The specific interaction of embryonal cells with the extracellular matrix (ECM) is one of the principal forces influencing embryonal development (Hay, 1984; Trinkaus, 1984). We used a muscle satellite cell line (MM14dy) to determine the relationship between locomotory response to laminin and the expression of specific cell surface binding sites for it.

Time lapse videomicroscopic analysis was used to study the locomotory response and radioligand binding assays and cell attachment assays were used to follow the expression levels of binding sites for laminin and its subfragments E8 and El –4. We report here the novel finding that the ability of MM14dy to locomote over laminin diminishes and finally vanishes as the cells differentiate. The simultaneous drop in expression of binding sites for laminin is interpreted as being of potential significance during development and repair.

During development myoblasts repeatedly alter their interaction with their environment. Arising from stationary replicating cells in the somitic dermomyotome, they invade the peripheral mesenchyme and terminally differentiate and fuse to form static syncytial myotubes (Chevalier, 1979; Christ et al. 1983; Mayne & Sanderson, 1985). These processes of activated migration, replication and terminal differentiation are reiterated during muscle repair by a quiescent stem cell population. These ‘satellite’ cells reside within the muscle basement membrane (BM), a specialized extracellular matrix (ECM) (Vracko & Benditt, 1974; Gulati et al. 1983; Cossu et al. 1980; Bischoff, 1986).

We have been studying the effects of ECM components on the behaviour of skeletal muscle myoblasts. During development and repair the quantities and types of these components changes dramatically. In particular, we are interested in the effects induced by the BM protein laminin and the interstitial component fibronectin (Kühl et al. 1985; Goodman et al. 1987; Öcalan et al. 1988). Although both proteins promote myoblast attachment in vitro, the behaviour of myoblasts is modulated by laminin, which, in contrast to fibronectin, stimulates primary murine myoblasts and the satellite cell line MM14Dy to proliferate, locomote and differentiate into nonreplicating myotubes (Kühl et al. 1985; Foster et al. 1987; Goodman et al. 1987; Risse et al. 1987; Öcalan et al. 1988). Such myotubes no longer respond to growth factors (Okazaki & Holtzer, 1966; Linkhart et al. 1981) and have down-regulated the relevant receptors (Lim & Hauschka, 1984). After terminal differentiation, the ability of myoblasts to respond to laminin is lost.

Laminin can be dissected by proteinases into several distinct regions that can mediate cell attachment to otherwise non-adhesive substrates, in particular E8, a 35 nm length of the long arm and El –4, the three short arms (Timpl et al. 1983; Edgar et al. 1984; Aumailley et al. 1987; Goodman et al. 1987). Cellular interactions with laminin and other extracellular matrices (ECMs) can be mediated by specific binding sites at the cell surface (Aumailley et al. 1987; Hynes, 1987; Timpl & Dziadek, 1987). For laminin, radioligand binding and cell attachment studies strongly suggest that there are two distinct classes of cell binding sites for the proteolytic fragments E8 and El –4 (Aumailley et al. 1987; Goodman et al. 1987), each distinct from receptors for fibronectin (Hynes, 1987; Gehlsen et al. 1988).

We report here that the ability of MM14dy myoblast satellite cells to locomote in vitro over laminin surfaces diminishes as they differentiate. Since this loss of function is accompanied by a reduction in the number, but not the affinity, of available specific binding sites for laminin, we developed the hypothesis that myoblast locomotion might be regulated through the specific expression of such cell surface binding sites.

Cell culture of primary murine myoblasts, of the differentiation competent satellite cell line MM14dy and of the Rugli cell line, and their use in attachment and locomotion assays has been described in detail elsewhere (Kühl et al. 1985; Goodman et al. 1987; Öcalan et al. 1988). The biochemistry and biology of MM14Dy in tissue culture is very similar to that of primary murine myoblasts. They replicate and differentiate with similar kinetics to primary cells, producing characteristic myogenic markers including desmin and skeletal muscle myosin. The differentiated cells fuse to give syncytial myotubes, often containing the contractile apparatus (Hauschka et al. 1979; Linkhart et al. 1982). Thus MM14Dy provide a good model for myoblast differentiation.

