Changes in cell surface antigens during in vitro lizard myogenesis

DEVELOPMENTAL

BIOLOGY

97,313-328

(1983)

Changes in Cell Surface Antigens during in Vitro Lizard Myogenesis MICHAEL Department

F. MARUSICH’

AND SIDNEY

of Biological Sciences, Northwestem

B. SIMPSON,

JR.

Univemity, Evanston, Illinois 60201

Received July 26, 1981; accepted in revised form December 80, 1962 Indirect immunofluorescence has been used to examine surface antigens of lizard myogenic cells during in vitro differentiation. At least two developmental stage-specific surface alterations have been identified. One of these is a compositional change and involves the appearance of a cell-surface antigen(s) as the cells differentiate. This antigen(s) (Ag1422) is muscle specific and is characteristic of some rounded-up Go myosin-positive myocytes, all stretched-back, G,, myosin-positive myocytes, and all identifiable myotubes. The antigen is not found on proliferating myoblasts, extended Cl1(myosin-negative) cell-cycle-competent myoblasts or newly differentiated rounded-up, Go myosin-positive myocytes. Pretreatment of cells with neuraminidase, trypsin, or proteinase K indicates the antigen is not present in “masked” form on normally nonreactive cells. Proteinase K is effective in the removal or destruction of the antigen, indicating it is at least partially protein in nature. The antigen is expressed in a similar developmental stage-specific fashion on early-passage myogenic cells taken from both adult lizard tail regenerates and embryonic muscle. The antibodies identifying Ag1422 can be removed by adsorption with homogenates of mature skeletal muscle. Therefore, Ag1422 is not an artifact due to in vitro conditions or the expression of a transformation antigen unique to the continuous culture line. The second alteration is an apparent restriction in the mobility of surface components (antigens and lectin receptors). Upon treatment with multivalent ligands, undifferentiated myosin-negative myoblasts exhibit rapid patching and capping of cell surface components while well-differentiated myocytes and myotubes do not. This mobility restriction is evident after the appearance of Ag1422. Treatment with cytochalasin B (15 pg/ml) and/or colchcine (100 ),L%f)does not alter the restricted mobility of surface components seen on differentiated cells. Therefore, neither microfilaments nor microtubules seem to be involved in the mobility restriction. These observations are discussed in relation to current views of myogenesis. INTRODUCTION

Numerous developmental studies have, in recent years, emphasized the role of the cell surface in cell recognition, cell aggregation, morphogenesis, and the regulation of cell proliferation (see reviews in Moscona, 1974; Poste and Nicolson, 1976; and Subtelny and Wessells, 1980). Hood et al. (197’7), drawing upon the wealth of information from studies of the immune system, have formulated the Area Code Hypothesis, which proposes that cell-surface molecules are involved in specific recognition phenomena during cellular differentiation. Specifically this hypothesis proposes that cell-surface (area-code) molecules provide cells with distinct surface addresses or phenotypes. Furthermore the hypothesis proposes that these molecules provide the basis for cellcell recognition, cell-cell interactions, and also serve as receptors for diffusible differentiation signals. The first postulate of this hypothesis states that there should be a “progressive display of specific combinations of areacode molecules on the surface of cells during development” (Hood et ah, 1977). In vitro differentiation of skeletal muscle provides an ‘Present address: Department Eugene, Ore. 97403.

of Biology, University

of Oregon,

excellent developmental system to further test the first postulate of the Area-Code Hypothesis. The differentiation of vertebrate skeletal muscle involves the progression of cells through an orderly sequence of cell proliferation, cell-cell recognition, aggregation, fusion, and maturation of multinucleate myotubes (Konigsberg, 1963; Nameroff and Munar, 1976; Bischoff, 1978). A variety of studies have documented functional, morphological, and compositional changes that occur at the cell membrane during myogenesis. It has been shown that upon differentiation, cells undergo prefusion cellcell recognition and alignment (Nameroff and Munar, 1976), acquire the ability to fuse (Yaffe, 1971), and develop a postfusion block to further fusion (Bischoff and Holtzer, 1969). A changing cell-surface morphology as viewed by SEM has been described (Bayne and Simpson, 1975). Using freeze-fracture techniques, Kalderon and Gilula (1979) have correlated the appearance and location of intramembrane particle-free regions of the plasma membrane with fusion. Dynamic aspects of the plasma membrane have been observed to change as the cells differentiate. These include a transient increase in lipid fluidity at the time of fusion (Prives and Shinitsky, 1977; Herman and Fernandez, 1978; Elson and Yguerabide, 1979), and alterations in the mobility of surface antigens (Friedlander and Fischman, 1979) and con-

313 0012-1606/83 $3.00 Copyright All rights

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

314

DEVELOPMENTAL

BIOLOGY

canavalin A receptors (Furcht et aZ., 1977). Quantitative changes in surface gangliosides (Whately et al., 1976), surface carbohydrates (Winand and Luzatti, 1975), cellsurface lectins (Nowak et al, 1976; Mir-Lechaire and Barondes, 1978; Kobilar et aZ., 1978), and various proteins accessible to 1251-lactoperoxidase-catalyzed iodination (Hynes et al., 1976; Moss et aL, 1978; Pauw and David, 1979) have been described. The appearance and distribution of an integral membrane protein, the acetylcholine receptor (Patrick et al, 1972; Anderson et al., 1977), and a surface-associated protein, acetylcholinesterase (Fluck and Strohman, 1973; Hall, 1973), have been investigated and described in detail. Until recently, the postdifferentiation appearances of AchR and AchE were the only qualitative compositional surface alterations reported. However, Friedlander and Fischman (1977,1979) have reported the detection of a cell-surface antigen which is apparently unique to undifferentiated chick myoblasts. Additionally, while the present work was in progress, several other reports describing qualitative surface changes during myogenesis have been published. Thus, Pauw and David (1979), working with a myogenic rat cell line, reported an increase in the number of proteins labeled by ‘251-lactoperoxidase-catalyzed iodination as these cells differentiate. At least one of these ‘251-labeled proteins was reported to undergo a transient increase in accessibility, temporally correlated with the prefusion alignment of myoblasts. Also working with a myogenic rat cell line, Lee and Kaufman (1981) have developed a series of monoelonal antibodies against cell-surface components, some of which are present on myoblasts but absent or greatly reduced on myotubes. Similarly, Walsh and Ritter (1981) have shown that the Thy-l antigen is expressed only on proliferating human myoblasts and not on myocytes or myotubes. We utilized an immunological approach toward the identification of putative “area-code” molecules during in vitro myogenesis of a continuous cell line of lizard myoblasts. In this communication we present immunohistochemical evidence that at least two types of developmental stage-specific surface alterations occur during in vitro lizard myogenesis. MATERIALS

