Acetylcholine receptor clusters are associated with nuclei in rat myotubes

DEVELOPMENTAL

BIOLOGY

115,35-43 (1986)

Acetylcholine Receptor Clusters Are Associated with Nuclei in Rat Myotubes JANET M. BRUNER*,’AND SHERRYBURSZTAJN’ Departments

of *Pathology

and Neurology

and Cell Biology/Program

Received April

in Neuroscience,

12, 1985; accepted in revised form

Baylor

College of Medicine,

Houston,

Texas 77030

December 16, 1985

Clustered and diffuse acetylcholine receptors are present in cultured myotubes. These clustered AChRs represent regions of myotube membrane containing high receptor density. We have studied the distribution of the AChR clusters and nuclei to determine whether there is an association in the distribution of nuclei beneath AChR clusters. AChR clusters were visualized with a-bungarotoxin conjugated to tetramethylrhodamine (aBTX-TMR) and the nuclei were stained with bisbenzimide which binds specifically to DNA. This double label procedure, and the computerized analysis of the data allowed us to determine the distribution of nuclei and AChR clusters in the same myotube. During early stages of myotube development the nuclei formed aggregates which were comprised of 4 to 10 nuclei in close apposition to one another. This association of AChR clusters with nuclear aggregates was greatest at Day 4 after plating. As the number of nuclear aggregates associated with clusters decreased the number of nuclei in the aggregates also decreased and the AChR clusters decreased in size as well as number. At all time points examined, the concentration of myotube nuclei in the cells was 3 to 12 times higher beneath areas of AChR clusters than away from clusters. Our computerized analysis shows that there is an association of the AChR clusters with the nuclear region during myotube development. 0 1986 Academic

Press, Inc.

myoblasts contain diffuse, but not clustered AChRs. In During normal development and growth of skeletal cell culture, the AChR clusters appear after cell fusion, muscle the amount of DNA and nuclear content in- at about 3 days after plating. The AChR density in a creases in proportion to the muscle mass (Enesco and cluster after exposure to brain extract is similar in Puddy, 1964; MacConnachie et ab, 1964; Moss, 1968). This number to that reported for the neuromuscular junction growth is the result of fusion of mononucleated myo- of the adult endplate (Salpeter et aZ., 1982). However, blasts into myotubes which develop into mature myofi- the adult endplates differ from the extrajunctional and bers (Kelly and Zucks, 1969; Allbrook et ah, 1971; Car- embryonic receptors in several physiological and biodasis and Cooper, 1975). A similar developmental scheme chemical properties (Schuetze et al., 1978; Sakmann and has been shown to occur in cultured muscle cells (Stock- Brenner, 1978; Braithwaite and Harris, 1979; Bevan and dale and Holtzer, 1961; Bischoff and Holtzer, 1966; Yeoh Steinbach, 1977). The mechanism by which AChR cluset al, 1978). Once the myoblasts fuse and form myotubes, ters are formed and are maintained is not known. Our the nuclei contained within the myotube are not capable recent observations show that the newly synthesized of mitosis. Thus any changes in the distribution of nuclei AChRs are inserted into the cluster at a faster rate than within the myotubes can occur by redistribution of nuclei the receptors from the diffuse region of the plasma rather than cell division. In situations where there is a membrane (Bursztajn et aZ.,1985). These larger receptor need to generate additional nuclei, such as during muscle clusters may arise by movement of diffuse AChRs in the repair, the primary source of nuclei is thought to be the plane of membrane (Axelrod et al., 1976) and/or by the satellite cells (Reznik, 1976; Allbrook, 1981; Mauro, 1979) direct insertion of AChRs into the clusters (Fischbach et al., 1984). Only the nonclustered AChRs are capable first described by Katz (1961) and Mauro (1961). of lateral diffusion and clustered AChRs remain stable Embryologically, mononucleated myoblasts fuse, for hours (Axelrod et al, 1976). The formation of large forming a syncytium or myotube which ultimately deAChR aggregates can be induced by neurons (Anderson velops into the mature myofiber. The mononucleated et ak, 1977; Frank and Fischbach, 1979), as well as other noncellular materials such as a piece of nylon thread i Current address: Department of Pathology, M. D. Anderson Hos(Jones and Vrbova, 1974) or positively charged latex pital and Tumor Institute, 6723 Bertner, Houston, Tex. 77030. ’ To whom correspondence should be addressed. beads (Peng et al, 1981). These large AChR clusters are INTRODUCTION

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0012-1606/86 $3.00 Copyright All rights

0 1986 by Academic Press, Inc. of reproduction in any form reserved.

