Myogenesis in primary cell cultures from drosophila melanogaster: Protein synthesis and actin heterogeneity during development

Cell,

Vol. 13, 589-598,

April

1978, Copyright

0 1978 by MIT

Myogenesis in Primary Cell Cultures from Drosophila melanogaster: Protein Synthesis Actin Heterogeneity during Development Robert V. Storti, Sharon J. Horovitch, Matthew P. Scott, Alexander Rich and Mary Lou Pardue Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02139

Summary Muscle cell cultures from Drosophila melanogaster were obtained by plating dissociated gastrula stage embryo cells on protamine-treated culture dishes. The myogenic cells in these cultures fuse to form multinucleated pulsating cells by 15 hr after plating. An analysis of protein synthesis during myogenesis in these cultures, as measured by the incorporation of ?5-methionine and analyzed by two-dimensional polyacrylamide gel electrophoresis, showed profound changes in the pattern of protein synthesis. This analysis enabled us to identify three distinct classes of proteins. Class A proteins, the most abundant, are synthesized continuously throughout myogenesis; class B proteins are those proteins whose synthesis is initiated during myogenesis and continued throughout development; class C proteins are those synthesized at specific times during development. In addition, three forms of actin have been identified in these cultures. Actin I, which shows increased synthesis concomitant with the myogenic development in these cultures, is apparently a muscle-specific form of actin. Actin II, the predominant “cytoplasmic” form of actin in the nonmuscle Schneider cell line 2, is also the major form in the gastrula cultures before differentiation begins. Synthesis of this actin continues in the myogenic cultures. Actin III is a rapidly turning over form of actin which does not accumulate in either the Schneider cells or the myogenic cultures. Introduction Differentiating muscle cells carry out a developmental program which includes the synchronous fusion of mononucleated myoblast cells to form multinucleated myotubes, the elaboration of the myofibrillar apparatus for muscle contraction, and the reorganization of the surface membranes for controlled excitation. With the advent of cell culture techniques which enable a meaningful biochemical analysis of this process in vitro, myogenesis has become an attractive system for studying the regulation of cellular differentiation. Seecof and his collaborators (Seecof and Unanue, 1968; Seecof, 1971; Seecof et al., 1971) have

and

described a useful system for studying myogenesis in primary cultures of Drosophila melanogaster. These cultures, which are derived from early gastrula embryos (3-3.5 hr of age at 26+05”C), permit development in vitro of primarily nerve, muscle and “epithelial” cells. The myogenic cells, in particular, undergo a predetermined and relatively synchronous developmental sequence including a single mitotic division, cell elongation, aggregation, alignment, fusion (to form myotubes and muscle sheets) and pulsation. To characterize more accurately the biochemical changes that occur during myogenesis in these cultures, we have sought cell culture conditions which preferentially select for the attachment and growth of myogenic cells. The myogenic cultures which we have established undergo a developmental sequence very similar to that of the myoblasts in Seecof’s heterogeneous cultures. These culture conditions have permitted us to study an in vitro system of muscle differentiation which apparently parallels the developmental scheme in the intact organism. We report here a study of protein synthesis during myogenesis in these cultures as analyzed by high resolution two-dimensional polyacrylamide gel electrophoresis. This analysis has allowed us to identify the synthesis of three distinct classes of proteins specific to the developmental scheme. Included in this analysis is the identification of three different forms of actin, one of which appears to be specific to myogenesis. Results Myogenesis in Drosophila primary cell cultures as reported by Seecof et al. (1973) consists of a sequence of morphological and developmental changes similar to those normally occurring in myogenic cell cultures of vertebrates (Konigsberg, 1963; Yaffe and Feldman, 1964; Cox, 1968; Konigsberg, 1971; Buckingham et al., 1974; Du Prat et al., 1975). Upon plating, Drosophila gastrula cells settle, and within 30 min, they are firmly attached to the substrate. The cells of these heterogeneous cultures initially appear flat and polygonal and show no evidence of elongation or fusion. Little sign of differentiation is evident initially, except for a single cell division occurring between 5-7 hr (at 26 +- 05°C) (Seecof et al., 1973). This is followed by elongation, aggregation and alignment. Cell fusion is essentially complete by about 24 hr after plating. Before undertaking any biochemical analysis of this in vitro myogenic phenomenon, we set out to establish cell culture conditions which would allow selective attac.hment and growth of the myogenic