AND

METHODS

Cell Cultures This study utilized the myogenic lizard cell line M23SlO previously described by Bayne and Simpson (1975, 1977). This cell line is one of several which have been established from promuscle aggregates of the regenerating tail of the lizard Anolis carolinemis. The technique for initiation of primary cultures and establishment of cell lines from promuscle aggregates have

VOLUME

97, 1983

been described in detail (Simpson and Cox, 1967, 1972; Cox, 1968; Bayne and Simpson, 1975, 1977). Such lines can be routinely established and can be maintained in culture indefinitely (Simpson and Cox, 1972). By all available criteria these lines are not transformed (Simpson and Cox, 1972; Simpson and Bayne, 1979). Cells were routinely plated at 1 X lo4 cells per 75-em2 Falcon tissue culture flask and grown in 10 ml nonfusion medium (NM) consisting of 85% Ham’s FlO, 10% horse serum, 4% chick embryo extract, and 1% Gibco antibiotic-antimycotic mixture. Cultures were gassed with 5% Co2-95% air, sealed, and incubated at 31°C. Medium was changed after 4 days and then every 3 days. When plated at this density, well-separated colonies arose and by about Day 12 approximately 50% of the cells in each colony had assumed a rounded-up morphology. These rounded-up cells are in an extended G1 phase of the cell cycle and if left undisturbed will differentiate (synthesize myosin and permanently withdraw from the cell cycle) without entering S. However, upon subculture at low density they reenter the proliferative cell cycle and form new colonies (Bayne and Simpson, 1980). To subculture, the medium on such a culture was changed and the culture gently flushed by pipetting. This preferentially dislodged the rounded-up cells which were then counted and replated in new flasks. This subculture technique avoids the use of proteolytic enzymes or divalent cation chelators and selects for “normal” cells, that is, those which are capable of responding to environmental cues governing differentiation. To obtain fused myotubes, cultures were grown in fusion permissive medium (FM: nonfusion medium supplemented with CaC12 to yield a final calcium concentration of 2.1 mM) (Cox and Gunter, 1973). Alternatively, cultures grown in NM were switched to FM when the rounded-up cells began to stretch back out of aggregates and cultured l-3 days further. Myogenic cell lines were also established from either tails or limbs of 14-day embryonic Anoles (when incubated at 31°C Anolis eggs hatch about 30 days after laying). The techniques used were as previously described for the initiation of myogenic cell lines from promuscle aggregates of regenerating tails (see above). Production

of Antisera

Forty milliliters of preimmune serum was drawn over a period of several weeks. This was processed, as was all serum, by allowing the blood to clot at room temperature (RT) for 30 min, then overnight at 4”C, followed by centrifugation at 30009 for 15 min to remove the clot. After being heat inactivated for 30 min at 56°C the serum was stored at -76°C. All immunizations were performed using cells from

MARUSICH

AND

SIMPSON

Surface Changes during Mpgenesis

the M23SlO myogenic cell line. A single rabbit was immunized with well-differentiated cultures grown in FM. The cells were harvested by vigorous flushing, washed twice with PBS, suspended in PBS a nominal density of 1 X 10’ cells/ml, and then emulsified with an equal volume of Freund’s complete adjuvant. One-half of the emulsion was injected at multiple intradermal sites on one side of the back of the rabbit. Three days later the remaining emulsion (stored at 4°C) was injected on the other side of the rabbit’s back. Beginning 1 month later, 1 X lo6 viable cells in PBS were injected at 14-day intervals into the ear vein of the rabbit. The rise in antibody titer, as monitored by both cytotoxicity assays and indirect immunofluorescence, leveled off after four injections of viable cells. These titers were then maintained with injections of viable cells every 6 weeks. Antiserum was collected by drawing about 40 ml of blood 8-10 days after each injection. A total of 250 ml of antiserum was collected. Indirect Immunojluorescent Viable Cells

(IIF)

Labeling

of

Although rounded-up stage myogenic cells are selectively harvested by gentle flushing, myogenic cells of all stages can be collected with more vigorous flushing. Cell suspensions thus prepared contain >99% viable, single cells. Lung fibroblasts, liver cells, and chondroblasts were removed from primary cultures by incubation at 31°C for 10 min in PBS containing 2 mg/ml collagenase. The nonmuscle cells were then washed with NM and incubated in NM at 31°C for 4.5 hr in a spinner flask. Such suspensions contain >95% viable cells. All cell suspensions were treated identically from this point on. A sample of 2 X lo5 cells was pelleted, resuspended in 25 ~1 antiserum or preimmune serum, and incubated for 20 min at the desired temperature. The cell suspension was then diluted with 1 ml of 10% HS. Layered under this was 1 ml of 50% HS and then 1 ml of 100% HS. The cells were pelleted and the diluted rabbit serum and HS were drawn off. The cells were resuspended in 100 ~1 of GAR-FITC (fluorescein conjugated goat anti-rabbit IgG; IgG fraction; 1:20 dilution; Cappel Laboratories Inc.), incubated for 20 min at the desired temperature, and washed as above through layered HS. The cell pellet was then resuspended in l-2 drops of 100% HS, spread on acid-cleaned slides, and air dried. The smear was fixed in 95% ethanol for 5 min, washed 3 X 10 min in PBS, mounted in a buffered glycerine medium (90% glycerol, 15 mJ4 NaCl, 83 mM Tris, pH 9.7), and viewed. Staining titer was defined as the last dilution at which detectable staining was observed on all cells. We found that nonspecific fluorescence could be com-

315

pletely eliminated by “preadsorbing” both antiserum and preimmune serum with an equal volume of heatinactivated horse serum, and washing the test cells in horse serum immediately prior to antibody incubations. IIF

Labeling

of Fixed Cells

Cells were plated at 1.3 X 10z cells/cm2 on plastic Leighton tube coverslips (Costar) and grown to the desired stage. The coverslips with attached cells were then rinsed once in PBS and fixed for 30 min at RT with 2% paraformaldehyde in PBS. After being washed 4 x 10 min in PBS and for 5 min in 100% HS they were incubated in immune or preimmune serum for 30 min at 37’C. The cells were then washed 3 X 10 min in PBS, 5 min in 100% HS, and incubated in GAR-FITC for 30 min at 37°C. They were then washed 3 X 10 min in PBS, mounted as above, and viewed. Enzymatic