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DEVELOPMENTAL BIOLOGY

also found on muscle cells in the absence of any exogenous factors. However, these clusters persist for a short time, and unless the muscle is innervated, they disappear within a short time after plating. Our previous studies were designed to determine how these AChR aggregates are formed and maintained. In agreement with studies of others (Fischbach and Cohen, 1973; Englander and Rubin, 1982), we noticed that many of the AChR clusters appeared to be in a close proximity to the nuclei of the myotube. In this study we have quantitated the nuclei and their relationship to the AChR clusters in cultured rat myotubes using double label fluorescent studies. The analysis of these data was carried out using a computerized image analysis system. Our results show that during early myotube development the nuclei aggregate and these nuclei are associated with the AChR clusters. Later in development the nuclei are redistributed toward the periphery of the cell and at the same time the AChR clusters appear to disperse into small fluorescent speckles which we refer to as microaggregates. The relationship between the distribution of nuclei and AChR clusters is of interest in terms of understanding how the topography of nuclei may contribute toward the cluster formation and the assembly of AChRs at the postsynaptic membrane. MATERIALS

AND

METHODS

Culture condition and medium preparation. Primary cultures were prepared from hind limbs of neonate Sprague-Dawley rats, as described by Yaffe (1969). Dissociated muscle tissue was plated in Dulbecco-Vogt modified Eagle’s medium (DME) containing 10% (vol/ vol) horse serum (Grand Island Biological Co., Grand Island, N. Y.) and 0.5% chick embryo extract (vol/vol), 200 mM glutamine, and 40 mg/ml garamycin. Cells at a density of 1 X 106/ml were plated into Falcon 35-mm petri dishes (Falcon Labware, Oxnard, Calif.) containing collagen-coated glass cover slips (Gold Seal No. 1 thickness). Cultures were maintained at 3’7°C in an atmosphere of 95% air, 5% COz. Within 24-48 hr after plating, cells attach to substratum, divide, and fuse with one another, forming long multinucleated myotubes. The culture medium was changed every 2 days. Experiments were performed on Days 3-10 after plating. Localization of AChR clusters and nuclei. a-Bungarotoxin (aBTX) was conjugated to tetramethylrhodamine (TMR) as described by Ravdin and Axelrod (1977). Labeled aBTX was separated from free TMR by chromatography on Sephadex G-25 in 0.10 mM sodium phosphate, pH 7.0. The excluded fluorescent peak was placed on carboxymethyl CM-Sephadex C-50 cation exchange column and eluted with sodium chloride in sodium phosphate buffer. Optical densities and absorbence spectra were determined on a spectrophotometer. The specificity of the aBTX-TMR (5 X lo-’ M) was determined by in-

VOLUME 115. 1986

cubating myotubes with unlabeled aBTX (5 X lo-* M for 1 hr) followed by incubation in aBTX-TMR (1 hr at 37°C in DME). Less than 10% of surface AChRs are internalized in 1 hr (Bursztajn and Fischbach, 1984). No fluorescent label was seen in the toxin-blocked cultures. Cultures incubated in (uBTX-TMR showed intensely fluorescent clusters. Cells grown on glass coverslips were fixed in 1% paraformaldehyde (Bursztajn et ah, 1984). Cells were rinsed in phosphate buffer, incubated in cold (-2O’C) acetone to permeabilize the plasma membrane, and nuclei were labeled with bisbenzimide (100 pg/ml) in phosphate-buffered saline added directly to the mounting medium before inverting the fixed coverslip cultures on glass slides for observation. At pH 7.2, bisbenzimide binds specifically to DNA, producing light blue fluorescence of the nuclei (Schmued et al, 1982). The mounting medium consisted of p-phenylenediamine (1 mg/ml) in 90% glycerol adjusted to pH 8.0 with 0.5 M carbonate-bicarbonate buffer. This modification is thought to prevent rapid fading of fluorescence during observation and photography (Johnson and Nogueira Araujo, 1981). Epi-illumination with BP546 + FT580 + LP590 filters allowed visualization of the red fluorescence of rhodamine. A broad band exciter filter (BP365/11, beam splitter FT395, barrier filter LP397) allowed visualization of the blue fluorescence of bisbenzimide in the same cells. Double exposure photographs were made using 3M Colorslide 640-T film or Kodak Tri-X film. Quantitation of receptor clusters and nuclei. Nuclear aggregates were defined as groups of four or more cell nuclei with nuclear membranes in direct contact. AChR clusters were defined as rhodamine-fluorescent patches measuring 40-900 pm’. The relationship between AChR clusters and nuclei in the same cell was quantitated using a Zeiss Videoplan Image Analysis System. A camera lucida and a digitizing tablet connected to a computer were used to trace the areas of myotubes and clusters. Point counts of nuclei were made with the same apparatus using separate channels to record myotubes with and without clusters, nuclei associated with or remote from clusters, aggregated and single nuclei. The following specific parameters were measured in multiple random fields on each slide: (a) myotube area (pm2), (b) area of each cluster in an individual myotube (pm2), (c) number of nuclear aggregates in myotube, (d) number of nuclei per nuclear aggregate, (e) number of non-aggregated or diffusely distributed nuclei per myotube. Differentiation was made between nuclear aggregates overlapping AChR clusters and those which did not. Distinction was also maintained between myotube segments which contained AChR clusters and those without clusters. Experiments were run in duplicate for each time point and a minimum of 30 myotube segments with AChR clusters were counted for each slide.