Cell 590

cells from the mixed gastrula cell populations. It has been observed that myogenic cells adhere poorly, if at all, to glass coverslips, while neural and “epithelial” cell types settle and develop on glass. Thus it seemed possible that the surface requirements of myogenic cells might differ from those of other cell types in our culture system. Our approach to cell selection, therefore, was to attempt to modify the surface onto which the cells adhered. McKeehan and Ham (1976) have used polyamine monolayers to enhance adhesion of normal human and chick fibroblasts to plastic tissue culture dishes by increasing the positive surface charge of the substrate. We prepared monolayers of several polyamines: polylysine, gelatin and protamine. Of these three, protamine preferentially enhanced the attachment of Drosophila myogenic cells and supported the fusion of resulting myocytes to myotubes and muscle sheets. Plating early Drosophila gastrula cells on protamine monolayers yields relatively pure myogenic cultures, while other cell types show little or no adhesion and development. Characteristics of Myogenic Cultures Plated on Protamine Following initial plating, the cultures consist primarily of flat, mononucleated polygonal cells (Figure 1A). During the next 24 hr, the cells undergo a dramatic sequence of morphological changes which culminate in the formation of mature multinucleated myotubes and muscle sheets, both of which pulsate. The myotubes seen in these mature cultures (>I5 hr) generally contain from 2-25 nuclei per cell, the large majority having IO-15 nuclei per cell (Figure 1 B). As a measure of the myogenic development in these cultures, we have determined the proportion of nuclei found in multinucleated cells at different times after plating (Figure 2). These counts were made on all the cells found in monolayer regions of the cultures where cell boundaries could be determined unambiguously. We cannot accurately measure fusion in the cell clusters which are scattered through the cultures; cells in these clusters, however, do begin to show one or more foci of pulsation at about the time such activity is seen in monolayer regions. As seen in Figure 2, prior to 4 hr in culture, fewer than 3% of the nuclei are found in multinucleated cells, but the proportion of such nuclei increases rapidly for the next 20 hr. The majority of the multinucleated cells seen prior to 8 hr contain 2-6 nuclei per cell. By 22 hr in culture, >80% of the nuclei seen are in multinucleated cells, and the majority of these cells containin IO15 nuclei. The remainder of the cell population, which remains uninucleate, may consist of non-

Figure Stage

1. Cells from Primary Myogenic Embryos of Drosophila Melanogaster

(A) Myogenic culture 5 hr after culture 24 hr after plating (600X). toxylin as described in Experimental

Cultures

of

Gastrula

plating (750X); (B) myogenic Cells were stained with hemaProcedures.

myogenic cells, myogenic cells arrested in development, or myogenic cells which are not normally programmed to undergo cell fusion. Protein Synthesis in Myogenic Cultures Myogenesis in cultures of vertebrate muscle has been shown to involve qualitative and quantitative changes in protein synthesis concomitant with fusion of mononucleated myoblast cells into multinucleated myotubes (Paterson and Strohman, 1972; Yaffe and Dym, 1972; Paterson, Roberts and Yaffe, 1974; Emerson and Beckner, 1975; Whalen, Butler-Browne and Gros, 1976). We have observed similar but more dramatic changes in protein synthesis in Drosophila myogenic cultures during the developmental sequence. These changes were analyzed by the incorporation of %-methionine into protein during 1 hr periods at various times after the initiation of the culture. The proteins synthesized during the 35S-methionine pulses were analyzed by high resolution two-dimensional polyacrylamide gel electrophoresis. Protein separation by this technique is dependent upon differences in overall net change in the isoelectric focusing dimension and differences in size in the SDS (sodium

Protein 591

Synthesis

during

Myogenesis

in Drosophila

‘0°1

‘.O

90

4

I

.9

;

? .a w +++ .7 !a

IO t

01

/

f

I

4

8 hours

I 12 after

I 16

I 20

I 24

plotlng

Figure 2. Index of Cell Fusion and Actin of Primary Myogenic Cell Cultures

Synthesis

during

Growth

The percentage of nuclei in multinucleated cells was determined as described in Experimental Procedures. I, II and Ill refer to Drosophila actins I, II and Ill, and are described in the text. The ratio of actin synthesis at each time was determined from the data of Table 1.

