Fusion of chick embryo skeletal myoblasts: Role of calcium influx preceding membrane union

DEVELOPMENTALBIOLOGY 82,297~30'7 (1981)

Fusion of Chick Embryo Skeletal Myoblasts: Role of Calcium Influx Preceding Membrane Union JOHN D. DAVID,~ WILLIAM M. SEE AND CHRYS-ANNHIGGINBOTHAM Division

of Biological Sciences, University

Received November

of Missouri,

19, 1979; accepted in revised

Columbia,

Missouri

65211

form August 22, 1980

Extracellular calcium is required for normal progression through the initial recognition-alignment phase of myogenesis. We have found that a net calcium influx into fusion-competent myoblasts is a requisite step in membrane fusion, in addition to the previously demonstrated requirement for extracellular calcium. This conclusion is based on the following evidence: (1) a measurable increase in net calcium influx occurs just prior to fusion; (2) ethylene glycol his@-aminoethyl ether) N,N-tetraacetic acid can block the fusion process at a stage after alignment but prior to membrane union; (3) the calcium ionophore A23187 induces precocious fusion; and (4) the calcium channel blocker D600 inhibits cell fusion but not alignment. Based on ultrastructural analyses of other investigators, we suggest a mechanism whereby the increase in intracellular calcium could trigger membrane union between two aligned, aposed, fusion-competent myoblasts. INTRODUCTION

The functional element of differentiated skeletal muscle, the nondividing multinucleate myotube, is formed by cytoplasmic fusion of mononucleated precursor cells, myoblasts. A majority of freshly isolated myoblasts, plated at low density, go through at least one round of DNA replication before fusion (Yaffe, 1971), with both plating density and medium composition determining the time of onset of fusion (Konigsberg, 1971; White and Hauschka, 1971). Once multinucleate cells appear, they rapidly form elongated myotubes as fusion progresses throughout the culture. Under appropriate culture conditions, fusion involves 60-80s of the cells, never reaching 100% even when the culture is initiated with a pure clone .of myogenic cells. Fusion is associated with a cessation of DNA synthesis and the accumulation of muscle-specific proteins (Stockdale and Holtzer, 1961). The fusion of mononucleate myoblasts is almost certainly a multistep process, including at a minimum the following separable compenents: (1) cell migration, recognition, and alignment, and (2) membrane fusion leading to cytoplasmic continuity (Nameroff and Munar, 1976; Knudsen and Horwitz, 1977). Both components, which we shall call recognition-alignment and fusion, have been studied extensively, although not always separately. Ultrastructural studies, necessarily a static treatment of a dynamic process, have suggested that fusion is the result of the partial disappearance of the plasma membrane between two adjoining cells (Lipton i To whom reprint requests should be sent. 297

and Konigsberg, 1972; Rash and Fambrough, 1973; Shimada, 1971; Kalderon and Gilula, 1979). Whether it is preceded by the formation of gap junctions, or particledepleted regions adjoining the plasma membranes, is a matter of debate (Rash and Staehelin, 1974; Rash and Fambrough, 1973; Kalderon et aZ., 1977; Kalderon and Gilula, 1979). Kalderon and Gilula (1979) have proposed a model for myoblast membrane fusion based on ultrastructural analyses in which they suggest that cytoplasmic particle-depleted vesicles, closely aposed to the plasma membrane, fuse with the plasma membrane thus generating particle-depleted plasma membrane domains which they argue are an essential component of the fusion process. Biochemical (Hynes, 1976; Moss et al., 1978; Pauw and David, 1979) and immunological (Friedlander and Fischman, 1975) analyses of the myoblast cell surface have revealed consistent alterations in cell surface proteins prior to fusion. In particular, Pauw and David (1979) observed several consistent alterations in the proteins accessible to lactoperoxidase-catalyzed iodination during the periods of alignment and fusion. Such alterations in the plasma membrane protein complement are consistent with the observation that the strength of cell-cell adhesions increases with time in culture (Knudsen and Horwitz, 1977). A number of agents which might be expected to alter either membrane structure and integrity or cell-cell adhesions are effective inhibitors of myoblast fusion. An increase in membrane fluidity apparently precedes myoblast fusion (Herman and Fernandez, 1978; Prives and Shinitzky, 1977) and perturbations which are ex0012-1606/81/040297-11$02.00/O Copyright All rights

0 1981 by Academic Press, Inc. of reproduction in any form reserved.

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DEVELOPMENTALBIOLOGY

pected to alter fluidity alter the recognition and fusion processes (van der Bosch et al., 1973; Knudsen and Horwitz, 1978). Furthermore, phospholipase C treatment (Nameroff et al., 1975) does not affect normal alignment but does inhibit membrane union, while direct (Shainberg, 1969; Nameroff and Munar, 1976) or indirect (Paterson and Strohman, 1972) reductions in extracellular calcium level inhibit the recognition-alignment and thus also the fusion processes. The involvement of Ca2+ in the fusion of biological membranes is an almost universal phenomenon (reviews, Papahadjopoulos et al., 1978, 1979). Although a direct role for Ca2+in the distinct process of membrane union during myogenesis has not been documented, an absolute requirement for Ca2+in membrane fusion during fertilization (Schackmann et al., 1978), cellular secretion (Miller and Nelson, 1977), virus-induced erythrocyte fusion (Volsky and Loyter, 1978), and acetylcholine release in presynaptic nerve endings (Douglas, 1974) has been demonstrated. In this communication we present evidence that a net calcium movement into fusion-competent myoblasts is a requisite step in myoblast membrane fusion, this in addition to the requirement for extracellular calcium in the recognition-alignment process. Our data complement the previous ultrastructural analyses of Kalderon and Gilula (1979) and lend biochemical support to their model of myogenic membrane fusion. MATERIALS

