 1.1 vs kinesin for pN 5–6 A ersnaieiae ftetdmoue.Nce eesandwt ocs y;iae r aiu rjcin of projections maximum are images dye; Hoechst with stained were Nuclei myotubes. treated of images Representative ) , 10 m imtr rael.The organelle. diameter) m m o lrt) ( clarity). for m D endsac ewe daetnce (mean nuclei adjacent between distance Mean ) ercutdt h ula neoetruhohrbinding other through envelope nuclear the to may or domain, recruited different to dynein a bind between be through mediated not interaction be brain did The may mouse nesprins here. dynein been and used although from construct 2009), have -2 kinesin nesprin al., dynactin and the et to X. nesprin-1 and (Zhang, with contribute Dynein lysate al., precipitate nucleus. also et to nesprin-2, (Schneider the shown may chain and to nesprin-1 light recruitment kinesin-1 kinesin however, data to between 2011); Our binding 2010). interaction by Fridolfsson, mediated the and (Starr lamina nuclear confirm nuclear cytoskeleton the outer the connect 3), to with (Fig. known nesprins proteins KASH with membrane interactions through part, I5 id otencerevlp nmoue,a es in least at myotubes, in envelope nuclear the to binds KIF5B 5 10%). ytbswr rnfce ihete iN + siRNA either with transfected were Myotubes 6 ... NV,*** ANOVA, s.e.m.; m i it;ls hn8 ftedata the of 8% than less width; bin m P , .0) ( 0.001). E Histogram ) n 5 42–55 Journal of Cell Science per ob o-nfrl itiue,bs lutae by p150 illustrated subunit best dynactin the distributed, for non-uniformly staining also be the complex motor to dynein The rotation. appears generation nuclear force to lead of may on areas that asymmetrical KIF5B create of could distribution the envelope non-uniform on the sites A that binding occupied. kinesin suggesting are the of envelope evenly, all not more tail conditions normal envelope the GFP–KIF5C under the the at whereas decorates distribution patchy, construct Kinesin appears of speed envelope translocation. and network. nuclear direction and the microtubule dictate rotation would local forces nuclear the all of on sum net force muscle The exerting proper by for dynamics essential 2007). al., is et Emery-Dreifuss Q. muscle (Zhang, and/or with skeletal function positioning patients in nuclear in that anchorage and suggesting found nesprin-1 dystrophy, 2007; both been muscular in al., mutations have et Moreover, X. nesprin-2 2010). (Zhang, al., muscle anchorage et in and Zhang critical positioning both are nuclear mice nesprin-2 for knock-out and from nesprin-1 Evidence 2010). that al., Zhang suggests of et 2001; Randles al., course 2005; et Zhang al., the 2000; et over al., et modulated (Apel development isoforms myotubes muscle spliced expression C2C12 relative alternatively the and with of nesprin-2, fibers and nesprin-1 muscle both express skeletal Mature partners. n oaiyo h irtbl ewr semdl i.7). Fig. 2011). model, (see dynamics al., network by microtubule and influenced et envelope, the are nuclear of dynamics the polarity (Parton to nuclear and bound the transport motors that by likely both cargo the is given it are direct polarity Therefore, especially microtubule to in movement, biases subtle sufficient nuclear a even that that of finding possible recent direction is with correlates It net growth cell. microtubule overall the the of nuclear of direction of the end direction in bias in one net the toward nuclear bias myotubes, was We many to movement overall myotube. in the that slight contribute of noticed end also a one nucleus toward exhibited directed individual growth microtubule myotubes an always Most on and of motors is dynamics. the dynamic nucleus surface how are influence given nuclear the significantly microtubules a the will these surrounding which on changing, of network between invaginations the all found within therefore or also lie Most are and surface. 1985) microtubules al., nuclei et However, adjacent Tassin surrounding 2). 