dodecylsulfate) gel electrophoresis dimension. After electrophoresis, the gels were stained with Coomassie blue to locate stable proteins accumulated during myogenesis, and then fluorographed to identify ?S-labeled proteins synthesized during the pulse. Fluorograms of two-dimensional gels depicting the more abundant proteins synthesized in myogenic cultures during 1 hr intervals at the various times after plating are shown in Figure 3. Approximately 150-200 proteins synthesized in these cuitures are resolved by the gels. The most striking feature of the fluorograms is the abundance of three specific proteins present at all stages of myogenesis. These proteins are labeled I, II and Ill in Figure 3 (3 and 18 hr). The identification and characterization of these proteins are discussed below. The remainder of the proteins shown here fall into three discrete classes. The first class consists of those proteins synthesized continuously throughout myogenesis. These proteins are present early in development (Figure 3; 3 and 6 hr) and continue to be synthesized at the latest times studied (Figure 3; 18 hr). The majority of the proteins synthesized fall into this class. On close inspection, however, it becomes obvious that during the intermediate stages of myogenesis, there are qualitative and quantitative

changes in the synthesis of several time-specific classes of abundant proteins. For example, an examination of the proteins synthesized at later times of development shows the appearance of a second class of proteins, those for which synthesis is initiated during the developmental sequence and apparently continues in the differentiated culture. The more obvious of these new proteins are indicated by arrows in Figure 3 (18 hr). This class of proteins represents an increase of approximately 20% in the number of new protein species synthesized during myogenesis. These proteins are not found in 3 or 6 hr cultures (Figure 3; 3 and 6 hr). Finally, there is a third class of proteins whose synthesis appears to be tightly coupled to specific stages of development. For instance, the prominent proteins indicated by arrows in Figure 3 (8 hr) are not synthesized in 3 hr cultures. One of these proteins first appears in 6 hr cultures, and all reach a maximum rate of synthesis in 8 hr cultures. These proteins are not seen, or are in greatly reduced amounts, in 15, 16 or 18 hr cultures. The period of maximum synthesis of these proteins coincides with the early stages of fusion (when ~25% of the nuclei are seen in multinucleated cells). Another major protein, indicated by an arrow in Figure 3 (15 hr), reaches its maximum rate of synthesis at 15 hr in culture, at the time when >50% of myocyte nuclei are found in multinucleated myotubes. Much less label is incorporated into this protein before or after 15 hr. It should be emphasized that the proteins observed here represent only the more abundant proteins synthesized and detected by our conditions of fluorography, and represent a minimum estimate of changes that occur. Nevertheless, it is clear that Drosophila myogenesis involves a substantial change in the types of proteins synthesized. We have grouped the proteins into three distinct classes. Class A proteins are proteins synthesized continuously during the entire developmental sequence. Class B proteins are those whose synthesis first appears some time after plating and continues throughout myogenesis. Class C proteins are proteins whose synthesis appears to be restricted to limited intervals during myogenesis. Since we have not yet identified these proteins, we can only speculate that they are probably regulatory and structural components required for cellular differentiation and myofibrillar structure and function. Actin Characterization in Drosophila Cell Cultures The two most abundantly labeled proteins in 3 hr cultures, designated II and III in Figure 3 (3 hr) (also present in the other cultures) resemble the actin

Cell 592

PH

Figure 3. Time Course of Protein Labeled Polypeptides Synthesized

5

Synthesis during Differentiation of Primary Myogenic during a 1 Hr Pulse and Separated by Two-Dimensional

PH

Cell Cultures: Fluorograms Gel Electrophoresis

of %-Methionine-

A single homogenate of Drosophila gastrulae was subdivided into equal aliquots, each of which was maintained at 26°C and labeled for 1 hr at the indicated time after plating. Total cell homogenates, each containing 64,000 cpm, were loaded onto 130 mm isoelectric focusing gels for first dimension electrophoresis. Fluorography was for 12 days. I, II and Ill indicate actins. Arrows indicate some of the proteins which show significant changes in relative intensity of synthesis during the developmental sequence (see text).