AND METHODS

Skeletal Muscle Cell Cultures Cultures were prepared by a modification of the procedure of Konigsberg (1971). Twelve-day-old white leghorn chick embryo thigh muscle was dissected free of skin and bone, minced, and incubated for 10 min at 30°C in 0.05% trypsin (1:250; DIFCO) in Puck’s saline G (GIBCO) with gentle agitation. The supernatant was discarded. The tissue clumps were rinsed with Pucks saline G and reincubated for 10 min at 30°C in 0.05% trypsin (1:250; DIFCO) in Puck’s saline G with gentle agitation. The supernatant from the second incubation was adjusted to 20% horse serum to neutralize the trypsin and the cells were collected by centrifugation. The cell pellet was resuspended in Dulbecco’s modified Eagle’s medium (DME;2 GIBCO) and filtered through four layers of lens paper. The cell suspension was “preplated” at 8 X 10” cells/l50-mm noncollagen-coated tissue culture dish in DME + 10% preselected horse ’ Abbreviations used: DME, Dulbecco’s modified Eagle medium; EBSS, Earl&s balanced salt solution; EGTA, ethylene glycol bis(Paminoethyl ether) N,hP-tetraacetic acid; DMSO, dimethyl sulfoxide.

VOLUME82, 1981

serum (GIBCO) + 3% extract + 1% embryo antibioticantimycotic solution (GIBCO) and incubated at 37°C in a 5% CO2 atmosphere. After 50 min, the plates were gently rocked to dislodge any settled but nonattached cells and the supernatant withdrawn with a pipet. Cells were then plated at 0.4 X lo5 tells/35-mm collagen-coated tissue culture dish in the above medium. The culture media was changed at 24 hr and every 48 hr thereafter unless specifically noted in the experimental protocol. Cultures prepared in this fashion were routinely composed of greater than 95% bipolar, spindle-shaped, presumptive myoblasts. Chick embryo extract was prepared from decapitated 12-day-old white leghorn chick embryos. The embryos were homogenized for a total of 1.5 min in an equal volume (w/v) of Earle’s balanced salt solution (EBSS; GIBCO) at the highest speed of an Osterizer blender. The resulting homogenate was incubated for 1 hr at 4°C and then stored at -20°C overnight. The thawed extract was clarified by centrifugation at 44,400~ for 60 min. After removal of the surface lipid layer, the supernatant was sterilized by filtration through a 0.2-pm filter and stored at -70°C. All steps in the procedure were performed at 0-5°C except where noted. Measurement of Cell Fusion Plates to be scored for cell fusion were rinsed two times with Puck’s saline G and fixed for 20 min in 0.1 M Na2P04(pH 7.4)-4% formaldehyde. The fixed cultures were cleared in Carnoy’s solution (4:1, ethanol:acetic acid) for 60 min, stained in Harris hematoxylin solution (So-H-20; Fischer Scientific) for 20 min, and developed with 1% NH40H for 10 min. The plates were rinsed with water and stored wet until counted. Mononucleate cells were classified as myoblast or fibroblast based on direct microscopic examination of their morphology, cells being designated as myoblasts only if they had a marked bipolar, spindle-shaped morphology. Cell fusion was determined by direct microscopic examination at a total magnification of 300X. Cells were considered fused only if there was clear cytoplasmic continuity and at least three nuclei were present in each myotube. Each data point represents counts of greater than 20 randomly selected fields and greater than 400 total nuclei. Under these conditions, the standard deviation between several separate 400 nuclei-20 field counts of the same plate was never higher than * 20%. Percentage fusion = number of nuclei in myotubes/total number of nuclei in single cells and myotubes. All experiments were run in duplicate, and unless otherwise indicated, data points represent the average of the duplicates.

DAVID, SEE, AND HICGINBOTHAM

Calcium I&x

and Myoblast Fusion

299

Net 45Ca2+Injbux Net 45Ca2+influx was determined by a modification of the procedure of Putney et aZ.,(1978). Cultures to be assayed were incubated in complete medium +33 &i/ml 45CaC12(20.7 mCi/mg; New England Nuclear) for 1 hr at 37°C in a 5% CO2 atmosphere. The medium was removed and the plates rapidly rinsed twice with Puck’s saline G and once with LaC&-saline (5.4 mM KCl-137 mM NaCl-1 mM LaCL-6.1 mM glucose). LaC&Saline (1.5 ml) was added to each 35-mm dish and the dishes were rocked gently (Lab-Line platform rocker, 20” displacement) for 5 min at 30°C. The buffer was removed and the plate rinsed twice with LaC&-saline at room temperature. The culture was harvested by lysis in 0.2 ml 1 mM Tris (pH 7.4)-0.1% SDS. The lysate was added to a glass scintillation vial to which 1.0 ml tissue solubilizer (NCS; Amerscham/Searle) was subsequently added. The vials were tightly capped and stored in the dark overnight. Samples were counted following the addition of 10 ml scintillant (Omnifluor-toluene; New England Nuclear). All time points represent the average of triplicate samples. The net 45Ca2+influx was normalized to the number of nuclei as determined in parallel cultures. The ratio of nuclear number or DNA mass to cytoplasmic volume (as estimated by the mass ratio of DNA to total cellular protein) did not change until 96 hr after the initiation of culture, at which time fusion was complete and contractile protein synthesis was well underway. The number of nuclei per plate and the percentage fusion were determined using parallel cultures fixed and stained as described above. The LaC& rinse removes surface-bound Ca2+. The length of treatment needed to displace surface-bound Ca2+ was determined experimentally using the above protocol. Surface-bound Ca2+ (LaC&-displacable Ca2+) represents 28% of the 45Ca2+recovered in the absence of LaCl+ DSOO, and Treatment with EGTA, ~42323287, Gramicidin S

Ethylene glycol bis(P-aminoethyl ether)N,N’-tetraacetic acid (EGTA; Sigma Chemical Co.) was prepared as a 50 mM stock solution at pH 7.0. Since different batches of horse serum and embryo extract have slightly variable Ca2’ concentrations, the optimal EGTA concentration in the media varied somewhat and was titered at each change of serum or embryo extract. An EGTA level (usually 1.83 mM) was chosen that produced maximal inhibition of fusion with minimal effect on cell growth and adhesion to the substratum. The Ca2+ionophore A23187 was a gift of Dr. Robert Hamill, Lilly Research Laboratories. The ionophore was prepared as a 20 mM stock solution in 100% dimethyl