BRUNER AND BURSZTAJN

Associatim

RESULTS Distribution

of Aggregates in Myotubes

Cultured rat myoblasts divide, fuse, and form branched myotubes 2 days after plating. Myoblasts are

of Acetylcholine

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mononucleated and association of cell nuclei into groups of four or more nuclei occurs after fusion, at a time when rapid growth of the myotubes takes place. Nuclear aggregates (defined as four or more nuclei touching one another) appear by Day 3 at about the time of, or just

FIG. 1. The topographical distribution of nuclei during the development of myotubes. (A) Three days after plating of myoblasts the nuclei are centrally located in the cell and in close proximity to one another (arrow). (B) Seven days after plating the nuclei are present in the periphery of the cell (arrow) and nuclear aggregates are no longer seen. X480.

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DEVELOPMENTALBIOLOGY

preceding, the formation of small AChR clusters which we have identified with CZBTX-TMR and fluorescent microscopy. Three to four days after plating, the nuclear aggregates are centrally located and under the phase contrast microscope in permeabilized cells, the nuclear membranes appear to touch one another (Fig. 1A). The number of nuclei remains constant after this time point as cell division does not occur after cell fusion. Seven days after plating, myotubes develop definite cross striations and nuclei tend to lie peripherally, simulating the appearance of mature skeletal muscle (Fig. 1B). We have studied the development of nuclear aggregates from the myoblast stage through the time of formation of mature myotubes (myotubes containing cross striations). Nuclear aggregates appear at the time of myoblast fusion and the AChR clusters appear 1 day after fusion of myoblasts has taken place. It is also during this period that the synthesis of new AChR receptors, and total surface receptors increases (Devreotes and Fambrough, 1975; Narula and Bursztajn, 1983). The number of nuclei per square micrometer of myotube segment, both aggregated and non-aggregated, is highest (Fig. 2A) at 3 days after plating. This concentration of nuclei declines with the corresponding rapid growth of the myotube segments. Five days after plating the number of nuclei per myotube segment did not change for myotube segments containing nuclear aggregates. However, in myotube segments containing non-aggregated nuclei, the number of nuclei per square micrometer of segment increased (Figs. 2A and B). The decrease in the distribution of the aggregated nuclei appears to be due to an increase in the myotube segment area (Fig. 2B). The number of nuclei per nuclear aggregate increases up to 4 days after plating the cells (Fig. 3). After this period, the ratio of nuclei per nuclear aggregate decreases in myotubes containing AChR clusters and in those cells in which AChR clusters are absent. This decline in the number of nuclei per aggregate correlates with the increase in the number of non-aggregated or diffusely distributed nuclei. Association of AChR Clusters with Nuclear Aggregates The AChR clusters in cultured rat myotubes appear on the third day after plating, and it is at this time point that the number of clusters per square micrometer of the myotube is greatest (Fig. 4A). After the initial myotube growth the number of AChR clusters per square micrometer of segment remained relatively constant. The size of the AChR clusters increased up to 5 days after plating, reaching a maximum of 600 pm2.The AChR cluster size declined until Day ‘7 (Fig. 4B) reaching a plateau at about 300 lrn2 through Day 10 (Fig. 4B). Despite the relative stability in cluster size and concentration in those myotube segments containing AChR clusters, the number of clusters in culture declined as many

VOLUME115.1986

A Distribution Nuclei

of Aggregated During Myotube

and Non-Aggregated Development

B Changes

in Myotube During

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Area

Segment

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6 7 in Cult”,.

