localization of myosin ii a and b isoforms in cultured neurons

3661Journal of Cell Science 108, 3661-3670 (1995)Printed in Great Britain © The Company of Biologists Limited 1995JCS6971

Localization of myosin II A and B isoforms in cultured neurons

M. William Rochlin1, Kazuyuki Itoh2, Robert S. Adelstein2 and Paul C. Bridgman1*1Department of Anatomy and Neurobiology, Washington University Medical School, 660 S. Euclid Ave, Box 8108, St Louis, MO63110, USA2Laboratory of Molecular Cardiology, National Heart, Lung and Blood Institute, NIH, Bethesda, MD 20892, USA

*Author for correspondence

Tension generated by growth cones regulates both the rateand the direction of neurite growth. The most likelyeffectors of tension generation are actin and myosins. Weare investigating the role of conventional myosin in growthcone advance. In this paper we report the localization ofthe two most prominent isoforms of brain myosin II ingrowth cones, neurites and cell bodies of rat superiorcervical ganglion neurons. Affinity purified polyclonal anti-bodies were prepared against unique peptide sequencesfrom human and rat A and B isoforms of myosin heavychain. Although each of these antibodies brightly stainednonneuronal cells, antibodies to myosin heavy chain Bstained neurons with greater intensity than antibodies tomyosin heavy chain A. In growth cones, myosin heavychain B was most concentrated in the margin bordering thethickened, organelle-rich central region and the thin, actin-rich peripheral region. The staining colocalized with actinbundles proximal and distal to the marginal zone, thoughthe staining was more prominent proximally. The trailingedge of growth cones and the distal portion of the neuriteoften had a rimmed appearance, but more proximalregions of neurites had cytoplasmic labelling. Localizing

MHC-B in growth cones previously monitored duringadvance (using differential interference contrastmicroscopy) revealed a positive correlation with edges atwhich retraction had just occurred and a negative correla-tion with lamellipodia that had recently undergone pro-trusion. Cell bodies were brightly labelled for myosin heavychain B. Myosin heavy chain A staining was dimmer andits colocalization with filamentous actin bundles in growthcones was less striking than that of myosin heavy chain B.Growth cones stained for both myosin heavy chain A andB revealed that the two antigens overlapped frequently, butnot exclusively, and that myosin heavy chain A lacked theelevation in the marginal zone that was characteristic ofmyosin heavy chain B. The pattern of staining we observedis consistent with a prominent role for myosin heavy chainB in either generating tension between widely separatedareas of the growth cone, or bundling of actin filaments,which would enable other motors to effect this tension.These data support the notion that conventional myosin isimportant in growth cone advance and turning.

Key words: myosin II, neuron, growth cone, cytoskeleton

SUMMARY

INTRODUCTION

The neuronal growth cone is a highly dynamic structure thatgoverns axon assembly and pathway selection during devel-opment and following nervous system trauma. Although theinteractions between the growth cone and the extracellularenvironment are complex, a crucial and instructive conse-quence of this interaction has been discerned: tension developsin axons resulting from growth cone exploration, and thistension regulates both axon formation rate and the direction ofoutgrowth (pathway selection) (Heidemann et al., 1991;Lamoureux et al., 1989). We are studying the molecular mech-anisms underlying tension production by growth cones.

Actin and myosins are abundant in growth cones (Lewis andBridgman, 1992; Miller et al., 1992; Espreafico et al., 1992;Ruppert et al., 1993) and their interaction is capable ofproducing tension in vitro. There are at least eleven classeswithin the myosin superfamily (Sellers and Goodson, 1995)and three are known to be represented in vertebrate brain,

myosins I, II, and V. Myosin I has been demonstrated to moveactin filaments along a phospholipid substratum (Zot et al.,1992), has been localized to Golgi vesicles in intestinal epi-thelial cells (Fath and Burgess, 1993), and tends to be localizednear the plasma membrane in growth cones (Lewis and P. C.Bridgman, unpublished observation). These data suggestinvolvement of myosins I in vesicle transport and linkage ofactin to the plasma membrane. Myosin V has a lipid bindingdomain (Cheney et al., 1993) and a primitive myosin V, myo2,is essential for vesicle transport in Saccharomyces (Johnstonet al., 1991). Furthermore, in the dilute (myosin V) mutantsthat have the most severe phenotype i.e. death followingseizures beginning 3 weeks after birth, brain developmentappears to be normal (Strobel et al., 1990), arguing against acritical role for this myosin in gross aspects of growth conenavigation. Thus, myosin V, like myosin I, is a good candidatefor a vesicle transporter. Whether these two myosins also con-tribute to tension production remains to be determined.

Myosin II is the conventional, two-headed myosin that forms

3662 M. W. Rochlin and others

Fig. 1. Western blot of fetal SCG extract showing the presence ofMHC-A and MHC-B. Protein extracts were prepared from SCG cellsfollowing overnight culture. The extract was loaded into a single laneon a 5.5% SDS-PAGE gel, and transferred to a nitrocellulosemembrane. After Ponceau staining, the nitrocellulose was cut intostrips and immunostained for MHC-A and -B. MHC-Bimmunoreactivity (arrowhead, lane B) is greater than that of MHC-A(arrowhead, lane A). The theoretical molecular mass for the MHCbands is approximately 225 kDa, although both migrate at 200 kDa.MHC-A was detected with an antibody prepared against humanMHC-A (see Materials and Methods).

