Activity-dependent accumulation of basal lamina by cultured rat myotubes

DEVELOPMENTAL

BIOLOGY

97,123-136

(1983)

Activity-Dependent Accumulation of Basal Lamina by Cultured Rat Myotubes JOSHUAR. SANES'ANDJOHN C.LAWRENCE,JR. Department

of Physiology

and Biophysics and Department of Pharmacology, School of Medicine, St. Louis, Missouri 63110

Received July 1, 1982; accepted in revised

fm

Washington

University

December 13, 1982

Myoblasts from 20-day rat embryos fuse and differentiate in culture to form spontaneously active myotubes. The myotubes acquire an extracellular matrix that includes a patchy basal lamina (BL) and a layer of fibrils that runs among and above the cells. Several antibodies that bind to muscle fiber basement membrane in vivo were used to study the organization of the extracellular matrix and the effect of muscle activity on the accumulation of its components. Light and electron microscopic immunohistochemical methods showed that the composition and organization of myotube BL in vitro resemble those seen in viva. Antibodies that bind to both synaptic and extrasynaptic muscle fiber BL in viva stain the entire myotube BL in vitro, while antisera that bind preferentially to synaptic BL in vivo stain small patches of myotube BL, which are usually associated with regions rich in acetylcholine receptors. The effects of activity on accumulation of BL were studied by comparing control myotubes to myotubes paralyzed with tetrodotoxin or lidocaine. Immunohistochemical and lz51-antibody binding experiments with three antisera that stain the entire BL showed that paralyzed myotubes accumulate less BL than active myotubes. The effects of activity and inactivity are reversible: new BL forms if toxin is removed from cultures and BL is lost if active myotubes are paralyzed. Thus, accumulation of BL by myotubes is dependent, at least in part, on activity. In contrast, the number of patches stained by synapse-specific BL antibodies is increased in inactive cultures. Thus, immunologically distinguishable components of BL are differentially affected by activity.

of adult muscle fibers is both structurally and functionally specialized. Skeletal muscle fibers are ensheathed by a basement As part of a study of how the basement membrane membrane, which includes a collagenous basal lamina regulates and is regulated by neuromuscular interac(BL) closely applied to the muscle cell’s plasma mem- tions, we have asked whether the formation of BL by brane and an overlying layer of collagen and other fi- cultured myotubes is influenced by activity. Many elecbrils called reticular lamina (Sanes, 1982a; Sanes et al., trical, contractile, and metabolic properties of muscle 1978). These basement membrane sheaths survive mus- are activity dependent, and it is clear that one imporcle damage in adults, and serve as a scaffold to orient tant way in which motor nerves regulate the maturathe regeneration of new myotubes (Vracko and Benditt, tion and maintenance of muscle fibers is by modulating 1972). Recent experiments have shown that the base- their levels of electrical and/or contractile activity. Thus, ment membrane also plays several roles in the for- several effects of innervation or cross-innervation can mation and function of the neuromuscular junction be mimicked by direct electrical stimulation of dener(Sames, 1982b). For example, when nerve and muscle vated muscle, and many effects of denervation can be regnerate after injury to form new synapses, the dif- mimicked by pharmacological paralysis of innervated ferentiation of both pre- and postsynaptic membranes muscle (reviewed in Harris, 1981;Lsmo, and Westgaard, is influenced by the small fraction of the BL that runs 1976; Purves, 1976). Since cultured myotubes are sponthrough the synaptic cleft between nerve and muscle taneously active, we grew them in the absence or pres(Bader, 1981; Burden et al., 1979; Sanes et al., 1978). ence of the action potential blocker, tetrodotoxin (TTX) Immunohistochemical studies have provided evidence (Catterall, 1980) to modulate their activity. This stratthat synaptic and extrasynaptic BL differ at a molecular egy has previously been used to show that accumulation level (Sanes, 1982a; Sanes and Hall, 1979):Thuq the BL of acetylcholine receptors (AChRs) (Cohen and Fischbath, 1973; Shainberg et aZ.,1976), acetylcholinesterase (Reiger et aL, 1980; Rubin et al., 1980), and myosin ’ To whom all correspondence should be addressed: Department of (Strohman et al., 1981) by muscle cells is regulated by Physiology and Biophysics, Washington University School of Mediactivity. To monitor the accumulation of extracellular cine, St. Louis, MO. 63110. INTRODUCTION

123 0012-1606/83 $3.00 Copyright All rights

0 1983 by Academic Press. Inc. of reproduction in any form reserved.

124

DEVELOPMENTAL BIOLOGY

matrix, we used a variety of antisera that bind to muscle fiber basement membrane in vivo (Sanes, 1982a; Sanes and Hall, 1979). Some of these antibodies bind to synaptic BL and thus define “synaptic antigens”; some bind to both synaptic and extrasynaptic BL, recognizing “shared antigens,” and some bind to reticular lamina. Using these antibodies for light and electron microscopic immunohistochemistry and in a lz51-antibody binding assay, we found that (a) cultured myotubes accumulate an extracellular matrix, including a BL that contains both shared and synaptic antigens; (b) less BL accumulates on paralyzed than on active myotubes; and (c) shared and synaptic antigens are differentially regulated by activity. By demonstrating that the metabolism of muscle BL is regulated by muscle activity, we provide a link between two factors-activity and BLknown to play important roles in long-term neuromuscular interactions.