Pretreatment

of Viable Cells

The effect of several degradative enzymes on antigen expression was examined using viable cells as targets. For trypsin digestion, 1 X lo6 cells were suspended in 1 ml PBS containing from 0.1 mg/ml to 2.5 mg/ml trypsin (1:250; Gibco) and incubated at 31°C for 20 min. Digestion was terminated by addition of an equal volume of 50% HS containing Trasylol (FBA Pharmaceuticals) at a concentration of 1000 kallikrein inactivator units (KIU)/ml. Cells were then washed three times through layered HS containing 500 KIU/ml. Proteinase K digestion was performed on 1 X lo6 cells in 1 ml PBS containing from 0.1 to 2.0 mg/ml proteinase K (type XI, from Tritirachium album; Sigma). After a 30-min incubation at 31”C, the digestion was stopped and the cells were washed as described for trypsinized cells. Neuraminidase digestion was performed on 5 X lo6 cells in 0.1 ml PBS containing 5 units/ml of neuraminidase (type VI from Clostridium ioerfringens; Sigma). Cells were then incubated for 30 min at 31°C suspended in 1 ml 10% HS, and washed through layered HS. In each case, following enzymatic treatment and washing, the cells were processed for IIF as described above for viable cells. IdentiJicatiw

of Myosin Containing Cells

Cells previously surface labeled with GAR-FITC and fixed with 95% ethanol were washed 3 X 10 min with PBS, rinsed in 100% HS, and incubated for 30 min at 37°C with a rabbit anti-lizard skeletal muscle myosin antiserum previously produced in this laboratory (Bayne and Simpson, 1977). After washing 3 X 10 min in PBS and for 5 min in 100% HS the cells were incubated for

DEVELOPMENTALBIOLOGY VOLUME97.1983

316 30 min at 37°C IgG fraction; The cells were as above, and

in GAR-RH (rhodamine-conjugated GAR 1:20 dilution; Cappel Laboratories Inc.). then washed 3 X 10 min in PBS, mounted viewed.

washing solutions contained cytochalasin B and/or colchicine at the same concentrations as in the original treatment. Fluorescence Microscopy and Photography

Con A-FITC

Labeling

of Viable Cells

Viable myogenic cells were collected as above. Cells, 1 x 106, were incubated for 20 min at the desired temperature in 0.1 ml PBS containing 35 pg/ml concanavalin A-FITC (Calbiochem-Behring Corp.). Samples were incubated either in the presence or in the absence of 0.2 M cu-methyl-D-mannoside (Aldrich Chemical Co., Inc.). The cells were then washed through layered HS, mounted, and viewed. Immunoadsorption

Particulate fractions of tissue homogenates were used as immunoadsorbents. The preparation and use of all adsorbents were performed at 4°C. The tissues were minced in PBS, blended in a Sorval Omni Mixer, and centrifuged for 20 min at 39,OOOg. The pellet was then washed three times with 20 vol of PBS. One volume of washed, packed homogenate was suspended in 1 vol of appropriately diluted antiserum plus 5 vol PBS and incubated for 2 hr with occasional agitation. After centrifugation at 39,OOOg for 20 min the supernatant was saved and the pellet washed with l-2 vol of PBS. The supernatants were combined, and if desired, adsorbed further. The final supernatant was concentrated to the original antiserum volume by ultrafiltration (10,000 MW cut-off, immersible separators, Millipore Corp.). Myogenic cells used for immunoadsorption were collected by flushing. Cells were resuspended at the desired concentration in antiserum, incubated for 2 hr, and removed by centrifugation. In some instances, bound antibody was removed and recovered from used immunoadsorbents as described by Goldschneider and McGregor (1973). Using this procedure, at least 40-50s of the bound antibody can be recovered functionally intact. Pretreatment with Colchicine and/or

Cytochalasin B

Myogenic cells were grown to the desired stage in 75cm2 flasks. For controls, cells from one-half of each flask were removed and processed immediately for IIF labeling. The media were replaced and supplemented with stock solutions of cytochalasin B (5 mg/ml in DMSO; Sigma) and/or colchicine (4 mg/ml in PBS; Sigma) to give the following final concentrations: (1) 15 pg/ml cytochalasin B (31 PM); (2) 40 pg/ml colchicine (100 pLM); and (3) 15 pg/ml cytochalasin B + 40 pg/ml colchicine. All treated cultures were incubated at 31°C for 3 hr, harvested by flushing, and processed as previously described for IIF labeling. All subsequent incubation and

We used an American Optical Fluorstar microscope equipped with a 50-W mercury arc lamp, fluorcluster filters, and epifluorescence optics. Fluorescein fluorescence was observed using BG12 excitation, 500-nm dichroic and OG515 barrier filters, Rhodamine fluorescence was observed using IF 530-nm excitation, 540-nm dichroic, and 06570 barrier filters. Fluorescence was recorded either in black and white on tri-X film push developed to an ASA of 2000 or in color on Ektachrome 400. In all cases where specimens were to be compared, exposure times as well as developing and printing conditions were identical. RESULTS Antiserum

1422

Cells flushed from 15-day cultures of differentiated muscle grown in FM were used as immunogens to produce antiserum 1422 (AS1422). This antiserum exhibited a high titer against cell-surface antigens as judged both by indirect immunofluorescence (1:1024) and by complement-mediated cytotoxicity (1:64). We employed sequential immunoadsorption to examine the complexity of AS1422. Removal of Antibodies to Tissue Common Antigens

IIF labeling of viable cells revealed that AS1422 bound extensively to the cell surface of a variety of nonmuscle cell types from Anolis. When a 1:8 dilution of this antiserum was adsorbed five times with an equal weight of packed lizard liver homogenate, all reactivity to surface antigens on liver cells, lung fibroblasts, chondroblasts, lymphocytes, and erythrocytes was removed. This liver homogenate-adsorbed AS1422 demonstrated muscle-specific IIF-staining and stained myogenic cells regardless of developmental stage. However, the cell surfaces of differentiated cells stained intensely while those of proliferation stage (undifferentiated) cells stained weakly, (Fig. 1). Adsorption of Muscle-Specific AS1422 to Developmental Stage Speci$city

The slight reactivity of muscle-specific AS1422 to proliferation stage myoblasts was removed by adsorption with a total of 4 X lo* viable proliferation stage cells/ ml. This last adsorption step yielded an antiserum that was both muscle specific and developmental stage specific, i.e., it stained only differentiated myotubes and