filaments via interactions between the coiled-coil tail domainsof different molecules (Warrick and Spudich, 1987). Structuraldifferences between brain myosin II and other types ofnonmuscle myosin were initially proposed by Burridge andBray (1975). Recently, three isotypes of myosin II that differ inthe primary sequences of their heavy chains have been foundin vertebrate brain (Murakami and Elzinga, 1992; Sun andChantler, 1991). Two isoforms that are prominently expressedin mammalian brain are designated myosin heavy chain A andB (MHC-A and MHC-B) (Simons et al., 1991). Alternativesplicing of the MHC-B message gives rise to three additionalisotypes (Itoh and Adelstein, 1995). Prior to the discovery ofthese isoforms, Bridgman and Dailey (1989) examined the dis-tribution of myosin II in rat superior cervical ganglion (SCG)neurons. However, this work was carried out with an antibodyprepared against platelet myosin, the heavy chain of which isexclusively MHC-A (Murakami et al., 1991). We subsequentlylearned that this antibody will recognize MHC-B inhomogenates of neural tissue and also on western blots, albeitwith a lower apparent affinity than that for MHC-A (see Milleret al., 1992). We undertook the current study to determine thedistribution of each myosin II isoform individually. During thecourse of our study two groups reported the localization ofmyosin II isoforms in growth cones (Cheng et al., 1992; Milleret al., 1992). Both groups emphasized the localization ofmyosin II to the leading edge of the growth cone periphery, andCheng et al. proposed a role for myosin IIB, which appears tobe the predominant neuronal isoform, in leading edgeextension. The distribution we had observed differed fromtheirs and did not suggest a role in protrusion. To investigatethis further, we fixed growth cones during live observation, per-mitting us to identify sites that were undergoing protrusion orretraction at the time of fixation, and localized myosin IIB. Ourobservations support a role for myosin IIB in retraction of thegrowth cone periphery, but not in protrusion. The distributionsof both myosin II A and B in growth cones are consistent withroles for these myosins in tension production by growth cones.

MATERIALS AND METHODS

Antibody preparationInitially two peptides, GKADGAEAKPAE and SDVNETQPPQSE,were synthesized based on the derived amino acid sequences at thecarboxyl-terminal end of human macrophage MHC-A (Saez et al.,1990) and T-cell MHC-B (Phillips et al., 1995), respectively. Thepeptides were conjugated to keyhole limpet hemocyanin (Cal-biochem) with glutaraldehyde (Sigma grade II) and rabbits wereimmunized and bled by Berkeley Antibody Company (CA). Bothantibodies were purified on peptide antigen columns prepared bycoupling 20 mg of the appropriate peptide to 25 ml of Affi-gel 15(Bio-Rad). Antibodies were then further purified on a Protein Acolumn (Pierce). The final concentration of both antibodies wasbetween 0.7 and 1.0 mg/ml. During the course of this study, welearned that the corresponding fragment for the rat MHC-B isidentical to that in the human, but the rat MHC-A differs from thehuman by 3 amino acids (K.I., unpublished observation). Wetherefore prepared an antibody against the carboxy terminus of ratMHC-A, GKADGADAKATE. The species specific anti-MHC-Ayielded immunoblots and immunofluorescence that was indistin-guishable from that of the anti-human MHC-A. The anti-MHC-A usedis indicated in each figure legend.

The dye Cy3 was directly conjugated to affinity purified anti-MHC-

B using a kit available from Biological Detection Systems. A molarratio of 5 (Cy3:anti-MHC-B) was obtained.

Western blottingSCG cells were plated onto laminin coated plastic culture dishes aspreviously described (Bridgman and Dailey, 1989). After overnightgrowth, the dishes were washed with 37°C L15 medium, and thentransferred to a cold surface, whereupon the L15 was immediatelyexchanged for ice-cooled 11% TCA. The precipitated cellularmaterials were scraped off of the dish, centrifuged, washed twice with1 ml of cold acetone, dried and prepared for SDS-PAGE using con-ventional methods. We used a low percentage of acrylamide (5.5%)so as to increase the completeness of the electric transfer to nitrocel-lulose. Following transfer, the membrane was stained with Ponceaureagent (Sigma) and the borders of the lanes were marked. Lanes werecut into strips and labelled for MHC immunoreactivity with thefollowing dilutions of affinity purified antibodies: MHC-A, 1:1,000;MHC-B, 1:2,000. Antibodies were detected using the EnhancedChemiluminescent Method (Amersham).

DIC observationSCG explants were cultured on coverglasses overnight as describedpreviously (Lewis and Bridgman, 1992), and mounted onto a chamberdesigned to permit perfusion during DIC observation (Berg andBlock, 1984). Advancing growth cones were imaged using aNewvicon camera and time lapse video enhancement (averaging 4background subtracted frames every 5 seconds). Glutaraldehydefixative (37°C, see Immunofluorescence, below) was perfusedthrough the chamber, usually during protrusion of a portion of theleading edge. During perfusion, fine focus became difficult, but it wasnonetheless possible to observe the fixation process by continuousimaging. Within seconds of initiating perfusion, the growth conesdeveloped a flattened, coarser appearance and regions near the baseof growth cone that had appeared smooth developed circumscribedregions with a reverse shadow cast morphology. No further changesin their appearance occurred, from which we inferred that fixation hadbeen largely completed in the initial seconds. These cultures wereprocessed as described below for immunofluorescence observation of

3663Myosin II A and B in neurons

anti-MHC-B and β-actin staining. To enable overlapping of theimmunofluorescence images (which were obtained with a CCDcamera) and the DIC images, the image distortion due to theNewvicon camera was digitally compensated. To ascertain whether agiven region of the growth cone perimeter was undergoing protrusionor retraction, changes in the position of the edge were determined overthe 2-frame (10 second) period preceding the fixation.