VOLUME 97,1983

Antiserum to GP-2, a noncollagenous protein first isolated from cultured cells (Chung et aZ., 1979) and later shown to be a subunit of laminin (Sakashita et aZ.,1980; Timpl et al., 1979), was the gift of Dr. A. E. Chung, Department of Biological Sciences, University of Pittsburgh. Antibody to human plasma fibronectin was provided by Dr. John McDonald, Department of Medicine, Washington University Medical Center (McDonald and Kelley, 1980). Antiserum to a high salt-soluble fraction rich in collagenous peptides (HSP) was prepared in a rabbit, as detailed by Sanes (1982a). Antiserum to solubilized, whole bovine lens capsule, prepared by Sanes and Hall (1979) stains synaptic BL intensely but extrasynaptic BL little if at all. This serum is designated antidS (for junctional specific) in accordance with nomenclature recently proposed by Silberstein et al. (1982). Antiserum to a basement membrane collagen-rich fraction from rat muscle (antiMBMC) was prepared and absorbed with connective tisMETHODS sue from synapse-free portions of rat skeletal muscle as described previously (Sanes and Hall, 1979) to reveal Muscle Cultures antibodies that specifically stain synaptic BL. The abSkeletal muscle cells were dissociated from the fore- sorbed serum is anti-JS2. Anti-JSl and anti-JS2 reclimbs of 20-day embryonic rats and cultured essentially ognize different antigens (Sanes and Hall, 1979). as described previously (Lawrence and Catterall, 1981). Antiserum to a collagenous fraction from bovine lens Cells used for binding assays were grown in collagen- capsule (anti-LCC) was prepared by Sanes and Hall coated wells of plastic multiwell tissue culture dishes (1979). Like anti-MBMC, anti-LCC contains antibodies (Costar, Cambridge, Mass.; l.&cm-diameter wells) at that bind specifically to synaptic BL. To use anti-LCC 36°C in a humidified atmosphere of 5% COz and 95% and anti-MBMC as probes for antigens present in both air. For morphological studies, cells were grown on col- synaptic and extrasynaptic BL, we took advantage of lagen-coated polystyrene cover slips (Lux Scientific Co., the fact that different lots of serum from the same rabNewbury Park, Calif.). The growth medium consisted bit vary in the amount of synapse-specific antibody they of 10% horse serum, 5% newborn calf serum, 50 units/ contain. For experiments reported here, we used bleeds ml of penicillin G, and 10 pg/ml of streptomycin in of anti-LCC and anti-MBMC which could be shown by Dulbecco’s modified Eagle’s medium. Sera were incu- absorption to have low titers of anti-JSl or anti-JS2bated at 56°C for 30 min before use. Medium was re- i.e., immunofluorescent staining (see below) of synapses placed at 4-day intervals. From Day 4 to Day 8, medium by the sera was barely detectable at a dilution of 1:50 was supplemented with.10 PM cytosine arabinoside to after absorption with synapse-free skeletal muscle conlimit growth of fibroblasts (Fischbach, 1972). [3H&eucine nective tissue, while both synaptic and extrasynaptic (0.2 &i/ml; New England Nuclear, Boston, Mass.) was BL were stained well by unabsorbed sera at a dilution added to cultures used for radioligand binding assays. of 1:400. Thus, these sera detect predominantly shared TTX (Calbiochem, La Jolla, Calif.; lop5 M final concen- (synaptic and extrasynaptic) antigens. tration except where noted) or lidocaine (Sigma, St. Preimmune sera were used as controls. Louis, MO.; 1 mM final concentration) was added to some Second antibodies. Goat anti-rabbit IgG (GAR) and cultures. its fluorescein and horseradish peroxidase conjugates were obtained from Cappel Laboratories, Cochranville, Antisera Pennsylvania. ‘%I-GAR was prepared by nonenzymatic Antibodies to muscle fiber basement membrane. To iodination of GAR using chloramine-T and Na-‘%I (New study the extracellular matrix of cultured myotubes, we England Nuclear), and was separated from free lz51by used a variety of antibodies that have previously been chromatography on Sephadex G-25 (Garvey et al, 1980). shown to stain rat muscle fiber basement membrane in Histology vivo (Sanes, 1982a; Sanes and Hall, 1979). The portions Light microscopy. For light microscopy, the antibody of the basement membrane to which the antisera bind binding was detected by an indirect immunofluoresare indicated in Table 1; their sources are as follows:

SANES AND LAWRENCE

cence method. Muscle cells grown on coverslips were rinsed, incubated for 30 min in antiserum or control serum (diluted l:lOO-1:200), washed 15 min, incubated for 30 min with a mixture of fluorescein-GAR (1:50) and rhodamine-a-bungarotoxin (prepared as described by Ravdin and Axelrod, 1977), and washed 15 min. The buffer used for incubations and washes contained 130 mM NaCl, 5 mM KCl, 1.8 mM CaClz, 0.8 mM MgS04, 5 mM glucose, and 50 mM N-2-hydroxyethylpiperazineN-2-ethanesulfonic acid [Hepes], pH 7.2, plus 10 mg/ml bovine serum albumin (BSA). Cultures were then fixed for 10 min with 1% paraformaldehyde in 150 mM NaCl buffered to pH 7.2 with 20 mM Hepes, washed, rinsed in saline without BSA, mounted (Johnson and Araujo, 1981), and viewed by epi-illumination with filters selective for rhodamine or fluorescein. Electron microscopy. For conventional electron microscopy, cultures on coverslips were rinsed with saline, fixed for 1 hr in a mixture of 0~0~ and potassium ferrocyanide (Goldfischer et al., 1981), dehydrated in ethanol, rinsed in propylene oxide, and embedded with Araldite. Thin sections were stained with uranyl acetate and lead citrate. We used an immunoperoxidase technique to study antibody binding at the ultrastructural level. Cultures were incubated with antisera, washed, and incubated with peroxidase-GAR (l:lOO), and washed again as described above for immunofluorescence. Then the cultures were fixed for 0.5 hr in 1% glutaraldehyde, 110 mM NaCl, 5 mM CaClz, 30 mM Hepes, pH 7.2, washed in saline with 10 mM glycine, incubated for 0.5 hr in a mixture of p-cresol, diaminobenzidine, and HzOz (Streit and Reubi, 1977), rinsed in 0.1 M cacodylate buffer, pH 7.2, refixed in 1% 0~0~ in cacodylate, dehydrated, and embedded. Thin sections were examined without further staining. Binding