Protein 593

Synthesis

during

Myogenesis

in Drosophila

purified from adult chick skeletal muscle (pl 5.72) in both molecular weight (44,000 daltons) and isoelectric points (pl 5.77 and 5.84). Because of their relative abundance, their similarity to chick muscle actin in isoelectric point and molecular weight, and the analogy with actin synthesis in vertebrate cells, it was considered probable that one or both of these proteins were actin. Initial studies to confirm this identity were made by examining protein synthesis in a stable continuous suspension culture line derived from the Schneider cell line 2 (Schneider, 1972; Lengyel, Spradling and Penman, 1975). Figure 4A shows a proteins fluorogram of %-methionine-labeled synthesized during a 1 hr pulse label. Like 3 hr myogenic cells, these cells contain two prominent proteins with isoelectric points and molecular weights similar to those of chick muscle actin (Figure 4A, II and Ill). A convenient method for preliminary characterization of actin utilizes its ability to bind to DNAase I-agarose (Lazarides and Lindberg, 1974). The binding to DNAase I has been shown to be specific for actin. Accordingly, 35S-methionine-labeled protein cytoplasmic extracts were prepared and tested for their affinity to DNAase I-agarose. Figure 4B shows that the only proteins retained by affinity chromatography to DNAase I, as determined by two-dimensional gel electrophoresis, were the two presumptive actins. This result strongly supports the tentative identification of these two proteins as different forms of actin. The small amount of radioactivity at the approximate position of actin I in Figure 48 is due to nonspecific “sticking” of radioactivity to the chick muscle actin added as a marker prior to electrophoresis and used to identify the precise positions of actins II and III. It is not seen when chick muscle actin is not added to such preparations. When the Coomassie blue staining of the presumptive actin from these cells was examined, however, only one major protein was detected in the actin region of the gel (Figure 4C). This protein had an isoelectric point of 5.77 and was coincident with the more acidic (II) of the two %S-methioninelabeled presumptive actin proteins. The ?S-methionine-labeled presumptive actin (III) at pl 5.84 had no Coomassie blue stain associated with it, suggesting that this actin is short-lived and does not accumulate. The same staining pattern was found in myogenic cultures such as those shown in Figure 3. In none of these cultures was there detectable Coomassie blue stain coincident with presumptive actin III. The unstable nature of actin III was further supported by analyzing the pattern of proteins synthesized in post-fusion cultures labeled for a 24 hr period starting after 18 hr in

I3

”

-

*

5”

Figure 4. Two-Dimensional Extracts from Schneider

Gel Electrophoresis Line 2 Cells

of Total

Cellular

First dimension isoelectric focusing was in a 130 mm gel for (A) and (C) and a 220 mm gel for (B). (A) %-methionine-labeled polypeptides synthesized during a 1 hr pulse. Autoradiography was for 2 days. (B) %-methionine-labeled proteins retained by DNAase-agarose affinity column chromatography of a total cytoplasmic extract of Schneider line 2 cells. The actins were eluted with 3.0 M guanidine-HCI. A sample of 22,000 cpm was eiectrophoresed and autoradiography was for 3 days. (C) Coomassie blue-stained proteins of the gel in (A). II and I11indicate actins.

culture. During this long-term 35S-methionine labeling period, the labeling of presumptive actin III was greatly reduced in amount relative to presumptive actin II (not shown).

