Increase in intracellular calcium induced by the polycation-coated latex bead, a stimulus that causes postsynaptic-type differentiation in cultured Xenopus muscle cells

DEVELOPMENTAL

BIOLOGY

126,63-70

(1988)

Increase in Intracellular Calcium Induced by the Polycation-Coated Bead, a Stimulus That Causes Postsynaptic-Type Differentiation Cultured Xenopus Muscle Cells DING-LIANG Laboratories

for

Cell Biology, Department University of North

H.

ZHU AND

BENJAMIN

of Cell Biology and Anatomy, Chapel Hill, North

Carolina,

Accepted

November

Latex in

PENG’ and the Neurobiology Carolina 27599

Curr-kulum,

4, 1987

The polycation-coated latex bead is a potent stimulus for the induction of postsynaptic-type differentiation in cultured Xenon myotomal muscle cells. Specializations characteristic of the neuromuscular junction, such as clusters of acetylcholine receptors and other postsynaptic-specific proteins, develop at the bead-muscle contact. Previous studies have shown that a deprivation of extracellular calcium inhibits the effect of the beads in causing the development of these specializations. This suggests that an increase in intracellular Ca2+ .is a necessary condition for the development of this specialization. In this study, we tested whether an increase in intracellular calcium is observable upon the bead-muscle contact. The measurement was carried out on cells loaded with the fluorescent calcium indicator furaAM by digitized video microscopy. When polycation-coated beads were added to cells, an increase in intracellular calcium concentration in the range of 5 to 57% of the resting level was observed within 10 set after bead-muscle contact. Afterward, the calcium level gradually returned to the resting level with a time course of about 3 min. Uncoated beads, which do not induce the formation of acetylcholine receptor clustering, failed to elicit this calcium transient. Removal of extracellular calcium as well as blocking calcium channels with 50 &verapamil also suppressed this transient induced by the polycation-coated beads. Both treatments are known to suppress the formation of receptor clusters by these beads. These results suggest that the polycation-coated beads cause an influx of calcium by increasing the membrane conductance to this ion. This process may underlie the signaling of the postsynaptic differentiation. 0 1988 Academic Preaa, Inc.

INTRODUCTION

and Loring, 1985). However, how the muscle cell responds to these stimuli is unknown. The analysis of postsynaptic differentiation has been facilitated by a simple model in cultured Xenoms muscle cells. Polycation-coated latex beads can mimic the nerve in effecting a postsynaptic-type differentiation (Peng et aZ., 1981; Peng and Cheng, 1982). At the beadmuscle contact, AChRs are concentrated into clusters. This event is accompanied by the accumulation of acetylcholinesterase within the cleft between the bead and muscle, the assembly of postsynaptic-specific proteins in the cortex of the cell facing the bead, and the development of a set of ultrastructural specializations which resemble those seen at the NMJ (Peng, 1987). In addition, the beads also cause a dispersal of the preexisting AChR clusters at sites both near and far away from the stimuli (Peng, 1986). Thus, the polycation-coated beads can reproduce both the local and the global effects of innervation. This investigation was prompted by our finding that the formation of AChR clusters induced by polycationcoated beads can be suppressed by deprivation of extracellular calcium, either by bathing the cultures in Ca’+-free medium or by the application of Ca2+channel

During formation of the neuromuscular junction (NMJ), the interaction between nerve and muscle results in a highly localized differentiation process at the site of contact. This is exemplified by the formation of acetylcholine receptor (AChR) clusters in the postsynaptic membrane, which is also accompanied by the accumulation of acetylcholinesterase, and the development of the postsynaptic cytoskeletal specialization (reviewed in Froehner, 1986; Moody-Corbett, 1986; Peng and Poo, 1986; Rubin and Barald, 1985). In addition to these local processes, innervation also causes global changes in the muscle cell, which are reflected by suppression of the AChR site density and elimination of AChR clusters in the extrasynaptic regions of the cell (Kuromi and Kidokoro, 1984; Kidokoro and Brass, 1985; Patrick et aZ., 1978; Peng and Poo, 1986). Experimental evidence has shown that the program for the postsynaptic development can be activated by certain molecules endogenous to the nervous tissues (reviewed in Salpeter i To whom correspondence should be addressed at Department Cell Biology and Anatomy, University of North Carolina, CB# 108 Swing Bldg., Chapel Hill, NC 27599

of 7090,

63

0012-1606/88 Copyright All rights

$3.00

0 1988 by Academic Press, Inc. of reproduction in any form reserved.