125

Activity and Muscle Basal Lamina

Assays

“‘I-Antibody binding. Antibody binding was quantitated in a two-step process. First, cultures were incubated for 30 min at 23°C with preimmune serum or antiserum, diluted 1:lOO in Krebs-Ringer phosphate buffer (KRP) containing 10 mg/ml BSA. The cells were then washed twice at 0°C with KRP-BSA (3 ml), and incubated at 0°C with sufficient lz51-GAR to bind all of the rabbit antibody present. After 2 hr, the cells were washed three times at 0°C with KRP-BSA and suspended in 0.4 NNaOH. A y counter was used to measure lz51 and 3H was determined using a liquid scintillation counter after correcting for spillover of lz51. The amount of protein in representative cultures washed in KRP without BSA was determined by the method of Lowry et al. (1951), and the amount of radioactivity from

[3H]leucine present in each culture was used to correct replicates for variations in the amount of protein recovered (Lawrence and Catterall, 1981). Specific binding of antibodies was calculated by subtracting the amount of “‘1 bound to cultures incubated with preimmune serum from the amount observed with antiserum. We were not able to determine the absolute number of antibody binding sites in our cultures because limited supplies of antisera prevented use of saturating concentrations. However, control experiments with each antiserum demonstrated that the amount of specific binding observed was directly proportional to the total amount of protein present in the cultures. This indicates that antibodies were present in excess, although at subsaturating concentrations. Therefore, for any single antiserum, changes in the amount of specific binding with age or treatment provide an index of changes in the number of exposed antigenic sites per culture. 1251-a-Bungarotoxin binding. Cells were incubated with 20 nM lz51-a-bungarotoxin (New England Nuclear) at 36°C in KRP-BSA in the absence or presence of 500 nM a-bungarotoxin. After 2 hr, cells were washed with KRPBSA, suspended in 0.4 N NaOH, and the amount of radioactivity present as lz51 and 3H was determined. The values presented for specific binding were obtained by subtracting the amount of lz51 bound in the presence of 500 nM a-bungarotoxin from that observed in the absence of unlabeled toxin. RESULTS

Cultured Myotubes Assemble a Basement Membrane Myoblasts begin to fuse 2 days after they are plated, and fusion is essentially complete by Day 5. Electron microscopy shows that the cultures assemble an elaborate extracellular matrix, including a felt-like BL and a network of extracellular fibrils that run above and among the cells in a pattern without obvious organization (Fig. la). Although patchy and incomplete, the BL of cultured myotubes is morphologically similar to the BL of adult muscle fibers, and the network of fibrils resembles in some respects the reticular lamina seen in viva (Fig. lb). To probe the composition of the extracellular matrix assembled in vitro, we used a variety of antisera previously shown to stain muscle fiber basement membrane in vivo (Table 1). Eight to fourteen days after plating, cultures were incubated with antiserum or preimmune serum, then stained with a mixture of fluorescein-GAR and rhodamine-a-bungarotoxin, and finally examined with fluorescent illumination, using fluorescein- and rhodamine-selective filters. cY-Bungarotoxin binds specifically and with high affinity to AChRs; thus rhodamine-bungarotoxin can be used to reveal

126

DEVELOPMENTAL BIOLOGY

VOLUME 97, 1988

aptic BL in vivo, and recognize shared antigens, stained large areas of the surface of cultured myotubes (Fig. 2a). Staining by anti-GP-2 and anti-LCC was largely confined to the myotube surface, while anti-MBMC also stained the fibrillar network that runs between and above the cells. Second, two antisera-anti-JSl and antiJSB-that bind to synaptic but not to extrasynaptic BL in vivo, and thus recognize synaptic antigens, stained small (about lo-50 pm long), discrete patches on the myotube surface (Figs. 2b and c). Most (over SOW) of these patches coincided with regions of high AChR density, as revealed by double staining with rhodamine-abungarotoxin (Fig. 2d), and most AChR-rich regions were stained by antibodies to synaptic BL. Finally, two antisera-anti-fibronectin and anti-HSP-that bind in large part to reticular lamina in vivo, stained the extracellular, fibrillar network in the cultures, but did not detectably stain the myotube surface (Figs. 2e, f). Thus, molecules antigenically related to a variety of components of muscle fiber basement membrane are present in vitro, and the staining patterns seen in vitro reflect the distribution of the various antigens in vivo. Electron microscopy of immunoperoxidase-stained FIG. 1. Cultured myotubes accumulate a basement membrane. (a) cultures showed that antisera to shared (Fig. 3a) and Electron micrograph of a cross-sectioned myotube from an 11-day synaptic (Fig. 3b) antigens stained the myotube BL. The control culture fixed with OsOl and ferrocyanide. For comparison, b patterns of staining seen in the electron microscope reshows a similarly fixed, cross-sectioned fiber from an adult rat insembled those seen by immunofluorescence: antisera to tercostal muscle. Arrowheads point to BL. Bar is 0.5 pm. shared antigens stained the entire myotube BL, while antiserum to the synaptic antigen, anti-JSl, stained AChR-rich patches of membrane (e.g., Anderson et aZ., some patches of BL intensely, while leaving other segments of BL unstained. The electron microscopic dem1977; Sanes and Hall, 1979). onstration that these antisera selectively and reliably Antisera stained cultures in one of three general patstain BL justified their use in studies of the effect of terns. First, three antisera-anti-GP-2, anti-LCC, and activity on accumulation of BL. anti-MBMC-that bind to both synaptic and extrasynTABLE 1 ANTISERA THAT STAIN MUSCLE FIBER BASEMENT MEMBRANES Staining Synaptic sites