FIG. 1. Myoblast fusion in presence of A23187 and D600. O--O, Control culture; A - - - A, A23187 added at 4’7 hr (1 m; A -. -. - A, D600 added at 24 hr (50 w&I); 0, D600 added at 24 (30 I1M), 48 (60 &f), and 72 hr (90 plK). Average of three experiments.

sulfoxide (DMSO; Sigma Chemical Co.). This stock was diluted, with vigorous vortexing, 1:2000 with Dulbecco’s modified Eagle’s medium to prepare a lo-pM working solution just before use. The working solution was immediately added to complete medium to provide a final concentration of 1 NM A23187. The Ca2+ channel blocker D600 (cu-isopropyl-cw-[(NmethykN-homoveratryl)-a-aminopropyl]-3,4,5,-trimethoxyphenylacetonitrite hydrochloride) was a gift of Dr. E. B. Kirster, Knoll Pharmaceutical Company. The reagent was prepared as a 240 mM stock solution in 100% DMSO. The D600 stock solution was diluted, with vigorous vortexing, to 2.4 mM with Dulbecco’s modified Eagle’s medium, and then added to complete medium. The monovalent cation ionophore Gramicidin S (Sigma Chemical Co.) was prepared as a 20 mM stock solution in 100% DMSO. Just prior to use, an appropriate aliquot of the initial stock was added, with vigorous vortexing, to Dulbecco’s modified Eagle’s medium to provide a 10X working solution. The working solution was immediately added to complete medium (1:lO dilution). RESULTS

Kinetics of Fusion in Standard Cultures The kinetics of fusion in cultures seeded at 0.4 X lo5 tells/35-mm dish under standard culture conditions (Dulbecco’s modified Eagle’s medium + 10% horse serum + 3% chick embryo extract) are presented in Fig. 1. Routinely, greater than 95% of the cells which attached exhibited the bipolar morphology characteristic of presumptive myoblasts (Konigsberg, 1963). The remaining cells exhibited an extended, flattened, fibro-

300

DEVELOPMENTALBIOLOGY

I

20

40

60

(10

100

120

140

Hours FIG. 2. Cell growth rate in the presence of A23187 and D600. 0, control culture; A, A23187 added at 4’7 hr (1 WI!); A, D600 added at 24 hr (50 p&f’); 0, EGTA added at 24 hr (1.8 mM). Data at 68, 72, 77, 96, and 116 hr represent average of three experiments. Other points represent average of two experiments.

blast-like morphology. Alignment in these cultures was extensive by 40-48 hr. Fusion was initiated at 53-55 hr, half-complete at 74 hr, and essentially complete by 9098 hr after initial plating. Although some cell division continued, the rate of cell division decreased as fusion progressed (Fig. 2). Effect of the Calcium Ionophore 423187 on Fusion The first indication that calcium transport might be a requisite step in myoblast fusion came from an analysis of the effect of the divalent cation ionophore A23187 (Reed and Lardy, 1972a,b) on myoblast fusion. The addition of 1 WIT A23187 to cultures 47 hr after initial plating resulted in a precocious increase in their rate of fusion compared to control cultures (Figs. 1 and 3). Fusion was initiated earlier in the A23187 culture,

VOLUME82,1981

and the extent of fusion was higher in the A23187 culture throughout the period of massive fusion. The final extent of fusion, however, in both control and A23187 cultures was similar, as was the number of nuclei per plate (Figs. 1 and 2). The latter point is important in that it is well documented that cell density affects the timing of the fusion process, and we wanted to be sure that any effect on fusion that we observed with A23187 was not secondary to an increase or decrease in the growth rate. Although there may be slight variations in cell density with various treatments, dramatic variations in cell density are required to markedly shift the time course of fusion (Fig. 4). Thus, it appeared that the ionophore treatment had shifted the time course of fusion forward 6-8 hr, without affecting the general shape of the curve. The divergence in the extent of fusion in A23187 and control cultures was greatest at 68 hr when fusion was approximately 50% complete in the A23187 culture (Figs. 1 and 3, Table 1). Thus, 68 hr was chosen as a reference point for future comparisons of ionophore A23187treated and control cultures. The monovalent cation ionophore, Gramicidin S, had no effect on fusion at concentrations from 0.1 to 2.0 &f. Myoblasts can be caused to fuse precociously by treatment with ionophore A23187 in the interval from 9 hr prior to initiation of normal fusion to 1 hr prior to initiation of normal fusion (Fig. 5). The earlier the treatment, the more marked the effect. Treatment either for longer than 12 hr or initiated after the normal onset of fusion, was inhibitory. Effect of EGTA and the Calcium Channel Blocker 0600 on Fusion EGTA blocks myoblast fusion completely and reversibly (Paterson and Strohman, 1972). The same effect is achieved by removing calcium from the medium prior to culture (Shainberg et al., 1969). Morris and Cole

FIG. 3. Myoblast fusion in presence of A23187. A23187 was added at 48 hr (1 &f). Cultures were fixed and stained at 68 hr. (a) Control culture. (b) A23187-Treated culture. Magnification 500X.

301

Calcium IT@UXand Myoblast FuAm

DAVID, SEE, AND HIGCINBOTHAM

I+ oar :I Ix’.’ I i : I

60.

i

I

cn 3

;R’

I 0 i 1,;

Fusion

Plating Density ctllt/Platc)

---

0.2 x 105 0.4 x 105

1.0

0.6~10~

20-

80

60

66 hrr

0.9OtO.06 1.26t0.05

Index at 72 hrr

0.64+0.06

96 hrs a96f0.01

1.0

1.0

1.24+0.16

1.04+0.02

100 100

Hours FIG. 4. Effect of initial plating density on myoblast fusion. Cultures were seeded at 0.2 X lo5 cells/plate, 0 - - - 0; 0.4 X lo5 cells/plate, o----O; and 0.8 X lo5 cells/plate, X -. -. - X. Percentage fusion was determined as described. Fusion index = (% fusion at an initial density of 0.2 or 0.8 X lo5 cells/plate)/(% fusion at an initial density of 0.4 X lo5 cells/plate). Average of three experiments.