64

DEVELOPMENTALBIOLOGY vOLUME126,1988

blockers (Peng, 1984). Consistent with this finding is the fact that nerve-induced AChR clustering is also inhibited by Ca’+-free condition (Henderson et al, 1984). In view of the ubiquitous role of the calcium ion as both primary effector and second messenger in the regulation of many cellular processes, these findings suggest the importance of Ca2+ in postsynaptic differentiation at the NMJ. To further understand the role of Ca2+ in this process, it is necessary to find out whether the intracellular Ca2+ level in muscle increases when the stimulus for postsynaptic differentiation is presented. In this study, we examined the change in intracellular Ca2+ when cultured Xenopus muscle cells were treated with polycation-coated latex beads by using the fluorescent Ca2+ indicator fura- (Tsien et al, 1985). We report here that an increase in intracellular Ca2+ is rapidly elicited by the beads and it is abolished by Ca2+-free medium or by the Ca2+ channel blocker verapamil. MATERIALSANDMETHODS Cell Cultures

and Latex Beads

Myotomal muscle cells were isolated from stage 20 to 22 Xenopus laevis embryos as described previously (Peng and Nakajima, 1978). They were cultured on 18 X 18 mm2 glass coverslips in Danilchick’s solution (Keller et al, 1985; 53 mM NaCl, 15 mM NaHC03, 4.5 mM K-gluconate, 1 mM MgS04, 1 mM CaC12, 18.1 mM Na-isothionate, 4.45 mM Na2C03, 5 mM bicine, pH 8.3), supplemented with 10% L-15 (Leibovitz) medium, 1% fetal bovine serum, and 1% garamycin. Polystyrene latex beads with a diameter of 4.5 pm (Polysciences, Warrington, PA) were washed with 70% ethanol and incubated with a 1 mg/ml solution of polyornithine overnight. After washing with 0.1 M phosphate buffer, they were resuspended with culture medium. Uncoated beads were used as control. Calcium Measurement

Fura- AM was dissolved in dimethyl sulfoxide at a concentration of 1 mg/ml as a stock solution. Aliquots of this solution were stored at -80°C. The loading solution was made by adding the stock solution to the experimental medium (53 mM NaCl, 4.5 mM K-gluconate, 1 mM MgS04, 1 mM CaC12, 4.45 mM Na2C03, 5 mM bicine, 10% L-15 medium, 1% fetal bovine serum, and 1% garamycin) to give a final fura- AM concentration of 10 pM. Cultures were incubated in this solution for 30 min at 23°C in the dark. After loading, the cultures were washed with the experimental medium extensively for 30 min and attached with Lubriseal (Arthur Thomas, Philadelphia, PA) to the bottom of a plastic plate which

had a center hole 8 mm in diameter. The chamber volume was approximately 200 ~1. A Zeiss IM-35 inverted microscope equipped with a Nikon 100X UV-F glycerin objective was used for fluorescence measurement. Illumination was provided by a 75 W xenon lamp through a quartz condenser and vertical illumination unit. Fluorescence images of the cells, which were excited at 340 or 380 nm and were detected at >520 nm, were recorded with a MTI-Dage ISIT video camera. The imaging system used in this study was described previously (DiGuiseppi et al., 1985; Lemasters et al, 1987). For each data acquisition, 64 video frames were digitized and averaged. The final images at the dual excitation wavelengths were stored and analyzed with a PDP 11/23 computer. The imaging area consisted of 256 X 256 pixels at a resolution of 8 bits. The digital images were processed by background subtraction and noise reduction through low- and highthresholding. The ratio of the 340 and 380 nm images was then calculated for each pixel. The mean gray level of the ratio image of the cell was then calculated. The gray level of the ratio image was calibrated with respect to Ca2+ concentration according to the method of Grynkiewicz et al. (1985). In short, standard solutions with known concentrations of free Ca2+ were prepared by mixing EGTA and Ca2+-EGTA buffer solutions [lo0 mM KCl, 10 mM 3-iV-morpholinopropanesulfonic acid (MOPS), pH 7.21. Fura- pentapotassium salt (1 PM) was then added to the standard solutions. The ratio of the fluorescence intensities at excitation wavelengths 340 and 380 nm for each Ca2+ concentration was measured and used to construct the calibration curve. By interpolating the mean gray level of the ratio image of the cell within the calibration curve, the calcium concentration was determined. Chemicals