Immunogen

Name of antiserum

of muscle fiber basement membrane Extrasynaptic regions

Basal lamina

Reticular lamina

+

+

+

+

Bovine lens capsule collagen

+

+

+

-

Anti-GP-2

GP-2, a subunit

+

+

+

-

Anti-JSl

Solubilized

+

-

+

-

Anti-JS2

MBMC, serum absorbed*

+

-

+

-

Anti-fibronectin Anti-HSP

Human plasma fibronectin High salt soluble collagenous muscle (HSP)

+ -

+ +

+ ?

+ +

Anti-MBMC

Rat muscle basement

Anti-LCC

membrane

collagen

of laminin

whole bovine lens capsule

proteins

from

” Results summarized from Sanes (1982a), Sanes and Hall (1979), and unpublished electron microscopic studies of anti-LCC and anti-MBMC. b Absorbed as described under Methods. Sera from different pools were used for anti-MBMC and for anti-JS2; see Methods.

FIG. 2. Antibodies to muscle fiber basement membrane stain cultured rat myotubes. (a) Myotube from an &day culture that was incubated with anti-MBMC and fluorescein-GAR and photographed with fluorescein optics. Most of the myotube surface is stained, as are fibrils that run between cells. This antiserum stains both synaptic and extrasynaptic BL in viwo. (b-d) Myotube from an 11-day culture that was incubated with antiJS1, fluorescein-GAR, and rhodamine-bungarotoxin, and photographed with Nomarski (b), fluorescein (c), and rhodamine (d) optics. The fluorescein antibody-stained patch occurs at a region of high AChR density. This antiserum stains synaptic but not extrasynaptic BL in viva. (e,f) Myotube from an 11-day culture incubated with anti-HSP and fluorescein-GAR and photographed with fluorescein (e) and phase optics (f). Extracellular fibrils are stained, but the myotube surface is not. Anti-HSP stains reticular lamina in extrasynaptic regions in viva. Bar indicates 50 pm in a-d, and 150 pm in e andf.

128

DEVELOPMENTALBIOLOGY

FIG. 3. Antisera that stain muscle fiber BL in viva stain myotube BL in vitro. Electron micrographs of cross-sectioned myotubes from 11-day cultures. Cultures were incubated with anti-LCC (a), anti-J241 (b), or preimmune serum (c), and peroxidase-GAR, then fixed, stained for peroxidase, and refixed with osmium. Sections were not stained further. The stained patch in b was identified en bloc in the light microscope+ then mounted and sectioned for electron microscopy. An invagination that resembles a junctional fold is covered by and filled with stained BL. Such invaginations were rarely seen in unstained areas. a and c are from control, b from a TTX-treated culture. Bar is 0.5 pm.

Accumulation

of3L

Is Inhibited

by Paralysis

Some myotubes in our cultures twitched occasionally as early as Day 4, and the vast majority of myotubes contracted vigorously and spontaneously by Day 7. To determine if the accumulation of BL depends on activity, we compared spontaneously active myotubes to TTXparalyzed myotubes. The concentration of TTX we used, 10 PM, was sufficient to block all visible contractile activity (Fig. 5b) and to reduce batrachotoxin-stimulated =Na+ influx b y over 75% (J. C. Lawrence, unpublished data). To monitor the accumulation of BL, cultures were

VOLUME9'7,1983

incubated with one of the three antisera that stained the full extent of the myotube’s BL-anti-GP-2, antiLCC, or anti-MBMC-and then with GAR conjugated to fluorescein for light microscopy, to peroxidase for electron microscopy, or to lz51 for binding assay. For each of the three antisera, all three methods showed that the accumulation of BL by cultured myotubes is activity dependent. The effect of activity was seen most dramatically by the indirect immunofluorescence method. Figure 4 compares myotubes from active and TTX-treated 11-day cultures stained with anti-GPZ and fluorescein-GAR. The antibody stained most of the surface of the control myotube (Figs. 4a and b), but only a few patches on the surface of the paralyzed myotube (Figs. 4c and d). Double-labeling with rhodamine-bungarotoxin showed that some of the antibody-stained patches on paralyzed myotubes were coincident with clusters of AChRs (Figs. 4e and f). Similarly, staining of the myotube surface by anti-LCC and by anti-MBMC was markedly reduced in paralyzed compared to control cultures, and stained patches on paralyzed myotubes often coincided with AChR-rich regions. Thus, activity stimulates (or paralysis inhibits) the accumulation of BL by cultured myotubes. The effect of paralysis on the fraction of the myotube surface covered by BL was confirmed and quantified by electron microscopy. Active and paralyzed cultures were sectioned perpendicular to the plane of the cover slip, randomly selected myotubes were photographed (5-15 from each of 3 blocs per culture), and a computerized planimetry system (Cowan and Wann, 1973) was used to measure the fraction of the perimeter covered by BL. Usually, BL was stained with antiserum and peroxidase-GAR, since conventionally stained BL was sometimes faint and difficult to measure. In one experiment with anti-LCC, an average of 42.6 + 3.3% (+_SEM, n = 40) of the surface of control myotubes was covered by BL, while only 16.3 + 3.5% (n = 25) of the surface of paralyzed myotubes was similarly stained (P < 0.001 by Student’s

tures well ically less than

t test).