Preparation of differentiated MM14dy cells

Rounded phase bright sparse MM14dy cells (sMM14; Fig. 1A proliferative, no myotubes visible (Kühl et al. 1985)) were maintained by passage when a cell density of ≈5 ×104 cells per 10cm collagen-coated dish was reached (every 2 –3 days). Intermediate density MM14dy cells (iMM14; Fig. IB) were established by similar passages of sparse MM14dy cells but the density was allowed to rise to ≈1 × I06 cells per dish. After two to three passages from this density, the cells had assumed a more flattened morphology. 5–10% of the nuclei were in multinucleate cells. Normally, for each population medium was changed daily. Dense MM14dy cells (dMM14; Fig. 1C) were produced by allowing third passage iMM14dy cells to reach a cell density of >2·5 × 106 per 10 cm plate. Intermediate density MM14dy cells due for passage were grown for a further 4–5 days, without change of medium. Occasionally fusion was promoted by addition of 5 mw-EGTA to these cultures at day 2, and removal on day 3. >40% of the nuclei were in myotubes. The viability of the cells was >95% as estimated by dye exclusion. The three cell populations are shown in Fig. 1.

Fig. 1

The appearance of MM14dy cells during differentiation. MM14dy cells were cultured (see Materials and methods) to produce sparse (A), intermediate (B) and dense (C) populations. Note the discrete rounded cells in the sparse population, the flattened morphology of the intermediate population, containing multinucleate cells (arrowhead), and the myotubes in the dense population (arrows).

Fig. 1

The appearance of MM14dy cells during differentiation. MM14dy cells were cultured (see Materials and methods) to produce sparse (A), intermediate (B) and dense (C) populations. Note the discrete rounded cells in the sparse population, the flattened morphology of the intermediate population, containing multinucleate cells (arrowhead), and the myotubes in the dense population (arrows).

Radioligand binding assays

Cells in monolayer culture were removed from the substrate and allowed to recover as for attachment assays (Goodman et al. 1987) before washing in Krebs–Ringer saline and radioligand binding using laminin, E8 and El–4 (Aumailley et al. 1987). Assays were performed on 105 cells. Cells removed from the substratum with EDTA (0·05% in PBS) behaved the same as trypsin-treated cells in binding, locomotion and adhesion assays (not shown). For each concentration of ligand (assays in triplicate), nonspecific binding was assessed in parallel by addition of 100-fold molar excess of unlabelled ligand. The nonspecific binding (10–30% of specific) was routinely subtracted from the total binding. It was always possible to saturate the (competable) binding sites. Scatchard plots of the data gave best fits that appeared to be monophasic, indicating uniform populations of binding sites in each case. Addition of 125-fold molar excess of FN did not compete for binding of either laminin, E8 or El–4 (not shown).

We assessed the specificity of the binding assays by measuring the binding of 125I-laminin, E8 and El–4 to primary murine myoblasts and Rugli cells. These cells can attach and spread both on laminin and on E8 substrates but only Rugli attaches to El–4 (Goodman et al. 1987). Both cell types bound laminin and E8 specifically and with saturable kinetics (Table 1) and Rugli but not myoblasts also bound El–4. Thus cell attachment to laminin and its subfragments correlated in general with expression of appropriate cell surface binding sites (Aumailley et al. 1987; Goodman et al. 1987).

Table 1

The binding of laminin and fragments to primary mouse myoblasts and Ru glioblastoma cells

The binding of laminin and fragments to primary mouse myoblasts and Ru glioblastoma cells
The binding of laminin and fragments to primary mouse myoblasts and Ru glioblastoma cells