ImmunofluorescenceCultured SCG cells were fixed and permeabilized by one of twomethods.

Method 1, glutaraldehyde fixationCultures were perfused with 37°C fixative (0.25% glutaraldehyde in100 mM cacodylate (pH 7.4) with 5 mM CaCl2 and 10 mM MgCl2).After 10 minutes, cultures were washed 3× with fixative buffer

Fig. 2. The distributions of MHC-A, MHC-B,and F-actin in SCG growth cones fixed by twomethods, glutaraldehyde (A-D) or freezesubstitution (E-G). Note that the brightness ofthe anti-rat MHC-A staining has beensubstantially enhanced relative to MHC-B (seetext). (A,E) MHC-A fluorescein; (B)rhodamine-phalloidin (rendered in grayscale)(C,F) Cy3-conjugated anti-MHC-B.(D,G) Overlay. (A-D) MHC-A staining (A) ismost intense in the C-domain, but colocalizeswith MHC-B (C) along F-actin bundles in themarginal zone (filled arrowheads). MHC-Afoci on such bundles did not always colocalizewith MHC-B (open arrowheads). In the C-domain, MHC-A foci often did not colocalizewith MHC-B (arrow), nor did it appear tocolocalize specifically with F-actin. DimMHC-A staining along F-actin bundles in theP-domain was observed (open arrows). (E-G) In this frozen, freeze-substituted growthcone, MHC-A (E) is more uniformlydistributed in puncta throughout the C-domainand marginal zone than in glutaraldehyde fixedgrowth cones (compare with A). The MHC-Apuncta colocalize well with MHC-B in themarginal zone (arrowhead), at edges thatappear to have been undergoing retraction (*),and within the C-domain (arrow). MHC-Bstaining was occasionally found in the absenceof MHC-A staining (open arrowhead).Bar, 5 µm.

without aldehyde. For phalloidin labelling, cultures were then simul-taneously permeabilized and labelled with 100 µl of PBS containing0.02% saponin and 133 nM rhodamine-phalloidin (Sigma). (Whenlabelling of actin was carried out with anti-β-actin, phalloidin wasomitted from the permeabilization buffer, and coverslips wereprepared as described in the next paragraph.) After 10 minutes,cultures were washed 3× in PBS and mounted for fluorescence pho-tomicroscopy in Vectashield mounting medium (Vector Laborato-ries). One corner of the coverglass was marked with a permanentmarker so that the orientation could be determined following theantibody staining. After photographing about 30 growth cones percoverglass, the coverglasses were prepared for antibody staining asdescribed below.

To quench unreacted glutaraldehyde moieties, decrease autofluo-rescence, and eliminate the phalloidin staining, cultures were treatedwith 1% OsO4 in PBS for 5 minutes at 4°C, washed exhaustively (7×)


Fig. 3. Confocal images of MHC-A and MHC-B staining in neuronalsomata. (A) MHC-A staining with an antibody prepared againsthuman MHC-A (see Materials and Methods) is found along theventral surface (attachment site) of a soma. Linear arrays of stainingcan be discerned (arrowhead). (B) MHC-B staining along the ventralsurface of a soma. The arrowhead indicates a linear array of MHC-Bstaining. The MHC-A staining was again much dimmer than that ofMHC-B. Bar, 10 µm.

in PBS at room temperature, then treated for 30 minutes with 5% β-mercaptoethanol (Pierce) in PBS (prepared within 20 seconds of use),and washed as before. Cultures were then blocked with PBS contain-ing 8 mg/ml BSA, 0.5% fish gelatin (v/v), and 5% normal goat serum(v/v). Antibodies were diluted into PBS containing 20% blockingbuffer. The following primary antibody concentrations were used:anti-rat MHC-A, 1:500; anti-human MHC-A, 1:7,200; anti-MHC-B,1:3,150; anti-Cy3-coupled MHC-B, 1:500; monoclonal anti-β-actin,1:1,800 (Sigma), fluorescein goat anti-mouse 1:400; Cy3 goat anti-rabbit, 1:800; fluorescein goat anti-rabbit monovalent Fab, 1:140; Cy5goat anti-mouse 1:200. All secondary antibodies were obtained fromJackson Research except fluorescein goat anti-mouse (MolecularProbes). For phalloidin stained coverslips, growth cones previouslyphotographed were relocated and rephotographed.

Method 2, freeze substitutionCultures were rapid frozen by immersion in liquid nitrogen-cooled50% ethane, 50% propane, and then stored in liquid nitrogen. Substi-tution and fixation were performed by transferring coverslips into acontainer of 0.5% paraformaldehyde in methanol cooled to −80°C,followed by warming to −20°C. Coverslips were then transferred tophosphate buffered saline (PBS) at 22°C, blocked and labelled asdescribed above, except that higher concentrations of anti-MHC couldbe used (anti-rat MHC-A, 1:100; anti-human MHC-A, 1:400; anti-MHC-B, 1:175).

For labelling of both MHC isoforms in the same growth cones, cov-erslips were labelled first with rabbit anti-MHC-A (and in some casesmouse anti-β-actin), next with a monovalent fluorescein anti-rabbit(and Cy5 goat anti-mouse), and finally with Cy3-conjugated rabbitanti-MHC-B.