(1979) have proposed that calcium, known to be required for alignment, may also be required in the process of membrane fusion itself. In support of that hypothesis, we found that although EGTA added at 24 hr blocked myoblast alignment, addition at 48 or 64 hr blocked cell fusion, although much of the alignment process was already complete at that time (Table 1, Fig. 6). Addition at either time resulted in continued cell proliferation (Fig. 2). Although the EGTA data supported the role of extracellular calcium in fusion itself, it did not provide

any information regarding transport of calcium across biological membranes. In order to extend the observations obtained with the ionophore A23187, we utilized the calcium channel blocker D600 (Kohlhardt et al., 1972). D600 at concentrations from 30 to 50 pM was an effective inhibitor of myoblast fusion (Table 1, Figs. 1 and 6). Myoblast alignment was unaffected (Fig. 6). The total cell number in DGOO-treated cultures was initially somewhat depressed (Fig. 2) although not to an extent sufficient to reduce the extent of fusion (Fig. 4). The nonfused cells did not become postmitotic, but contin-

TABLE 1 EFFECTOF A23187, D600, AND EGTA ONMYOBLASTFUSION” Fusion indexb Reagent

Time of addition (hr)

Time of removal (hr)

68 hr

72 hr

96 hr

-

-

-

1.00

1.00

1.00

A23187

49

-

1.79 + 0.26 (9)

1.41 + 0.20 (4)

D600 D600 D600 D600 D600 D600 D600 D600

0

-

-

-

24 48 72 96 0 0 0

-

-

0.28 f 0.06 (10) -

-

-

-

48 72 96

-

-

0.79 f 0.23 (2) 0.54 * 0.15 (2) 0.36 k 0.13 (3)

EGTA EGTA EGTA EGTA

24 48 64 72

-

-

-

-

0.06 0.20 0.34 0.73

-

0.36 0.48 0.43 0.58

+ 0.13 (3) rt 0.05 (5)

k 0.17 (2) 2 0.17 (2) 0.96 + 0.19 (2)

+ 0.04 + 0.14 AZ 0.06 k 0.18

(2) (2) (2) (2)

a Reagents were added at the following concentrations: A23187, 1 /.&$ D600, 30& EGTA, 1.8 mM. Numbers in parentheses indicate the number of separate experiments averaged. b Fusion index = % fusion with experimental treatment/% fusion in control culture.

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DEVELOPMENTALBIOLOGY

-8

-4

0

+4

Hours

FIG. 5. Period of myoblast sensitivity to A23187. Abcissa is time of addition of A23187 (1 lrlK) relative to initiation of fusion at 55 hr. Fusion measured at 68 hr. Average of two experiments. Fusion index = % fusion with experimental treatment/% fusion in control culture.

ued to divide at the rate typical of prefusion nyoblasts (Fig. 2). D600 treatment did not alter the myoblast/ fibroblast ratio during the course of the experiment, as judged by gross cellular morphology (Fig. 6). Maintenance of maximal inhibition beyond ‘75hr was achieved only with the addition of sufficient excess D600 to compensate for the continuing increase in cell number. Under the latter conditions, fusion was maintained below 10% in D600 cultures while it reached 65’70% in control cultures (Fig. 1). The primary effect of D600 occurs in the interval between 48 and 96 hr in culture. Addition of D600 at any time between 0 and 48 hr was equally effective in blocking fusion. Addition of the drug at later times resulted in an increased level of fusion at 96 hr (Table 1). Addition of D600 at the onset of culture, and removal at 48 hr resulted in nearly normal fusion, while removal at 72 or 96 hr resulted in a progressively decreased fusion index (Table 1). In order to obtain consistent inhibition by D600, in all subsequent experiments D600 was added between 0 and 24 hr in culture. If D600 was indeed acting by blocking calcium channels, its effect should be reversed by the addition of excess calcium to the medium (Kolhardt et al., 1972). Added calcium did reverse the D600 inhibition, and at 11.8 PMtotal calcium in the medium the extent of fusion in control and DGOO-treatedcultures was identical (Table 2). The interpretation of this experiment is complicated in myoblast cultures since: (1) excessive calcium releases myoblasts from the substratum, and thus by itself reduces the extent of fusion; and (2) high calcium concentrations themselves inhibit fusion (Table 2). We were able to compensate for cell-density-related abberations by plating cultures destined for treatment with 11.8 PMcalcium at 10 times the normal cell density (Table 2, experiment 2). After treatment, the number of nuclei/plate in control and treated cultures were nearly identical. As judged by gross cellular morphology, all cultures described in Table 2 had nearly identical myoblast/fibroblast ratios of 95/5. We cannot,

VOLUME82, 1981

however, eliminate the possibility that high calcium concentrations selectively remove DGOO-sensitive cells from the plates. EGTA reduced the extent of A23187-stimulated fusion (Table 3), as expected if the ionophore acts by promoting transport of calcium across the plasma membrane. That D600 would also reduce the extent of A23187-stimulated fusion (Table 3) was not expected. The latter result could either indicate: (1) that D600 can directly block both natural calcium channels and ionophore-stimulated calcium transport; or (2) that D600 can block the ionophore stimulation of myoblast fusion by a mechanism other than a direct disruption of ionophore-stimulated calcium transport. Net Calcium Iqfiux in Myoblast Culture The EGTA inhibition of membrane fusion implicated extracellular calcium in the fusion process, while the A23187 stimulation and D600 inhibition of fusion implicated the movement of calcium across a biological membrane, presumably the plasma membrane. We therefore measured the net influx of 45Cathroughout the development of a control culture (Fig. 7). Shortly before the initiation of fusion, the net calcium influx @pm 45Ca accumulated/104 nuclei/hr) began to increase. By 65 hr (when fusion was 30% complete) the net calcium influx was doubled. It remained constant for 20-25 hr and then began to increase again, more dramatically this time. The latter increase corresponds to the expected time of appearance of sarcoplasmic reticulum in these cultures. Effect of A23187 and 0600 on Net Calcium &flux Jensen and Rasmussen (1977) measured the intracellular calcium content of lymphocytes stimulated with A23187 and found that the initial high levels of cellular calcium induced by the ionophore were not maintained. After 17 hr of incubation with A23187, cellular calcium levels had declined to the control level, either because of metabolism of the ionophore itself, or a time-dependent redistribution of the ionophore within the cells. We found a similar effect (Table 4). The ionophore exerted a maximal effect within the first hour after addition. Four hours after addition the net calcium influx had returned to the control value and 8 hr after addition the net calcium influx was below control values. To link the ionophore-stimulated net calcium influx to the ionophore-stimulated fusion process, we performed the experiments outlined in Table 5. Concentrations of ionophore above 0.3 PM were required to observe either stimulated calcium influx or stimulated fusion. Beyond 0.3 pM ionophore, the relative net cal-