Cell 594

Time Course of Actin Synthesis during Myogenesis in Culture An examination of protein synthesis during development of the myogenic culture shows, in addition to the two presumptive actins present in all the Drosophila cells studied, the gradual appearance of a new major protein with molecular weight and isoelectric point (pl 5.70) similar to those of chick skeletal muscle actin (Figure 3; 18 hr, I). The synthesis of this presumptive actin (actin I) is coincident with cell fusion (Figure 2). A comparison of the total radioactivity incorporated into actin I and the incorporation into actins II and III during a 1 hr pulse of myogenic cultures shows that the relative rate of synthesis of actin I increases by a factor of 4-5 during the culture period (Table 1). During this same period, the relative rates of synthesis of actins II and III change little, if at all. A concomitant increase in the accumulation of Coomassie bluestained protein was also noted (not shown). This increase in stained material indicates that actin I is a relatively stable component of the differentiating cultured cells. A fusion-related form of actin has been shown to be synthesized during myogenesis in vertebrate cells (Garrels and Gibson, 1976; Storti, Coen and Rich, 1976; Whalen, et al., 1976; Rubenstein and Spudich, 1977). By analogy, this suggested that the new protein in the Drosophila myogenic culture might also be a form of actin specific to myogenesis. The identity of these three presumptive actins from myogenic cultures was confirmed by a comparison of peptides generated by partial chymotryptic digestion of each of the proteins. 17 hr after plating, myogenic cultures were labeled for 3 hr in the presence of ?S-methionine. After electrophoresis, the position of the presumptive actins was located by autoradiography, and the individual polypeptides were cut from dried gels. The polypeptides were eluted by electrophoresis, chick muscle actin was added as a carrier and as an internal Coomassie blue-stained marker, and the

Table Hr after Plating

1. Incorporation

of ?S-Methionine

Actin

I

into Actin

Actin

II

in 1 Hr Periods

Actin 383

Ill

samples were partially digested with chymotrypsin under identical conditions. Each digest was then separated into individual peptides by pH 3.5-10 isoelectric focusing in the first dimension and by SDS slab gel electrophoresis in the second dimension. The results of these experiments are shown in Figure 5. A comparison of the fluorograms of the three presumptive actins shows that these three protein digests have several major 35S-methioninecontaining peptides in common. These common peptides include three major polypeptides with molecular weights of 44,000, 41,000 and 36,000 daltons. Moreover, these three polypeptides were also the major polypeptides of the Coomassie bluestained chick muscle actin carrier co-electrophoresed in the same gels. Similar high molecular weight polypeptides are obtained when actin is partially digested with trypsin (not shown). Several of the lower molecular weight polypeptides are also common to all three Drosophila actins and to chick muscle actin. The lower molecular weight polypeptides that co-electrophoresed with Coomassie blue-stained chick muscle actin are indicated by arrows. These similarities between Drosophila and chick muscle actin indicate a significant evolutionary conservation of the molecule between the two phyla (~50% of the 35S-methionine-containing Drosophila peptides were coincident with Coomassie blue-stained chick actin peptides). A control digest consisting of a 55,000 dalton molecular weight Drosophila protein of unknown identity and unlabeled chick muscle actin was performed to demonstrate the specificity of the partial digestion and to demonstrate that no nonspecific “sticking” of radioactive peptides to cold carrier peptides present in excess occurred. This control digest (Figure 5C) showed no similarity to any of the Drosophila actin digests, nor did the unknown Drosophila protein show any homology to the Coomassie blue-stained chick actin. For simplicity and to avoid possible confusion

during

Myogenesis I

III

I

0

ii

(II + Ill)

0.29

0.80

0.16

3

136

477

6

296

510

749

0.58

1.47

0.24

8

563

801

735

0.70

0.92

0.37

15

1144

1023

586

1.12

0.57

0.71

16

513

544

411

0.94

0.76

0.54

18

787

708

541

1 .I1

0.76

0.63

The actin spots were cut from dried gels shown in Figure 3, swelled in 0.5 ml of 90% NCS solubilizer (Amersham) at 37°C for 24 hr and counted in 10 ml toluene Liquifluor scintillant. A similar cut from a blank area of the gel was used to determine the background of 50 cpm which has been subtracted from each experimental value.

Protein 595

Synthesis

during

Myogenesis

10

Figure

5. Partial

in Drosophila

3

PH

Chymotryptic

Digestion

Fingerprint

of %-Methionine-Labeled

3

PH

10

Peptides

of Drosophila

Actins

I, II and Ill

Details are given in Experimental Procedures. Drosophila actin samples I, II and Ill contained 10,500, 25,000 and 7,500 cpm, respectively. (C) is an unknown Drosophila protein with a pl of 5.5 and a molecular weight of approximately 55,000 daltons. (C) contained 12,600 cpm. Fluorography was for 1 month. The numbers refer to molecular weights x 1000 daltons. Peptides indicated by arrows were coincident with Coomassie blue-stained chick muscle actin peptides co-digested and electrophoresed in the same gel.