317

FIG. 1. Muscle-specific IIF staining with AS1422 following adsorption with 5 vol of packed Anolis liver homogenate. Test cells were myogenic cells from a 15-day culture grown in NM. (a) FITC fluorescence detecting muscle-specific surface antigens; (h) RH fluorescence detecting cytoplasmic skeletal muscle myosin; and (c) phase contrast. AS1422 thus adsorbed reacts with myogenic cells of all stages. Myosin (+) cells (arrows, b) exhibit bright, diffuse surface fluorescence (a). Myosin (-) cells (b) exhibit weak, patchy surface fluorescence (a). x1890.

mononucleate, myosin-positive myocytes (Figs. 2-5). This antigen(s), found only on postdifferentiation, myogenie cells has been designated Ag1422. As a further test of the efficacy of our adsorption procedures, we recovered the antibodies directed against “common” antigens and tested their IIF-staining specificities. Antibodies directed against tissue-common surface antigens were recovered from the liver homogenates that were used in the adsorption of AS1422 to muscle specificity. The recovered immunoglobulins reacted, as expected, with viable liver cells, fibroblasts, blood cells, and myogenic cells of all developmental stages. Likewise, antibodies directed against “musclesurface antigens were recovspecific, stage-common” ered from the proliferation stage cells used to adsorb muscle-specific AS1422 to differentiated stage specificity. Here too, the recovered antibodies reacted with viable myogenic cells of all developmental stages but did not react with the cell surfaces of viable non-muscle cells. Although the myogenic “line” M23SlO has been shown, by a variety of criteria, to be nontransformed (Simpson and Bayne, 1979), we wished to demonstrate that the differentiation antigen(s) (Ag1422), detected by AS1422, was not a transformation antigen. All reactivity of the differentiated stage-specific AS1422 to differentiated cultures of M23SlO can be removed by a 3X adsorption (w/w) with homogenates of mature Anolis skeletal muscle. Additionally, Agl422 is expressed in a differentiated stage-specific manner by embryonic muscle grown in vitro (see below). Therefore, Ag1422 is not a transformation antigen. Summary

of AS1.4~2 Adsorptions

AS1422 contains antibodies which can be used to demonstrate the presence of the following classes of cell surface antigens in lizard myogenic cultures: (1) a dif-

ferentiation stage-specific, muscle-specific antigen(s) (designated Ag1422); (2) a stage-common, muscle-specific antigen(s); and (3) a stage-common, tissue-common antigen(s). Although some use has been made of the antibodies directed against the common antigen(s) (see below) we have concentrated our efforts toward a description of the stage-specific antigen(s), Ag1422. Temporal

Expression

of Ag1422

Using AS1422 adsorbed to differentiated stage specificity we examined a staged series of cultures ranging from early proliferation (all undifferentiated) to welldifferentiated (>80% myosin-positive myocytes) in order to map the appearance of Ag1422 during myogenesis. Differentiated cells were defined as those cells that had synthesized detectable levels of skeletal muscle myosin as monitored by IIF. Previous studies have shown that lizard myogenic cells that have synthesized myosin have irreversibly withdrawn from the proliferative pool (Bayne and Simpson, 1977). For each time point, cells grown in NM were collected by flushing and labeled in suspension with the differentiated stage-specific AS1422, followed by GAR-FITC. The cells were then spread on slides and fixed in 95% ethanol. Ethanol fixation preserved antibodies bound to surface antigens and rendered the cells permeable to antibodies directed against lizard skeletal muscle myosin (Bayne and Simpson, 1977). Antibodies bound specifically to skeletal muscle myosin were visualized using a second antibody, GAR-RH. Fluorescence due to GARFITC bound to surface antigens was easily distinguished from the GAR-RH fluorescence which identified skeletal muscle myosin (see Figs. 1, 2, 6, and 7). Preimmune treated cells exhibited negligible background staining when viewed with fluorescein filters. Thus, low levels of surface-bound GAR-FITC could be accurately scored as positive. Background stain viewed with rho-

318

FIG. 2. Sequence of appearance of Ag1422 on developing myogenic cells. Cells were grown in NM and treated to demonstrate both cellsurface Ag1422 (FITC) and skeletal muscle myosin (RH). All myosin (-) cells are Ag1422 (-). Some cells (arrows) in the 14- and 17-day cultures are both myosin (+) and Ag1422 (-). Note that young 14-day Ag1422 (+) cells exhibit antibody-induced redistribution of the antigen while some of the “older” E-day Ag1422 (+) cells and the majority of the 1’7-day Ag1422 (+) cells exhibit a restriction in antibody-induced redistribution of the antigen. X1890.

damine filters was low, but noticeable. Although this presumably resulted in a higher threshold for GARRH-positive staining, positives were still easily dis-

cerned from negatives. For each time point, duplicate cultures were tested separately and at least 400 cells from each culture were scored. The experiment was rep-

319

FIG. 3. IIF staining of M23SlO myogenic cells grown in NM, prefixed with paraformaldehyde and reacted with differentiation stage-specific AS1422. First antiserum: AS1422 diluted 1:8, 5X Anolis liver absorbed, followed by adsorption with 4 X 10’ proliferation stage myogenic cells/ml. Second antiserum: GAR-FITC. Fluorescein fluorescence (a-c). Phase contrast (d, e and f). Photographs are of cells from different regions of the same 16-day culture. Flattened, proliferation stage cells (a) are nonreactive. Some rounded-up cells (b) express Ag1422, while others (arrows, b), are negative. Stretched-out cells express Ag1422 (b and c). X1305.

licated three times with uniform results. The results of one experiment are presented in Fig. 5. Representative photographs of the four classes of cells observed are shown in Fig. 2. As can be seen in Fig. 5, small numbers of myosin (+) cells were first observed in Days 11 and 12 of culture. This corresponds to the time during which rounded-up cells begin to accumulate in cultures. By Day 13 or 14, when large aggregates of rounded-up cells are present, myosin (+) cells constituted approximately 70% of the cell population. The myosin (+), rounded-up cells stretched back onto the culture dish between Days 15 and 1’7, and cultures of this age exhibited a reduced rate of increase in myosin (+) cells. Only cells that had synthesized detectable levels of skeletal muscle myosin exhibited surface staining for Ag1422. While it is apparent from Fig. 5 that with time the majority of the myosin (+) cells also became positive for Agl422, the increase in cells that were positive for both Ag1422 and myosin lagged behind the increase in myosin (+) cells by approximately 24 hr. Thus the appearance of Ag1422 at the cell surface occurs some time after cells have synthesized detectable levels of skeletal muscle myosin. When paraformaldehyde-fixed, staged colonies were tested for the presence of Agl.422, the number of cells

positive for this antigen(s) agreed with the previous data obtained from viable cells tested in suspension. Furthermore, the distribution and morphology of Ag1422 (+) cells within each colony were consistent with expectations based on the invariant association of Ag1422 with myosin (+) cells and the previously established (Bayne and Simpson, 1977) distribution of myosin (+) cells within the colony. All myotubes, all stretchedback mononucleate myocytes, the majority of roundedup cells in aggregates, and occasional single roundedup cells outside of aggregates stained positively for Ag1422 (Figs. 3 and 4). Flattened and bipolar mononucleate cells, the majority of single, rounded-up cells outside aggregates, and an occasional rounded-up cell within aggregates (Fig. 3) were negative for Ag1422.