DAVID, SEE, AND HIGGINBOTHAM

Calcium IqfCux and Myoblast Fusion

303

FIG. 6. Myoblast fusion in presence of EGTA or D600. EGTA and D600 were added at 1.8 mM and 30 &f, respectively. (a) EGTA added at 24 hr, culture fixed at 96 hr; (b) EGTA added at 64 hr, culture fixed at 96 hr; (c) D600 added at 24 hr, culture fixed at 96 hr; (d) control culture fixed at 96 hr; (e) D600 added at 24 hr, culture fixed at 120 hr; (f) control culture fixed at 120 hr. Magnification 500x.

influx increased with increasing concentrations of A23187. At 2.0 pM and beyond, however, the ionophore directly or indirectly promoted detachment of cells from the culture dish. Since fusion is a cell-densitydependent process (Fig. 4), the fusion index dropped accordingly. In Table 6 we present the results of an experiment designed to link the presumed effect of D600 on net calcium influx with the demonstrated D600 inhibition of fusion. As expected, D600 blocked net calcium influx, although not completely even at 50 pM. Since other investigators (Kohlhardt et al., 19’72) have found 5 pM D600 sufficient to almost completely block net calcium movement, we assume that either chick embryo myoblasts have a higher concentration of calcium channels cium

in their plasma membrane or a lower affinity for D600, or D600 is being absorbed by some component of the medium and thus the effective concentration is well below 50 pM. The latter possibility is eminently reasonable based on the complexity and relatively undefined nature of the culture medium, and the fact that the maximally effective dose of D600 varied with the batch of embryo extract. In any case, the D600 inhibition of net calcium influx and the D600 inhibition of fusion increase coordinately with increasing D600 concentration. DISCUSSION

It has been well documented (Shainberg, 1973; Paterson and Strohman, 1972; Papahadjopoulos, 1978,

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DEVELOPMENTALBIOLOGY

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TABLE 2 Ca2+ REVERSALOF THE D600 INHIBITION OF FUSION Percentage fusion

Nuclei/field

Ca*+ added to medium (mW

Control

Experiment 1” 0 2 4 6 10

51.6 zk 6.7 51.8 + 4.7 38.3 k 8.0 25.9 + 4.5 26.0 e 3.5

4.6 f 8.7 + 16.5 3~ 14.6 f 22.3 k

0.9 1.3 2.0 3.1 3.9

18.5 f 2.4 12.3 + 1.9 11.6 -+ 1.0 5.0 zk 0.6 6.1 + 0.7

13.9 k 9.2 r 7.3 f 5.5 k 4.2 +

Experiment 2* 0 10

56.6 k 7.2 34.6 + 7.0

7.6 + 1.1 32.0 + 11.1

15.5 * 1.5 14.9 + 1.8

13.4 f 1.7 12.7 k 2.6

D600

D600

Control

1.1 2.2 1.3 0.8 0.6

Note. D600 was added at 18 hr to 50 a. Fusion and total nuclei per field were determined at 72 hr. Nuclei/field represents the average of at least 400 nuclei in 30-40 fields. Initial Ca*+ in Dulbecco’s modified Eagle medium = 1.8 m&f. Additional Ca2+ was added as CaClc at 0 hr. Average of two experiments. ’ Cells plated at 0.4 X lo5 ceils/35-mm dish. * Dishes without added Ca2+seeded at 0.4 x lo5 tells/35-mm dish; dishes with 10 m&f Ca2+added to medium seeded at 4.0 X 10’ tells/35mm dish.

1979) that extracellular calcium is required for normal progression through the initial recognition-alignment phase of myogenesis. Our conclusion that a subsequent increased rate of net calcium influx is also a normal and absolutely essential step in myoblast fusion is based on four separate lines of evidence. (1) There is a measurable increase in the net calcium influx just prior to observable membrane fusion. (2) Depletion of extracellular calcium by EGTA can halt the fusion process at a stage after alignment is completed but prior to actual membrane union. (3) The calcium inophore A23187 induces precocious fusion in treated cultures. (4) The calcium channel blocker D600 is a potent inhibitor of cell fusion, although myoblast alignment apparently proceeds normally.

Since much of our evidence is drawn from experiments utilizing the drugs A23187 and D600, it is essential to demonstrate that they are indeed acting as described. The divalent cation inophore A23187 has been shown to increase the permeability of a number of natural membranes to calcium and magnesium ions (Reed and Lardy, 1972a,b). Addition of A23187 has been shown to induce calcium-dependent increases in sperm capacitation (Schackmann et al., 1978), histamine release (Cochrane and Douglas, 1974), exocytotic secretion in the pancreas (Eimerl et al., 1974), and human lymphocyte stimulation (Jensen and Rasmussen, 1977). In most of these cases, A23187 has also been shown to cause a rapid increase in the uptake of calcium.

TABLE 3 EFFECT OF EGTA AND D600 ONA23187-STIMULATED MYOBLAST FUSION

Reagent’

Fusion index*

A23187 EGTA A23187 + EGTA D600 A23187 + D600

1.00 1.42 f 0.09 0.03 + 0.03 0.10 f 0.09 0.36 i 0.08 0.45 f 0.16

a Reagents were added at the following times and concentrations: A23187,l r&i, 47 hr; EGTA, 1.8 mM, 24 hr; D600,30 FM, 24 hr. Average of four experiments. Cultures were terminated at 68 hr. *Fusion index = % fusion with experimental treatment/% fusion in control culture.