1974; cell, Fig. (Warren, the (see nuclei of located long-axis centrally the the along array bidirectional dynamics. nuclear influence further to that likely imbalances also An force is to 5A). motors lead directed (Fig. oppositely nuclei of distribution adjacent uneven between accumulation enhanced epooeta ula-on oosifunenuclear influence motors nuclear-bound that propose We h irtbl ewr nmoue om dynamic a forms myotubes in network microtubule The Glued hc shows which , erae oiiyi I5-eiin ytbsmyinhibit is may nuclei myotubes The of KIF5B-deficient organelles. rotation large in these the mobility of dynamics that decreased the fluidize hypothesize that study to observation We essential translocation recent our nuclear rotation. with inhibited a line substantially and in more in is KIF5B of This myonuclei loss 2012). of al., 6). et (Fig. aggregation (Metzger myotube central the in function caused of kinesin-1 midline of loss the to Similarly, leading near KIF5B, aggregation of depletion nuclear upon disrupted severely most was r tcatc ittdb h raiainaddnmc fthe of dynamics and organization the by dictated stochastic, a are time. create over aggregation enhancing processes further and rotation these translocation nuclei nuclear and reducing the further Together, thereby for through, navigate network to shown). cytoskeletal 4G the complex (Fig. not of more model progressively disorganization this data local with and and consistent cytoskeleton, clumping microtubule nuclear of between correlation bipolar local direct degree a the less observed becomes we Further, nuclei disordered; and increasingly aggregated 9). the unipolar surrounding Movie network more material microtubule the supplementary becomes toward 4H; areas nuclei (Fig. aggregate cell in to of organization microtubule begin depleted and nuclei myotube, the as the of outward, center depleted, oriented bidirectional where is plus-ends activity ends with a kinesin myotube kinesin at polarized 2011), of significant are of most loss al., microtubules be to Since development et expected myotubes. be the would Zaal in nuclei Thus array 1985; to 1985). the microtubule al., al., from contribute et component. et (Tassin part actively directions in (Tassin feed-forward all in nucleated nuclei a polymerizing are of have myotube this surface in the may movement in of process Microtubules driver dominant the the as be system, to appear appears dynein-driven length. which surface cell the of jams, along sites Loss traffic discrete at nuclear spacing. aggregates also cause localized nuclear is might dynein proper driver, mobility primary suggests for the This be myotube. needed may the kinesin of still length although were the nuclei that dynein along the of distribute although to Loss nuclei, able center. of cell aggregation the caused at factor also aggregation critical a nuclear be to may leading nuclear myotubes of kinesin-depleted past loss the moved Thus, in of they hypothesis. rotation both this as with rotated or consistent events one another, passing one recordings, in cell involved including network live nuclei microtubule the path, In dense nucleus. their the the even in surrounding or obstacles myofibrils, nuclei, around other moving from nuclei ula yaisi eeoigmoue 4167 myotubes developing in dynamics Nuclear h itiuino uliaogtelnt fCC2myotubes C2C12 of length the along nuclei of distribution The hs epooeta h yaiso h ulii hssystem this in nuclei the of dynamics the that propose we Thus, nuclear the from translocation and rotation motor-driven While ln h egho h myotube. the of length nuclei the of along dispersal abnormal inappropriate causes and myotubes aggregation from KIF5B either or of dynein Depletion of translocation. speed and microtubule and rotation local direction the the of determine polarity network the on and motors nucleus opposing a of distribution links. and other number or nesprins The through nucleus