The sources of the chemicals used in this study were as follows: polyornithine was from Sigma (St. Louis, MO), fura- was from Molecular Probes (Eugene, OR), ionomycin was from Calbiochem (La Jolla, CA), and verapamil was a kind gift of Knoll Pharmaceutical (Whippany, NJ). RESULTS Intracellular Myotomal

Calcium Concentration Muscle Cells

of Xenopw

The mean Ca2+ concentration of individual muscle cells as computed by the ratio imaging method varied from 10 to 500 nM. This variation was obvious even when cells within the same culture were examined. Thus, it appears to be an intrinsic property of these

ZHU AND PENC

Ca”

cells. Figure 1 is a histogram of the Ca2+ level in 213 cells. Its peak is at a Ca2+ concentration of 100 nM, 90% of the cells had Ca2+ concentration below 300 nM. The average of the mean Ca2+ concentration of these 213 cells was 166 nM (SEM, 8 nM). Two examples of the ratio image are shown in Figs. 4A and 4D. No consistent difference in the intensity of the ratio image was seen across the bulk of the cytoplasm, except at the thin peripheral area of certain cells. As shown in Fig. 4A, the intensity in this area was often lower than that of the rest of the cell. This indicated a lower Ca2+ concentration at the thin peripheral region of the cell. However, the Ca2+ concentration there showed an immediate elevation following stimulation by polycation-coated beads as discussed below (Fig. 4B). Thus, it is unlikely that this inhomogeneity in Ca2+ concentration was due to imaging artifacts. To test whether our method would enable us to detect a change in intracellular Ca2+, we studied the effect of agents known to alter the level of this ion in cells. First, we tested the effect of ionomycin, which is a Ca2+ ionophore. This compound should equilibrate the intracellular and extracellular Ca2+ concentration and thus should cause an increase in intracellular Ca2+ level. A total of three experiments was performed with similar results. Figure 2 summarizes the results of one experiment. Since ionomycin caused cell contraction at concentrations in the micromolar range, we adopted a stepwise application procedure. After each application, fura- images were taken at 2 min and again at 5 min before the next dose. As shown in Fig. 2, a significant increase in Ca2+ level was observed when the ionomycin concentration reached 1 PM and it was doubled at 2 pM. The threshold Ca2+ concentration at which the cell started contraction could not be determined by the ratio imaging method, since movement causes images at the two wavelengths to be out of register.

am? AChR

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In an attempt to decrease the intracellular Ca2+ level, we tested the effect of chelation of extracellular Ca2+ by stepwise bath application of Ca2+-free medium containing varying amount of EGTA. As shown in Fig. 3, the intracellular Ca2+ concentration was unaffected when the concentration of EGTA was below 3 mM. However, a sharp decrease was observed when its concentration reached 5 mM. At this concentration of EGTA, the external Ca2+ concentration was calculated to be less than 50 nM, which was one-sixth of the intracellular concentration. This decrease was probably due to an efflux of Ca2+ out of the cell. Since the sarcoplasmic reticulum is the major Ca2+ storage site within skeletal muscle, the fura- fluorescence could also come from Ca2+ within this organelle. To know whether our method detected Ca2+ changes in the cytoplasm rather than within the sarcoplasmic reticulum, we examined the effect of caffeine which

3 c

300

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FIG. 2. Effect of ionomycin on the Ca2+ concentration of a single cell. Ionomycin was added in steps. After each addition, the Ca2+ level was determined at 2 min (open bars) and at 5 min (solid bars).

20-

Caklum

@Ml

^r 100 -s 1.0 2 rao b5

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65

Clustering

(nM)

FIG. 1. Histogram of intracellular free Ca2+ concentration tured Xenopus muscle cells. A total of 213 cells were scored.

in cul-

of EQTA

FIG. 3. Effect of Gas+ chelation on the intracellular Caa+ concentration of a single cell. Medium containing EGTA was added in steps. After each addition, the Ca‘+ level was determined at 2 min (open bars), 5 min (solid bars), and 25 min (hatched bar after application 6).