40

60

80

100

120

I

HOUrS

FIG. 7. Net 45Cainflux in myoblast culture. Average of four experiments. 0, % fusion; 0, 45Cainflux.

DAVID, SEE, AND HIGCINBOTHAM

Calcium IT&X

and Myoblast

305

Fusion

TABLE 4

TABLE 6

EFFECT OF IONOPHORE A23187 ON &Ca INFLUX

COORDINATE EFFECT OF D600 ON NET 45CaINFLUX AND FUSIONS

Hours after A23187 addition”

cpm 45Caaccumulated/104 nuclei/hr Control

A23187

Relative net %a influx*

D600 (LclK) 0 3

0

94 + 13

1 2 4 8

98+ 9 110 f 15 104 f 10 112 f 8

321 k 109 177 f 160 f 117 + 63f

53 26 7 11

3.4 1.8 1.5 1.1 0.6

’ Ionophore was added at 47 hr to 1 pM final concentration. At the indicated times, 45Cawas added to ionophore-treated and control cultures. One hour later, the plates were harvested as described. * Relative net 46Cainflux = net 45Cainflux of experimental/net 45Ca influx of control. Average of three experiments.

Percentage inhibition of net %a influx

10 30 50

Percentage inhibition of fusion

0 -1.0 15.8 32.4 44.5

+ 1.5

xi 2.9 f 6.5 + 8.4

0 6.9 17.0 58.3 73.2

+ 2.4

f 4.5 k 7.0 + 2.3

’ D600 was added at 18 hr. Fusion was measured at 72 hr. Net %a influx was measured at ‘72 hr as described. Average of three experiments (except for 3 WM D600).

tubes, thus it apparently does not affect events involved in generating fusion competency. A23187 can increase membrane permeability to Mp, and to a much lesser extent permeability to monovalent We have demonstrated that A23187 treatment leads cations. Although we do not know whether A23187 into a transitory increase in net calcium influx. The trancreased permeability to ions other than calcium in myository nature of the increase is fully compatible with blasts, several experiments argue against such perthe rather limited period of sensitivity to the drug. Jenmeability changes affecting fusion. Gramacidin S, a sen and Rasmussen (1977) found a similar transitory rise in calcium uptake when lymphocytes were treated monovalent cation ionophore, has no effect on myoblast with A23187. They argued that the biphasic nature of fusion. The extent of the A23187 effect is abolished by the uptake curve was due to a time-dependent redis- EGTA, and severely reduced by D600. Finally, calcium tribution of ionophore within the cells. Our data would influx and fusion respond coordinately to ionophore not allow us to distinguish this possibility from simple treatment. D600, a methoxy derivative of verapamil, blocks caltime-dependent inactivation of the drug. In either case, cium uptake in cardiac and smooth muscle (Kohlhardt A23187 can apparently only act upon fusion-competent et al., 1972), islets of Langerhans (Malaisse et al., 1973), cells, and this competency is not conferred upon the and lymphocytes (Jensen et al., 1977), presumably due cells until roughly 5-8 hr prior to the normal initiation to competition with calcium for a common receptor or of fusion. It is important to note that A23187 treatment special carrier system. It has been recently noted, howdoes not increase the final percentage of nuclei in myoever, that the (+) isomer of D600 blocks sodium channels in cardiac tissue while the (-) isomer blocks the TABLE 5 calcium channel in the same tissue (Galper and CatCOORDINATE EFFECT OF IONOPHORE A23187 ON NET 45Ca terall, 1979). INFLUX AND FUSION’ Although our experiments were performed with a racemic mixture of the (+) and (-) isomers of D600, it A23187” Relative net Fusion Nuclei is extremely likely that D600 was affecting fusion by 45Cainflux” indexd fielde (PLIK) blocking calcium channels rather than by any effect on 0 1.0 1.0 9.7 2 1.4 sodium transport. We have already noted that Gram0.1 0.95 f 0.28 0.98 k 0.14 10.2 f 1.2 icidin S has no effect on fusion. More importantly, D600 0.3 0.94 AZ0.14 1.08 + 0.07 9.5 + 0.4 reduced the net calcium influx in control cultures and 0.6 5.4 f 0.9 1.53 + 0.13 8.2 _+ 1.8 reduced the extent of A23187-stimulated fusion. The 1.0 6.2 f 1.7 1.49 f 0.15 8.4 f 2.0 2.0 inhibitory effect of D600 on fusion was reversed by in11.6 f 1.3 1.13 + 0.11 4.4 k 1.5 creasing external calcium. Finally, D600 reduced the net calcium influx and inhibited fusion coordinately. ’ Average of two experiments. * Ionophore A23187 was added at 47 hr. The increased calcium influx would presumably in‘Net 45Cainflux was measured, as described, in the first hour after crease, at least transiently, the intracellular calcium addition of the ionophore. Relative net 45Cainflux = net 45Cainflux concentration. The extracellular calcium concentration of experimental/net 45Cainflux of control. is 1.8 mM, while the free intracellular concentration in d Fusion index determined at 64 hr. eNuclei/field represents the average of at least 400 nuclei in 30-40 mature skeletal muscle is estimated at 0.1-l pM (Baker, fields. 1975). Although the overall increase in Ca2+influx that