the to bound myotubes. nuclear developing the in in dynamics opposing motors of dynein roles and the kinesin depicting Model 7. Fig. Drosophila mro also embryos oosare Motors Journal of Cell Science loohr-ojgtdscnayatbde eefo oeua Probes Molecular Jackson from from were antibodies Inc. Laboratories, anti-chicken were ImmunoResearch goat Cy2-conjugated antibodies and (Invitrogen) secondary fluorophore-conjugated os 21 ybat Aeia yeClueCleto,Mnsa,VA, Manassas, 37 Collection, at Culture maintained Type were (American CRL-1772) myoblasts C2C12 treatment drug Mouse and transfections culture, Cell University 5 16573; Akhmanova, ID (Gene (Anna KIF5B mouse GFP-EB3 against directed were and oligos YFP- siRNA University), Utrecht). London), Medical College Upstate EGFP-Nesprin2 King’s Medicine), University Shanahan, Hamamatsu of Setou, of (Mitsutoshi Linstedt, University tail School Gleeson, GFP-KIF5C (Adam Diego), (Joseph San mCherry-GPP130 DsRed1-Centrin-2 California University), University), Mellon Columbia Carnegie pGFP-emerin Worman, and pRFP-Lmna (Howard provided: generously were constructs following The Reagents Methods and Materials exercise decreased of Similarly, significantly 2010). distribution al., with et abnormal (Zhang myofiber capacity correlates exhibit function. the which mice, muscle nuclei, affects null in transcriptional likely nesprin-1 distances which sufficient Indeed, 2003), transport al., ensure et minimize (Bruusgaard to and necessary the capacity under is nuclear proper place that proposed in positioning been anchored has It become myofibers. in fluidsarcolemma will a continues, nuclei differentiation for distributed as allows myotube; the This the surface. in nuclei its of on distribution motors as of well complement as the nucleus individual an surrounding network microtubule 4168 nlss el eegono olgncae 1-cm collagen-coated on grown were cells analyses, nDS oihbtmoirlcontraction. myofibril inhibit to DMSO in 50 of presence the n wthdt MMwt ltmxad1%hresrmfr37days. coated 3–7 Instruments) for Precision on serum grown World were horse (FluorDish, cells 5 10% imaging, dishes with live-cell and glass-bottomed For hours. mm glutamax 24 50 with every replaced DMEM was to Medium to serum]. grown bovine were switched fetal myoblasts and 10% differentiation, and myogenic glutamax induce with To (DMEM) medium Eagle’s modified CUUACCAUU-3 CAACAGACAUGUCGCAGUU-3 yaismyb seta o rprfbrrpi n subsequent and repair fiber function. where proper muscle this for dystrophies, essential nuclear be for that frequently, muscular may occurs dynamics likely required the damage is muscle are like it induced mechanically and rotation diseases 2011), and In al., positioning. et translocation Li nuclear Lichtman, 2010; and both al., (Rich et myofiber the Terada of 1989; periphery fiber the a During at contributing positioned fiber, (Charge injury. injured nucleus the after with new themselves fuse cells repair myogenic repair, to fibers muscle function. muscle normal normal that for decreased suggesting required again is with distribution 2012), nuclear correlates defects al., et positioning which (Metzger locomotion development, nuclear muscle display during MAP7 mutant expressing ottaseto.Moue eetetdwt ooaoea 0o 20 or 10 37 hours at at 72 min imaged nocodazole 30 and for with control differentiation DMSO treated of or (Sigma-Aldrich) were induction after Myotubes cells days noted, of post-transfection. with otherwise 4 concentration Unless final performed transfected (Invitrogen). a RNAiMax transfected were were at Lipofectamine were Dharmacon) using transfections nM cells Scientific 50 (Thermo knockdown, DNA duplexes RNAi siRNA Inc.). For with (Invitrogen). Pella, 2000 (Ted Lipofectamine film embedding GAAAUCAACUUGCCCGAUAUU-3 ln 2 ilpr;SK,Acm,at-I MB68 ilpr) anti- Millipore), (MAB1618, anti-DIC Abcam), SUK4, (MAB1614, anti-KHC Millipore; p150 Brady), H2, Scott Cruz from clone Santa (63–90, 1/2 (R-325, anti- anti-KLC Abcam) anti-DHC (ab15705, Sigma-Aldrich), Inc.), KIF5B anti- Labs, Inc.), Aves Biotechnology, (GFP-1020, anti-GFP Serotec), CAAACCUGUUU-3 ula yaismyas otiuet h blt fskeletal of ability the to contribute also may dynamics Nuclear Glued m g/cm B isine) n niGPH(A 44 ba) Alexa Abcam). 