Expression of Agl&?i? on Embryonic Myogenic Cells

Lizard

Cells derived from embryonic lizard limb or tail musculature were grown in NM, harvested after stretchingback out of aggregates, and then tested for both Agl422 and myosin. The results (Table 1) were similar to those seen when comparable cultures of M23SlO cells were tested. The antiserum labeled cells stage specifically, as Ag1422 (+) cells were invariably also myosin (+), and

DEVELOPMENTALBIOLOGY VOLUME97,1983

320

and reacted with differentiation stage-specific AS1422. FIG. 4. IIF staining of myogenic cells grown in FM, prefixed with paraformaldehyde The first and second antisera were the same as in Fig. 3. (a, b) Fluorescein fluorescence. (c, d) Phase contrast. (a, c) Cells of line M23SlO. (b, d) Cells of line L2E4B2 derived from embryonic limb muscle. In both examples the myotube expresses Ag1422 while surrounding undifferentiated cells are unreactive. X1305.

myosin (-) cells were invariably Ag1422 (-). Moreover, some myosin (+), Ag1422 (-) cells were observed. This suggests that the same temporal relationship exists between the appearance of myosin and Ag1422 in both cultured embryonic myogenic cells and M23SlO cells. Embryonic muscle was also allowed to differentiate in FM, and then tested after paraformaldehyde fixation. As with the case with M23SlO cells, myotubes were invariably Ag1422 (+) while spread out proliferating myoblasts were negative (Fig. 4). Expression

of Agl422

after Enzymatic

Digestion

There have been several reports of masked or cryptic cell-surface antigens which were only expressed after proteolytic (Burger, 1971; Hayry and Defendi, 1970; Friedlander and Fischman, 1977) or neuraminidase (Grothaus et aZ., 1971) digestion of otherwise unreactive cells. We were interested in determining whether or not such pretreatment could expose Ag1422 on undifferentiated myogenic cells. Mixed suspensions of differentiated and undifferentiated viable cells were treated with trypsin, proteinase K, or neuraminidase as described under Materials and Methods. The results are shown in Table 2. In each case, the cells remained viable after treatment and the rel-

ative proportions of differentiated and undifferentiated cells remained unchanged. No treatment resulted in the expression of Ag1422 on myosin (-) cells. Therefore undifferentiated cells do not possess Agl.422 masked by surface components which are susceptible to trypsin, neuraminidase, or proteinase K digestion. Similarly, neither neuraminidase nor trypsin treatment resulted in a reduction of the number of intensity of Ag1422 (+) cells. Therefore, the antigenic determinants(s) responsible for the detection of Ag1422 do not involve sialic acid residues or trypsin-sensitive proteins. However, treatment with proteinase K (a nonspecific protease) resulted in a drastic reduction in the number of Ag1422 (+) cells and significantly lowered the fluorescence intensity of the remaining positives. Since the percentage of myosin (+) cells remained the same before and after treatment, the reduction in Ag1422 (+) cells must have been due to the removal or degradation of Ag1422. We conclude that Ag1422 is at least partially protein in nature. Restriction in Mobility after D$$erentiation

of Cell-Surface

Components

We have used antibody-induced redistribution of surface antigens as a probe to monitor developmental changes in the relative mobility of myogenic cell surface

MARUSICH AND SIMPSON

surface

antigens. Unadsorbed AS1422 was used in these experiments to ensure labeling of surface antigens on cells of all developmental stages. The native distribution of these surface antigens is uniform on cells of all stages. This has been determined by labeling either viable cells in suspension at 0-4’C or cells prefixed with paraformaldehyde. In both cases, all cells showed uniform surface fluorescence (Fig. 6). When viable cells are labeled at RT, the surface antigens of undifferentiated cells are redistributed into discrete clusters separated by antigen-free regions (Fig. 7). However, after identical treatment, most myosin (+) cells exhibit a diffuse surface fluorescence indistinguishable from that seen at 0-4°C (Fig. 7). These differences were observed on cells grown in either NM (Fig. 7) or FM (Fig. 8). These findings indicate that the mobility of surface components is significantly restricted after the cells differentiate. In order to more precisely determine when this mobility restriction occurs we examined a staged sequence of cultures grown in NM ranging from early proliferation stage (all undifferentiated) to stretchedout (>‘75% differentiated). The results are summarized in Fig. 9 and representative photographs of the three classes of cells observed as presented in Fig. 7. First, it should be noted that myosin (-) cells always show antibody-induced redistribution, regardless of culture age. Second, a restriction in the mobility of surface components is not observed until sometime @er myosin (+) cells appear. The majority of the first myosin (+) cells that appear still exhibit antibody-induced redistribution of surface components (Fig. 9). The number of myosin (+), restriction (+) cells increases over the next several days until by Days 15-17, the majority of myosin (+) cells are also restriction (+). Therefore, the restriction in surface antigen mobility occurs sometime after the appearance of detectable levels of myosin. Although we have scored the cells as either restriction (+) or (-), in reality a gradual transition is seen. Ideally, we would have liked to identify the time of the initial alteration as well as its end point. In practice, however, it soon became obvious that it was impossible to reliably score the intermediate stages. Cells exhibiting noticeable patching were scored restriction (-) and only those that showed completely uniform surface fluorescence were scored as restriction (+). We recognize that the error involved in this scoring procedure would tend to bias the data in favor of the restriction in mobility occurring after the appearance of myosin. However, since the degree of patching on many myosin (+) cells (especially “young” myosin (+) cells) is comparable to that seen on myosin (-) cells, we feel confident that the overt change in surface antigen mobility does not occur until after detectable levels of myosin appear.

changes during

321

kfyogenesis

I

I

I

14

1

16

Age of Cutture in Days FIG. 5. Developmental time of appearance of Ag1422 and skeletal muscle myosin. hlyogenic cells (M23SlU) were grown in NM and treated as described in the text to demonstrate both surface-bound Ag1422 (FITC) and cytoplasmic skeletal muscle myosin (RH). The filled circles (solid line) represent the percentage of cells which were myosin (+) at each day tested, The open circles (dashed line) represent the percentage of cells which were positive for both myosin and Ag1422 (note: this includes all of the Ag1422 (i-) cells, but only some of the myosin (+) cells). Lines are drawn through the average of the values obtained with two separate, simultaneously processed sets of cultures. Cells expressing Ag1422 appear about 24 hr after the appearance of myosin (+) cells. The majority of the first myosin (+) eelIs to appear are Ag1422 (-), By Days 15 and 17, however, the majority of myosin (+) cells also express detectable levels of Ag1422.