Similar

results

stained with anti-GP-2 as from

cultures

that

were

obtained

from

cul-

(Fig. 9) or anti-MBMC were

as

not immunocytochem-

stained. In general, the effect of activity seemed striking when assessed electron microscopically when estimated by immunofluorescence. This dif-

ference

may

reflect

the ability

of electron

microscopy

to reveal small uncoated areas on cells rich in BL and small patches of BL on cells poor in BL. In any event, light and electron microscopic results both clearly demonstrate that the accumulation of BL is suppressed in paralyzed myotubes. Knowing that anti-GP-2, anti-LCC, and anti-MBMC bind in large part to myotube BL, we were able to study

SANES AND LAWRENCE

Activity

and Muscle

Basal

Lam&a

129

FIG. 4. Accumulation of BL by cultured myotubes is activity dependent. H-day control (a,b) and TTX-paralyzed (c-f) cultures were stained with anti-GP-2, fluorescein-GAR, and rhodamine-bungarotoxin, and photographed with Ruorescein (a, c, e), phase (b,d), or rhodamine (f) optics. Anti-GP-2 binds to a large fraction of the surface of spontaneously active control myotubes, while only a few patches are stained on chronically paralyzed myotubes. Arrowhead in c marks region shown at higher magnification in e and f. This paralysis-resistant antibodystained patch (e) coincides with an AChR-rich area (f). Bar indicates 50 Frn for a-d, 20 pm for e and f.

accumulation of BL further by measuring the binding of “‘1-GAR to cultures that had been incubated with one of these antisera. Results presented in Fig. 5 and Table 2 confirm that accumulation of BL is depressed by TTX, and also provide three lines of evidence that the effects of TTX are indeed attributable to inhibition of spontaneous activity. First, the dependence of BL accumulation on the concentration of TTX (Fig. 5a) par-

alleled the concentration dependence of paralysis by TTX (Fig. 5b). Second, TTX caused an increase in the number of AChRs (lz51-bungarotoxin binding sites) over the same range of concentrations that led to a decrease in antibody binding (Fig. 5a). The dependence of AChR level on activity has been well documented in cultured myotubes (Cohen and Fischbach, 1973; Shainberg et al., 1976) as well as in viva (Lramo and Westgaard, 1976).

130

DEVELOPMENTALBIOLOGY

VOLUME 97, 1983

a 2400 -

” 0 L/

’ 0

1

I

I

,i

I

300 0

10-S

10-l

Tetrodotoxin ‘?iy

I

0

1

10-7 Tetrodotoxin

1

10-6

I

I

10-s

(Ml

FIG. 5. (a) TTX-treated muscle cells accumulate fewer anti-LCC binding sites but more AChRs than controls. Skeletal muscle cells were cultured for 2 weeks, with varying concentrations of TTX present in the growth medium for the last 6 days. Cells were then incubated with ‘2SI-bungarotoxin to estimate numbers of AChRs or with anti-LCC and ‘%I-GAR to estimate antibody binding sites. Values have been corrected for nonspecific binding as described under Methods; bars show SE of 4 cultures per point. (b) Dose-dependent inhibition of spontaneous contractile activity by TTX. For each concentration of TTX, a field of view was randomly chosen in each of 12 cultures, and the percentage of myotubes that contracted within 30 set was determined. Data in a and b are from different experiments.

Finally, a second paralytic agent, the local anesthetic lidocaine, had effects indistinguishable from those of TTX on accumulation of BL and AChRs (Table 2). Thus, binding assays, like light and electron microscopic immunohistochemical methods, show that accumulation of BL by myotubes is regulated by electrical activity. Eflects of TTX on BL Are Age Dependent and Reversible Results presented thus far were obtained from 8- to 14-day cultures in which control myotubes were freTABLE 2 LIDOCAINEMIMICSTHE EFFECTSOFTTX ONANTIBODY AND (Y-‘~ I-BUNGAROTOXINBINDING Percentage of control binding 10 pM Tetrodotoxin Anti-MBMC and ‘%I-GAR Anti-LCC and lz51-GAR Anti-JSl and “‘I-GAR ‘%I-Bungarotoxin

1 mM Lidocaine

5

71*

3

51 + 10 94 zk 11

58+ 94+

5 8

60+

256 +

8

297 + 19

Note. Cells were cultured for a total of 13 days. Growth medium was supplemented with 10e5M TTX or 1 mM lidocaine during the last 5 days. Cultures were then incubated with antiserum and ‘%IGAR or with r&I-bungarotoxin and values for specific binding of ‘%I were determined as desribed under Methods. The results are presented as percentages of binding to cells which were cultured without TTX or lidocaine.

quently striated and almost invariably active. When we examined younger cultures, we found that myotubes accumulated a considerable amount of BL during their first week in culture, and that this accumulation was not detectably influenced by TTX. Figure 6 shows the binding of anti-LCC to control and TTX-treated cultures of various ages. Antibody binding in control cultures (expressed per milligram of protein) increased for 8-11 days before reaching a plateau that was maintained for at least 1 l/2 weeks more. TTX had no detectable effect on the accumulation of anti-LCC binding sites for the first week, thereafter binding decreased for a week to reach a low plateau that was then maintained. Qualitatively similar results were obtained using antiMBMC. The loss of antibody-binding sites from paralyzed cultures during the second week does not represent a general toxic effect of TTX: total protein per culture and incorporation of [3H]leucine into protein were similar for control and TTX-treated cultures at each time point, and the number of AChRs (measured with ‘251-bungarotoxin) increased during the second week in paralyzed cultures, but dropped in control cultures (not shown). Studies with the immunofluorescence method, using anti-GP2, anti-LCC, and anti-MBMC, were consistent with results obtained by ‘%I-antibody binding. Some portions of the myotube surface were stained on Day 4; interestingly, AChR-rich patches were often stained by these antisera on myotubes that were otherwise