9484, (mAb anti-GAPDH and Biosciences), (BD ora fCl cec 2 (17) 125 Science Cell of Journal 2 a alclae,tp B isine) o immunofluorescence For Biosciences). (BD 1 type collagen, tail rat 9 ,admuedni ev hi GnakN_328 5 NM_030238; (GenBank chain heavy dynein mouse and ), m -ezlpoun upoaie(T;Tci Bioscience) Tocris (BTS; sulphonamide N-benzyl-ptoluene M 9 .Piayatbde nld anti- include antibodies Primary ). ´ n unci 04.Teence are nuclei These 2004). Rudnicki, and 9 cabe oto:5 control: scrambled , ˚ /%CO C/5% 9 cabe oto:5 control: scrambled , a KS rsde 8–4)(Catherine 483–542) (residues -KASH a atnn(osp agr SUNY Sanger, (Joeseph -actinin 2 ngot eim[Dulbecco’s medium growth in Drosophila ˚ .Moue eeiae in imaged were Myotubes C. a 2 a 9 tbln(L/,AdD (YL1/2, -tubulin -GAACGAAUGUCG- atnn(ln EA-53, (clone -actinin qae fACLAR of squares 9 -GACCGAUAAU- , uatlarvae mutant 0 confluence 80% m g/ml 9 9 - - rprduigHMlssbfe 5 MHPS 5m al MEDTA, mM 1 NaCl, mM 25 HEPES, mM [50 MgCl were buffer mM lysates 1 lysis myotube HEM C2C12 using (Amersham). prepared 4B glutathione–Sepharose to binding niomna hme t37 at chamber environmental oehrdgtlyi ooiyt bancmoieiae of images every composite obtain frame to Volocity 1 in of digitally together rate ( a myotube at each 0.5 of z-plane of 40 depth step-size at single were entire acquired were a the analyses myotubes fixed encompassing in of GFP–EB3 Images 100 taken seconds. times. 3 were Apochromatic Images exposure were an fluorophore (Nikon). minimize each using of to z-series performed movies, consecutively dual-color For taken minutes. 15 for 0.5 minute a with Where (0.5 obtained dye Elmer). 33342 (Improvision/Perkin Hoechst with Software labeled Analysis was DNA Image noted, 3D Volocity and meso pcrmtcojcie Nkn na niomna hme e to set chamber environmental an in (Nikon) 37 objectives apochromatic immersion el eeicbtdwt rmr n eodr nioisa oe hnstained then noted as (0.5 antibodies Fixed 33342 secondary noted. Hoechst and as fixed acetone:methanol primary with 1:1 with were or incubated myotubes EDTA were mM Alternatively, cells 1 X-100. with methanol Triton ice-cold 0.1% with with permeabilized and Aolimrinojciewt 1.6 a Leica 63 with a Apochromatic objective on an oil-immersion performed using was NA microscope imaging (Sigma- epifluorescence oil analyses, inverted mineral with translocation DMI6000B overlaid nuclear HEPES Glutamax, mM mM For 25 2 Aldrich). with and DMEM serum, horse Phenol-Red-free 10% in (Gibco), performed was imaging cell Live Microscopy DE3 BL21 in Karakesisoglou expressed Iakowos were and 2011) Noegel al. Angelika et (Schneider from constructs GST-Nesprin2 assays GST-pulldown n dmG edik,MthwG ryadMcalF Howland F. scripts. Michael Matlab and of Bray G. development assistance, Matthew for technical Hendricks, for G. Tokito Adam and Mariko acknowledge gratefully We Acknowledgements for standards binned nuclei and as of assessed served visually subset then was nuclei a nuclei remaining These 30 in the rotation. into of quantified rotation of Software first as range comparison Analysis was wide in rotation velocity a Image nuclear exhibiting of angular analyses 3D positions. For myotubes, the chromocenter Matlab. find treated in tracked Volocity implemented the to were from using used routines speed Analysis was translational automatically time and analysis velocity component were angular over Principal bright nuclei Elmer). XYZ rotation, (Improvision/Perkin nuclear individual in quantify reflects in To tracked reported nuclei. chromocenters velocity non-moving mean Hoechst-dye-positive and Microsystems); tracked translocating manually (Leica was both nucleus software each LAS-AF of centroid using the translocation, nuclear assess To analysis Image nlsswr efre sn rpPdPimV. Gaha otae Inc.). statistical Software, and (GraphPad graphs V5.0 Prism All of GraphPad calculated. percent using were a performed as length were position myotube myotubes myotube analyses nuclear the the unbranched and along along only nuclei distance linearized adjacent XY; the was between in myotubes. position distances Nuclear myotube the and analysis. the length of the in and in long-axis ends nucleus included myotube the each were the (ImageJ). of of to position frames centroid the regard defining the by pseudo-colored with assessed was sequential evaluated distribution Nuclear three was direction of Comet images overlay resulting yae eeicbtdoengta 4 at overnight incubated were Lysates daetaeswr banda 40 at obtained were areas adjacent ( myotube each of depth the encompassing Z-series, imaging. pnigDs ofclwt io iMcocp qipdwt F,a PFS, with equipped Microscope Ti 40 Nikon and a stage, with min. motorized Confocal 180 or Disk 60 for Spinning sec 30 every taken were images Phase-contrast Microsystems). ueSga etPc hmlmnsetSbtae(hroSinii)and MD). Rockville, Scientific) (NIH, by ImageJ (Thermo with enhanced performed Substrate was was Densitometry Chemiluminescent film. Chemiluminescence SDS-PAGE to by Pico exposed analysis. separated West was immunoblot protein SuperSignal to total equal subjected the (Pierce), using and measured kit was assay protein protein total (Roche); BCA inhibitors protease and X-100 Triton (Invitrogen). reagent antifade Gold ProLong ih4 Mguahoe H75 n nlzdb etr blotting. western by analyzed and 7.5, eluted pH were glutathione, proteins mM Interacting beads. 40 GST–Sepharose with to coupled proteins fusion ORCA-R ytbsgono CA imwr ie ih4 aaomleye(PFA) paraformaldehyde 4% with fixed were film ACLAR on grown Myotubes loecneiaigwspromdo eknEmrUtaiwVox UltraView Elmer Perkin a on performed was imaging Fluorescence iN ncdw a sesdb yigclsi E yi ufrwt 1% with buffer lysis HEM in cells lysing by assessed was knockdown siRNA ˚ .Dgtliae eeaqie ihaHmmtuECDC105 camera C9100-50 EMCCD Hamamatsu a with acquired were images Digital C. ˚ aeois h ieto fGPE3cmt a uniidfo the from quantified was comets GFP–EB3 of direction The categories. 2 hreculddvc aeauigLSA otae(Leica software LAS-AF using camera device charge-coupled 2 H70pu .%Tio -0 n rtaeihbtr (Roche)]. inhibitors protease and X-100 Triton 0.1% plus 7.0 pH , m .Fraayi fncerdsrbto,zsre (1 z-series distribution, nuclear of analysis For m. m tpsz.Iae eetkna aeo -eisper z-series 1 of rate a at taken were Images size. step m 6 m /l n one n4 mgascvrlp with coverslips glass mm 40 on mounted and g/ml) 13 A 60 NA, /1.30 ˚ .Dgtliae eeaqie ihaHamamatsu a with acquired were images Digital C. 6 ih2%oelp mgswr stitched were Images overlap. 20% with 6 ˚ iheulaonso uiidGST purified of amounts equal with C 6 6 anfe LiaMcoytm)i an in Microsystems) (Leica magnifier 14 A n 100 and NA, /1.49 .9N i-meso objective oil-immersion NA 1.49 , 15–30 m 6 /l o 0mnpirto prior min 20 for g/ml) .coli E. ,60 m , )wr ae ta at taken were m) 6 , 1000 15–30 r100 or 6 n uiidby purified and 14 Aoil- NA /1.49 6 m -tp of z-step) m 1000 6 m Z-series . ) were m), m 6 m. 1.4 Journal of Cell Science atn .M,Hmlo,R . al . ag . uln .F,L,W,Ohkura, W., Lu, F., C. Cullen, L., Yang, G., Ball, S., R. Hamilton, M., R. Parton, eze,T,Gce . u . ao,B,Fle,E . ihrsn .E,Gomes, E., B. Richardson, S., E. Folker, B., Cadot, M., Xu, V., Gache, T., Metzger, atoi . oubr,M,Cpni . aad,N . en,V,Solni K., Scotlandi, V., Cenni, M., N. Maraldi, C., Capanni, M., Columbaro, E., P. Mattioli, H. Hauri, and H. Reggio, J., Suhan, A., Mehta, D., A. Linstedt, i . e,Y n hmsn .J. W. Thompson, and Y. Lee, Y., Li, G., S. Young, K., Reue, L., C. Stewart, G., L. Fong, L., Vergnes, T., R. Lee, Y., J. Ji, M. Han, and R. J. Sanes, L., G. Ackerman, A., D. Starr, A. M., R. D. Grady, Starr, and N. H. Fridolfsson, E., Y. Goldman, R. I. M., Konigsberg, S. and G. Baylor, W. Cooper, S., Hollingworth, A., J. Dantzig, A., Cheung, L. E. Holzbaur, and Charge M. Tokito, M., S. Antony, L., J. Ross, P., J. Caviston, E. Ralston, R. and C. Capers, J. K. Zaal, E., Bugnard, K. Gundersen, and K. Kollstad, M., Ekmark, K., Liestøl, C., J. Bruusgaard, upeetr aeilaalbeoln at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.108688/-/DC1 online months. available 12 material after Supplementary release for AR- PMC T32 in and [grant Deposited GM-07229 Health M.H.W.]