Cell locomotion assays

Cells were cultured and removed from the substrates as described above for adhesion and ligand binding assays. After allowing the cells to recover, they were plated in 5 ml of MM14 medium (Hams F10 (Gibco) supplemented with 1·4mm-CaC12, 15% horse serum, 5% fetal calf serum, 1% chick embryo extract, and 100i.u. ml−1 penicillin 100 μg ml−1streptomycin) at 2×103 cells cm−2 onto 25 cm2 flasks (Falcon, 3013E; first coated with 2μg ml−1 laminin, then residual protein-binding sites were blocked with 10 mg ml−1 heat treated BSA). During cell attachment the flasks were equilibrated with CO2 (≈60 min) before sealing and transfer to an inverted microscope (Zeiss ICM35) thermostatted to 37°C and time-lapse video-microscopy. The camera (Newicon-VTE1001; VTE, Garching, W. Germany) output was recorded on a National Panasonic 8030 video recorder. A day-date generator recorded actual cell time on the film and recordings were continued for at least 12 h. In order to digitize the films, cell centres (taken as position of the nucleus) were tracked using a transparent computer–controlled digitizer tablet (Scriptel SpA series: 1212T; PCP Pfalzgraf, Hamburg) mounted against the monitor and the paths of the cells, stored as a list of x–y coordinates and times, subsequently analysed by a PASCAL computer program, RUNA (SLG & R. Merkl, unpublished). Briefly, RUNA runs down the coordinate list calculating pythagorean distances between points, if the distance is greater or equal to a preselectable gating distance the cell is considered to have moved. In this way an itinerary is built up for each cell path which is used for further analysis.

The ‘wind-rose’ display (Fisher et al. 1988) starts all the cells locomoting from the same x–y coordinate (0,0). In this way, the differences in locomotion between populations can be more readily appreciated than in absolute displays of the cell paths. The values presented represent typical cell movements averaged over 5–10 h periods.

Satellite cells and primary myoblasts proliferate and differentiate similarly in culture (Cossu et al. 1980; Lim & Hauschka, 1984; Bischoff, 1986; Foster et al. 1987; Öcalan et al. 1988). Myoblasts differentiate in response to depletion of medium-derived growth factors and in response to factors that they secrete (Zalin, 1979; Linkhart et al. 1981; Bischoff, 1986). Thus relatively poorly differentiated populations can be maintained in sparse well fed cultures while differentiated highly fused populations are achieved by deprivation of growth factors and allowing the population density to rise (Linkhart et al. 1981; Lim & Hauschka, 1981). An arbitrarily defined intermediate stage is achieved by regular feeding of cultures passaged at moderate densities (see Fig. 1 and Materials and methods). We studied three aspects of the interaction of the murine satellite cell line MM14Dy with laminin as the cells differentiated: their ability to locomote over laminin substrates, their binding of radiolabelled laminin and its fragments and their ability to attach to laminin substrates.

As MM14dy satellite cells differentiate they lose the ability to locomote over laminin substrates

In vivo, myoblasts are a motile population and become localized during development to specific regions in the embryo, where they become surrounded by a basement membrane (BM). During repair of muscle, the satellite cells once again become locomotory within the BM. We therefore studied the effect of the typical BM adhesion factor, laminin, on myoblast locomotion. We used quantitative videoanalysis to measure the velocity of myoblasts migration over laminin substrates. The paths of typical populations are shown in Fig. 2 and described in Table 2. Primary myoblasts locomoted extensively over laminin with a mean speed of ≈65μm h−1, sparse MM14dy cells locomoted almost as fast as the primary cells, at an average speed of ≈55 μm h−1. Intermediate density MM14dy cells locomoted more slowly than the primary and sparse populations, at ≈30 μm h−1, but MM14dy cells derived from a dense population were only poorly locomotory, they moved at a mean speed of only ≈5μm h−1,ie. essentially in the background of the measurement technique. We show elsewhere that although myoblasts attach with similar kinetics and affinities to FN, they are unable to significantly locomote over FN substrates (Öcalan et al. 1988; and Goodman et al. unpublished). The sparse cells dynamically and rapidly extended narrow growth-cone-like lamellipodia to become highly polarized, before rapidly detaching the cell body and rushing into the extended pod. Cells from the dense population were very flattened and expressed broad actively ruffling fibroblastlike lamella.