with multiple actins reported in other systems, we have named these three Drosophila actin species I, II and III (in order of increasing basicity). Discussion Myogenesis in primary cell cultures from Drosophila is very similar to the developmental sequence observed in vertebrate myogenic cultures. Unlike vertebrate cultures, however, myogenesis in these Drosophila cultures appears to be quite synchronous and essentially complete by 24 hr. This is a distinct advantage for analysis of biochemical differentiation over short periods of time. Indeed, the degree of synchrony in the cultures is reflected by the discrete quantitative and qualitative changes in protein synthesis that we observe during development. The developmental sequence reported here

shows much more pronounced changes in the pattern of protein synthesis than have been observed in primary cell cultures from vertebrates (Whalen et al., 1976; I?. V. Storti, unpublished observations). As a result of the striking nature of these changes in protein synthesis (Figure 3), we have been able to detect three distinct classes of proteins. Our classification scheme is based on the time and duration of synthesis of these proteins during the developmental sequence. We have called the first and most abundant class of proteins class A proteins. These proteins are synthesized continuously throughout myogenesis and are present at each stage of development in Figure 3. As a group, these proteins are probably responsible for maintaining general cellular structure and function as so-called “household” proteins.

Cell 596

A second class of proteins, class B proteins, includes those proteins which are not observed in 3 or 6 hr cultures, but whose synthesis is initiated during myogenesis and continues indefinitely. These proteins are indicated in Figure 3 (18 hr) and account for approximately 20% of the protein species synthesized in 18 hr cultures. The third class of proteins, which we have called class C proteins, are those synthesized at specific times during myogenesis but are not found in mature cultures. This class contains the smallest number of detectable proteins (Figure 3; 6, 8 and 15 hr). Unfortunately, myofibrillar proteins of Drosophila and dipterans in general are not well characterized. We can only speculate that at least some of these proteins, particularly class B and C proteins, are specialized proteins required for regulation and control of myogenesis, as well as structural proteins involved in myofibrillar structure and function. We have identified three of the most prominent proteins observed in these cultures as actin. Two of these actins occur in both myogenic (II and III in Figure 3; 3 hr) and nonmyogenic cells (II and III in Figure 4) and probably represent nonmuscle forms of actin. The third form, actin I, appears to be muscle-specific, since synthesis is concomitant with myogenic fusion. We have not found actin I in nonmuscle cells, such as those of the Schneider line (Figure 4), early blastulae or salivary glands (unpublished observations). A fusion-related form of actin in Drosophila myogenic cultures has also been observed by E. Fyrberg and J. Donady (personal communication). Multiple forms of actin and fusion-related forms of actin have been reported in muscle and nonmuscle cells of vertebrates (Garrels and Gibson, 1976; Storti et al., 1976; Whalen et al., 1976; Rubenstein and Spudich, 1977). On the basis of protein stability, it appears that actin III in our cultures may represent an unstable form of actin. The role of this unstable form of actin is unclear; unstable minor forms of actins, however, have been reported for rat (Garrels and Gibson, 1976) and have also been observed in chick (R. V. Storti, unpublished observations). The unstable actins have half-lives of <2 hr in rat and <0.5 hr in chick. The role of these actins in the cell is still unclear; thus to avoid any unwarranted correlation with the multiple actins of vertebrate cells, we have adopted the nomenclature I, II and III (in order of increasing basicity) for the Drosophila actins. In summary, actin synthesis in Drosophila appears to be similar to that of vertebrates in that there are multiple forms of actin. One of these actin forms appears uniquely in muscle cells. Experimental