Fluorescence images were obtained with a cooled charge-coupleddevice (CCD). Digitized images were particularly useful for aligningthe triple-stained images and brightness/contrast adjustment. To aligntriple-stained images of growth cones, the actin staining was used asthe reference, and the myosin images were individually positioned soas to align edge foci with the F-actin bundles at the edge of the growthcone. For the triple-stained non-neuronal cell images, precisecolocalization of the MHC isoforms was evident, and used to positionthe MHC images, and the actin staining was positioned next. Inaddition, these images revealed that the fluorescence associated withthe rhodamine-phalloidin obtained from Sigma was not completelyeliminated by the oxidation/reduction treatment. The Cy3 anti-MHC-B labelling appeared to be elevated in the periphery of phalloidinstained growth cones, but not in the periphery of β-actin stainedgrowth cones. To subtract this staining, the elevation in peripheralCy3 anti-MHC-B staining was estimated and the grayscale values ofthe original phalloidin image were factored down so as to be approx-imately equal to the estimated elevation in Cy3 MHC-B staining. Thefactored down grayscale values of the phalloidin image were then sub-tracted from the Cy3 anti-MHC-B image.

RESULTS

MHC-A and B are both present in SCG cellsAntibodies prepared against isoform specific regions in thecarboxy terminus of MHC-A and MHC-B recognize bands onwestern blots that comigrate with skeletal muscle myosin (Fig.1). The band identified as MHC-A migrates slightly morerapidly than MHC-B, and these bands separate if the durationof SDS-PAGE is extended (not shown; see Kawamoto andAdelstein, 1991). The antibodies do not cross-react at all onwestern blots (Maupin et al., 1994; Phillips et al., 1995). Thedistributions of the two major types of myosin II were examinedby immunofluorescence light microscopy. MHC-B labelling

was bright in both neurons and non-neuronal rat superiorcervical ganglion (SCG) cells, whereas MHC-A staining wasbright in non-neuronal cells, but much dimmer in neurons.

MHC distribution in growth cones and neuritesBy directly conjugating Cy3 to anti-MHC-B, we were able tocolocalize both myosin II isoforms in the same growth conesand non-neuronal cells (see Materials and Methods). In growthcones, MHC-B staining, though less intense than when asecondary antibody was used, could easily be detected throughthe eyepieces of our fluorescence microscope, but MHC-A wasbarely visible. The staining pattern of MHC-A could not bedetermined without the use of a sensitive CCD camera andimage processing software to enhance brightness and contrast.The brightest MHC-A foci were approximately of the sameintensity as the dim MHC-B foci detected in CCD images ofthe growth cone periphery. To compare the distribution ofMHC-A and MHC-B staining (Fig. 2) the peak brightnesses of


the two MHC images were normalized, i.e. the MHC-Astaining intensity was amplified relative to that of MHC-B.Two methods of fixation were used for the immunostaining.Glutaraldehyde fixation enabled excellent labelling of actin,but the MHC staining usually appeared to have a higher back-ground than growth cones fixed by freeze substitution.Although all but the most intense actin staining is compro-mised by freeze substitution, the myosin staining appearsbrighter and areas of increased fluorescence path length are notsites of increased diffuse staining. We will refer to four regionsof well-spread growth cones: the peripheral domain (P-domain) is composed of the F-actin-rich lamellipodia, thecentral domain (C-domain) refers to the more proximal,thickened, organelle-rich region (see Bridgman and Dailey,1989), the marginal zone refers to a transition region betweenthe P- and C-domains, and the base of the growth cone refersto the proximal-most portion of the growth cone that has notcompleted differentiation into a segment of neurite.

The pattern of staining of MHC-A depended more stronglyon the fixation conditions employed than did that of MHC-B.In glutaraldehyde fixed growth cones MHC-A staining (Fig.2A) is brightest in the C-domain. In contrast, the brightness ofthe staining in freeze substituted growth cones was eventhroughout the C-domain and marginal zone (Fig. 2E). It ispossible that the elevation of C-domain labelling in the glu-taraldehyde fixed cells (which was also evident for MHC-Bstaining, compare Fig. 2C with Fig. 2F) represents non-specificlabelling. Staining foci of the two isoforms overlapped fre-quently in growth cones prepared by both methods (Fig. 2D,G,orange), but not exclusively (Fig. 2A,C,D, arrow; E-G, openarrowhead). Perhaps because of the higher background associ-ated with the glutaraldehyde fixed growth cones, colocaliza-tion of MHC-A and -B foci in the C-domain is more easilyobserved in the freeze-substituted growth cones (Fig. 2E-G,arrow). The brightest staining of the B isoform was associatedwith F-actin bundles that also had MHC-A staining foci (Fig.2, filled arrowheads). The MHC-B staining appeared to have aslightly more peripheral localization than the MHC-A staining,owing to the elevation of the MHC-B staining in the marginalzone. MHC-B was typically elevated at sites that appeared tohave undergone substantial retraction (Fig. 2C,D,F, asterisks).MHC-A labelling, while present at these sites, was not alwayselevated (Fig. 2E, asterisks). In neurites, MHC-A staining wasoften uniform, lacking the rim-like quality often observed forMHC-B staining (see below). In summary, while MHC-A and-B were both concentrated in the C-domain, MHC-A had amore uniform distribution and lacked the marginal zoneelevation characteristic of MHC-B.