. T32 of to E.L.F.H., Institutes 053461 to National GM087253 the PO1 by number supported was work This Funding ey .R n ozar .L. E. Holzbaur, and R. J. Levy, C. C. Hoogenraad, and M. Kuijpers, N. Hirokawa, and S. Terada, A., Harada, Y., Tanaka, Y., Okada, Y., Kanai, and M. Tokito, 3rd, W., H. Schroeder, L., J. Ross, E., Perlson, G., A. Hendricks, L. L. Rubin, and L. R. L. I. Englander, Konigsberg, and G. W. Cooper, Steinhardt, and M. J. Scholey, M., J. Alderton, G., Liao, L., R. Morris, Q., G. Bi, R. J. Sanes, and M. R. Grady, M., R. Lewis, D., E. Apel, O. M. Steinmetz, and A. Akhmanova, References umr .T,Msed . ihmn .W n ae,J R. J. Sanes, and W. J. Lichtman, T., Misgeld, T., T. Kummer, M. Setou, and Y. Konishi, .adDvs I. Davis, and H. eurdfrseea ucefunction. muscle skeletal for K. M. required Baylies, and R. E. eitdrcuteto U1t h ula neoedrcsncerpstoigin positioning nuclear directs G. envelope Lattanzi, nuclear the and muscle. to human S. SUN1 Squarzoni, of L., recruitment mediated Merlini, T., M. Marino, fatp IeryGlimmrn protein. membrane Golgi early targeting II pH-sensitive type saturable for a evidence of GPP130/GIMPc: of overexpression and J. b1. Lammerding, lamin and of M. absence C. Shanahan, Q., Zhang, F. II. A. myosin Straight, muscle and J. T. Mitchison, differentiation. muscle during nucleation ucin r xlie ydgnrto n eeeaino ucefbrsget at segments fiber muscle of regeneration synapse. and the degeneration by explained are junctions fibroblasts. motile of progression transport. vesicle bidirectional L. E. Holzbaur, junction. neuromuscular the at USA nuclei muscle anchor proteins nuclei. of migrations bidirectional the mediate envelope culture. in fibers muscle in movement nuclear April Philadelphia, Association, Culture Tissue Meeting, 27. Annual 9-10. 9th the of Program regeneration. transport. USA vesicle Sci. dynein/dynactin-mediated facilitates Huntingtin Cytol. studied mice normal of fibres muscle the in vivo. nuclei in of distribution spatial and Number exocytosis. regulated A. R. needn omto fatplgclycmlxpssnpi apparatus. postsynaptic complex topologically a Biol. of formation independent development. axons. to domain neurons. motor in enriched kinesin 6384. neuronal novel a KIF5C, Rec. Anat. neuromuscular the at nuclei synaptic with associated junction. protein Klarsicht-related and tips. microtubule of fate the controls network ,S .adRdik,M A. M. Rudnicki, and B. S. ´, 102 164 7 19) iei-admoi-rvnseso eil erimn o Ca2+- for recruitment vesicle of steps myosin-driven and Kinesin- (1997). 559-566. , .Physiol. J. 4359-4364. , 1077-1087. , .Bo.Chem. Biol. J. 104 140 16) utnceto fseea ucei vitro. in muscle skeletal of Multinucleation (1960). .Neurosci. J. 10045-10050. , hso.Rev. Physiol. o.Cl.Neurosci. Cell. Mol. 195-205. , elDahDiffer. Death Cell a.Neurosci. Nat. 21) oo oriainvaatgo-a ehns drives mechanism tug-of-war a via coordination Motor (2010). a.Cl Biol. Cell Nat. 551 21) A--eedn retto rdeto dynamic of gradient orientation PAR-1-dependent A (2011). .Bo.Chem. Biol. J. .Cl Biol. Cell J. 467-478. , 275 20) uui yoiainnvgtsteknsn1motor kinesin-1 the navigates tyrosination Tubulin (2009). 31 21) A n iei-eedn ula oiinn is positioning nuclear kinesin-dependent and MAP (2012). 