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FIG. 2. (A) The numberof nuclearaggregates per Frn’ of myotube segment, and the number of nuclei which showed a diffuse distribution were quantitated using computerized image analysis. The area of 262 myotube segments were measured. The bars denote f SEM. (B) After initial rapid growth, myotube segment area remains relatively constant for those containing both clustered and diffuse AChRs through Day 10 in culture. Myotube segments (270 f SEM) were counted and segment area was determined in myotubes containing and lacking labeled AChRs (TMR-(YBTX) on Days 3-10 in culture.

BRUNER

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myotubes AChR

0

myotubea AChR

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5

4 Days

in

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Association

BURSZTAJN nlth clustera

wlthout clusters

‘I

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Receptors

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clusters and on myotubes without AChR clusters using a computerized analysis system (Fig. 6). This analysis allowed us to directly correlate the distribution of nuclei in relation to AChR clusters. At all the time points examined, the concentration of myotube nuclei was 3 to 12 times higher beneath areas of AChR clusters than away from clusters (Fig. 7). This high concentration of nuclei appeared to arise from their distinct aggregation beneath AChR clusters. As the number of nuclear aggregates associated with clusters decreased, the number of nuclear aggregates not associated with clusters increased. The number of nuclei and nuclear aggregates which were associated with AChR clusters depended upon the stage of myotube development.

Cultur.

FIG. 3. The ratio of the number of nuclei to nuclear aggregates decreases with time in culture for aggregates both associated with and remote from AChR clusters. However, the decline is far more rapid for aggregates near clusters.

more myotubes did not contain any AChR clusters, but only diffusely distributed AChRs. The relationship between AChR clusters and nuclear aggregates at early stages of development (Figs. 2A and 4A) led us to examine more closely the distribution of the nuclear aggregates and the AChRs clusters during myotube development. For this purpose a double label technique was utilized and myotubes were examined from Day 3 through 10 days after plating. To visualize AChR clusters, cultures were labeled with aBTX-TMR and the nuclei were stained with bisbenzimide (pH 7.2), which binds specifically to DNA. Cells were examined under fluorescent epi-illumination, and by alternating between narrow-band green excitation and wide-band uv excitation filters, we were able to determine the distribution of nuclei and AChR clusters in the same myotube. The myotubes a few days after plating were well developed, and the distribution of AChR clusters and nuclei was readily visualized and quantitated. Figure 5 shows an example of this colocalization procedure. At 4 days after plating, the majority of AChR clusters appear to be superimposed over nuclear aggregates (Figs. 5A and B), however, at 8 days after plating, such association was no longer observed (Figs. 5C and D). At the later stages of myotube development the nuclei are no longer located beneath the clusters, but rather are present at the periphery of the myotube (Fig. 1B). At these later stages of development, many of the AChR clusters are no longer intact, but instead intensely fluorescent speckles, which we termed “microaggregates,” surround the nuclei (Figs. 5E and F). We have mapped the distribution of AChR clusters and the number of nuclei beneath clusters on the myotubes. Similar mapping was carried out at least two microscopic fields away from

DISCUSSION

In cell culture mononucleated myoblasts divide rapidly, fuse, and give rise to multinucleated myotubes. As early as the myoblast stage, diffuse AChRs are present (Smilowitz and Fischbach, 19’78),however, AChR clus-

3

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FIG. 4. Distribution and size of AChR clusters. (A) After the initial myoblast fusion and myotube growth early in development, the number of AChR clusters per pm2 of segment (in segments containing clusters) remains relatively constant. (B) AChR cluster size reaches a maximum on the fourth day in culture, then decreases. Each time point represents a mean f SEM of 60 AChR clusters.

BRUNER

AND

BURSZTAJN

Association

of Acetylcholine

Receptors

with

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41

FIG. 6. The association of nuclei with AChR clusters was quantitated using a computerized analysis system. An example of the procedure is shown in this print out. The AChR clusters are encircled by solid lines and dots represent nuclei.