Schultz and obtained as a gift from Dr. Marilyn Sanders, was used throughout. Flies were maintained at 21°C and allowed to lay eggs for 3 hr periods on standard cornmeal-agar medium coated with autoclaved yeast paste to encourage egg laying. Asynchrony in the development of eggs was minimized by discarding all eggs laid during the first hour of the egg-lay period; in addition, the low temperature at which the eggs were laid slowed the early development of the embryos and thus helped to synchronize the cultures derived from these embryos. Embryos were gathered by gently scraping the fly food surface with a rubber policeman. They were suspended in 30% sucrose to remove most of the yeast, and then rinsed with distilled water into a nylon net using a Buchner funnel under mild aspiration. Rinsed embryos were dechorionated [30-60 set in 1 :I 5% hypochlorite (chlorox)/95% ethanol], surface-sterilized (0.05% HgCl,/70% ethanol) and rinsed in Ringer’s solution (Seecof, 1971). Embryos were staged visually using reflected light. The ventral furrow stage of gastrulation is desirable for plating. Any obvious blastulae or neurulae were removed with sterile forceps. Preparation of Culture Dishes for Muscle Enrichment 1 ml of a sterile 0.1 mglml protamine solution was spread over the surface of the 35 and 60 mm Falcon plastic tissue culture petri dishes to be used for cell culturing. After 5 min, the protamine solution was removed with a sterile Pasteur pipette, and the petri dishes were rinsed twice in autoclaved glass-distilled water and used immediately. Plating of Gastrula Cells Two cell plating procedures were employed. The first, used primarily for pilot experiments, was based on the procedure originated by Seecof and Donady (1972). Gastrulae were selected with sterile forceps and punctured with a sterile, siliconized micropipette (with a 50 p diameter tip) attached to a mouthpipette apparatus. The cell contents were withdrawn from the vitelline membrane and dispersed in 2 ml of growth medium (Seecof and Donady, 1972) in a Falcon plastic tissue culture petri dish (35 mm) freshly coated with a monolayer of protamine. About 100 embryos could be plated this way while still maintaining relatively good synchrony (total plating time was about 30 min). A homogenization procedure was devised to scale up the culturing procedure. Dechorionated gastrulae were transferred to a Thomas AA Teflon-pestle homogenizer containing culture medium and were homogenized with five strokes. The homogenate was poured into a sterile Falcon capped test tube and spun at low speed in a clinical centrifuge for 3 min to pellet vitelline membranes, unbroken embryos and debris. The supernatant was poured into a 60 mm protamine-coated Falcon tissue culture petri dish, and the cells were allowed 10 min to settle. The medium was replaced and the cultures were incubated at 26°C in a humid atmosphere. Index of Cell Fusion Seven 20 embryo cultures were plated and allowed to develop for the desired time. The cultures ware then rinsed in Ringer’s solution and fixed for 24 hr in phosphate-buffered saline containing 10% formalin. The cultures were subsequently dehydrated in 70% ethanol for 20 min and 95% ethanol for 20 min, and then stained in hematoxylin for 20 min. Nuclear counts ware obtained by methodically scanning the petri dishes from end to end, and counting the nuclei from all cells whose boundaries were clear and unambiguous. (Nuclei of cells in tight clumps could not be unambiguously counted. Each such clump, however, was commonly observed to demonstrate l-5 foci of pulsation in mature cultures.) Each point in Figure 2 represents approximately 1400 nuclei.

Procedures

Collection of Drosophila The P2 line of Oregon-R,

melanogaster Gastrulae a line originally isolated

by Dr. Jack

Labeling of Myogenic Cell Cultures with %-Methionine Cell cultures were rinsed twice in Ringer’s solution and labeled in 2 ml of met (-) medium containing 200 &i/ml

then 35S-

Protein

Synthesis

during

Myogenesis

in Drosophila

597

methionine, 700-1200 Ci/mmole was performed at 26°C F 0.5”C.

(Amersham-Searle).

