myogenic cells express multiple myosin isoforms

Myogenic cells express multiple myosin isoforms

CLAIRE WELLS 1, DEBORAH COLES1, ALAN ENTWISTLE 2 andMICHELLE PECKHAM 1�1Molecular Biology and Biophysics Group, The Randall Institute, King's College London, 26±29 Drury Lane, London, WC2B 5RLand 2Ludwig Institute for Cancer Research, 91 Riding House Street, London, W1P 8BT, UK

Received 2 October 1996; revised 9 December 1996; accepted 12 December 1996

Summary

In vivo and in vitro, proliferating motile myoblasts form aligned groups of cells, with a characteristic bipolar morphology,subsequently become post-mitotic, begin to express skeletal myosin and fuse. We were interested in whether members ofthe myosin superfamily were involved in myogenesis. We found that the myoblasts expressed multiple myosin isoforms,from at least ®ve different classes of the myosin superfamily (classes I, II, V, VII and IX), using RT±PCR and degenerateprimers to conserved regions of myosin. All of these myosin isoforms were expressed most highly in myoblasts and theirexpression decreased as they differentiated into mature myotubes, by RNAse protection assays, and Western analysis.However, only myosin Iá, non-muscle myosin IIA and IIB together with actin relocalize in response to the differentiativestate of the cell. In single cells, myosin Iá was found at the leading edge, in rear microspikes and had a punctatecytoplasmic staining, and non-muscle myosin was associated with actin bundles as previously described for ®broblasts.In aligned groups of cells, all these proteins were found at the plasma membrane. Co-staining for skeletal myosin II, andmyosin Iá showed that myosin Iá also appeared to be expressed at higher levels in post-mitotic myoblasts that hadbegun to express skeletal myosin prior to fusion. In early myotubes, actin and non-muscle myosin IIA and IIB remainedlocalized at the membrane. All of the other myosin isoforms we looked at, myosin V, myosin IX and a second isoform ofmyosin I (mouse homologue to myr2) showed a punctate cytoplasmic staining which did not change as the myoblastsdifferentiated. In conclusion, although we found that myoblasts express many different isoforms of the myosinsuperfamily, only myosin Iá, non-muscle myosin IIA and IIB appear to play any direct role in myogenesis.

Introduction

In the embryo, in chick and mammals, myoblastsmigrate out from the somites into the limb budswhere they form muscle ®bres. In mice, this processoccurs in day 9 embryos. In the limb bud, themyoblasts proliferate, form parallel arrays of cellsand eventually fuse into multinucleated muscle®bres at about day 11±12 (reviewed in Miller,1992). Similar processes are recapitulated by satellitecells in the growth and regeneration of muscle ®bres,in young and adult animals. Proliferating satellitecells are known to move large distances within amuscle and even into other adjacent muscles (Watt etal., 1994). These processes can also be recapitulatedin tissue culture, and it has been demonstrated thatwhen proliferating motile myoblasts become com-mitted to fusion, they withdraw from the cell cycle,begin to express skeletal myosin heavy chain and

then fuse to form multinucleated myotubes (Andres& Walsh, 1996).

Whilst the biochemical and electrophysiologicalevents that are involved in skeletal muscle myogen-esis have been reasonably well characterized, andthere are many studies on the assembly of musclespeci®c proteins during myo®brillogenesis, muchless is known about the basis of myoblast motilityand aggregation. Myoblasts do not express musclespeci®c myosin (skeletal myosin II), and it is likelythat other members of the myosin superfamily areinvolved in their motility as in other cell types. Forexample, ®broblast locomotion is likely to arise fromactin and at least one myosin (Conrad et al., 1989;Maupin et al., 1994; Verkhovsky et al., 1995;Mitchison & Cramer, 1996). Postmitotic cell spread-ing of ®broblasts also requires myosin, as thisprocess is inhibited by BDM, (butanedone mono-xime) which inhibits myosin ATPase (Mitchison &Cramer, 1996). Myosin I and non-muscle myosin II

Journal of Muscle Research and Cell Motility 18, 501±515 (1997)

0142±4319/97 # 1997 Chapman & Hall

�To whom correspondence should be addressed.

also play a role in the extension of the growth coneof neurones (Rochlin et al., 1995).

Myosin I isoforms have been found in almostevery eukaryotic cell studied (reviewed in Sellers etal., 1996). However most cells have more than oneisoform, which appear to have overlapping func-tions (reviewed in Ostap & Pollard, 1966). Somemyosin I isoforms have been demonstrated to havea speci®c function such as myosin IC in acantha-moeba, which provides the force that enables thecontractile vacuole to expel water from the cell(Doberstein et al., 1993). In mammalian brushborder, myosin I crosslinks the plasma membraneand the core of actin ®laments in the intestinalmicrovillus (see Heintzelman et al., 1994). In othersystems, several myosin I isoforms have beenshown to contribute to a single function, such asmyosin I isoforms in Dictyostelium that are in-volved in pinocytosis (Novak et al., 1995; Jung etal., 1996).

Apart from myosin I and myosin II, there are 12or more other classes of myosin, whose functionsare mostly unknown, that make up the myosinsuperfamily (Sellers et al., 1996). Moreover, it hasbeen demonstrated that a single cell type cancontain multiple members of the myosin super-family. For example, human and pig cell linescontain six different classes of myosins (Bement etal., 1994). All of these myosins share a commonmotor domain, but are highly divergent in otherregions of the molecule. It therefore appears likelythat they are generally involved in many differentkinds of motile activity within the cell. Some ofthese myosins may be involved in cell signallingsuch as myosin IX, which contains an active GAP(GTPase activating protein) domain in its tail thatcan interact with rho and cdc42 (Reinhard et al.,1995; Wirth et al., 1996).