MHC distribution in neuronal cell bodiesConfocal microscopy was used to examine the distribution ofMHC staining in neuronal cell bodies fixed by freeze substitu-tion. MHC-A staining (Fig. 3A) was again dimmer than thatof MHC-B (Fig. 3B). The lower level of diffuse staining withinthe cell body (vs MHC-B), made it easier to discern the patternof cortical MHC-A staining (Fig. 3A, ventral surface). MHC-A staining on the dorsal surface was difficult to detect (notshown). Lack of dorsal cortical staining with anti-MHC-A wasalso observed by Maupin et al. (1994) in fibroblasts, but thedorsal surface of non-neuronal cells in our SCG cultures was

intensely stained for MHC-A. The dorsal (not shown) andventral (Fig. 3B) surfaces of the cell bodies had linear arraysof MHC-B staining (arrowhead), suggesting that corticalmyosin II was organized into filaments. The arrays werethicker and longer on the ventral surface than on the dorsalsurface. It was difficult to determine whether MHC-B stainingwas organized into filaments within the cytoplasm due to thehigh level of diffuse staining. The distribution of actin matchedthat of the MHC-A and -B in the cell bodies (not shown).

The distributions of MHC-B and MHC-A in non-neuronal cellsUnlike growth cones, non-neuronal SCG cells stained brightlyfor both MHC-A and MHC-B (Fig. 4A,C, respectively).Following freeze substitution fixation, the large bundles of F-actin (e.g. stress fibers) present in non-neuronal cells can bediscerned with anti-β-actin staining (B). The non-neuronal cellshown in Fig. 6 was at the front of the advancing halo ofneurites emanating from an SCG explant. The MHC-A stainingpattern of the non-neuronal cells (A) could easily be discernedthrough the eyepieces, but not that of the two small growthcones that had grown over the non-neuronal cell (open arrow-heads). The staining patterns of MHC-A and -B within the non-neuronal cells were similar but not identical. In some non-neuronal cells, MHC-A appeared to be distributed closer to theleading edge than MHC-B (A,B,D, small triple arrow), andMHC-B appeared more concentrated than MHC-A toward thenucleus (A,B,D, large triple arrow). This was observed previ-ously for human melanoma cells (Maupin et al., 1994). Giventhe striking difference in the ratio of MHC-A:MHC-B stainingbetween growth cones and non-neuronal cells, we investigatedthe relationship between MHC-B staining, F-actin, and growthcone dynamics more closely.

Precise colocalization of MHC-B and F-actin ingrowth conesMHC-B colocalized extensively with F-actin, in particular F-actin bundles. The actin staining looked similar regardless ofwhether we used rhodamine-phalloidin (Fig. 5A,B, rendered ingreen) or an antibody that recognized β-actin (Fig. 6I). Long,radially disposed F-actin bundles typical of forming lamel-lipodia within the P-domain are often dotted with dim MHC-B foci (Figs 5A, 6J, open arrowheads). MHC-B was almostalways found along the subset of these bundles that extend intothe marginal zone. Bundles that terminate at widely separatedareas of the leading edge appear to converge and interminglein the marginal zone, consistent with the possibility thatmyosin II crosslinks these bundles (Figs 5A,B, 6J). The C-domain often had bright MHC-B staining that colocalized withF-actin bundles, but at a lower density than in the marginalzone (Figs 5A, 6G,I,J). The C-domain was also associated withan increase in the diffuse MHC-B staining that could not becorrelated with F-actin staining. The edges of the C-domain(the sides and trailing edges of the growth cone), were sites ofintense MHC-B staining that colocalized with thick F-actinbundles (Figs 5A, 6G,I,J, short filled arrows).

Although neurites were brightly labelled for MHC-B (Figs5A, 6G-J, small open arrows), colocalization of MHC-B andF-actin bundles was difficult to establish in neurites. Thelabelling often appeared to be brightest along one or both edgesof the neurite, suggesting a concentration of MHC-B in the


Fig. 4. Triple immunofluorescence of MHC-A,MHC-B, and β-actin in a non-neuronal SCGcell. (A) Anti-rat MHC-A (fluorescein); (B) β-actin (Cy5, rendered in grayscale); (C) MHC-B(Cy3); (D) overlay. MHC-A is much brighter innon-neuronal cells than in growth cones. Twosmall growth cones (open arrowheads) that havegrown over the non-neuronal cell are not visiblylabelled for MHC-A when the brightness isadjusted to optimize non-neuronal staining.Although non-neuronal cells are also morebrightly labelled for MHC-B than growth cones,the growth cones do have MHC-B levels thatare comparable to the non-neuronal cell (in thiscase, less than usual MHC-B is present in thegrowth cones). In this non-neuronal cell, MHC-A is more concentrated along peripheral F-actinbundles, whereas MHC-B is more concentratedcentrally. Nonetheless, there is a strikingoverlap of the two MHC isoforms at bothlocations (triple arrow sets). Bar, 5 µm.

cortex. However, in neurites that had varicosities along theirlength, the staining was brightest at the varicosities, suggest-ing that MHC-B is also present in the cytosol.