Exprtbon

of Agl&?

prior to Mobility

Restriction

To determine the temporal relationship between the observed restriction in antigen mobility and the expression of Ag1422, staged cultures were labeled for both Ag1422 and myosin as previously described. We found that Ag1422 was redistributed into patches on the surfaces of the majority (80%) of young Ag1422 (+) cells (Figs. 10 and 2). By Day 15, however, less than 20% of all Ag1422 (+) cells were capable of undergoing antibody-induced redistribution. Therefore, Ag1422 is expressed prior to the overt restriction of lateral mobility in the cell membrane. Generality of the Restriction compcment Mobilitf#

is! Surface

All surface components tested reveal the same basic alteration in mobility upon differentiation. Additionally, all unadsorbed antisera raised against cells of earlier developmental stages can be used to label cells and

322

DEVELOPMENTAL

BIOLOGY

VOLUME

97, 1983

FIG. 6. Uniform native distribution of cell-surface antigens. (a and b) Proliferation stage myogenic cells (M23SlO) prefixed with AS1422 diluted 1:8, followed by GAR-FITC. (a) Fluorescein fluorescence. (b) Phase contrast. X1305. The distribution of antigens is uniform. (c-e) Viable, proliferation stage myogenic cells (M23SlO) were treated on ice with AS1422 diluted 1:128, GAR-FITC. After mounting and ethanol fixation, the cells were reacted with antimyosin antiserum followed by GAR-RH. (c) fluorescence. (d) Rhodamine fluorescence. (e) Phase contrast. X1890. The cells are all myosin (-) (d). At low temperature the exhibit noticeable antibody induced redistribution of surface antigens (c).

show the same distribution and redistribution patterns (data not presented). Therefore, the phenomenon appears to be the result of a general change in the basic structure or fluidity of the plasma membrane of these myogenic cells. Specific components tested include Ag1422, tissue-common antigens, muscle-specific, stagecommon antigens, and Con A receptors (Table 3). Furthermore, early-passage embryonic myoblasts exhibit a similar developmental alteration in mobility of surface antigens (Table 3). Thus the phenomenon is not unique to cells of the continuous culture line M23SlO. Eflect of Cytochalasin B and Colchicine Immobilized Surface Components of Diflerentiated Cells

on

There is growing evidence that cytoskeletal elements can exert transmembrane control over the mobility and distribution of certain surface receptors (Edelman, 1976; Nicolson. 19’79). We have asked if such transmembrane ~-

and labeled the surface followed by Fluorescein cells do not

control is responsible for the mobility restriction which occurs during lizard myogenesis. Cytochalasin B and colchicine are known to disrupt microfilaments and depolymerize microtubules, respectively (Weber et al, 1976; Wilson, 1975). Therefore, if microfilaments and/or microtubules were involved in anchoring and restricting the mobility of surface components after cell differentiation, treating the cells with these drugs should restore the original mobility. Well-differentiated cultures grown in NM were used for each experiment. Cells treated with cytochalasin B and/or colchicine, as well as untreated cells, were prepared and labeled as described under Materials and Methods. The results (Table 4) show that the proportion of cells with restricted surface component mobility remained the same or increased slightly. Therefore, the mobility restriction seen in differentiated cells is not altered by these drugs. These results suggest that the restriction in mobility is not mediated by microfilament or microtubule anchorage.

323

FIG. ‘7. Sequence of appearance of myogenic cells exhibiting a restriction in lateral mobility of cell-surface antigens. The cells were grown in NM, flushed from the substance, and treated live, in suspension, at RT, with a 1:128 dilution of AS1422, followed by GAR-FITC. Following fixation the cells were reacted with anti-myosin antiserum, followed by GAR-RH. All myosin (-) cells exhibit patching characteristic of antibody-induced surface antigen redistribution. Many of the first myosin (+) cells that appear at 14 days of culture also exhibit patching of surface antigens (arrows). Most myosin (+) cells in older (15- and 17-day) cultures exhibit a uniform distribution of surface antigens, indicating a restriction in lateral mobility of cell surface antigens. All myosin (+) cells which retain their stretched-out morphology throughout the labeling protocol exhibit a uniform distribution of cell surface antigens (arrow, Day 17 cells). X1890.

VOLUME 97, 1983

DEVELOPMENTAL BIOLOGY

324 EXPRESSION

TABLE 1 OF Ag1422 ON EMBRYONICLIZARD MYOGENIC CELLS’

Cells

Percentage myosin(-) Ag1422(-)

Percentage myosin(-) Ag1422(+)

Percentage myosin(+) Ag1422(-)

Percentage myosin(+) Ag1422(+)

Tail (T2E4R3) Limb (L2E4B2)

27 38

0 0

38 33

35 29

(LEmbryonic myogenic cells grown in NM were flushed from 15-day cultures, then labeled to demonstrate both Ag1422 (FITC) and skeletal muscle myosin (RH).

DISCUSSION We have demonstrated three classes of cell-surface antigens which are expressed during in vitro lizard myogenesis: (1) a differentiated stage-specific, musclespecific antigen(s) (Ag1422); (2) stage-common, musclespecific antigen(s); and (3) stage-common, tissue-common antigen(s). These three classes of surface antigens are not unique to the continuous culture line used in this study. The tissue common antigen(s) are shared with nonmyogenic cells of adult Anolis and with embryonic myogenic cells. The stage-common, muscle-specific antigens are shared with mature skeletal muscle of adult Anolis (embryonic cells were not tested). The differentiated stage-specific, muscle-specific antigen(s) are shared with mature skeletal muscle of adult Anolis and with embryonic muscle. The differentiated stage-specific antigen(s) Ag1422 is not present on proliferation stage myoblasts or rounded, extended G1, myosin-negative, proliferation-competent myoblasts. Agl422 first appears on rounded, Go, myosinpositive, proliferation-incompetent myocytes and continues to be expressed on stretched out mononucleate myocytes in NM and multinucleated myotubes in FM. Ag1422 is trypsin and neuraminidase insensitive, but can be removed by proteinase K. There appears to be a lag of 24 hr between the time when the rounded Go cells have synthesized detectable amounts of myosin and the appearance of detectable levels of Agl422. Although rounded, Go, myosin-positive lizard myocytes also display acetylcholine receptors and acetylcholinesterase (data not included) it appears unlikely that either of these molecules is a component of the stage-specific Agl422 that we have demonstrated. Patrick et al (1973) have reported that rabbits producing precipitating antibodies to the acetylcholine receptor from Electrophorus developed an autoimmune response resulting in flaccid paralysis and death. The rabbit that