Myogenic cells are a specialized cell type that arecommitted to fusion and differentiation into musclecells. We expected that they would only express asubset of the myosin superfamily and that thesecould be important in the process of myogenicdifferentiation. However, we show here that theyexpressed at least ®ve different classes of myosinfrom sequence analysis of cloned RT±PCR productsas previously described (Bement et al., 1994). Welooked at their relative abundance in myoblasts anddeveloping myotubes by Western analysis. Weexamined the distribution of the majority of thesemyosin isoforms by immuno¯uorescence in singleand aligned myoblasts, and in early myotubes, todetermine if there was any obvious indication oftheir functions in the cells. We used conditionallyimmortal myogenic cells (H2kb-tsA58; Morgan et al.,1994) for this study, as these cells appear to behaveas primary myoblasts, but have the advantage that

we could use pure myogenic clones, which couldeasily be proliferated for several months in culturewithout losing their myogenicity. This is an advan-tage over primary cultures which contain ®broblastsas well as myoblasts, and traditional muscle celllines which are spontaneously immortalized andhave altered behaviour compared to primary cul-tures.

Materials and methods

Growth and differentiation of H2kb-tsA58 myogenic cells

Conditionally immortal H2kb-tsA58 myogenic cells wereisolated from 1±2-day-old H2kb-ts6 mice as described(Morgan et al., 1994). Cells were plated out at clonaldensities in DMEM with 0.11 g lÿ1 sodium pyruvate,4.50 g lÿ1 glucose, 3.7 g lÿ1 sodium bicarbonate, supplemen-ted with 20% foetal calf serum (FCS), 2% chick embryoextract (CEE), 100 ìg mlÿ1 penicillin=streptomycin and 20units mlÿ1 of murine recombinant gamma interferon(IFNã, Gibco) and maintained at the temperature of338 C, at greater than 95% humidity and 5% CO2. Cloneswere picked, expanded, and frozen down at passage 3after they were characterized as myogenic by differentiat-ing a subset at 398 C in the absence of gamma interferon, inthe supplemented DMEM now containing 5% FCS, and 1%CEE. Single clones were recovered and expanded in ¯asksfor experiments.

Time lapse phase stepping interference microscopy

Cells were trypsinized and plated onto acid cleaned glasscoverslips coated with 0.01% gelatin at a density of2 3 104 cmÿ2. They were incubated in CO2 independentmedium (Gibco) containing 20% FCS, 2% CEE, 4 mM

glutamine and 100 ìg mlÿ1 penicillin=streptomycin at378 C for ,4 h prior to ®lming to allow the cells to spread.The coverslip was then sealed into a small chamber, with asmall air bubble to buffer the medium, on the stage of aHorn type transmitted-light interference microscope, mod-i®ed for automatic phase-shifting and visualized with aminiature video camera connected to a frame grabber andprocessor board in a PC. Further details of the Hornmicroscope, microscope set up and interference recordingswere as described (Dunn & Zicha, 1994, 1995; Zicha &Dunn, 1995).

RNA extraction and RT±PCR

RNA was extracted using RNAzol (Biogenesis, UK) basedon the method of Chomczynski and Sacchi (1987). Brie¯y,cells were scraped off 15 cm dishes and homogenized with2 ml of RNAzol B (mix of guanidinium thiocyanate andphenol) on ice. The RNA was extracted from thehomogenate by adding 0.1 volume of chloroform, leavingthe mix on ice for 5 min, spinning for 15 min at 12 000 3 g(48 C) and transferring the aqueous phase containing theRNA to a fresh tube. The RNA was precipitated with anequal volume of isopropanol for 15 min at 48 C. Sampleswere centrifuged at 12 000 3 g (48 C) for 15 min. The RNApellet was washed with 75% ethanol, dried and dissolvedin 1mM EDTA, (pH 7) and stored at ÿ808 C.

502 WELLS et al.

Approximately 1±2 ìg of isolated RNA was used tomake cDNA. The cDNA was mixed with diluted 53AMVRT buffer (Promega), 1mM dNTP mix (Pharmacia),20nM random 9mer primers, and oligo-dT (18mer),RNasin (Promega), and AMVRT enzyme (Promega).Reaction mixes (20 ìl) were incubated at 428 C for 1 h,then stored at ÿ208 C before use. One to 2 ìl was thenused in each PCR (20 ìl). Three different sets ofampli®cations were carried out. First, an initial ampli®ca-tion was made using primers 1 and 2 (Table 1). Analiquot of 2 ìl from this PCR was then used in a newreaction with primers 2 and 3 (Table 1). This reactiongave a single discrete band of ,195 bp. Second, an initialampli®cation was made using primers 4 and 5. An aliquotof 2 ìl from this PCR was then used in a new reactionwith primers 6 and 7 (Table 1). This reaction gave a singlediscrete band of ,325 bp. Both of these bands wereexcised from the gel, the DNA extracted (Mermaid kit,Amersham) and directly sequenced. All the aboveampli®cations used 2.5 mM Mg2�, TAQ polymerase (Ap-plied Biosystems) and 30 cycles of 1 min 958 C, 1 min358 C, and 1 min 728 C.

Finally, an initial ampli®cation was made using primers8 and 9 (Table 1). The reaction was separated on anagarose gel. A gel slice corresponding to molecularweights 100±220 bp was excised from the gel, placed ina 0.5-ml eppendorf tube with a hole in the bottomplugged with glass wool, placed inside a 1.5-ml eppen-dorf tube, and brie¯y centrifuged (2 min, 14 000 3 g). Fiveìl of the liquid fraction recovered was used in a secondPCR with primers 8 and 9, which produced broad bands

in the range of 150±200 bp. TAQ polymerase (AppliedBiosystems) and 2.5 mM Mg2� were used in both ampli-®cations. The ®rst ampli®cation consisted of 5 cycles of1 min 958 C, 1 min 358 C and 1 min 728 C followed by 25cycles of 1 min 958 C, 1 min 558 C and 1 min 728 C. Thesecond ampli®cation consisted of 30 cycles of 1 min 958 C,1 min 558 C and 1 min 728 C.