66

DEVELOPMENTALBIOLOGY V0~~~~126,1988

causes a release of Ca2+ stored in this organelle into the cytoplasm (Weber, 1968). Caffeine, at a concentration of 3 mM, caused a more than twofold increase in intracellular Ca2+. This indicates that our method predominantly measured the cytoplasmic free Ca2+ concentration and not the sequestered Ca2+. In conclusion, these experiments showed that changes in cytoplasmic Ca2+ concentration in response to ionophore, deprivation of external Ca2+, and caffeine followed the expected directions in cultured Xenow muscle cells. This proves the validity of using fura- and ratio imaging method for subsequent measurements. Increase in Intracellular Calcium upon the Addition Polycation-Coated Beads

of

In these experiments, the resting intracellular Ca2+ concentration of the cell to be monitored was first measured. Polyornithine-coated beads were then added to the culture. After the bead suspension was applied, the cell was monitored continuously under bright-field optics until a bead-muscle contact was established. Prior to contact with the cell, beads showed Browniantype movement. But this movement ceased abruptly when they came into contact with the cell. Thus, the cessation of their movement was used as a marker of bead-muscle contact. Once this happened, Ca2+ concentration was measured immediately. There was approximately a lo-set interval between the time of bead-muscle contact and the first measurement. Thereafter, the Ca2+ concentration was determined at lo-set intervals for a total period of 3 min. Upon the bead-muscle contact, an immediate increase in intracellular Ca2+ concentration was observed. Two examples are shown in Fig. 4. In the first, the periphery of the cell had a low Ca2+ concentration before the addition of beads (Fig. 4A). Within 10 set after the beads came into contact with the cell (Fig. 4C), the intensity of the fluorescence ratio image at this thin peripheral area showed an obvious increase (Fig. 4B). Figure 4D shows the ratio image of another cell before the bead addition and Fig. 4E shows the same cell after the bead-muscle contact (Fig. 4F). The image in Fig. 4D is dimmer and more punctate than that in Fig. 4E. This is due to the fact that most pixels in Fig. 4D had low gray levels while those in Fig. 4E all had high gray levels. Thus, both examples show an increase in intracellular Ca2+ upon bead stimulation, although this increase was not localized only at the contact site. The mean Ca2+ concentration of the cell in an area of 256 X 256 pixels, which encompassed the bead-muscle contact, was calculated from the ratio image at each time point. Figure 5 shows two examples of this analysis. In all cases, there was an immediate rise in intra-

cellular Ca2+ level so that the first measurement after the bead’s landing on the muscle already showed an elevated, and in most cases the peak, concentration. This was usually followed by a gradual decline to the resting level during the next several minutes (open circles in Fig. 5). In one case (solid circles in Fig. 5), a highly transient rise in Ca2+, lasting less than 20 set, was also observed. This type of serial measurements were carried out on 13 cells treated with polycationcoated beads. The results are summarized in Table 1. In each case, the Ca2+ levels before the addition of the beads and at 10 and 170 set after the establishment of the bead-muscle contact are shown. The increase in Ca2+ level at 10 set was between 5 to 57% of the resting level, with an average of 24% for these 13 cells. This increase over the nonstimulated state is statistically significant (Table 1). At 170 set, it declined to a value not statistically different from that of the resting level (Table 1). To rule out the possibility that mechanical stimulation by the beads was the cause for the increase in Ca2+ level, we performed measurements on cells treated with uncoated beads, which also stick to the cell surface as shown by both light and electron microscopy (Peng and Chen, 1982). We found that these beads did not cause an increase in intracellular Ca2+. The results of one experiment are shown in Fig. 5 (open triangle). They show that there was no statistically significant change following the addition of uncoated beads. This conclusion was confirmed in nine additional experiments involving uncoated beads. In another experiment, we examined the effect of negatively charged polycarboxylate beads and found that they too failed to elicit a change in intracellular Ca2+ (d a t a not shown). Thus, mechanical contact by the beads alone does not result in a change in intracellular Ca2+ level. Eflects of Calcium

Deprivation

and Calcium

Antagonist

To determine whether the bead-induced increase in intracellular Ca2+ level was due to an influx of extracellular Ca2+, we conducted measurements in Ca2+-free medium containing 0.4 mM EGTA. At this concentration, the chelator itself did not cause a decrease in intracellular Ca2+ (Fig. 3). The results of one experiment are shown in Fig. 5 (open squares). Together with data from three other experiments, they show that there was no significant increase in Ca2+ in response to the bead treatment under this condition. In addition, we examined the effect of the Ca2+ antagonist verapamil on the bead-induced increase in Ca2+ level. Verapamil inhibits the influx of Ca2+ by blocking the Ca2+ channels in the plasma membrane (Fleckenstein, 1983; Hagiwara and Byerly, 1981). When polyornithine-coated beads were