SANES

1000 o$

’

02466

’

’

’

AND

LAWRENCE

1

I

I

II

14

21

Activity

Days in culture 6. The effect of TTX on binding of anti-LCC depends upon the age of cultured muscle cells. Cells were grown in culture for periods of up to 3 weeks. The growth medium of some cultures was supplemented with 1O-5M TXX from Day 0 (triangles) while other cells were first exposed to TTX after 8 days in culture (squares). Control cells (circles) received no TTX. At the times indicated, cells were incubated with anti-LCC or normal serum prior to incubation with ‘%I-GAR. Specific binding was determined as detailed under Methods. Bars indicate SE of four cultures per point. FIG.

poorly stained. Many myotubes were evenly stained on Day 6 (Figs. 7a and b). No differences between control and TTX-treated myotubes were apparent on Day 4 or Day 6. Both control and TTX-treated myotubes were more intensely stained on Day 8 than on Day 6, but in some sets of cultures (four of six experiments) control myotubes were stained marginally more intensely than paralyzed myotubes (Fig. 8). By Day 11, the effect of TTX was pronounced: control myotubes were intensely and paralyzed myotubes poorly stained (Figs. 7c and d; and see above). Thus, the initial accumulation of components of BL does not appear to require activity. After about 1 week in culture, however, accumulation of BL becomes activity dependent; this is roughly the time when the myotubes become spontaneously active. The reversibility of the effects of activity and inactivity was studied by adding TTX to active myotubes or removing it from paralyzed myotubes during the second week of culture. Active myotubes were paralyzed in less than a minute when TTX was added, and paralyzed myotubes contracted spontaneously within a few hours after TTX-containing medium was rinsed off and replaced with control medium. Whether accumulation of BL was assayed by lZ51-antibody binding (Fig. 6), by immunofluorescence (Fig. 7), or by electron microscopy (Fig. 9), similar results were obtained: New BL ap-

and Mu.wle

Basal

Lamina

131

peared when paralyzed cultures were freed of toxin, and BL was lost when active cultures were paralyzed. These results were obtained whether TTX was added or removed on Day 8 (Figs. 6 and 9), when both active and paralyzed myotubes bear comparable amounts of BL, or on Day 11 (Fig. 7), when the effect of paralysis is already marked. Effects were seen within 3 days of adding or removing TTX, the shortest interval tested. In one experiment, TTX was added to half of a group of 50-day cultures; 5 days later, the paralyzed myotubes had far less BL than active myotubes, as assessed by immunofluorescence. The fact that BL is lost when TTX is added late shows that accumulation of BL is a dynamic process that represents a balance between the synthesis (or binding) and the degradation (or release) of its components.

Synaptic BL Antigens Persist m Paralyzed Myotubes Two antisera-antiJS1 and anti-JS2-that selectively stain synaptic BL in wivo stain small patches on cultured myotubes that often coincide with AChR-rich regions (Fig. 2). The number of AChR-rich clusters on cultured myotubes (as well as the total number of AChRs) is increased by paralysis (Cohen and Fischbach, 1973). We therefore asked whether the number of antiJSl- and anti-JS2-stained patches would be greater in paralyzed than in control myotubes, or whether synaptic, like shared BL antigens would be suppressed by paralysis. As shown in Fig. 10, anti-JSl and fluoresceinGAR stained severalfold more patches on TTX- or lidocaine-paralyzed myotubes than on control myotubes. Similar results have been obtained for anti-JS2 (not shown). Furthermore, AChR- and antigen-rich patches are, on average, somewhat larger on paralyzed than on active myotubes. The number of patches stained by antibody and by rhodamine-bungarotoxin increased roughly in parallel in paralyzed myotubes, and the correspondence between them changed little if at all. Thus synaptic antigen-rich patches of BL increase in size and number under conditions that suppress the accumulation of total BL. We have so far been unable to detect effects of paralysis on binding of synapse-specific antibodies using ‘%I-antibody (Table 2). It is possible that paralysis causes a redistribution of antigen, such that new clusters are formed at the expense of a diffuse, low-density pool. It is also possible, however, that the lz51-binding assay is not yet sensitive or specific enough to detect the small change in binding (a difference of a few percent of the myotube surface, at most, between control and paralyzed cultures) that occurs. Experiments to distinguish among these and other possibilities are in progress. However, regardless of the outcome of this work, it is clear that the two synaptic BL antigens we have studied

FIG. 7. Effects of activity on accumulation of BL appear late and are reversible. Myotubes cultured with or without TTX were stained with anti-MBMC and fluorescein-GAR, and photographed with fluorescein (a-f) and phase (a’-f’) optics. Myotubes from g-day cultures are moderately well stained, whether they are grown under control conditions (a) or in the continuous presence of TTX (b). By II days, spontaneously active myotubes in control cultures (e) are stained far more intensely than paralyzed myotubes in TTX-treated cultures (d). Addition of TTX to control cultures on Day 11 leads to substantial loss of antigens stained by anti-MBMC by Day 14 (e), while removal of TTX from paralyzed cultures on Day 11 leads to marked increase in staining by anti-MBMC by Day 14 (f). Bar is 56 pm.

of BL rich in these antigens) differ from the shared antigens in their dependence on activity. (or patches