To clone the ampli®ed products, 1 ìl from the reactionwas added to linearized TA vector (Invitrogen) andligated overnight at 148 C following the manufacturer'sinstructions. The ligation was transformed into `one shot'cells (Invitrogen), plated out onto LB plates containingampicillin (100 ìg mlÿ1) and X-gal (40 ìg mlÿ1) and grownovernight at 378 C. White colonies were picked, grownovernight at 378 C in superbroth (32 g Tryptone (Difco),20 g yeast extract (Difco), 10 g lÿ1 MOPS, Na salt) contain-ing 100 ìg mlÿ1 ampicillin. Plasmid DNA was preparedby alkaline lysis, phenol chloroform extraction andethanol precipitation (Sambrook et al., 1989). To sequencethe DNA inserts, the plasmid DNA was further puri®edby PEG precipitation (7.5% PEG, 8000, 0.4 M NaCl), alkalidenatured (0.2M NaOH) and sequenced by dideoxynu-cleotide DNA sequencing using a Sequenase kit (Amer-sham, UK) and vector sequence primers (T7 and SP6).

RNase protection assays

RNase protection probes were generated using subclonedPCR products. Antisense [32P] UTP labelled RNA weretranscribed from plasmids using T7 or SP6 RNA poly-merase. A glyceraldehyde 3-phosphate dehydrogenase

Table 1. List of primers used to amplify members of the myosin superfamily.

primer 1, forward. CG GGA TCC GGA� GCA� GGA� AA=GA ACA G`BMATP'(GESGAGKT)primer 2, reverse TTG A=GTA A=GAA A=GAT A=GTG A=GAA A=GTT A=TCT=G TTC`RUNHIF'(QYFIHFNRE)primer 3, forward CC GGA TCC C=TTA� GAA=G GCA� TTC=T GGA� AAC=T GCA� AAA=G AC`LEAFGNA'primer 4, forward AT ATC TAG AAG CTT CTG GAI GCI TTT GGI AAT GCC AA`LEAFGNA2'(LEAFGNA)primer 5, reverse AT AGA ATT CAT CGA TGG CTT GAT GCA CCT GAT`YIRCIKP2'(YIRCIKP)primer 6, forward AT ATC TAG AAG CTT CAG GII TAI TAI GCC CGT GAQAYYARDprimer 7, reverse ATA GAA TTC ATC GAT G=ACA IAT GAT ICG ATT GTT GAAFNNAIICprimer 8, forward GGI GAG=A A=TG=CI GGI GCI GGI AAG=A ACGESGAGKTprimer 9, reverse IGT C=TTT IGC A=GTT ICC A=GAA IGC C=TTC IAA=GLEAFGNA

Myosin degenerate primers used to amplify cDNA. For each primer the direction of ampli®cation, the name, and the equivalent aminoacid sequence (in brackets) is given, where it is different from the name. Primers 1, 2 and 3 were a generous gift from Dr M. Titus,Duke University. Primers 4, 5, 6 and 7 were a generous gift of Professor P. Chantler, Royal Vetenary College, London. Primers 8 and 9were constructed by King's College oligo service at the Randall Institute, based on those described previously (Bement et al., 1994). A�indicates that at this position the base was either A, C, G, or T. I represents an inosine that was used where there was potentialdegeneracy in the coding sequence.

Myosin superfamily and myogenesis 503

(GAPDH) loading control was produced by SP6 transcrip-tion of a 220 bp SfaN1-HindIII fragment of coding sequencecloned in pGEM 2 (Smith et al., 1992). Probes werehybridized to 10 ìg of total cellular RNA, digested withRNase T1 (Ausubel et al., 1989) and separated on 6%polyacrylamide gels, using end labeled pBluescript II SK�digested with Hpa II as size markers. Approximateprotected sizes were expected to be: 66 bp for GAPDH;141 bp for myosin IX, and 160 bp for myosin VII.

Protein and Western analysis

Cells were harvested from ¯asks either using a scraper orby trypsinization, and were centrifuged (1000 3 g, 5 min)in media to pellet the cells. The pellet was washed withPBS, centrifuged (12 000 3 g 1 min) and the dry cell pelletwas resuspended in modi®ed Laemmli buffer (63 mM

Tris=HCI, pH 6.8, 2% SDS, 10% glycerol, 5% â-mercap-toethanol, 6 M urea, 0.005% Bromophenol Blue) added togive 44 mg cell pellet mlÿ1 of Laemmli buffer. Sampleswere heated to 808 C for 1 min, and passed through a 21gauge hypodermic needle to shear the DNA.

Samples were analysed by performing one-dimensionalgel electrophoresis in a Mini Protean II Electrophoresis Cell(Bio-Rad Laboratories, UK) in a 7.5% polyacrylamide slabgels. Samples were run on the gel immediately, orfollowing brief storage at ÿ208 C. The gels were eitherstained with 0.05% Page Blue to visualize the proteins, ortransferred to nitrocellulose (BA85, Schleicher & Schull,supplied by Anderman, UK) for Western analysis. Proteinswere transferred for 1 h at 100 V in transfer buffer (25 mM

Tris, 192 mM glycine, 20% methanol, 0.05% SDS), using aMini Protean II Blotting apparatus containing an ice pack.The blot was incubated in blocking buffer (100 mM glycine,50% (w=v) dried milk (Marvel), 10% (w=v) ovalbumin and5% foetal calf serum) at 48 C overnight.