FIG. 7. The concentration of nuclei associated with AChR clusters is greatly increased in early development. Computerized analysis of AChR cluster association with nuclei. Nuclei directly associated with acetylcholine receptor clusters (AChRs) and nuclei remote from clusters were counted in myotube segments. Nuclei in myotube segments which did not contain AChR clusters were also counted. The number of nuclei/pm’ of area (?SEM, bars) is plotted for 175 segments including 121 clusters.

ters are not seen on myoblasts. These appear on fused myotubes at the time when an increase in the specific activities of several enzymes and the accumulation of contractile proteins (Schubert et al., 1973; Bennett et al., 1979; Gard and Lazarides, 1980; Holtzer et al., 1984) occurs. The AChR clusters are regions of high AChR density and do not represent areas of the cell with extensive membrane folding (Vogel and Daniels, 19’76;Bursztajn and Fischbach, 1984; Bursztajn et aZ.,1985). The surface membrane staining pattern using a-bungarotoxinhorseradish peroxidase (aBTX-HRP) shows no ultrastructural distinction between the surface membrane which contains AChR clusters and the one in which AChR clusters are absent (Bursztajn et al, 1985). However, freeze fracture studies revealed a great abundance of intramembrane particles at the sites of membrane containing high density of AChRs (Yee et al., 1978; Heuser and Salpeter, 1979).

At the time of fusion the majority of nuclei are centrally located in the myofiber, and a significant number of these appear in clusters which we termed nuclear aggregates. Previous studies have indicated an association of clusters with nuclei (Fischbach and Cohen, 1973; Englander and Rubin, 1982), others have reported a high abundance of nuclei at the cell periphery (Sytkowski et aZ.,1973), and still others have found no distinctive cytological correlation (Hartzell and Fambrough, 1973). Our studies, which utilized computer analysis, show that during the early period (3-4 days) of myotube formation, many of the nuclei are associated with AChR clusters. The mechanism by which AChR clusters are formed and are maintained is not known. Lateral mobility of the diffusely distributed AChRs have been shown to contribute to the AChR cluster formation (Stya and Axelrod, 1983). Direct insertion of newly synthesized AChRs also contributes to the formation of AChR clusters

FIG. 5. AChR clusters are associated with nuclei during myotube development. A double-labeled fluorescence procedure was utilized which allowed us to determine the association of AChR clusters with nuclei. Cells were labeled with a-BTX-TMR and with bisbenzimide (100 rg/ml) which binds specifically to DNA at pH 7.2. By interchanging excitation filters, both AChR clusters and nuclei were observed on the same cell. (A) Four-day-old culture showing a large AChR cluster. (B) Nuclei in the same region of the cluster are readily revealed. Note that the nuclei are touching and overlapping one another beneath the AChR cluster. (C and D) Eight-day-old culture. Nuclear aggregates (D) are no longer found to be associated with AChR cluster (C) and the nuclei are present at the periphery of the cell. (E and F) Nine-day-old culture. Intact AChR clusters are infrequently found, but instead intensely fluorescent speckles (microaggregates) are present (E). Note that some of the nuclei (F) appear to be surrounded by the microaggregates. White lines indicate the borders of the cells. X480.

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DEVELOPMENTALBIOLOGY

(Fischbach et ak, 1984). Our observation that newly synthesized AChRs are more rapidly inserted into clusters (Bursztajn et aZ., 1985), raises the possibility that this nuclear AChR cluster topography may facilitate the accumulation of AChRs in the plasma membrane. This hypothesis is further supported by our data which shows that during early development of the myotubes a definite pattern of nuclear aggregation is observed and at the same time an increase in the number of AChR clusters occurs. This association between nuclear aggregates and AChR clusters decreases as the myotube matures, develops cross-striations and the nuclei move toward the periphery of the myotube. Later in development (10 days) the number of nuclei per aggregate decreases with a corresponding increase in the number of single nuclei. These observations suggest that the single nuclei moved from the nuclear aggregates towards the periphery of the cell. The close association of nuclei with AChR clusters early in development and continued greater concentration of nuclei beneath clusters at all stages of development, correlating with rapid increase in AChR cluster size, implies a vital role for the nucleus in the regulation of AChR synthesis and insertion into plasma membrane. In this respect it is of interest to note that Sanes and Merlie (1984) using cDNA probe encoding for AChR a subunit have found that the levels of mRNA are higher at the synapse than in the extrasynaptic region. If the AChR clusters are to be considered as a model system of the assembly of postsynaptic membrane, then we would expect that an increase in the accumulation of nuclei would result in an increase in the AChR mRNA level in these myotubes. Post-translational modification of AChR subunits occurs rapidly (Merlie et al, 1981, 1982), and the entire process of AChR synthesis to insertion into the plasma membrane takes about 3 hr (Devreotes and Fambrough, 1975; Merlie et al., 1982). The AChRs synthesized during the time when nuclear aggregates are present probably follow the path of least distance. Thus after synthesis on the rough endoplasmic reticulum they are somehow transported for glycosylation to the Golgi apparatus (Devreotes and Fambrough, 1975; Bursztajn and Fischbach, 1984). Although the route of transport of this glycoprotein from the Golgi apparatus to the plasma membrane is not known, coated vesicles are involved in this transport process (Bursztajn and Fischbach, 1984). The Golgi apparatus and coated vesicles which are associated with the Golgi cisternae lie in close proximity to the nuclei (Bursztajn, unpublished observation). This intracellular topography of the organelles involved in the processing of the AChR may allow for the direct insertion of the AChRs into the plasma membrane which leads to the observed association of nuclei with AChR clusters. The increase in the