Table 2

The locomotion of myoblasts over laminin substrates

The locomotion of myoblasts over laminin substrates
The locomotion of myoblasts over laminin substrates
Fig. 2

Wind-rose displays of locomoting myoblasts described in Table 2. Primary mouse myoblasts (A), sparse MM14Dy (B), intermediate MM14Dy (C) and dense MM14Dy cells (D) were filmed with a 20 s time lapse, and analysed during migration over laminin substrates. The cells paths were plotted from the series of x–y coordinates. Minimum time resolution is ≈2min, space resolution ≈5μm (SLG and Merkl, unpublished). The points from which the cells began their run have been transformed so that they all appear to have started at the same x–y coordinate. The axes run from –400 μm to +400 μm with ticks at 100 μm intervals. In (A) 14 cells have run on average 6h 52 min and covered on average 478μm. For the other plots the figures were (B), 14 cells, 6h 13min, 361μm, (C) 11 cells, 8h 33min, 297μm and (D) 10 cells, 10h 12 min, 57 μm.

Fig. 2

Wind-rose displays of locomoting myoblasts described in Table 2. Primary mouse myoblasts (A), sparse MM14Dy (B), intermediate MM14Dy (C) and dense MM14Dy cells (D) were filmed with a 20 s time lapse, and analysed during migration over laminin substrates. The cells paths were plotted from the series of x–y coordinates. Minimum time resolution is ≈2min, space resolution ≈5μm (SLG and Merkl, unpublished). The points from which the cells began their run have been transformed so that they all appear to have started at the same x–y coordinate. The axes run from –400 μm to +400 μm with ticks at 100 μm intervals. In (A) 14 cells have run on average 6h 52 min and covered on average 478μm. For the other plots the figures were (B), 14 cells, 6h 13min, 361μm, (C) 11 cells, 8h 33min, 297μm and (D) 10 cells, 10h 12 min, 57 μm.

As MM14dy satellite cells differentiate laminin-specific cell surface binding sites are down-regulated

On preterminally differentiated sparse MM14dy cells the number of both laminin and laminin fragment E8 binding sites per cell was similar (±20%) and about twice the number of sites found on primary murine myoblasts (Tables 1,3). The number of sites decreased as the population differentiated. The affinity of the sites as determined by Scatchard plots remained quite constant during differentiation and corresponded to the affinity of the sites on both primary skeletal muscle myoblasts and Rugli glioblastoma. Scatchard plots of the data were linear and monotonic (Fig. 3). On sparse MM14dy cells (Fig. 3A) there were ≈35000 sites per cell, on dense MM14dy cells (Fig. 3C) ≈8500, both with affinities of 2–4 nM (Table 3). The behaviour of the binding sites for laminin fragment El–4 was more complex. Sparse MM14dy cells bound detectable amounts of El–4 molecules ≈1500 per cell, but intermediate density MM14dy cells bound significantly more, ≈5500 molecules per cell. Both dense MM14dy cells and primary myoblasts (Table 1,3) bound few El–4 molecules (i.e. <500 per cell, the limit of resolution of the assay). The binding affinities agree with previously published values for laminin, E8 and Pl (cf. El–4) binding (Terranova et al. 1983; Albini et al. 1986; Aumailley et al. 1987).

Table 3

The binding of laminin and fragments to differentiating MM14 cells

The binding of laminin and fragments to differentiating MM14 cells
The binding of laminin and fragments to differentiating MM14 cells
Fig. 3

Binding of radiolabelled laminin to sparse MM14dy, intermediate density MM14dy and dense MM14dy. The main figures shows the background-subtracted saturation curves for the binding of 125I-laminin to sparse MM14dy cells (A) intermediate density MM14dy cells (B) and dense MM14dy cells (C). Insets show the same data plotted according to Scatchard. Specific saturable binding was observed on each cell. Binding assays were performed on 105 cells as described in Materials and methods and the legend to Table 1.

Fig. 3

Binding of radiolabelled laminin to sparse MM14dy, intermediate density MM14dy and dense MM14dy. The main figures shows the background-subtracted saturation curves for the binding of 125I-laminin to sparse MM14dy cells (A) intermediate density MM14dy cells (B) and dense MM14dy cells (C). Insets show the same data plotted according to Scatchard. Specific saturable binding was observed on each cell. Binding assays were performed on 105 cells as described in Materials and methods and the legend to Table 1.