To identify speci®c proteins on the blots, the blots werehybridized to antibodies diluted to 1=2000 in blockingbuffer for 1 h at room temperature. The blots werewashed three times for 5 min each in PBS containing0.05% Tween. They were then hybridized with dilutedsecondary HRP conjugated secondary antibody (1=1000;Amersham, UK) for 1 h, washed for 5 min each in PBScontaining 0.05% Tween. The blot was blotted ontoWhatmann paper brie¯y, and antibody binding wasvisualized using chemiluminescence (ECL kit, Amersham).

Immuno¯uorescence and confocal microscopy

Myoblasts were grown on acid-washed glass coverslipscoated with rat-tail collagen. Rat tail collagen was madefrom collagen ®bres teased from rat tails, dried anddissolved in glacial acetic acid diluted 1=1000 with sterilewater. One gram of dried collagen was dissolved in 300 mlof diluted acetic acid. Coverslips were coated with a 1=10dilution of the stock collagen solution for 30 min±1 h andthen allowed to air dry under UV illumination. Todifferentiate the myoblasts, the cells were switched to398 C and the ã-interferon was removed.

Cells were either ®xed in a 50:50 mixture of 4%paraformaldehyde dissolved in PBS (pH 7.2) and mediumfor 20 min or they were ®xed in methanol at ÿ208 C.Paraformaldehyde (PFA) ®xation was used when it was

essential to preserve actin structure as methanol ®xationdestroys it. After PFA ®xation, cells were permeabilizedwith 0.2% triton X-100 for 5 min and immediately usedfor immunostaining. Two per cent PFA ®xation is verygentle, and it is possible that some cytosolic proteinscould be lost. However, the ®xation protocol used issuf®cient to retain almost all of the small rapidly diffusinggreen ¯uorescent protein in rhabdomyosarcoma cells(Alan Entwistle, personal observation) and if any washoutof protein occurs it is most probably very small.Furthermore, the same staining patterns for myosin wereobserved whether we used PFA ®xation, or methanol®xation. To achieve good immunostaining it was impor-tant to ®x and stain the cells on the same day.

Primary antibodies were diluted 1=100, except wherestated below, into PBS containing 1% BSA. The coverslipswere incubated with the primary antibody for 1 h,washed ®ve times in PBS containing 1% BSA, incubatedwith the secondary antibody for 1 h, washed three timesin PBS containing 1% BSA, then twice in PBS. Thecoverslips were blotted on tissue paper and mounted inglycerol containing an antifading agent 2.5% w=v solutionof 1,4 diazabicyclo-2.2.2. octan (DABCO-Sigma). The edgesof the coverslips were sealed with nail varnish.

The primary antibodies used were rabbit polyclonalantibodies; Tu 29 (anti-myr 1, rat homologue to mamma-lian myosin Iá; Ruppert et al., 1993), Tu 49 (anti-myr2,myosin I), Tu 55 and Tu 66 (anti-myr5, myosin IX) (all ofwhich were generous gifts of Dr Martin BaÈhler, TuÈ bin-gen), anti-myosin V (kind gift of R. Cheney, Chapel Hill)and anti non-muscle myosin IIA and IIB (HA and HB, akind gift of Professor R. Adelstein, NIH). An anti-skeletalmyosin monoclonal antibody A1025 (Cho et al., 1994) wasused at 1=10 dilution of hybridoma supernatant (agenerous gift of Dr Hughes). Secondary antibodies usedwere anti-mouse FITC (Dakopratt, UK) at 1=20 dilution,anti-rabbit FITC (Vector UK) at 1=100 dilution, and amouse biotinylated antibody (Calbiotech). A tertiary anti-body, RITC conjugated streptavidin (Molecular Probes,OR, USA), was used in conjunction with the mousebiotinylated antibody. Finally, actin was localized usingeither anti â-actin (Sigma) or rhodamine-conjugatedphalloidin (Molecular Probes).

Laser scanning confocal microscopy

An MRC 500 confocal visualization system (Bio-Rad,Hemel Hempstead, UK) mounted over an in®nity cor-rected Axioplan microscope ®tted with a 310 eyepiece andeither a 340 NA0.75 dry or a 363 NA 1.4 oil immersionobjective (Zeiss, Germany) was con®gured for simultane-ous confocal laser scanning, quantitative ¯uorescence(BoÈgler et al., 1993), polarized-light and differential inter-ference contrast microscopy (Entwistle, 1992). The compo-nents were aligned and used as described previously(Entwistle & Noble, 1992, 1994) employing ®lter set A forthe detection of FITC emissions or ®lter sets B and E(Entwistle & Noble, 1992) to distinguish between FITC andRITC, using a detector pinhole with diameter of ' 4 opticalunits (OU), (Wilson, 1990), reduced to ' 1.5 OU forre¯ection imaging. Image ®les, a matrix of 768 3 512 pixelsrecording the average of 96±256 frames scanned with a

504 WELLS et al.

repetition frequency of 1 Hz, were generated by collectingat least 14 bits of data per pixel and saving the eight mostsigni®cant bits for either an individual ®le or a group of®les whose intensities were to be compared.

Image processing and presentation

FITC and RITC emissions were either displayed separatelyin monochrome or together in green and red respectivelywhere of regions overlap appear yellow. Gamma correc-tion factors of 1.2, 1.2, 1.4, 1.6 and 2 were applied togreyscale, yellow, green, red and blue encripted data,respectively, immediately prior to printing and making theprinted reproductions as similar as possible to the imagesfound on a 24-bit colour monitor viewed with ambientindirect lighting. When it was essential that the readerperceived relatively feint objects, additional gamma shapedcontrast altering look-up tables were used to make thelower intensities more visible which inevitably compressedthe contrast in more intense regions. Views generated byconfocal laser scanning differential interference contrastwere presented in blue. As DIC was used to demonstratethe presence of cell bodies which did not stain immuno-¯uorescently, DIC signal that coincided with ¯uorescentemissions was suppressed by eliminating, magenta, cyanand greyscale contributions, the presence of whichdistorted the appearance of the distribution of ¯uorescentemission. Full colour images were generated by makingmontages of single image ®les using Photoshop (AdobeInc., CA, USA) and printing them on an Proslide 35Slidewriter (Agfa, MA, USA).