Correlation of MHC-B staining and motile eventsoccurring at the time of fixationTo determine the relationship between growth cone dynamicsand the distribution of MHC-B, we used DIC microscopy toobserve advancing growth cones (Fig. 6A-E), fixed them

Fig. 5. (A,B) Overlays of MHC-B staining (Cy3,red) and rhodamine-phalloidin-labelled F-actin(rendered in green) in a wide growth cone. Thisgrowth cone possesses a long trailing edge whichis brightly labelled for MHC-B (A, double arrow).The breadth of the neurite clarifies the rim-like,presumably cortical MHC-B staining along theneurite (open arrow). F-actin bundles that coursefrom the leading edge through the marginal zoneare weakly labelled in the P-domain, but stronglylabelled in the marginal zone and P-domain (A,open arrowheads). The 4× magnification of asection of A shown in B illustrates the closeassociation between MHC-B foci and F-actin inthe marginal zone and C-domain. The openarrowhead in B corresponds to the openarrowhead in the marginal zone in A. Bar, 5 µm.

during their advance (Fig. 6F), and stained them for MHC-Band β-actin (Fig. 6G,I). The following associations emergedfrom our live observations (Table 1). During growth coneadvance, lamellipodia and filopodia undergoing protrusionhave very little MHC-B (Fig. 6G,H, long arrow). If a leadingedge lamellipodium was retracted to the marginal zone, MHC-B was elevated at this leading edge (n=4), but the leading edgesof partially retracted lamellipodia were not sites of intenseMHC-B staining (n=3, including Fig. 6G,H, o).


Fig. 6. The distribution of MHC-B and β-actin in an SCG growth cone fixed during DIC observation. (A-E) Live observation prior to fixation.(F) Following fixation. (G) Anti-MHC-B staining (Cy3). (H) Superimposition of the MHC-B staining on the DIC image of the fixed growthcone. (I) β-actin staining (fluorescein). (J) Overlay of MHC-B and β-actin staining. (A-E) The central (C-) and peripheral (P-) domains aremarked in A (C and P, respectively). The dotted line indicates the marginal zone. During this 2.8 minute series, growth cone advance occurredat an average of 36 µm/hour. Sites of retraction (o) and protrusion (+) are indicated. The last frame prior to arrival of the fixative front is E. Dueto focus drift during fixative perfusion, the best image of the fixed growth cone was obtained following completion of fixation. (G-J) Thedistribution of MHC-B (G,H,J, red) and β-actin (I,J, green) at the time of fixation. Regions undergoing protrusion are only weakly stained forMHC-B (long arrow), but many of the foci line up along F-actin bundles that extend into the marginal zone (open arrowheads). Sites of partialretraction within the P-domain are also weakly stained (o), but retraction to near the marginal zone is associated with elevated MHC-B staining(*, short arrows). The most intense MHC-B labelling is in the marginal zone, where F-actin bundles from the P-domain appear to converge(filled arrowhead). Cortical MHC-B staining within the distal neurite is also present (open arrow). Finally, a long filopodium that terminates onanother growth cone (out of field) appears to be under tension and is associated with elevated MHC-B staining (convex arrowhead). Bar, 5 µm.


Table 1. Myosin II B localization in growth cones fixedduring DIC microscopic observation

I. Staining interior to the growth cone perimeterMarginal

C-domain zone P-domain

15/16 16/16 0/16

II. Perimeter stainingBase Trailing Rear/side Leading Leading edgeedge edge filopodia edge filopodia

7/16 10/16 9/16 0/16 3/1615/16* 14/16*

III. Staining at sites of retraction, protrusion or stasisSites of retraction

Leading edge Leading edgeBase Trailing lamellipodia lamellipodia Tensileedges edges (partial) (complete) filopodia†

20/30 18/20 0/2 4/4 5/5

Sites of protrusion

Trailing Leading Stationaryedges edge trailing edges

1/5 0/16 4/5

Live observation of 16 growth cones prior to fixation enabled us toascertain whether motile events were occurring at the time of fixation.Localization at the indicated sites refers to the presence of intensely stainingfoci comparable to those indicated by the filled arrowhead and short arrows inFig. 6G,H. For parts I and II, the denominator value is the number of growthcones considered. For part III, the denominator value is the number of edgesexamined. In all but one case, neurite formation, i.e. retraction at the base ofthe growth cone, was observed up to the time of fixation. In another case, dueto the large size of the growth cone only one trailing edge could be observed,and in another case, only one trailing edge was present due to thedisplacement of the neurite to one side of the trailing edge of the growth cone.

*The top value is the incidence of bright staining on both sides of theindicated structure, the bottom value is the incidence on at least one side.

†A tensile filopodium is one that appeared to be under tension because its tiphad remained in contact with another cell or debris on the coverslip, andbecause it remained straight during growth cone advance away from the object.

The sides of the growth cone are sites at which protrusionand retraction occur in adjacent areas, often perpendicular tothe direction of advance. If retraction had occurred just priorto fixation, MHC-B staining was found close to the side edge(e.g. Fig. 6G,H, asterisk) but if the growth cone was spreadinglaterally, the associated protrusion was only weakly stained, asobserved for leading edge lamellipodia.

As neurite formation occurs, the trailing edge of the growthcone moves forward and inward, and trailing edge filopodiaoften appear to be dragged along prior to being retracted. Thetrailing edges of the growth cone were typically a site ofintense MHC-B staining and trailing filopodia were labelledmore intensely than leading edge filopodia (Fig. 6H, comparethe staining of filopodia between the asterisk and the short,filled arrow with that of leading edge filopodia). Due to the V-shape of the rear edge of the growth cone in Fig. 6, it is difficultto categorize it as the trailing edge vs the side. In Fig. 5A, awider growth cone illustrates the trailing edge staining (doublearrow). Occasionally filopodia on the side or base of the growthcone appeared to be anchored to other neurites or debris on thesubstratum (Fig. 6G,H, convex arrowhead). These structuresappeared to be under tension because they were straight alongtheir length, and, in some cases, deflected neurites. Such tensileelements were more brightly stained for MHC-B than averagefilopodia.