DISCUSSION

The Form&ion of Extracellular Cultured Myotubes

Matrix by

The extracellular matrix assembled by cultured myotubes displays considerable order. Each of three anti-

sera that stain synaptic and extrasynaptic BL in viva, and recognize “shared” antigens, stain essentially all myotube BL in vitro. Two antisera that recognize “synaptic” antigens in vivo bind to small patches of BL on cultured myotubes, many of which are coincident with AChR-rich regions. Silberstein et al. (1982) have also recently shown that synaptic BL antigens accumulate in small AChR-associated patches on muscle cells in

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FIG. 8. An intermediate stage in the loss of basement membrane antigen from the surface of paralyzed myotubes. An &day culture grown in the continuous presence of ‘ITX was stained with anti-MBMC and fluorescein-GAR, and photographed with fluorescein optics. Numerous small patches on the surface are stained; compare with the more extensive staining of the myotube from a sibling control culture, shown in Fig. 2a. Paralyzed myotubes from 6- and U-day cultures are shown in Figs. 7b and d. Bar is 20 pm.

aneural cultures. Two antisera that bind largely to reticular lamina in viva, bind to extracellular fibrils in the culture, but not to BL. Similarly, extracellular fibrils but not BL are stained by anti-fibronectin in human myotubes (Walsh et al., 1981) and by anti-collagen V in chick myotubes (Sasse et aZ., 1981). Anti-MBMC, which intensely stains both BL and reticular lamina in viva, stains both in vitro as well. One anomaly is that anti-fibronectin binds to BL as well as to reticular lamina in vivo, but does not stain BL in culture. In general, however, our results and those of others show that the organization of extracellular matrix in vitro mirrors that observed in vivo to a considerable extent. Myotubes in control cultures accumulate BL over a large fraction-up to one-half-of their surface. In contrast, several previous ultrastructural studies have reported that BL is sparse on myotubes cultured from frog, rat, and chick (Betz, 1976; Burrage and Lentz, 1981; Fischbach et ab, 19’74;Lipton, 1977; Vogel and Daniels, 1976; Weldon and Cohen, 1979). In several instances, areas that bear BL have been found to have a thickened plasma membrane and/or to be rich in AChRs (Burrage and Lentz, 1981; Vogel and Daniels, 1976); in nervemuscle cocultures, BL and AChRs are both preferentially associated with regions of nerve-muscle contact (Frank and Fischbach, 1979; Nakajima et aL, 1980; Weldon and Cohen, 1979). In a recent abstract, Daniels et al. (1981) reported that laminin and other basement membrane components codistribute with AChRs on cultured myotubes. Such components are present throughout muscle fiber BL in vivo (Sanes, 1982a), and we find them to be present on a large fraction of the surface of mature, control myotubes in vitro (Figs. 2, 4, and 7). These components are, however, preferentially associated with AChR-rich patches in immature (4-6 days in culture) or paralyzed myotubes (Figs. 4e, f). Thus, the restricted distribution of BL reported previously

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Lamina

may be due in part to the use of relatively immature or inactive myotubes. On the other hand, we do find that synaptic BL antigens are preferentially associated with AChR-rich regions. Thus, in vitro as in vivo, these or similarly distributed antigens are more likely than BL in general to be involved in interactions with AChRs or other components of the postsynaptic membrane. We have measured accumulation of components of BL by cultured myotubes, but have not yet studied their synthesis or sources. The presence of patches rich in synaptic BL antigens and the increase in the number of such patches upon paralysis demonstrate that the presence of nerves is not required for their assembly (see also Silberstein et aL, 1982). However, the cultures we used are made from 20-day embryos, in which muscles have already been innervated. Therefore, it remains

35

30

t

r 5

0 :

-

-

t

-B

CONTROL CONTROL -TTX

TTX

TTXCONTROL

FIG. 9. Electron microscopy shows the dependence of BL accumulation on activity. Four sibling U-day cultures were incubated with anti-GP-2 and peroxidase-GAR, fixed, stained for peroxidase, embedded, and cross-sectioned. The fraction of the myotube surface covered by peroxidase-stained BL (virtually equivalent to total BL) was calculated from measurements of electron micrographs of randomly selected myotubes. Spontaneously active myotubes in the control culture were more fully covered with basement membrane than paralyzed myotubes grown in the continuous presence of TTX. In cultures where TTX was added (CONTROL - TTX) or removed (TTX - CONTROL) on Day 8, myotubes bore intermediate amounts of BL. Each bar represents mean f standard error of measurements from 15 to 30 myotubes. Differences between control and TTX, control and control - TTX, and TTX and TTX - control are significant at P < 0.001, < 0.05, and < 0.001 respectively, by Student’s t test.

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The Eflect of Activity

J

FIG. 10. Anti-JSl, a synapse-specific antiserum in viva, stains more patches on paralyzed than on spontaneously active myotubes. ll-day cultures, grown in standard medium (CONTROL) or in the presence of TTX or lidocaine, were stained with anti-JSl and fluorescein-GAR and viewed with a 40X objective and 10X eyepieces, using phase and fluorescein optics. The number of myotubes that crossed each of 25 fields per culture and the number of fluorescein-stained patches per field were counted. Each bar represents the mean number of patches per myotube segment f standard error in 25 fields from a single culture. Control and TTX-treated myotubes were studied in two separate experiments, while lidocaine-treated myotubes were studied in a single experiment. The mean number of myotubes that crossed each field was 17.8, 14.0, and 14.0 for CONTROL, TTX, and LIDOCAINE cultures, respectively.