ZHU AND PENG

Ca”

and AChR

Clustering

67

FIG. 4. Changes in the fura- ratio image after the addition of polyornithine-coated latex beads. The left and right columns show images from two different cells. (A, D) The ratio image before the addition of beads. (B, E) The ratio image immediately after (10 set) the bead-muscle contacts were established. (C, F) Phase-contrast image of the cell showing the bead-muscle contacts (arrowheads).

added in medium containing a normal amount of Ca2+ and 50 PM verapamil, the bead-stimulated increase in intracellular Cazf level was suppressed as shown in Fig. 5 (filled triangles). The antagonist itself did not cause any change in the Ca2+ concentration during these mea-

surements. This result was confirmed in four additional experiments. From these results, we conclude that the bead-induced increase in intracellular Ca2+ comes mainly from an influx of Ca2+ from extracellular sources. The Ca2+

68

DEVELOPMENTAL

5 I J

BIOLOGY

130 120 HO too 00 -20

0

20

40

00

Tlmo

00

100

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140

100

100

(rccndrl

FIG. 5. Time course of the change in Ca2+ concentration of individual cells effected by the head application. First, the resting Ca2+ level was determined every 10 set during a 30-set interval. The beads were then applied at time 0. After bead-muscle contact was established, the measurements were carried out at lo-set intervals for a duration of 3 min. Open and solid circles, cells treated with polycation-coated beads. Open triangles, cell treated with uncoated beads. Squares, cell treated with polycation-coated beads in Ca’+-free medium. Solid triangles, cell treated with polycation-coated beads in medium containing 50 FM verapamil.

antagonist data also suggest the possible involvement of Caz+ channels in this process. DISCUSSION

We have shown that an immediate increase in intracellular Ca2+ accompanies the addition of polyornithine-coated latex beads to cultured Xenom myotomal muscle cells, which is known to be a stimulation for postsynaptic development. This increase can be blocked by a deprivation of external Ca2+ supply as well as by Ca2+ channel blocker verapamil. These results suggest that the polycations adsorbed on the beads cause an influx of Ca2+ into muscle cells. Despite its strong temporal correlation with the establishment of the beadmuscle contact, our results showed that the Ca2+ increase occurred diffusely and was not confined just to the contact area. A possible explanation is that Ca2+ rises abruptly at the site of activation, but it quickly diffuses throughout the cell. This may also explain the fact that the net increase in intracellular Ca2+ is relatively small. If the observed increase totally originates from Ca2+ influx at the bead-muscle contacts, the early change at those sites may be considerably higher. On the other hand, the stimulus could elicit a response, for example, a depolarization of the membrane (cf. Gould and Stephano, 1987; see discussion below), which spreads away from the contact site. This spread of signal would then cause a diffuse influx of Ca2+. Due to limitations in instrumentation, we were unable to measure Ca2+ concentration instantaneously upon the bead-muscle contact. Thus, these two possibilities remain to be distinguished.

VOLUME

126,1988

These results are consistent with our previous hypothesis that the effect of polycation-coated beads in inducing the AChR clustering is mediated by Ca2+ (Peng, 1984). Uncoated beads, which are ineffective in inducing AChR clustering (Peng et ab, 1981; Peng and Cheng, 1982), also do not cause a rise in intracellular Ca2+. Ca2+ antagonist or Ca2+ deprivation blocks both the bead-induced AChR clustering (Peng, 1984) and the rise in Ca2+ level. Thus, the rise in intracellular Ca2+ triggered by the polycation-coated beads is a necessary condition for AChR clustering. The rapidity of the bead-induced Ca2+ response suggests that this may be one of the earliest events in the signaling of AChR cluster formation. It remains to be seen whether Ca2+ alone is sufficient to activate AChR clustering. Previous studies have also shown that calmodulin inhibitors suppress the formation of AChR clusters (Peng, 1984; Tai and Connolly, 1986). Since the calcium-calmodulin interaction is one of the mechanisms that the cell uses for the rapid activation of many enzyme systems (Cheung, 1980; Means and Dedman, 1980), the effect of Ca2+ on postsynaptic differentiation may be mediated by calmodulin. In addition to AChR clustering, the elevation of Ca2+ also promotes the synthesis of acetylcholinesterase (Rubin, 1985), another important molecule in postsynaptic function. On the global level, the dispersal of ex-

TABLE CHANGE

1

IN CALCIUM CONCENTRATION BY POLYORNITHINE-COATED

AFTER STIMULATION BEADS

Calcium concentration WV’

Mean SEM Prob.