Cell attachment to laminin correlates with expression of cell surface laminin-binding sites

We wanted to determine whether the number of cell surface binding sites was related to the ability of MM14Dy to attach to laminin and its subfragments. In attachment assays to laminin and E8, sparse, intermediate and dense MM14Dy all attached. Sparse and dense cells require approximately the same coating concentrations for half-maximal adhesion, while intermediate density cells required lower concentrations. The shape of the attachment curves was different. Thus, the relationship between receptors and ability to bind the substrate was only semi-quantitative (Fig. 4).

Fig. 4

Cell attachment assays for MM14 populations. Assays were performed as previously described (Goodman et al. 1987) on sparse MM14dy cells (A) intermediate density MM14dy cells (B) and dense MM14dy cells (C). Error bars represent the standard deviation of 4 measurements. For the purposes of clarity, some error bars have been omitted; undisplayed errors were of the same order as those shown. Laminin substrates (Closed squares). E8 substrates (Crosses). El–4 (Diamonds).

Fig. 4

Cell attachment assays for MM14 populations. Assays were performed as previously described (Goodman et al. 1987) on sparse MM14dy cells (A) intermediate density MM14dy cells (B) and dense MM14dy cells (C). Error bars represent the standard deviation of 4 measurements. For the purposes of clarity, some error bars have been omitted; undisplayed errors were of the same order as those shown. Laminin substrates (Closed squares). E8 substrates (Crosses). El–4 (Diamonds).

Intermediate density MM14dy cells attached to El–4-coated substrates, while sparse, dense and primary myoblasts (Goodman et al. 1987) attached very poorly. This correlated with the change in numbers of El–4 binding sites on the cells (Table 3).

Thus, although in general cells able to bind the radioligand could attach to the ligand-coated substrates (Terranova et al. 1983; Albini et al. 1986; Aumailley et al. 1987) (Fig. 4), this correlation was not straightforward. Factors apart from receptor number alone may be involved.

The specific control of cell function by localized determinants of the extracellular matrix provides an attractive model for the regulation of embryonal development (see Hay, 1984; Trinkaus, 1984). We have considered the differentiation and locomotion of cells during skeletal muscle myogenesis as a model system. In vivo, myoblasts migrate to their sites of terminal differentiation and halt there; however, the controlling mechanisms for these processes are unknown. Recent evidence points at the involvement of the specific cell surface binding sites for the matrix components fibronectin and laminin (Horwitz et al. 1985; Hynes, 1987; Menko & Boettiger, 1987; Jaffredo et al. 1988).

It has previously been noted that myoblasts respond differently to laminin than to fibronectin (Kühl et al. 1985; Öcalan et al. 1988). This is most striking in their locomotory behaviour. In this study, we could demonstrate that both primary skeletal myoblasts and the MM14 satellite cell line are stimulated to locomote by laminin. There is a previously unreported correspondence between the ability of the cells to migrate over laminin and their expression of specific cell surface laminin-binding sites. Conditions that induced myoblast differentiation gave rise to cells that were unable to migrate over laminin substrates. Interestingly, this population had a drastically reduced number of binding sites for laminin, but the affinity of the sites remained unchanged. It is important to emphasize that these cells were not immotile;, they still produced ruffling lamellae and extended pseudopodia; but they couldn’t translocate over the substrate. Their motile machinery appeared intact, but the adapters that linked the machinery to the outside world, the cell surface binding sites for laminin were, as we show here, functionally deficient. We therefore hypothesize in the absence of counter evidence that there is a causal relationship between these observations.

We are not able formally to exclude that the high-density cultures necessary to induce the differentiation of MM14dy cells affected the number of laminin-binding sites independently of a differentiation-dependent effect on cell locomotion, however, this seems unlikely for two reasons. First, the stimulated locomotory response of myoblasts to laminin appears to be independent of their plating density, whether very high-density (‘race track’ assay) or very low-density (videomicroscopy) is used (this study; Öcalan et al. 1988). Second, at least for Rugli glioblastoma cells, the number of laminin-binding sites per cell does not significantly vary with culture density (SLG & VN, unpublished observations).