Results

Time-lapse digitally recorded interference microscopy ofmyoblast alignment

Single myoblasts are motile and form aligned groupsof cells with a de®ned geometry as shown by atypical sequence of time-lapse digitally recordedinterference microscopy recordings (Fig. 1). At thestart of ®lming, the myoblasts are sparse and activelymoving, with an average speed of 1 ìm minÿ1,similar to ®broblasts (Fig. 1A; Dunn & Zicha, 1995).After , 16 h, several groups of linear arrays ofspindle shaped cells have formed (Fig. 1B). Of the25 cells in the ®eld, only three cells remain as singlecells, and the rest are within aggregates (88%). Thesegroups of cells are also motile, and often the leadingcell in the group shows ruf¯ing behaviour (Fig. 1C).The cells in these groups are not post-mitotic as cellscan be observed moving to the edge of the groupwhere they divide (Fig. 1D), the daughter cellssubsequently rejoining the group. By the end of®lming (20 h) three separate groups (Fig. 1C) hadaggregated to form one single group (Fig. 1H). Onecell in one of the three groups appeared to activelyrecruit the other two groups to form this singlegroup (Fig. 1E±H).

Multiple myosin isoforms expressed in myoblasts

We found that at least ®ve different classes of themyosin superfamily were expressed in myoblasts.We performed RT±PCR using degenerate myosinprimers to conserved regions of myosin and myo-blast cDNA (Fig. 2). We used two rounds of PCRampli®cation, and subcloned the RT±PCR productsinto the TA vector (Invitrogen) for sequencing. Wesequenced the ampli®ed insert of 19 RT±PCRderived clones. It is likely that there other membersof the myosin superfamily that could be expressed inmyoblasts, and which were not found in this simplescreen.

From sequence analysis of the RT±PCR clones,we found seven different isoforms of myosin, ofwhich six were completely homologous to pre-viously described members of the myosin super-family (Fig. 2). The only divergent sequence wasthat for myosin VII which was most closely relatedto pig myosin VIIB (Bement et al., 1994). Thesequence showed ®ve amino acid differences outof 39 residues (87% conservation). In total, twoisoforms of class I myosins; myosin Iá (Sherr et al.,1993) and myosin I-SH3 (mouse homologue tomyr3, StoÈf¯er et al., 1995), two isoforms of class IImyosins; non-muscle myosin IIA and IIB (Simmonset al., 1991), one isoform of class V myosin (dilute,Mercer et al., 1991), one of class VII (Bement et al.,1994) and one of class IX (mouse homologue tomyr5; Reinhard et al., 1995) were found.

Levels of non-skeletal myosin isoforms decrease asmyoblasts differentiate

When we investigated the expression of the myosinisoforms described above, either by RNase protectionassays or by Western analysis, we found that all thenon-skeletal myosin isoforms were more abundant inmyoblasts than in mature myotubes (Figs 3, 4). Incontrast, the expression of skeletal myosin heavychain was low in myoblasts and increased as theydifferentiated into myotubes. Each of the antibodieswe used reacted with a single band on the Western(anti-myosin Iá showed three bands close together aspreviously described for this myosin; Ruppert et al.,1993), and were speci®c for the isoform tested. Therewas some expression of skeletal myosin heavy chainin the myoblast culture, as some of the cells havebecome post-mitotic and fusion competent. Myoblastcultures contain mixtures of single myoblasts andgroups of aligned cells, the relative proportions ofwhich depend on the density of the culture, but donot normally contain myotubes.

The pattern of changes in expression levels as themyoblasts differentiate suggest that these non-skeletal myosin isoforms are more important inmyoblasts and early myotubes than in mature


Fig. 1. Selected frames from an overnight recording (20 h) of myoblasts ®lmed using phase-stepping interferencemicroscopy, at 378 C. The appearance of the cells in these frames is due to a representation of their dry mass distributionby a grey scale as described (Dunn & Zicha, 1995) and is somewhat different from the more normal phase contrastimage. The cells were plated onto a gelatin coated glass coverslip and incubated for 24 h at 378 C before the recordingcommenced. (A) 0 s (Frame 1), (B) 15 h 19 min, (Frame 909), (C) 15 h 40 min, (Frame 940), (D) 16 h 40 min, (Frame 1000),(E) 18 h 20 min, (Frame 1100), (F) 19 h (Frame 1140), (G) 19 h 10 min, (Frame 1150), (H) 19 h 50 min, (Frame 1190). ScaleBar � 20 ìm.

506 WELLS et al.

Fig. 2. A summary of the sequences determined for theRT±PCR ampli®ed products produced using the myosinprimers described in Table 1 from mouse myoblast cDNA.The sequences are shown aligned with a sequence in thedatabase which was the best match. Sequences A±E,obtained using primers 8 and 9 (Table 1). Out of 19 RT±PCR clones using those primers, 50% were myosin IX, 20%embryonic myosin II, 20% myosin V, 10% myosin VII, 5%myosin IIA and 5% myr3. (A) best ®t to rat myosin I, myr3,residues 120±153, GenEMBL Acc. no. X74815; StoÈf¯er et al.,1995; (B) best ®t to pig myosin VIIB, residues 5±44,GenEMBL Acc. no. 29134; Bement et al., 1995, differencesbetween the two sequences are shown in bold (pig myosinVIIB has recently been reclassi®ed as myosin X); (C) best ®tto rat myosin IX, myr5, residues 247±279, GenEMBL Acc.no. X77609; Reinhard et al., 1995; (D) best ®t to mousemyosin 5, dilute, residues 171±204, GenEMBL Acc. no.X57377; Mercer et al., 1991; (E) best ®t to rat myosin IIA,residues 182±218, (RT±PCR product only partially se-quenced), GenEMBL Acc. no. U31463; Choi et al., 1996.Sequence F obtained using primers 1, 2 and 3 (Table 1);best ®t to rat myosin IIA, residues 234±251, GenEMBL Acc.no. U31463; Choi et al., 1996. Sequence G, obtained usingprimers 4, 5, 6, and 7 (Table 1); best ®t to mouse myosin Iá,residues 359±437, ??? are residues not sequenced, Gen-EMBL Acc. no. L00923 (Sherr et al., 1993).