produced AS1422 was repeatedly boosted with antigen over a 2-year period and it never exhibited signs of paralysis or muscle weakness. Additionally, we were unable to precipitate lz51-labeled bungarotoxin-bound

protein

from solubilized membranes of differentiated myogenic cultures using AS1422 and Staph A. Therefore, we feel confident that AgI422 is a develop-

stage

mental stage-specific surface antigen(s) other than the acetylcholine receptor. Likewise, AchE is a sensitive to trypsin digestion (Hall and Kelly, 19’71), whereas Ag1422

is not, thus indicating a lack of identity. Shortly following the synthesis of myosin and Ag1422, the rounded lizard Go myocyte undergoes a general restriction

in the lateral

mobility

of cell-surface

antigens.

This restriction is exhibited by tissue-common, musclespecific, and developmental stage-specific antigens, as well as Con A receptors (Figs. 2,7,9, and Table 3). From our data we conclude that Ag1422 is expressed prior to the restriction in surface antigen lateral mobility (Fig. 10). A similar restriction of surface antigen mobility has been reported to occur during the in vitro differentiation of embryonic chick myogenic cells (Friedlander and Fischman, 1979). This suggests that a postdifferentiation restriction in membrane protein mobility may be a general feature of vertebrate skeletal muscle development. However, contradictory results were reported by Furcht et al. (1977,1978), who used an approach similar to ours to monitor lateral mobility of Con A receptors during in vitro differentiation of a myogenic rat cell line: They reported that Con A receptors of myoblasts exhibited a restricted lateral mobility, while those on myotubes were capable of extensive redistribution (patching). The reason for the apparent differences obtained with lizard and chick cells on the one hand, and rat cells on the other, is unclear.

TABLE 2 EXPRESSION OF Ag1422 AFTER ENZYMATIC

DIGESTION=

Percentage myosin(-) Ag1422(-)

Percentage myosin(-) Ag1422(+)

Percentage myosin(+) Ag1422(-)

Percentage myosin(+) Ag1422(+)

None Neuraminidase (5 units/ml)

17.6

0

61.7

20.7

22.3

0

59.5

18.2

None

33.8

0

10.2

56.0

Trypsin (2.5 mg/ml)

35.6

0

12.8

51.7

16.7

0

21.6

61.7

22.9

0

33.0

44.0

16.0

0

64.8

19.3

Treatment

None Proteinase K (0.1 mg/ml) Proteinase K (2.0 mg/ml)

-

a The different enzyme treatments were performed on separate days using 14- to 16-day cultures. This accounts for the differences in the number of myosin-positive and Agl422-positive cells observed in the three experiments.

MARUSICH

AND

SIMPSON

FIG. 8. Mononucleate cells and a single multinucleate temperature with a 1:128 dilution of AS1422, followed by right. The myotube (m) and a large mononucleate cell (c) cells (arrows) exhibit extensive patching characteristic of

Surface

Changes during

325

Myogenesis

myotube from a 17-day culture grown in FM. Live cells were labeled at room GAR-FITC. Fluorescein fluorescence is shown on the left, phase contrast on the exhibit a uniform distribution of cell surface fluorescence. Small undifferentiated antibody-induced antigen redistribution. X1890.

A comparative survey of recent reports on cell-surface antigens of myogenic cells reveals that at least six classes of myogenic cell surface antigens have been identified. These include surface antigens which are (1) myogenic stage common and tissue common (Lee and Kaufman, 1981; present study); (2) myogenic stage common and muscle specific (Walsh and Ritter, 1981; present study); (3) undifferentiated stage specific, but not 100

Age of Culture

in Days

FIG. 9. Developmental time of appearance ‘of cells containing skeletal muscle myosin and cells exhibiting restricted lateral mobility of surface antigens. Myogenic cells (M23SlO) were grown in NM and processed for IIF as described in the legend to Fig. 7. The filled circles represent the percentage of cells which were myosin (+) on each day tested. The open circles represent the percentage of myosin (+) cells exhibiting a uniform distribution of cell-surface antigens, out of the total number of cells which were myosin (+) on each day tested. The open squares represent the percentage of myosin (-) cells exhibiting a uniform distribution of cell-surface antigens, out of the total number of cells which were myosin (-) on each day tested. Lines are drawn through averages of the values obtained with two separate, simultaneously processed cultures (at least 400 cells from each culture were scored at each time point). When myosin (+) cells first appear (Days 12 and 13) only a small percentage of them exhibit the uniform surface antigen distribution characteristic of a restricted mobility of cellsurface antigens. The majority of young myosin (+) cells exhibit a patchy distribution of surface antigens characteristic of antibodyinduced redistribution. However, in older cultures (Days 15 and 17) the majority of myosin (+) cells exhibit uniform labeling of cellsurface antigens. Regardless of culture age, myosin (-) cells never exhibit a restriction in lateral mobility of surface antigens.

“---o

IO

I’ I

12

I

I4

Age of Culture

I

I

I6 in Doyr

FIG, 10. Ag1422 appears prior to the restriction in lateral mobility of cell-surface antigens. Myogenic cells (M23SlO) were grown in NM and processed for IIF as described in the legend for Fig. 2. The open circles (dashed line) represent the percentage of cells which were Ag1422 (+) at each day tested. The filled circles (solid line) represent the percentage of diffusely stained, Ag1422 (+) cells out of the total number of cells which were Ag1422 (+) on each day tested. Lines are drawn through the average of the values obtained with two separate, simultaneously processed cultures. Note that only a minority of young (Days 13 and 14) Ag1422 (+) cells are diffusely stained, while the majority of older (Days 15 and 17) Ag1422 (+) cells exhibit a diffuse stain.