In almost all cases, at least one edge of the base of thegrowth cone had elevated MHC-B staining, and in 40% of thecases elevated MHC-B staining was observed on both sides(Table 1; Fig. 6G,H, short arrows). We re-examined the DICvideotapes to determine if there were any motile or morpho-logical correlates to the variability in the edge stainingobserved at the base. At the time of fixation two phenomenawere observed at the base of the growth cone: retraction offlattened lamellipodia towards the forming neurite, and recruit-ment of already thickened cytoplasm to form the cylindricalshape characteristic of the neurite. In six of eight cases inwhich one-sided staining was observed, that staining waslocated on a side that had just undergone lamellipodial retrac-tion, and the non-stained side had undergone further centripetalrecruitment of already thickened cytoplasm. However, basesthat had staining on both sides were undergoing recruitment ofthickened cytoplasm. Together, these data on the relationshipbetween MHC-B staining and changes in the perimeter of thegrowth cone establish a strong correlation between myosin IIB and sites of lamellar retraction, and a negative correlationwith sites of lamellar extension.

As described above, the most intense MHC-B labellingoccurred away from the growth cone perimeter, in the marginalzone (Fig. 6A, dotted boundary). Although the advance of themarginal zone is difficult to monitor, our DIC observationsindicated that this is the site at which the constitutive retro-grade flow (Forscher and Smith, 1988) appears to cease. Theconcentration of actomyosin IIB in this area (Fig. 6G,I,J, filledarrowhead) suggests that it is the focal point of myosin IIBactivity.

DISCUSSION

This paper reports that myosin II, in particular MHC-B, isabundant in neuronal cell bodies and neurites, and has a

striking distribution in growth cones. The distributions of themajor isoforms of myosin II have been published previouslyby other workers, but our results are different from theirs,provide a more extensive analysis of the association with actin(Cheng et al., 1992; Miller et al., 1992), show the stainingpatterns of the two isoforms in the same growth cone, and showthe relationship of this distribution to growth cone motility. Wereport that MHC-B is concentrated along F-actin bundlesthroughout the organelle-rich central domain (C-domain) ingrowth cones, and is especially bright in the margin betweenthis domain and the actin-rich peripheral domain (P-domain).Cheng et al. (1992) reported that MHC-B is most concentratedin the periphery of the growth cone. We also found MHC-Bstaining foci in this region, but they were present at a muchlower concentration than in the C-domain and marginal region.The myosin distribution that we observed also differed fromthat reported by Miller et al. (1992), who found that myosin IIwas concentrated throughout the P- and C-domains, excludingfilopodia. Colocalization of myosin II with F-actin bundlescould not be discerned in the above studies. This colocaliza-tion is clear in our high resolution two- and three-color images.In addition, staining for the two myosin isoforms in the same


growth cone established that they partially colocalize along F-actin bundles in growth cones. Finally, our observations ofgrowth cone behavior just prior to fixation revealed a positivecorrelation between sites of lamellipodial retraction and theconcentration of myosin.

The differences between our findings and the previouslyreported findings could result from the use of different popu-lations of rat peripheral neurons (the other groups used DRGcells, whereas we used SCG cells) or from differences in thefixation methods. The other investigators pre-fixed their cellsin −20°C methanol whereas we used either a rapid freezemethod (−190°C) followed by freeze substitution, or perfusionwith warm glutaraldehyde-cacodylate buffer. In our experiencewith SCG neurons, the fine structure of the growth cone isbetter preserved by our methods.

What do our data on the distribution of myosin II in advancinggrowth cones suggest about the dynamics of myosin II organiz-ation during growth cone advance? The motile events occurringin the marginal zone, where myosin IIB is most concentrated,are difficult to monitor. The presence of a low level of stainingdistal to this site, and a higher level proximal to this site, is con-sistent with rapid assembly of actomyosin bundles at the leadingedge of the marginal zone, perhaps in concert with retrogradeflow (or retraction) of F-actin rich structures, followed by dis-assembly/disruption as the C-domain advances (see McKenna etal., 1989). Recent work has shown that myosin filaments containfewer myosin molecules in the periphery than in the centraldomain (Verkhovsky et al., 1995) consistent with the possibil-ity that myosin filaments grow during centripetal transport. Thesharp increase in the size and intensity of MHC foci at themarginal zone suggests that myosin filament assembly is differ-entially regulated at this site. Since the advancing trailing edgeis also more intensely labelled than the C-domain, actomyosindynamics at this site may undergo a similar assembly/disassem-bly process. In contrast to the marginal zone, both assembly anddisassembly of actomyosin would occur in front of (central to)the direction of the retraction of the trailing edge.