Fig. 3. RNase protection assays for (a) myosin IX and (b) myosin VII. Lanes 1±5 show the results for myoblasts, � 12 h,� 24 h, � 72 h and � 7 days under differentiation conditions respectively. Comparison of the intensity of the bands forthe myosin isoform and the GAPDH loading control shows that in each case, myosin expression is highest in myoblastsand lowest in 7-day-old myotubes. Approximate protected sizes were predicted to be: 66 bp for GAPDH; 141 bp formyosin IX, and 160 bp for myosin VII.


myotubes. These myosin isoforms are expressed attheir highest levels in myoblasts and during the ®rst2 days under differentiation conditions duringwhich the majority of myoblasts fuse (Figs 3, 4).By 4 days, most of the myoblasts have fused intolong linear multinucleated myotubes, and the levelsof these myosin isoforms have decreased.

Localization of myosin isoforms in myoblasts, alignedmyoblasts and myotubes

To determine if any of these myosin isoforms wereinvolved in early myogenesis, we localized them byimmuno¯uorescence. In single motile myoblasts,myosin Iá was present in the leading lamellae ofthe cells and in the rear microspikes, where it co-localized with actin, but was not found at the plasmamembrane elsewhere. It also had a punctate stainingdistribution throughout the cytoplasm (Fig. 5A).Non-muscle myosins IIA and IIB (Fig. 5B, D) wereexcluded from the leading lamella, co-localized withactin bundles in the cell body, and showed adifferential localization close to the leading lamellaas previously described for ®broblasts (Conrad et al.,1995). Non-muscle myosin IIA was located justbehind the leading lamella together with actin in aregion of the cell that appeared to be adhering to thesubstratum (Fig. 5D), but IIB was excluded from thisregion (Fig. 5B). Myosin V (data not shown) andmyosin IX showed a punctate staining distributedthroughout the cytoplasm. Myosin IX was excludedfrom the lamellae (Fig. 5C) but myosin V wassometimes found at the leading edge of the lamella.

Proliferating myoblasts commonly form aggre-gates of aligned bipolar cells, and in these cellsnon-muscle myosin IIA and IIB, myosin Iá andactin all relocalized to the plasma membrane bothwhere cells were in apposition, and on the outeredge of the aggregate, where the cells are not incontact with other cells (Fig. 6A, D±F). Thisrelocalization was both speci®c, and was not a®xation artefact, as none of the other myosinsinvestigated [Myosin I (mouse analogue to ratmyr2), myosin IX and myosin V] relocalized to themembrane in aligned cells, but they continued toshow punctate staining distributed throughout thecytoplasm (Fig. 6B, C; data not shown). As well asits membrane staining, myosin Iá also continued toshow a puctate staining distribution throughout thecytoplasm. Co-staining for actin and myosin IIA(Fig. 6D, E), and for actin and myosin Iá (Fig. 7)showed that they were co-localized at the plasmamembrane. Therefore, non-muscle myosin II, myo-sin Iá and actin must be in close proximity witheach other at the plasma membrane.

Co-staining for skeletal myosin II and myosin Iá,showed that postmitotic, unfused cells which hadbegun to express skeletal myosin II also appeared toexpress higher levels of myosin Iá (Fig. 8A). Themajority of cells, including the aligned cells, inmyoblast cultures do not express skeletal myosin II,but a small proportion does and this proportionincreases during the ®rst day under differentiationconditions, as cell division ceases and the cellsbecome post-mitotic. After culture for 5 days underdifferentiation conditions, when most of the myo-

Fig. 4. A typical result for Western analysis of theexpression of embryonic skeletal myosin II, non-musclemyosin IIA (Ha), non-muscle myosin IIB (Hb) and myosinIá, in con¯uent myoblasts, and myoblasts=myotubes after1±14 days under differentiation conditions. The relativemolecular masses of each protein were as expected fromthe published data. No other bands were detected on theblot other than those expected, and shown here. Thespeci®cities of all the antibodies used have been demon-strated previously. At least two Western analyses werecarried out for each protein, which gave a similar result.An equivalent protein gel of the samples used in theseWestern analyses is shown below to demonstrate theapproximately equivalent loading of the samples. Relativemolecular mass standards are shown to the right handside.

508 WELLS et al.

blasts had fused into myotubes, the expression ofmyosin Iá was negligible, both in the myotubes andin the post-mitotic unfused cells (Fig. 8B). Skeletalmyosin II was assembled into the repeating sarco-meric pattern characteristic of myo®brils in themyotubes.

In myotubes, following culture for 3 days underdifferentiation conditions, non-muscle myosin IIAand IIB were found to remain at the plasmamembrane, although they could also be found inthe cytoplasm in stress ®bre-like structures (Fig. 9C,D). Actin also remained present at the plasmamembrane, in stress ®bre-like structures, and indeveloping sarcomeres (Fig. 9B). In contrast, myo-sins IX and V were not present at the plasmamembrane, or co-localized with actin stress ®bres,but showed a punctate staining distributed through-out the cytoplasm (Fig. 9A, data not shown).