84 14910-14919. , 31986-31995. , 209-238. , 20) yendie ula oaindrn forward during rotation nuclear drives Dynein (2008). ur Biol. Curr. 12 20) ellradmlclrrglto fmuscle of regulation molecular and Cellular (2004). .Cl Sci. Cell J. 4 138 18 48 83-88. , 559-567. , 21) etooe,mcouue n neuronal and microtubules Centrosomes, (2011). 21) hne naigmueneuromuscular mouse aging in Changes (2011). 282 15) eairo ybat ntsu culture. tissue in myoblasts of Behavior (1958). 1305-1315. , 18) ctlhln eetrcutrn and clustering receptor Acetylcholine (1987). 349-358. , 21) iei- n yena h nuclear the at dynein and Kinesin-1 (2010). Nature 20) ml-oeueihbtro skeletal of inhibitor small-molecule A (2002). 999-1008. , 20) rcigteed:adnmcprotein dynamic a ends: the Tracking (2008). 16) yaiso ygnssi vitro. in myogenesis of Dynamics (1961). 20015-20026. , elMtl Cytoskeleton Motil. Cell 20 o.Bo.Cell Biol. Mol. 20) eraiaino microtubule of Reorganization (2005). 697-702. , 121 484 a.Rv o.Cl Biol. Cell Mol. Rev. Nat. .Cl Biol. Cell J. 3187-3195. , 120-124. , 20) elnce pni the in spin nuclei Cell (2007). 20) ye1 dystrophin- a Syne-1, (2000). .Cl Biol. Cell J. 8 104 1073-1087. , .Neurosci. J. rc al cd Sci. Acad. Natl. Proc. .Bohs Biochem. Biophys. J. 87-95. , 21) rlmnA- Prelamin (2011). rc al Acad. Natl. Proc. 60 19) Sequence (1997). 1-13. , 20) Nerve- (2004). 191 20) Syne (2005). 9 309-322. , 115-128. , 20 6374- , (2007). (2003). (2000). .Cell J. agr .W,Wn,J,Fn . ht,J n agr .M. J. Sanger, and J. White, Y., Fan, J., Wang, W., J. Sanger, W. J. Lichtman, and M. Rich, E. Karsenti, and C. C. Diaz, F., Gerbal, V., Pizon, hn .Y,Lbte . uc,M,Nee,A .adKrnam E. Korenbaum, and A. A. Noegel, M., Munck, T., Libotte, Y., Y. Zhen, cnie,M,L,W,Nuan . rcnr . ozan . ogl .A and A. A. Noegel, J., Gotzmann, A., Brachner, S., Neumann, W., Lu, M., Schneider, Wehnert, D., Furling, M., Puckelwartz, A., C. Sewry, T., le Lam, N., K. Randles, hn,Q,Rgat,C . kpe,J . ot,N . arn .T,Roberts, T., D. Warren, F., N. Worth, N., J. Skepper, D., C. Ragnauth, Q., Zhang, hn,X,Li . un . u . hag . u . u .adHan, and R. Xu, T., Xu, Y., Zhuang, X., Wu, X., and Yuan, Y. K., Zhuang, T., Lei, Xu, X., S., Duan, Zhang, X., Ding, X., Yang, B., Zhu, R., Xu, X., Zhang, re,V,Fn,X,Had . aoil,C n eds A. Merdes, and C. Rabouille, R., H. Heald, L. X., Tsai, and Fant, G. V., J. Srsen, Gleeson, Z., Xie, D., M. Nguyen, R., Ayala, T., Shu, hn,Q,Sepr .N,Yn,F,Dve,J . ey,L,Rbrs .G., R. Roberts, L., Hegyi, D., J. Davies, F., Yang, N., J. Skepper, Peterson, Q., Y., Gu, Zhang, D., N. Dalton, S., Lange, T., L. Guo, Y., Liu, A., Felder, J., Zhang, H. R. Warren, W. J. Tsai, and E. G. Seale, B., R. Vallee, hn,T,Za,K . hrdn . et,A,Gnesn .G n Ralston, and G. G. Gundersen, A., Mehta, J., Sheridan, J., K. Zaal, T., Zhang, tr,D .adFiofsn .N. H. Fridolfsson, and A. D. Starr, T. Shimmen, al .J,Ri,E,Muai . hn,T,Mha . unr,E,Sartorelli, E., Bugnard, A., Mehta, T., Zhang, K., Mousavi, H. E., L. Reid, J., Tsai, K. and Zaal, H. Shih, A., B. Samuels, K., Sanada, Z., Xie, B. R. Vallee, and H. K. Bremner, W., J. Tsai, hn,Q,Btmn,C,Wrh .F,Dve,J . anr . ee,A., Feuer, C., Wasner, D., J. Davies, F., N. Worth, C., Bethmann, Q., Zhang, asn .M,Mr,B n onn,M. Bornens, and B. Maro, M., A. Tassin, M. W. Saxton, and E. W. Theurkauf, J., B. Cha, R., L. Serbus, eaa . a,Y . aao . hr,T,Hg,Y,Nki . miui K., Imaizumi, N., Nakai, Y., Higo, T., Ohira, F., Kawano, B., Y. Lan, M., Terada, ula yaisi eeoigmoue 4169 myotubes developing in dynamics Nuclear ucefibers. muscle 1009. nhrg ttencerenvelope. nuclear the at anchorage I. Karakesisoglou, myofibrils. of dynamics development. muscle during envelope nuclear the E. at G. Morris, isoforms and M. E. McNally, M., differentiation. muscle skeletal J. during EMBO myosin sarcomeric of organization and transport oocyte. Drosophila the in transport 194 cargo posterior directs microtubules ut-smrcpoenta id ai n mrna h ula neoeadforms and muscle. M. envelope skeletal nuclear C. in the Shanahan, at network and emerin subcellular and A. a lamin J. binds Ellis, that protein L., multi-isomeric