It is yet to be resolved whether in vivo myoblasts in fact utilize either laminin or fibronectin during embryonal migration or during repair, although the seminal study of Jaffredo (1988) indicates that these molecules play some role in the developmental process. It seems certain, however, that during muscle repair, satellite cells come into intimate contact with the laminin-containing BM of the necrotic fibre (Bischoff, 1986). It is thus an interesting coincidence that each of the necessary repair processes–adhesion, migration, profiferation and differentiation–are drastically accelerated by laminin and, excepting adhesion, inhibited by fibronectin. Excepting Öcalan et al. (1988), we have been unable to find any comparative reports on the motile ability in vitro of either murine or avian myoblasts on laminin and fibronectin substrates. Earlier, qualitative reports stated that avian myoblasts could move over fibronectin (e.g. Turner et al. 1983). Data presented recently in vivo (Jaffredo et al. 1988) used the anti-CSAT antibody, which, in binding to the )31 chain of integrin (Hynes, 1987; Marcantonio & Hynes, 1988), alters myoblast attachment both to laminin and to fibronectin (Horwitz et al. 1985) and possibly to other matrix components as well.

There is little quantitative data in the literature on the variation of laminin-binding sites with the differentiation state of cells. Here we show for the first time that as muscle satellite cells differentiate they down-regulate such sites. It is likely that the changes in binding site expression is greater than we report here. Due to population heterogeneity, both the fall in E8 and laminin sites (on i- and d-MM14dy cells) and the increase in El–4 sites (on iMM14dy cells) is probably understated; the intermediate density population was mixed and contained cells in sparse and dense states which have no El–4 receptors (Table 2); thus there must be a population of cells binding more El–4 than our per cell data suggest. The increase in El–4 receptors is an as yet inexplicable event. Primary myoblasts bore no El–4-binding sites and could not attach to El–4.

E8 competes directly with laminin for cell surface binding sites (D. Edgar, Martinsried, personal communication), which explains the similarities in numbers and affinities of E8 and laminin-binding sites that we observe. The simplest explanation for our observations is that a single receptor class of unknown identity binding the E8 region of laminin is being functionally removed (by inactivation or clearing) as MM14dy cells terminally differentiate, although a modification of a receptor to alter its specificity cannot be formally excluded. We will show elsewhere (Goodman et al. manuscript in preparation) that, in fact, all the locomotion-stimulatory activity of the laminin molecule resides within the E8 subfragment.

As occupancy of the binding sites for matrix molecules on the cell surface exerts a control over myoblast differentiation (Menko & Boettiger, 1987; Öcalan et al. 1988), their down-regulation as differentiation proceeds is of great interest. Furthermore, as myoblasts themselves synthesize and secrete both laminin and fibronectin (Kühl et al. 1982; Sanderson et al. 1986), the possibility of an autocrine regulation in vivo cannot be excluded.

Recent studies have suggested that fibronectin receptors are important during morphogenetic movement (Boucant et al. 1984a,b; Bronner-Fraser, 1986; Jaffredo et al. 1988). But less is known about the developmental significance of the laminin receptors. Interestingly, and supporting our hypothesis that laminin–cell interactions are important during embryonal migratory processes, an antibody directed against the laminin–heparan sulphate proteoglycan complex (INO) perturbs avian cranial neural crest cell migration in vivo (Bronner-Fraser &Lallier, 1988; see also Bronner-Fraser, 1986). Our data raise the intriguing possibility of an autoregulatory locomotion control. The down-regulation of specific cell surface binding sites for laminin occurs during myoblast differentiation and correlates with their inability to move over laminin, a major component of the basement membrane they actively produce during myogenesis. Our hypothesis is that a modulation of the numbers of laminin-specific cell surface binding sites can influence both how myoblasts move to specific embryonal sites, and how they halt and differentiate there.

We would like to thank Drs K. von der Mark and D. Edgar for support and stimulating discussion throughout this study, Dr G. Gerisch for access to videomicroscopy facilities and Rainer Merkl help with videomicroscopy and PASCAL. Drs D. Newgreen, P. Ekblom, W. Bertling and F. Watt provided invaluable comments on the manuscript. VN is C. J. Martin fellow of the National Health and Medical Research Council of Australia and the recipient of a fellowship from the Alexander von Humbolt foundation.

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