Discussion

Myoblasts express at least ®ve different classes of themyosin superfamily, classes I, II, V, VII and IX, all of

which were expressed at higher levels in myoblaststhan in mature myotubes. However, only a subset ofthese myosin isoforms, myosin Iá and non-musclemyosins IIA and IIB, may be involved directly inearly myogenic differentiation. This is suggested bythe striking relocalization of these isoforms, togetherwith actin, to the plasma membrane in alignedmyoblasts, in proliferating cultures. Furthermore,myosin Iá was more highly expressed in skeletalmyosin positive cells prior to and just after fusion.Non-muscle myosin and actin remained at theplasma membrane in early myotubes. None of theother myosin isoforms investigated showed a stain-ing pattern which suggested that they could beinvolved in early muscle differentiation. In myo-blasts, myosin Iá, non-muscle myosin IIA and IIBwere localized in regions consistent with a role forthese proteins in myoblast locomotion, as suggestedfor ®broblasts (Mitchison & Cramer, 1996).

Proliferating myoblasts commonly form stableaggregates of bipolar cells for some time prior tofusion, and we found that in these cells, myosin Iá,actin and non-muscle myosin are all enriched at the

Fig. 6. Immuno¯uorescence localization of actin, myosin Iá, myosin I (myr 2 homologue), non-muscle myosin IIA andIIB and myosin IX in aligned myoblasts, using confocal microscopy. (A) myosin Iá, (B) myosin I (mouse homologue tomyr2), (C) myosin IX, (D) actin, (E) non-muscle myosin IIA, (F) non-muscle myosin IIB. Figures (D) and (E) are the sameset of cells that were co-stained with actin (D) and myosin IIA (E) demonstrating the nearly complete co-localization ofthese two proteins in aligned cells. All the specimens in this ®gure were ®xed and stained with 2% PFA andpermeabilized with triton except (A), which was methanol ®xed. Scale bar � 12 ìm.


Fig. 7. Immuno¯uorescence localization of actin (red) and myosin Iá (green) in a group of aligned cells, using confocalmicroscopy. Cells were ®xed with paraformaldehyde and permeabilized with triton as described in Materials andmethods. Actin and myosin Iá are co-localized at the plasma membrane.

Fig. 5. Immuno¯uorescence localization of actin, and non-muscle myosins, myosin Iá, non-muscle myosin IIA and IIB,and myosin IX, in motile myoblasts using confocal microscopy. Each cell has been stained for actin (red) and the non-muscle myosin (green). (A) Myosin Iá (green), actin (red). (B) Non-muscle myosin IIB (green), actin (red). (C) Myosin IX(green), actin (red). (D) Non-muscle myosin IIA (green), actin (red). All the specimens in this ®gure were ®xed with 2%PFA and permeabilized with triton. Scale bar � 12 ìm (5A±C), 5 ìm (5D).

510 WELLS et al.

plasma membrane. This localization at this earlytime, when cells are not post-mitotic and have notbegun to differentiate is unlikely to be related toearly myo®brillogenesis. However, it is more likelythat these proteins are in¯uencing the cell shapeand alignment in these aggregates. The character-istic bipolar shape of these cells is probablyrequired to generate the geometric interactionsrequired between myoblasts to enable fusion intolinear myotubes (Clark et al., 1997). This bipolarshape could arise from the tension produced by theassociation of non-muscle myosin, actin and myosinIá with each other and with the cytoplasmic surfaceof the plasma membrane. Interestingly, myoblastalignment is impaired by a calmodulin antagonist

(Bar-Sagi & Prives, 1983), which could act byinhibiting myosin I activity.

Futher investigation needs to be carried out todetermine what initiates the relocalization of myosinIá, actin and non-muscle myosin II to the plasmamembrane in aligned cells, and where exactly, inrelation to the plasma membrane, these proteins arelocated. For example, non-muscle myosin IIA andIIB and myosin Iá could all bind directly to theplasma membrane through their tails, and to actinvia their catalytic head domains. Non-musclemyosin II can bind directly to the cytoplasmicsurface of the plasma membrane by binding tophosphatidylserine (PS; a major component of theplasma membrane found mainly on the cytoplasmic

Fig. 8. Immuno¯uorescence localization of myosin Iá (green) and skeletal myosin II (red) in myoblasts and myotubes,using confocal microscopy. (A) Myoblasts after 1 day under differentiation conditions, (B) myotubes after 5 days underdifferentiation conditions. Also shown is the DIC image obtained with the confocal microscope as described in themethods, which shows all the cells in the ®eld. It is apparent that cells that appear to be expressing higher levels ofmyosin Iá are those cells that have begun to express skeletal myosin II in (A). Both the specimens in this ®gure weremethanol ®xed. Scale bar � 12 ìm.


surface), or to phosphatidylinositol (PI; a moreminor component) via its COOH terminal region(Murakami et al., 1995). Non-muscle myosin couldbind to PS, or PI, in the lacunae, regions of lipidbilayer devoid of glycoproteins, which have beenfound in prefusion myoblasts (Fulton et al., 1981).Alternatively, myosin Iá could bind to the mem-brane via its tail, and bind to actin via its head, andthe non-muscle myosins could bind to actin, but notbind to the membrane.

Myosin Iá must be speci®cally recruited to theplasma membrane in aligned cells, as anothermyosin I isoform (mouse homologue to myr 2), isnot found at the plasma membrane. Myosin Iisoforms are normally associated with membranerich regions of cells, via a membrane bindingdomain in the tail (Conrad et al., 1989; Fukui etal., 1989; Baines & Korn, 1990; Hayden et al., 1990;Cheney et al., 1993). The localization of myosin Iá atthe plasma membrane in aligned cells could be aresult of transporting vesicles. Vesicles have beenfound at the plasma membrane in fusing myoblasts,and have been suggested to play a role in the fusionprocess (Kalderon & Gilula, 1977) and myosin I

isoforms have been linked with vesicle movement(Novak et al., 1995; Jung & Hammer, 1996; reviewedin Ostap & Pollard, 1996). This could explain thehigher expression levels of myosin Iá found inpostmitotic, pre-fusion myoblasts, that have begunto express skeletal myosin II and are just about tofuse. However, these vesicles have not been ob-served in other studies (Wakelam, 1985, 1988).