2

Ratio (%)

Cell

1

3

(2-1)/l

(3-1)/l

1

118

185

2 3 4 5 6

90 47 216 181 149

135 64 232 216 175

116

57

149 70 199 216 163

50 36 7 19

-2 66 49 -8

5 35 17

17

7

147

155

116

8 9

158 48

214 56

178 50

10 11

149 199

167 246

145

12

12 13

195 197

220 226

214 220

24 13 15

146 16

176 17

153 16

24 5
19 9

-21 13 4 -3 8 12 12 7 0.126

a 1, before the application of beads; 2,10 set after the bead-muscle contact; 3,170 set after the bead-muscle contact.

ZHU AND PENG

Ca’+ and AChR

trajunctional AChR clusters induced by polycationcoated beads or by the nerve may also be triggered by the increase in Ca 2f . Kuromi and Kidokoro (1984) have provided evidence that protease inhibitors can partially protect the extrajunctional AChR clusters from being dispersed by nerve innervation. Ca2’ may activate the proteases which degrade the cytoskeletal specializations which may be responsible for the stability of these clusters (Froehner, 1986; Peng and Phelan, 1984; Steinbath and Bloch, 1985). Furthermore, the synthesis of AChR also depends on Ca2+. A depletion of intracellular Ca2+ has been shown to result in a suppression of the AChR synthesis in rat myotubes (McManaman et al., 1981). Thus, an increase in Ca2+ level at the site of cluster formation may act locally to trigger the formation of the postsynaptic specializations as well as globally to effect a range of cellular activities involved in synaptogenesis. This study has demonstrated the effect of polycations on Ca2+ influx in Xenopus muscle cells. In addition, these molecules have been shown to exert a wide range of other biological effects. They, together with polyamines and proteins with highly basic sequences, such as histones and certain rcts oncogene proteins, are potent activators of several membrane-bound enzymatic systems. For example, they stimulate phosphorylation of membrane-bound proteins and lipids and activate the adenylate cyclase (Gatica et aZ., 1987; Vogel and Hoppe, 1986; Wolff and Cook, 1975). Whether these events need to be preceded by an increase in intracellular Ca2+ is not known. When applied locally or to the bath, polylysine causes a muscle-like contraction of the cortical cytoplasm in Xenopus oocytes, which can be blocked by Ca 2+ chelation (Gingell, 1970). This effect may be mediated by an increase in membrane permeability to Ca2+ (Gingell and Palmer, 1968). Consistent with this effect is our observation that contraction of muscle cells is often elicited when polyornithine or polylysine-coated beads make contact with Xenopus muscle cells (our unpublished data). This observation, in fact, implicates an increase in intracellular Ca2+ upon the bead-muscle interaction. In the eggs of the sea worm Urechis, Gould and Stephano (1987) have recently shown that the acrosomal protein from the sperm, which is highly enriched in basic amino acids lysine and arginine, induces an increase in membrane conductance to Na+ and perhaps also to Ca2+. This causes a depolarization of the egg in a manner similar to that induced by fertilization. Thus, highly basic proteinaceous molecules may have a general effect on biological membranes in opening ion channels. Whether molecules of similar characteristics are used by the nervous system to signal the synaptic differentiation remains to be elucidated.

69

Clustering

We are grateful for the help of Drs. Brian Herman and James DiGuiseppi in digital fluorescence microscopy. This work was supported by NIH Grant NS-23583 and the Muscular Dystrophy Association. REFERENCES CHEUNG, W. Y. (1980). Calmodulin plays a pivotal role in cellular regulation. Science 207,19-27. DIGUISEPPI, J., INMAN, R., ISHIHARA, A., JACOBSON,K., and HERMAN, B. (1985). Applications of digitized fluorescence microscopy to problems in cell biology. Bi0Technique.s 3,394-403. FLECKENSTEIN, A. (1983). “Calcium Antagonism in Heart and Smooth Muscle.” Wiley, New York. FROEHNER, S. C. (1986). The role of the postsynaptic cytoskeleton in AChR organization. Trends NeuroSci. 9,37-41. GATICA, M., ALLENDE, C. C., ANTONELLI, M., and ALLENDE, J. E. (1987). Polylysine-containing peptides, including the carboxyl-terminal segment of the human c-Ki-ras 2 protein, affect the activity of some key membrane enzymes. Proc. Nat1 Acad Sci USA 84, 324-328.