All labeling

Growth, Labeling and Fractionation of Tissue Culture Ceils The Schneider line 2 Drosophila melanogaster cultured cells (Schneider, 1972) have been adapted to spinner culture and defined medium (Lengyel et al., 1975). Cells are grown at 25°C in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 0.5% lactalbumen hydrolysate, MEM nonessential amino acids, 50 units per ml penicillin and 50 pg/ml streptomycin (all Gibco). These cells double about every 25 hr, growing at 2.5-6 x 106/ml. For labeling with 35S-methionine. cells were centrifuged at low speed and resuspended in the same medium without methionine or lactalbumen hydrolysate. The fetal calf serum used in this medium was dialyzed extensively against phosphate-buffered saline and sterilized by filtration. The cells were centrifuged after the wash and resuspended at 5-10 times their normal concentration in the same methionine-free medium. Cells were labeled using 50 pCi/ml 35S-methionine for 1 hr. Extracts were prepared from 100 ml of cells by washing the labeled cells in ice-cold phosphate-buffered saline and then resuspending the cell pellet in 2 ml of hypotonic lysis buffer [IO mM Tris, IO mM NaCl, 10 mM CaClz (pH 8.3)]. The cells were broken with a small Dounce-type glass homogenizer. The nuclei were broken by passing the extract several times through a 26 gauge needle. Unsolubilized material was pelleted by a IO min 5000 x g centrifugation. The supernatant was fractionated on a DNAase-agarose column as described by Lazarides and Lindberg (1974). When separate nuclear and cytopiasmic fractions were desired, nuclei were pelleted from the cell homogenate by a 1200 x g centrifugation for 4 min. No difference in the pattern of actins seen by two-dimensional gel electrophoresis was observed between cytoplasmic and nuclear extracts. Sample Preparation and Two-Dimensional Poiyacryiamide Gel Electrophoresis of Proteins from Myogenic Ceil Cultures Following labeling in ?S-methionine, primary cell cultures were washed with saline and drained. Cellular extracts were prepared by lysing the cells directly in 50 ~1 of isoelectric focusing buffer [9.5 M urea, 2% NP-40, 2% ampholines (80% pH 5-7, 20% pH 3.5 10, LKB), 0.5% SDS]. The culture plate was scraped thoroughly with a rubber policeman, and the extract was used directly for electrophoresis. Two-dimensional polyacrylamide gel electrophoresis was modified from O’Farrell (1975). 25-60 *I samples of extract were electrophoresed in 2 x 130 mm (or 2 x 220 mm) glass tubes on isoelectric focusing gels containing a pH 5-7 gradient. Samples were electrophoresed at 500 or 1000 V, respectively, for 16-20 hr. After electrophoresis, the isoelectric focusing gels were equilibrated for 30 min in 10% (w/v) glycerol, 0.1 M dithiothreitol, 0.0625 M Tris-HCI (pH 6.8), and either frozen in dry-ice ethanol and stored at -80°C or electrophoresed directly in the second dimension. Second dimension electrophoresis was in 12% SDS-polyacrylamide slab gels according to Laemmli (1970), except that the stacking gel contained 2.5 M urea as described previously (Storti et al., 1976). The addition of urea facilitated overlaying the stacking gel with 0.1% SDS. The isoelectric focusing gel was layered on top of the stacking gel and sealed with 1% agarose in equilibration buffer. The gels were electrophoresed and stained with Coomassie brilliant blue as described previously (Storti et al., 1976). The gels were either dried for autoradiography (Figure 4) or fluorographed (Figures 3 and 5) (Bonner and Laskey, 1974) using prefogged XR-5 x-ray film (Laskey and Mills, 1975). Partial Chymotryptic Digestion Fingerprint of Drosophila Actins Drosophila actins I, II and Ill were separated by two-dimensional gel electrophoresis. The 35S-methionine-labeled presumptive actins were located by autoradiography, cut from dried gels and eluted by electrophoresis as described previously (Storti and Rich, 1976). After addition of 20 pg of purified adult chick skeletal

muscle actin. the samples were dialyzed overnight at 5°C against water and then lyophilized to dryness. The resultant pellets were resuspended in 200 ~1 of IO mM Tris (pH 7.4), 0.5% SDS and digested with 4 yg ol-chymotrypsin (Worthington) for 10 min at room temperature. The digestion was stopped by freezing in dry ice-ethanol and the samples were lyophilized to dryness. The digest was resuspended in 25 ~1 of isoelectric focusing buffer, and the peptides were separated by two-dimensional gel electrophoresis as described above. The first dimension isoelectric focusing gel contained a 3.5-10 pH gradient, and the second dimension was in a 15% slab gel. After electrophoresis, the gels were stained with Coomassie brilliant blue and prepared for fluorography as described above. Acknowledgments We are grateful to James Donady for introducing us to the embryonic culture of Drosophila. R.V.S. is a fellow of the Muscular Dystrophy Society of America. S.J.H. is the recipient of an Imperial Oil Company of Canada graduate research fellowship. This work was supported by grants from the NIH to M.L.P. and A.R. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

October

3, 1977; revised

January

12, 1978

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