It is even more uncertain how actin becomesenriched at the plasma membrane of aligned cells. Itcould be recruited to the membrane by binding tomyosin Iá and non-muscle myosin II. However it isperhaps surprising that we did not ®nd actin®lament bundles in the cytoplasm in aligned cells,suggesting that they must be actively dissociated bysome unknown mechansim. Focal adhesions arealso mostly absent from the cell body of alignedcells (M. Peckham, personal observation). Thisperhaps demonstrates that cell±cell contact is moreimportant than cell±substratum adhesion in alignedcells prior to fusion. The actin cytoskeleton isknown to be important in fusion, because depoly-merisation of actin ®laments by Cytochalasin Binhibits fusion (Holtzer et al., 1975).

Fig. 9. Immuno¯uorescence localization of actin, non-muscle myosin IIA and IIB and myosin IX in 3-day-old myotubesusing confocal microscopy. (A) Myosin IX, (B) actin, (C) non-muscle myosin IIA, (D) non-muscle myosin IIB. All thespecimens in this ®gure were ®xed with 2% PFA and permeabilized with triton. Scale bar � 12 ìm.

512 WELLS et al.

To enable the myoblasts to fuse, it is likely thatactin, myosin Iá and non-muscle myosin II must becleared from the plasma membrane where cells arein contact. Two events linked to myoblast fusioncould be involved in this process. Protein kinase C(PKC) which can dissassemble non-muscle myosin®laments by phosphorylating the heavy chain at theCOOH terminus, is also known to be important inmyoblast fusion (reviewed in Wakelam, 1988).Protein kinase C is known to be activated duringfusion by an increase in diacylglycerol (DAG)following a breakdown of PIP (polyphosphoinosi-tides). However, a pre-requisite for non-musclemyosin ®lament disassembly by PKC is that non-muscle myosin is bound to PS, where the localconcentration is greater then 70 mol % (Moussavi etal., 1993; Murakami et al., 1995). These highconcentrations of PS could be present in the lacunaeobserved in fusing myoblasts (Fulton et al., 1980).Fusion is also accompanied by an increase incalcium levels, which can deactivate myosin Iá bydisassociating calmodulin, as demonstrated in invitro motility assays (Williams & Collucio, 1994).

The continued presence of actin and non-musclemyosin II at the plasma membrane in 3-day-oldmyotubes, suggests that actin and non-musclemyosin II are only removed from the membraneswhich have fused, and that actin and non-musclemyosin II must continue to play a role in earlymyogenesis in the myotube. Non-muscle myosinhas been suggested to play a role in earlymyo®brillogenesis both in myotubes (Fallon &Nachimas, 1980), and in spreading cardiomyocytes(Rhee et al., 1994). In contrast, myosin Iá iscompletely lost from 5-day-old myotubes, andunfused post-mitotic myoblasts after 5 days underdifferentiation conditions. This suggests that thisprotein does not play a role in myo®brillogenesis,but is only important in proliferating and aggregat-ing myoblasts.

The localization of myosin Iá, non-muscle myosinIIA, IIB and actin in motile myoblasts are consistentwith their role in cell adhesion and locomotion,similar to that described for ®broblasts and othercell types (DiBasio et al., 1988; Conrad et al., 1989;Maupin et al., 1994; Verkhovsky et al., 1995;Mitchison & Cramer, 1996). Myosin I and actinhave been suggested to provide a motive force forlamellipodial extension or retraction in migrating®broblasts (Conrad et al., 1989). Similarly, myosin Iámay provide a motive force for the extension of thegrowth cone (Lewis & Bridgman, 1996). The absenceof myosin II from the leading edge, and itsassociation with actin in the stress ®bres in thecortical regions, has suggested a role for myosin IIin movement of the cell body and in maintainingcell adhesion to the substrate (Conrad et al., 1989;

Verkhovsky et al., 1995; Mitchison & Cramer, 1996).The differential localization of non-muscle myosinIIA and IIB that we observe here is also similar tothat observed previously in ®broblasts (Maupin etal., 1994; Conrad et al., 1995).

In conclusion, although we found many differentisoforms of the myosin superfamily in myoblasts,the only isoforms that appear to play a direct role inearly muscle differentiation are myosin Iá and non-muscle myosins IIA and IIB. The localization ofboth myosin IX and myosin V did not changeduring myogenic differentiation, although the ex-pression levels were reduced in mature myotubes.The exclusion of myosin IX from the lamellae couldarise from a general exclusion of myosins other thanmyosin Iá or myosin V from this region. We wereunable to observe the perinuclear staining seen inhuman leucocytes suggested to arise from anassociation with the Golgi apparatus (Wirth et al.,1996). The roles of these other isoforms in myoblastsremain to be determined.

Acknowledgements

We are enormously grateful to Graham Dunn, DanielZicha and Paul Fraylich for their expert help in®lming the myoblasts using phase stepping inter-ferometry. We would like to thank Meg Titus, PeterChantler, Martin BaÈhler, Robert Adelstein, SimonHughes and Richard Cheney who generously gaveus reagents used in this work. We would also like tothank Martin BaÈhler, Simon Hughes and Peter Clarkfor comments on an earlier version of the manuscriptand Kate Kirwan for help in producing the ®gures.This work was supported by the Royal Society andthe MRC. Claire Wells is an MRC funded PhDstudent. Michelle Peckham is a Royal SocietyUniversity Research Fellow.

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myogenic cells express multiple myosin isoforms

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