The localization of the major myosin isoform in neuronsafforded by our methods suggests several hypotheses on therole of conventional myosin in effecting growth cone shapechanges. The DIC and fluorescence observations indicate astrong correlation between intense myosin II B staining fociand sites of lamellar retraction/retrograde flow (Table 1). Thisis consistent with the possibility that myosin II filamentassembly assists in cross-linking actin filaments as they aretransported centrally from the periphery. In the marginal zone,such cross-linking may result in connecting widely separatedregions of the growth cone, perhaps facilitating the integrationof actin-based force production throughout the growth cone.Cross-linking may also be an important step in the process thatleads to the apparent cessation of the retrograde flow at themarginal zone. In addition to this passive (i.e. non-motor-dependent) assembly of actomyosin II bundles, myosin II maycontract, and thereby oppose protrusion or destabilize existingprotrusions. This possibility is suggested by experiments inwhich myosin function was disrupted (De Lozanne andSpudich, 1987; Honer et al., 1988; Knecht and Loomis, 1987).The abundance of myosin II in the marginal zone and the C-domain may help restrict protrusion away from these regions,confining it to the forward leading edge (see Lee et al., 1994).Marginal zone actomyosin bundles may also be involved in

changing the direction of growth cone advance by contractingagainst, and thereby destabilizing, lateral lamellipodia. Indirectevidence consistent with acto-myosin bundles exerting tensionalong their axes was provided by the observation that filopodiathat appear to be deflecting an encountered neurite have moreacto-myosin staining than average filopodia. Alternatively, ifthe bundles do not contract, they could serve a purely struc-tural role, enabling an alternative motor mechanism to pull ona large strip of the growth cone cytoskeleton by pulling on aportion of the bundle.

How might myosin II, being concentrated in the C-domain,influence growth cone advance? The C-domain undergoes twoprocesses in which myosin II is likely to be involved: advance-ment and differentiation into an axon. These processes areprobably not identical, since elastic tension develops in axons,apparently as a result of axon formation lagging behind advanceof the growth cone C-domain (Heidemann et al., 1991). If thecytoskeleton is more stably substratum-linked toward themarginal zone than in the C-domain, which is consistent withthe higher apparent density of actomyosin bundles in themarginal zone, myosin II-mediated contraction would pull theC-domain forward (see Lee et al., 1994). A role for conven-tional myosin in overcoming substratum adhesion is implied bythe work of Jay et al. (1995), and a role for conventional myosinin trailing edge retraction has been suggested for fibroblasts(Small, 1989). Myosin II is at present the best candidate for thisretraction-based advance of the trailing edge of the growthcone. Myosin II is also often elevated along the rear edge of thegrowth cone, and the cortex of the base of the growth cone, con-sistent with a role in constricting the base of the growth coneinto a cylinder during axon formation. Such a role is predictedby the dependence of cleavage furrow constriction on conven-tional myosin function (Mabuchi and Okuno, 1977).

MHC-A colocalized with F-actin bundles and appeared to bepresent at a lower level than MHC-B. In chick brain (Kawamotoand Adelstein, 1991) and human brain (Itoh and Adelstein,1995), both protein and mRNA levels are higher for MHC-Bthan MHC-A (approximately 4:1). The distribution of MHC-Awas slightly different from that of MHC-B. Whereas MHC-Bwas clearly concentrated in the marginal zone, MHC-A wasevenly distributed throughout the C-domain and marginal zone.In contrast, non-neuronal cells derived from the SCG occa-sionally had MHC-A staining that was closer to the peripheraledge than MHC-B. The apparently low levels of MHC-Asuggest that myosin II-B generates most of the myosin II-mediated force in SCG neurons, but we cannot presentlyeliminate a crucial role for myosin II-A in this process (seeWaterston, 1989). An antibody (Biomedical Technologies, Inc.)prepared against platelet myosin II (MHC-A) was used in ourprevious work (Bridgman and Dailey, 1989), and also that ofCheng et al. (1992) and Miller et al. (1992). Cheng et al. (1992)found no cross-reactivity of the anti-platelet antibody withMHC-B, but this claim conflicts with both our unpublisheddata, and the immunoprecipitation data published by Miller etal. (1992). Thus, this paper is the first to report the distributionof myosin II A in neurons using an isoform specific antibody.

Myosin II staining in the neurite was typically as bright asin the growth cone C-domain, although it often had a periodicdistribution. Myosin II may serve a purely structural role,perhaps in maintaining the cylindrical shape of the neurite.Such a role is consistent with the finding that no changes in


neurite tension are observed in the absence of growth coneadvance in vitro (Lamoureux et al., 1989).

Cell bodies stained brightly for conventional myosin.Confocal microscopy revealed that cortical myosin II stainingwas organized into linear arrays. The long, thick arrays of acto-myosin II on the ventral surface may participate in adhesion ofthe soma to the substratum, in analogy to stress fibers in non-neuronal cells. Cortical actomyosin II may have functions inaddition to stabilizing the linkage of somata to the substratum.Maintaining the spherical shape of neurons presumablyrequires considerable surface tension. In addition, somasexhibit very little protrusive activity following completion ofprocess production. Both of these functions may be subservedby cortical tension that involves myosin II activity.

Our data on the distribution of conventional myosins inneurons differ from those obtained from previous investiga-tions and provide higher resolution information on the subcel-lular localization of each of the myosin isoforms, their associ-ation with F-actin, and their relationship to protrusion andretraction of the growth cone perimeter. The localization thatwe report suggests important roles for conventional myosin ingrowth cone navigation, axon assembly, and soma cortexhomeostasis. We are presently testing these hypotheses by per-turbing myosin II function in living neurons.

This work was aided by a grant from Paralyzed Veterans ofAmerica Spinal Cord Research Foundation (to M.W.R.) and by agrant from the NIH (to P.C.B.).

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(Received 21 March 1995 - Accepted 12 September 1995)

localization of myosin ii a and b isoforms in cultured neurons

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