possible that some influence of the nerve is required for accumulation of these molecules. Since fibroblasts are present in our cultures, at least initially, they may also play a role in the formation of the extracellular matrix. Lipton (1977) and Fischbach et al. (1974) have reported that chick myotubes form a BL when they are grown with fibroblasts, but not when they are cultured alone. In our cultures, fibroblasts might well be important in the early, activity-independent phase of BL accumulation that occurs before the myotubes become spontaneously active. On the other hand, myotubes that have been paralyzed for the first 2 weeks of culture can form BL if TTX is removed. Few fibroblasts are present at this time, since the cultures have already been treated with the antimitotic cytosine arabinoside. One possibility is that fibroblasts and active myotubes are alternative sources of some factors important for the assembly of BL. Finally, it is important to note that we have no evidence that components of BL are synthesized in the cultures; some might be provided by sera present in the growth medium, and assembled by the myotubes (see Hayman and Rouslahti, 1979; Ristelli et aZ., 1981).

on Accumulation

of BL

In order to study the effect of activity on accumulation of BL, we compared control cultures, in which myotubes twitched spontaneously, with paralyzed myotubes grown in the presence of TTX. We chose TTX because its only known direct effect is to block the voltage-sensitive sodium channels that generate action potentials (Catterall, 1980). Several observations show that TTX is not generally toxic to the cultured myotubes: Myotubes survive for at least 2 weeks in the presence of l10 PM TTX; control and TTX-treated cultures differ little in their total protein content or in the amount of rH]leucine they incorporate into protein; levels of AChRs are increased by TTX; synaptic and shared BL antigens are differentially affected by TTX; and effects of TTX on accumulation of BL are reversible. Additional results provide evidence that TTX affects BL by blocking spontaneous activity: First, lidocaine, a local anesthetic and effective paralytic agent, mimics the effects of TTX. Second, TTX inhibits accumulation of BL over the same range of concentrations that it blocks contractile activity and enhances accumulation of AChRs. Third, no effect of TTX on accumulation of BL (or AChRs) is seen until myotubes become spontaneously active. Thus, although we cannot rigorously exclude alternative mechanisms of action, we are reasonably confident that the effects of TTX on accumulation of BL are attributable to its paralytic action. The observation that less BL accumulates on paralyzed than on active myotubes places accumulation of BL in a category with several other aspects of myotube metabolism that have been shown to be activity dependent. For example, active muscles accumulate more acetylcholinesterase, fewer AChRs, and different types of contractile proteins than inactive muscles (references in Introduction). However, the way in which activity regulates BL accumulation remains to be determined. Activity might increase the rate of BL synthesis, decrease the rate of BL degradation, or alter the myotube surface in a way that enhances its ability to bind or polymerize components of BL. Precedents for the first two possibilities exist: activity decreases the rate of AChR synthesis without changing its degradation rate appreciably (Reiness and Hall, 1977), and decreases the rate of myosin degradation (Strohman et aZ., 1981). It also remains to be determined why small patches of BL that are enriched in synaptic antigens (Figs. 2, 3, and 9), also contain shared antigens (Fig. 4), and are preferentially associated with AChR’s (Figs. 2 and 4) are largely immune to the effects of paralysis and actually increase in size and number when myotubes are paralyzed. Perhaps the major questions raised by these results

SANES AND LAWRENCE

Activity

are whether activity regulates the accumulation of BL in Go, and what role(s) such regulation could play in neuromuscular development. As to the first question, it is clear that mature muscle fibers differ from cultured myotubes in that their BL survives prolonged denervation and consequent paralysis. However, it is possible that subtle changes in BL follow denervation, or that the fibrillatory activity that occurs in denervated muscle (Purves, 1976) is sufficient to prevent loss of BL. Alternatively, the initial formation of BL, but not its subsequent maintenance, might be activity dependent in viva. Experiments to test these possibilities are in progress. The differential regulation of synaptic and shared BL antigens by activity resembles in some respects the activity dependence of other membrane properties. When axons reinnervate denervated muscle, they preferentially contact muscle fibers at original synaptic sites, and their differentiation into nerve terminals at these sites occurs in both active and inactive muscle (Bixby and Van Essen, 1979; Frank et al., 1975). In contrast, extrasynaptic regions of the muscle fiber surface are refractory to innervation, and this refractiveness is maintained by muscle activity: axons will form synapses on extrasynaptic membrane in innervated, paralyzed muscle, but not in denervated, electrically stimulated muscle (Frank et al, 1975; Jansen et al, 1973). Similarly, the clustering of AChRs at synaptic sites is activity independent, while muscle activity suppresses the appearance of AChRs on extrasynaptic membrane (Anderson et al., 1977; Lomo and Westgaard, 1976). BL might be regulated in tandem with AChRs and susceptibility to innervation, with nonsynaptic components more and synaptic components less dependent on muscle activity for their accumulation. However, recent studies have shown that formation of nerve terminals and clustering of AChRs at synaptic sites is regulated by components of BL (Burden et ah, 1979; Sanes et al, 1978). The finding that activity modulates accumulation of BL raises the possibility that some activity-dependent properties of extrasynaptic membrane might also be regulated by components of BL. We thank Jeanette Cheney for exceptionally skillful technical assistance (including taking the micrographs in Fig. 3), Albert Chung and John McDonald for generous gifts of antisera, A. Chiu, D. Feldman, and D. Purves for helpful comments, and T. Woolsey for help with the computerized planimetry system. This work was supported by grants from NSF (to J.S.), NIH (to J.L.), and the Muscular Dystrophy Association (to J.L and J.S.). J. Sanes is a Sloan Fellow and an Established Investigator of the American Heart Association. REFERENCES ANDERSON, M. J., COHEN, M. W., and ZORYCHTA, E. (1977). Effects of innervation on the distribution of acetylcholine receptors on cultured muscle cells. J. Physiol. 268, 731-756.

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