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we detect is relatively small, it may be, and we believe is, localized to a small region of the cell and thus that region would experience a relatively great increase in calcium concentration. Furthermore, one of the characteristics of some calcium-mediated responses, such as synaptic transmitter release and muscle contraction, is that they respond as the fourth power of the calcium ion concentration (Rasmussen et al., 1972). Thus a very small change is sufficient to induce a marked response. Calcium normally enters a cell via a relatively specific calcium channel that is independent of membrane potential, or via a potential-dependent calcium permeability channel (Rasmussen and Goodman, 1977). Both channels may be activated by hormonal interaction with the plasma membrane. We do not know at this time which, if either, channel is involved in calcium flow into myoblasts. The increased intracellular calcium could, by either of two separate mechanisms, trigger or permit membrane union between two aligned, aposed, fusion-competent myoblasts. Volsky and Loyter (1978), examining virus-induced fusion of chicken erythrocytes, have proposed that the destabilization of cell shapes accomplished by dissociation of the “marginal” band of microtubules is a prerequisite for the induction of fusion in nucleated cells. They also demonstrated a virus-induced decrease in intramembraneous particle density during the first stages of the fusion process. The production of particle-free regions is important if fusion takes place between the lipid bilayers of two adjacent cells (Papahadjopoulos, 1978, 1979), and could require prior disassembly of cytoskeletal microtubules subadjacent to the plasma membrane. An increased intracellular calcium concentration, which can cause depolymerization of microtubules and inhibit their assembly (Poste, and Nicholson, 1976), could thereby destablize the myoblast morphology, and/or cause the passive or active produciton of regions free of intramembranous particles. Kalderon and Gilula’s (1979) observation of cytoplasmic, unilamellar, particle-free vesicles underlying fusion-competent myoblast plasma membranes suggests a second way to generate the particle-free, acidic phospholipid-enriched plasma membrane domains that may be necessary for membrane fusion (Papahadjopoulos, 1978,1979). They suggest that the vesicle membrane fuses with the plasma membrane to form a single particle-free bilayer. The particle-free bilayers of two adjacent myoblasts could then fuse. If the cytoplasmic vesicles were enriched in essential acidic fusogenic phospholipids (Portis et al., 1979; Papahadjopoulos 1978, 1979), their fusion with the plasma membrane would provide a mechanism to insert those phospholipids into the external surface of the myoblast membrane rapidly,

VOLUME82, 1981

and specifically, upon receipt of the appropriate developmental signal. We propose that the increase in intracellular calcium concentration triggers the fusion of these cytoplasmic vesicles with the overlying plasma membrane in the same manner as calcium triggers exocytosis of secretory vesicles in other cells (Miller and Nelson, 1977; Douglas, 1974; Rasmussen and Goodman, 1977; Lawson et al., 1977). One of the properties of a fusion-competent myoblast would therefore be the presence of these fusogenic vesicles. Indeed, Kalderon and Gilula (1979) found vesicles in EGTA-treated myoblasts, but no physical contact was observed between the vesicle and plasma membranes. This research was supported, in part, by grants from the Research Council of the Graduate School, University of Missouri-Columbia (Biomedical Research Support Grant RR07053, NIH), and Grant GM 25162 from the Public Health Service. We wish to thank Drs. Robert Hamill and E. B. Kirster for their gifts of A23187 and D600, respectively. REFERENCES BAKER, P. F. (1975). Transport and metabolism of Ca++ ions in nerve. Progr. Biophys. Mol. Biol. 24, 177-224. COCHRANE,D. E., and DOUGLAS,W. W. (1974). Calcium-induced extrusion of secretory granules in mast cells exposed to 48180 or the ionophores A-23187 or X-537A. Proc. Nat. Acad. Sci. USA 71,408412. DOUGLAS,W. W. (1974). Mechanism of release of neurohypophysial hormones: Stimulus-secretion coupling. In “Handbook of Physiology,” Section 7, “Endocrinology” (R. 0. Greep and E. B. Astwood, eds.) Vol. IV, pp. 191-224. American Physiological Society, Washington, D. C. EIMERL, S., SAVION, N., HEICHAL, O., and SELINGER,Z. (1974). Induction of enzyme secretion in rat pancreatic slices using the ionophore A23187 and calcium. J. Biol. Chem. 249,3991-3993. FRIEDLANDER,M., and FISCHMAN,D. A. (1975). Serological analysis of developing muscle cell surfaces. .I Cell. Biol. 67(2, Pt. 2), 124a. GALPER, J. B., and CATTERALL, W. A. (1979). Inhibiton of sodium channels by D600. Mol. Pharmacol. 15,174-178. HERMAN, B. A., and FERNANDEZ,S. M. (1978). Changes in membrane dynamics associated with myogenic cell fusion. J. Cell. Physiol. 94, 253-264. HYNES, R. 0. (1976). Viral transformation of rat myoblasts: Effects on fusion and surface properties. Develop. Biol. 48. 35-46. JENSEN, P., and RASMUSSEN,H. (1977). The effect of A23187 upon calcium metabolism in the human lymphocyte. Biochim. Biophys, Acta 468,146-156.

JENSEN,P., WINGER,L., RASMUSSEN,H., and NOWELL,P. (1977). The mitogenic effect of A23187 in hyman peripheral lymphocytes. Biochim.

Biophys.

Acta 496, 374-383.

KALDERON,N., EPSTEIN, M. L., and GILULA, N. B. (1977). Cell-to-cell communication and myogenesis. J. Cell Biol. 75, 788-806. KALDERON,N., and GILULA, N. B. (1979). Membrane events involved in myoblast fusion. J. Cell Biol. 81, 411-425. KOHLHARDT,M., BAUER, B., KRAUSE, H., and FLECKSTEIN,A. (1972). Differentiation of the transmembrane Na and Ca channels in mammalian cardiac fibers by use of specific inhibitors. Pjllegers Arch. 335,309-322.