P. Weissberg, G., R. UNE in rti oncigtencesadatncytoskeleton. actin and nucleus the connecting 115 protein giant a mice. NUANCE, in migration neuronal and neurogenesis during nucleus M. innervation. neuron motor M. Han, fseti-eetcnann rtista oaiet h ula ebaein membrane nuclear the to localize that tissues. proteins M. multiple C. Shanahan, spectrin-repeat-containing and A. of J. Ellis, L., P. Weissberg, al. et D. G. anchorage. Shelton, and P., positioning A. Mizisin, L., K. cones. growth and 355. neurons migrating in dynein cytoplasmic 979. ifrnito,eogto n fusion. and elongation differentiation, E. rtisfr nislbeprncermti uigmsl eldifferentiation. cell muscle during Biol. matrix Cell perinuclear insoluble an regulate form proteins to dynein cytoplasmic and LIS1 with positioning. pathway neuronal common cortical a in operates Ndel1 progress. TC og ope n Rei ie esssdsielc fnra microtubule normal of lack despite persists sites exit ER tracks. and complex Golgi E. MTOC, Ralston, and V. Biol. tissue. brain live in migration neuronal radial in dynein and r novdi h ahgnsso mr riusmsua ytoh n are and dystrophy muscular Dreifuss Emery integrity. of envelope pathogenesis al. nuclear et the for T. in critical D. Warren, involved A., are J. Mellad, 2 Q., Yi, D., C. Ragnauth, yokltnaemdae ySNKS ula-neoebridges. Biol. nuclear-envelope SUN-KASH Dev. by mediated are cytoskeleton Drosophila in streaming cytoplasmic kinesin-driven control oocytes. cytoskeleton actin the hshrlto fFKb d5i motn o irtbl raiain nuclear organization, microtubule for migration. important neuronal is and Cdk5 movement, by FAK of phosphorylation mice. mdx of fibers al. muscle et Y. soleus Adachi, N., Nishimoto, A., Ogura, etr uigmoeei nvitro. in myogenesis during centers 20) irtbl lsedbnigpoenE1i eesr o ucecell muscle for necessary is EB1 protein binding plus-end Microtubule (2009). 121-135. , 3207-3222. , 20) U12adSn/epi-/ opee onc etooet the to centrosome connect complexes Syne/Nesprin-1/2 and SUN1/2 (2009). 63 LSONE PLoS 550-566. , Development 20) ye1adSn- lycuilrlsi ynceracoaeand anchorage myonuclear in roles crucial play Syne-2 and Syne-1 (2007). .PatRes. Plant J. 24 10 26 3781-3792. , 20) h ldn hoyo yolsi temn:ffyyasof years fifty streaming: cytoplasmic of theory sliding The (2007). 28. , 17) irtblrognzto neogtn ygnccells. myogenic elongating in organization Microtubular (1974). 421-444. , Neuron .Cl Sci. Cell J. 6 21) oeua ehnsso etooeadcytoskeleton and centrosome of mechanisms Molecular (2011). e29057. , 21) h ed irtbls ygncrognzto of reorganization Myogenic microtubules? needs Who (2011). 132 3 120 677-688. , .Boe.Biotechnol. Biomed. J. 3743-3752. , Development 31-43. , u.Ml Genet. Mol. Hum. 114 Neuron 18) oo ev emnlls rmdegenerating from loss terminal nerve Motor (1989). 4485-4498. , el ise Organs Tissues Cells el o.Lf Sci. Life Mol. Cell. .Cl Biol. Cell J. Cell 44 21) neatosbtennce n the and nuclei between Interactions (2010). .Cl Sci. Cell J. u.Ml Genet. Mol. Hum. 21) epis u o u rtis switch proteins, sun not but Nesprins, (2010). 134 263-277. , 114 .Cl Sci. Cell J. 20) mrigrlsfrmoi Iand II myosin for roles Emerging (2009). 21) yncesrltdpoete in properties Myonucleus-related (2010). 901-908. , 18) aeo microtubule-organizing of Fate (1985). 21) epi sciia o nuclear for critical is 1 Nesprin (2010). 469-482. , 20) ulsbellrrlsfrLIS1 for roles subcellular Dual (2007). 19 2010 329-341. , 100 122 20) epis oe family novel a Nesprins: (2001). 35-46. , 858606. , 20) Microtubule-dependent (2005). 118 1401-1409. , 68 191 673-687. , 16 1593-1610. , rnsCl Biol. Cell Trends 2816-2833. , a.Neurosci. Nat. 248-259. , 20) epi- n - and Nesprin-1 (2007). 20) epi- sa is Nesprin-2 (2005). Neuron 21) sebyand Assembly (2010). e.Dyn. Dev. 20) Centrosome (2009). 20) yenand Dynein (2005). 20) eie732 Serine (2003). nu e.Cell Rev. Annu. 64 .Cl Biol. Cell J. 173-187. , .Cl Sci. Cell J. 239 10 19 (2002). (2004). .Cell J. 998- , 970- , 347- , BMC