VOLUME115, 1986

number of nuclei beneath the AChR clusters may come about due to their migration within the myotubes. It is also at the AChR clusters that the highest number of coated vesicles containing AChR are present (Bursztajn and Fischbach, 1984), suggesting that these vesicles are directly involved in the shuttle of the AChRs to and from the plasma membrane. In the developing myotube the aggregation of nuclei may allow for insertion of the coated vesicles bearing AChR either directly into the AChR clusters (Fischbach et al., 1984) or elsewhere in the plasma membrane. In the absence of muscle innervation AChR clusters do not persist and the nuclear aggregates are infrequently seen. The behavior of nuclei and AChR clusters in innervated muscle may provide further insight into the regulatory mechanism involved in the AChR expression at the postsynaptic membrane. We thank Clemond Woodard for technical assistance. This work was supported by grants from NINCDS (NS 17876) and a Research Career Development Award to S. Bursztajn. REFERENCES ALLBROOK,D. G., HAN, M. F., and HELMUTH, A. E. (1971). Population of muscle satellite cells in relation to age and mitotic activity. Pathology 3,233-243. ALLBROOK,D. G. (1981). Skeletal muscle regeneration. Muscle Nerve 4,234-245. 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 (London) 268, 731-756. AXELROD,D., RAVDIN, P., KOPPEL, D. E., SCHLESSINGER, J., WEBB, W. E., ELSON,E. L., and PODLESKI,T. R. (1976). Lateral motion of fluorescently labeled acetylcholine in membranes of developing muscle fibers. Proc N&l. Acad. Sci USA 73,4594-4598. BENNETT,G. S., FELLINI, S. A., TOYAMA,Y., and HOLTZER,H. (1979). Redistribution of intermediate filament subunits during skeletal myogenesis and maturation in vitro. J. Cell Biol. 82, 577-584. BEVAN,S., and STEINBACH,J. H. (1977). The distribution of a-bungarotoxin binding sites on mammalian skeletal muscle developing in vivo. J Physiol. 267, 195-213.

BISCHOFF,R., and HOLTZER,H. (1966). Mitosis and the process of differentiation of myogenic cells in vitro. J. Cell BioL 41,188-206. BRAITHWAITE,A. W., and HARRIS, A. J. (1979). Neural influence on acetylcholine receptor clusters in embryonic development of skeletal muscle. Nature (Lmdm) 279,549-551. BURSZTAJN,S., and FISCHBACH,G. D. (1984). Evidence that coated vesicles transport acetylcholine receptors to the surface membrane of chick myotubes. J. Cell Biol g&498-506. BURSZTAJN,S., MCMANAMAN,J., and APPEL, S. H. (1984). Organization of acetylcholine receptor clusters in cultures rat myotubes is calciumdependent. J. Cell Biol 98,507-517. BURSZTAJN,S., BERMAN,S. A., MCMANAMAN,J., and WATSON,M. L. (1985). Insertion and internalization of acetylcholine receptors at clustered and diffuse domains on cultured myotubes. J. Cell BioL 101,104-111. CARDASIS,C. A., and COOPER,G. W. (1975). An analysis of nuclei numbers in individual muscle fibers during differentiation and growth; satellite cell muscle fiber growth. J. Exp. Zool. 191, 347-358. DEVREOTES, P. N., and FAMBROUGH, D. M. (1975).Acetylcholine receptor turnover in membranes of developing muscle fibers. J. Cell Biol. 65, 335-358.

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MERLIE,J. P., ISENBERG,K. E., RUSSELL,S. D., and SANES,J. R. (1984). Denervation supersensitivity in skeletal muscle: Analysis with a cloned cDNA probe. J. Cell BioL 99,332-335. MOSS,F. P. (1968). The relationship between the dimensions of fibers

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