DAVID, SEE, AND HICGINBOTHAM KONIGSBERG,I. R. (1963). Clonal analysis of myogenesis. Science 140, 1273-1284. KONIGSBERG,I. R. (1971). Diffusion mediated control of myoblast fusion. Develop. Biol. 26, 133-152. KNUDSEN,K. A., and HORWITZ,A. F. (1977). Tandem events in myoblast fusion. Develop. Biol. 58, 328-338. KNUDSEN,K. A., and HORWITZ,A. F. (1978). Differential inhibition of myoblast fusion. Develop. Biol. 66,294-307. LAWSON,D. M., RAFF, M. C., GOMPERTS,B., FEWTRELL,C., and GILULA, N. B. (1977). Molecular events during membrane fusion. A study of exocytosis in rat peritoneal mast cells. J. Gel2 Biol. 72,242-259. LIPTON,H. B., and KONIGSBERG,I. R. (1972). A fine structural analysis of the fusion of myogenic cells. J. Cell Biol. 53, 348-364. MALAISSE,W. J., PIPELEERS,D. G., MAILAISSE-LAGAE, F., and ORCI, L. (1973). Effect of a verapamil derivative (D600) on calcium uptake and insulin release by isolated islets. Diabetologia 9, 80. MILLER, B. E., and NELSON,D. L. (1977). Calcium fluxes in isolated acinar cells from rat parotid: Effect of adrenergic and cholinergic stimulation. J. Biol. Chem. 252, 3629-3636. MORRIS,G. E., and COLE, R. J. (1979). Calcium and the control of muscle-specific creatine kinase accumulation during skeletal muscle differentiation in vitro. Develop. Biol. 69, 146-158. Moss, M., NORRIS,J. S., PECK, E. J., JR., and SCHWARTZ,R. J. (1978). Alterations in iodinated cell surface proteins during myogenesis. Exp. Cell Res. 113, 445-450. NAMEROFF,M., TROTTER,J. A., KELLER, J. M., and MUNAR, E. (1975). Inhibition of cellular differentiation by phospholipase C. J. Cett Biol. 58, 10’7-118. NAMEROFF,M., and MUNAR, E. (1976). Inhibition of cellular differentiation by phospholipase C. II. Separation of fusion and recognition among myogenic cells. Develop. Bio2. 49, 288-293. PAPAHADJOPOULOS, D. (1978). Calcium-induced phase changes and fusion in natural and model membranes. In “Membrane Fusion” (G. Poste and G. L. Nicolson, eds.), Cell Surface Reviews Ser., Vol. 5, pp. 766-790. Elsevier/North-Holland, Amsterdam. PAPAHADJOPOULOS, D., POSTE, G., and VAIL, W. J. (1979). Studies on membrane fusion with natural and model membranes. In “Methods in Membrane Biology” (E. D. Korn, ed.), Vol. 10, pp. 1-121. Plenum, New York. PATERSON,B., and STROHMAN,R. C. (1972). Myosin synthesis in cultures of differentiating chicken embryo skeletal muscle. Develop. Biol. 29, 113-138. PAUW, P. G., and DAVID, J. D. (1979). Alterations in surface proteins during myogenesis of a rat myoblast cell line. Develop. Biol. 70,2738. PORTIS,A., NEWTON,C., PANGBORN,W., and PAPAHADJOPOULOS, D. (1979) Studies on the mechanism of membrane fusion: Evidence for an intermembrane Ca’+-phospholipid complex, synergism with Me, and inhibition by spectrum. Biochemistry 18, 780-790.

Calcium

IT&X

and hfyoblast

Fusion

307

POSTE,E., and NICHOLSON,G. L. (1976). Calcium ionophores A23187 and X5478 affect cell agglutination by lectins and capping of lymphocyte surface immunoglobulins. Biochim. Biophys. Acta 425,148155. PRIVES, J., and SHINITZKY, M. (1977). Increased membrane fluidity precedes fusion of muscle cells. Nature (London) 268,761-763. PUTNEY, J. W., VAN DE WALLE, C. M., and LESLIE, B. W. (1978). Receptor control of calcium influx in parotid aeinar cells. Mol. Pharmacol. 14, 1046-1053. RASH, J. E., and FAMBROUGH,D. (1973). Ultrastructural and electrophysiological correlates of cell coupling and cytoplasmic fusion during myogenesis in vitro. Develop. Biol. 30.166-186. RASH, J. E., and STAEHELIN, L. A. (1974). Freeze-cleavage demonstration of gap junctions between skeletal myogenic cells in vivo. Develop. Biol. 36, 455-461. RASMUSSEN,H., GOODMAN,D. B. P., and TENEHOUSE,A. (1972). The role of CAMP and Ca2+in cell activation. CRC Crit. Rev. Biochem. 95-146. RASMUSSEN,H., and GOODMAN,D. B. P. (1977). Relationships between Caf* and cyclic nueleotides in cell activation. Physiol. Rev. 57,421516. REED, P. W., and LARDY, H. A. (1972a). A23181: A divalent cation ionophore. J. Biol. Chem. 247, 6970-6977. REED, P. W., and LARDY, H. A. (1972b). In “The Role of Membranes in Metabolic Regulation” (M. A. Mehlman and R. W. Hanson, eds.), pp. 111-118. Academic Press, New York. SCHACKMANN,R. W., EDDY, E. M., and SHAPIRO,B. M. (1978). The acrosome reaction of Strongylocentrotus purpuratis sperm: Ion requirements and movements. Develop. Biol 65,483-495. SHAINBERG,A., YAGIL, G., and YAFFE, D. (1969). Control of myogenesis in vitro by Caf+ concentration in nutritional medium. Exp. CelZ Res. 58,163-167.

SHIMADA, Y. (1971). Electron microscope observation on the fusion of chick myoblasts in vitro. J. Cell Biol. 48, 128-142. STOCKDALE,E. E., and HOLTZER,H. (1961). DNA synthesis and myogenesis. Exp. Cell Res. 24, 508-520. VAN DER BOSCH,J., SCHUDT,C., and PETTE, D. (1973). Influence of temperature, cholesterol, dipalmitoyl-lecithin, and Ca++ on the rate of muscle cell fusion. Exp. Cell Res. 82, 433-438. VOLSKY,D. J., and LOYTER,A. (1978). Role of Ca++ in virus-induced membrane fusion: Ca++ accumulation and ultrastructural changes induced by Sendai virus in chicken erythrocytes. J. Cell Biol. 78, 465-477. WHITE, N. K., and HAUSCHKA, S. D. (1971). Muscle development in vitro. Exp. Cell Res. 67, 479-482.

YAFFE, D. (1971). Developmental changes preceding cell fusion during muscle differentiation in vitro. Exp. Cell Res. 66, 33-48.