326

DEVELOPMENTAL TABLE

GENERALITY MOBILITY

SURFACE

Percentage Surface

component

Tissue-common antigens’

3

OF THE POSTDIFFERENTIATION OF CELL

myosin(-) restriction(-)

BIOLOGY

RESTRICTION

ANTIGENS

AND

LECTIN

Percentage myosin(-) restriction(+)

IN LATERAL RECEPTORS

Percentage

Percentage

myosin(+) restriction(-)

myosin(+) restriction(+)

43.8

0.0

17.6

38.7

55.1 23.4

0.0 0.0

4.3 11.8

40.6 64.8

26

0

23

51

34

0

12

54

Muscle-specific, stage-common antigens’ Con A receptors*’ Embryonic tail myogenie surface antiger& Embryonic myogenic antigen.9

limb surface

’ Cells from 14- to lbday cultures of M23SlO cells were tested with appropriately antiserum or Con A. The various components were tested on separate occasions in some variability in the number of differentiated cells.

‘Specificity of Con A-FITC binding of 0.2 Ma-methyl D-mannoside.

was verified

by the absence of binding

a Embryonic myogenic cells grown in NM were Rushed AS1422 was used to label surface antigens.

from

16.day

cultures.

adsorbed resulting

in the presence Unadsorbed

muscle specific (Lee and Kaufman, 1981; Walsh and Ritter, 1981); (4) undifferentiated stage specific, and muscle specific (Friedlander and Fischman, 1979; Marusich and Simpson, 1979); (5) differentiated stage specific, and muscle specific (present study); and (6) myogenic stage common, but quantitatively stage dependent (Chen, 1977; Lee and Kaufman, 1981). In addition to these antigenic changes, numerous other quantitative alterations and some qualitative changes in cell-surface components of differentiating myogenic cells have been identified using other approaches (see Introduction). Finally, the relative lateral mobility of a variety of myogenic cell surface components is subject to developmental regulation (see above, and Introduction). Together, these results provide additional support for the first postulate of the Area-Code Hypothesis (Hood et al, 1977). With respect to skeletal muscle, the available data suggest that there is a progressive display of stage-specific surface molecules (area-code molecules) during differentiation. With the exception of the actylcholine receptor, none of the reported developmental stage-specific surface components, including Ag1422, have an assigned function. However, attempts have been made to correlate their temporal appearance with known morphogenetic stages or biochemical events in myogenesis. The temporal appearance of Ag1422 suggests that it could be functionally involved in postdifferentiation events associated with (1) fusion, (2) the restriction in membrane fluidity, or (3) cytoskeletal rearrangements and/or cellsubstrate adhesivity changes involved in the conversion

VOLUME

97, 1983

of myosin-positive rounded myocytes into long attenuated bipolar myocytes. A functional role has not been established for the restriction in mobility which occurs during lizard and chick myogenesis. One possibility arises from the results of a recent freeze-fracture study of chick myogenesis (Kalderon and Gilula, 1979). They found that intramembrane-particle (IMP)-free regions were formed just prior to myoblast fusion, and that membrane fusion later occurred only in these areas. They suggested that the IMP-free regions are protein depleted or lipid enriched, and that fusion may be facilitated by fusogenie lipids found in these regions. It is possible that the lateral mobility of membrane proteins may be restricted in order to allow the formation of such IMPfree domains. It is significant that the lateral mobility of membrane proteins and lipids are apparently under separate controls (De Laat et al, 1980). Therefore, the seemingly contradictory reports of increased lipid mobility at the time of fusion (Prives and Shinitsky, 1977; Herman and Fernandez, 1978; Elson and Yguerabide, 1979) and decreased membrane protein mobility (Friedlander and Fischman, 1979; present study) may be reconciled. The protein mobility restriction may facilitate the formation of protein depleted domains while the lipids in these regions may exhibit an increased fluidity in response to separate controls. Although we have no evidence that either of the alterations described in this report subserve a particular function, we have directly demonstrated that these alterations define a distinct sequence of developmentally regulated postdifferentiation cell-surface phenotypes. Moreover, when considered in conjunction with previous studies of in vitro lizard myogenesis (see above), our results indicate that at least six stages or subpop-

LACK

OF EFFECT

TABLE OF CYTOCHALASIN

POSTDIFFERENTIATION FACE ANTIGENS’

Treatment

RESTRKXION

4 B AND/OR IN LATERAL

COLCHICINE MOBILITY

ON THE

OF CELL

SUR-

Percentage myosin(-)

Percentage my&n(-)

Percentage myosin(+)

Percentage myosin(+)

restriction(-)

restriction(+)

restriction(-)

restriction(+)

None

40.3 30.8

0.5 0.7

12 11.7

47.3 56.8

None CB 15 pg/ml + 40 pg/ml

47.1

0

13.7

39.3

eolchicine

39.9

0.4

7.6

52.1

43.9

0

14.6

41.5

34.3

0.5

14

51.3

CB 15 /.&ml

None 40 pg/ml

colchicine D 15-day cultures

of M23SlO myogenie

cells were used in each case.

MARUSICH

AND SIMPSON

Surface Changes during

327

M~ogemsis

by Grant PCM-7904200 from NSF and Grant AG-014’76 from NIH. This paper represents a portion of a thesis submitted by Michael F. Marusich to the Graduate School, Northwestern University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

FLATTENED CELLS PLUMP BIPOLAR CELLS ROUNDED MITOTICS

REFERENCES

A 8

‘unrestricted

I ,

myosln (+) Ag 1422 (+) lateralmobility

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rounded-up myosin (+ I Go(irreverslble) Ag 1422 unrestricted

Biol. 47, 237-256.

t-1 lateral

mobility

63

I

myosm (+) Ag 1422 (+) restricted lateral

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J / FIG. details.

11. Current

NON FUSION MEDIUM strmted mononucleated Ag 1422 (+) restricted lateral mobility

view

of in vitro

striated multinucleoted Ag 1422 (+) restricted lateral mobility

lizard

myogenesis.

See text

for

ulations exist during in vitro lizard myogenesis (Fig. 11). These are (1) rapidly cycling, spread out myoblasts; (2) rounded-up myoblasts which are in an extended G1, but are still proliferation competent; (3) rounded-up, Go, myosin (+), Ag1422 (-) myocytes; 4) rounded-up, Go, myosin (+) myocytes which are Ag1422 (+) and restriction (-); (5) rounded-up Go, myosin (+) myocytes which are Ag1422 (+) and restriction (+); and (6) stretched-out myosin (+) myocytes or myotubes, which are Ag1422 (+) and restriction (+). These phenotypes are common to myogenesis as it occurs in cells derived from both regenerating adult muscle and developing embryonic muscle. The expression of these developmentally regulated phenotypes have remained stable in myogenic line M23SlO through 6 years of continuous culture involving over 1500 cell generations. Work in progress is directed toward assessing the functional role of Ag1422 and defining the basis for the restriction in lateral mobility of cell-surface antigens. The of Mr.

authors wish to acknowledge the expert Mark T. Siragusa. The research reported

technical assistance here was supported

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