GINGELL,, D. (1970). Contractile responses at the surface of an amphibian egg. J. Embol. Exp. Mcwphol. 23,583-609. GINGELL, D., and PALMER, J. F. (1968). Changes in membrane impedance associated with a cortical contraction in the egg of Xenopus la&s. Nature (London) 217,98-102. GOULD, M., and STEPHANO, J. L. (1987). Electrical responses of eggs to acrosomal protein similar to those induced by sperm. Science 235, 1654-1656. GRYNKIEWICZ, G., POENIE, M., and TSIEN, R. Y. (1985). A new generation of Ca indicators with greatly improved fluorescence properties. J. Biol.

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HENDERSON, L. P., SMITH, M. A., and SPITZER, N. C. (1984). The absence of calcium blocks impulse-evoked release of acetylcholine but not de novo formation of functional neuromuscular synaptic contacts in culture. J. Neurosci 4,3140-3150. KELLER, R. E., DANILCHIK, M., GIMLICH, R., and SHIH, J. (1985). The function and mechanism of convergent extension during gastrulation of Xenopus laevis. J. Embryol. Exp. Morphol. 89(suppl.), 185-209. KIDOKORO, Y., and BRASS, B. (1985). Redistribution of acetylcholine receptors during neuromuscular junction formation in Xenopus cultures. J. Physiol. (Paris) 80,212-220. KUROMI, H., and KIDOKORO, Y. (1984). Nerve disperses preexisting acetylcholine receptor clusters prior to induction of receptor accumulation in Xenopus muscle cultures. Dev. Biol 103,53-61. LEMASTERS, J. J., DIGUISEPPI, J., NIEMINEN, A.-L., and HERMAN, B. (1987). Blebbing, free Ca*+ and mitochondria membrane potential preceding cell death in hepatocytes. Nature (London) 325.78-81. MCMANAMAN, J. L., BLOSSER,J. C., and APPEL, S. H. (1981). The effect of calcium on acetylcholine receptor synthesis. J. Neurosci. 1, 771-776. MEANS, A. R., and DEDMAN, J. R. (1980). Calmodulin-An intracellular calcium receptor. Nature (London) 285,73-77. MOODY-CORBETT, F. (1986). Formation of the vertebrate neuromuscular junction. In “Developmental Biology: A Comprehensive Synthesis” (L. W. Browden, Ed.), Vol. 2, pp. 605-635. Plenum, New York. PATRICK, J., HEINEMANN, S., and SCHUBERT, D. (1978). Biology of cultured nerve and muscle. Annu. Rev. Neurosd 1,417-443. PENG, H. B. (1984). Participation of calcium and calmodulin in the formation of acetylcholine receptor clusters. J. Cell Biol. 98, 550-557.

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PENG, H. B. (1986). Elimination of preexistent acetylcholine receptor clusters induced by the formation of new clusters in the absence of nerve. J. Neurosci 6,581-589. PENG, H. B. (1987). Development of the neuromuscular junction in tissue culture. CRC &it. Rev. Anat. Sci. 1,91-131. PENG, H. B., and CHENG, P.-C. (1982). Formation of postsynaptic specializations induced by latex beads in cultured muscle cells. J. Neurosci. 2,1’760-1774. PENG, H. B., CHENG, P.-C., and LUTHER, P. W. (1981). Formation of ACh receptor clusters induced by positively charged latex beads. Nature (London) 292,831~834. PENG, H. B., and NAKAJIMA, Y. (1978). Membrane particle aggregates in innervated and noninnervated cultures of Xenopus embryonic muscle cells. Proc. NutL Acad Sci. USA 75,500-504. PENG, H. B., and PHELAN, K. A. (1984). Early cytoplasmic specialization at the presumptive acetylcholine receptor cluster: A meshwork of thin filaments. J. CeU Biol 99,344-349. PENG, H. B., and POO, M.-M. (1986). Formation and dispersal of acetylcholine receptor clusters in muscle cells. Trends NeuroSci 9, 125-129. RUBIN, L. L. (1985). Increases in muscle Ca mediate changes in acetylcholinesterase and acetylcholine receptors caused by muscle contraction. Proc. NatL Acad Sci USA 82,7121-7125.

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