Association of the synaptic form of acetylcholinesterase with extracellular matrix in cultured mouse muscle cells

Cell, Vol. 29, 71-79,

May 1982,

Copyright

0 1982

by MIT

Association of the Synaptic Form of Acetylcholinesterase with Extracellular Matrix in Cultured Mouse Muscle Cells Nibaldo C. Inestrosa,* Laura Silberstein and Zach W. Hall Division of Neurobiology Department of Physiology University of California San Francisco, California 94143

Summary Myotubes of a mouse muscle-cell line (C2) synthesize in culture a 16s form of acetylcholinesterase that is normally found only in regions of adult mouse muscle that contain endplates. The 16s enzyme in C2 cell extracts has the properties expected of acetylcholinesterase forms that have a collagen-like tail. In intact cells, the active site of the 16s acetylcholinesterase is protected by a membrane-impermeable inhibitor, and this form of the enzyme can be removed by treatment of the cells with collagenase. Thus the enzyme is extracellular. Its extraction by high ionic strength solutions lacking detergent suggests that the 16s form is associated with the extracellular matrix by ionic interactions. Histochemical staining shows focal patches of acetylcholine&erase activity on the cell surface. Collagenase treatment, which removes only the 16s form, abolishes this staining pattern, indicating that the patches consist of the 16s enzyme. We conclude that the 16s enzyme in C2 myotubes occurs in focal patches on the cell surface, where it is associated with the extracellular matrix. Introduction There is increasing evidence that the extracellular matrix plays an important role in the coordination and direction of the activities of cells during the differentiation of tissues (Grobstein, 1967, 1975). The extracellular matrix has been implicated in the morphogenesis of glands (Bernfield and Banerjee, 1978) in the organization of epithelial sheets (Hay, 1977) in the regulation of cellular growth and proliferation (Gospodarowicz and Tauber, 1980; Vlodavsky et al., 1980) in cell migration (Le Dourain, 1975) and in cellular differentiation (Meier and Hay, 1975; Chen et al., 1978; Sieber-Blum et al., 1981). A recent, dramatic example is the demonstration that a component of the matrix, the basal lamina, can direct the differentiation of presynaptic and postsynaptic cells during the regeneration of skeletal muscle (Sanes et al., 1978; Burden et al., 1979; Bader, 1981). An understanding of how these influences are exerted at a molecular level requires identification of the components of the extracellular matrix, determination of their * On leave from: Laboratory Biology, Catholic University Chile.

of Neurophysiology, Department of Cell of Chile, P. 0. Box 114-D, Santiago,

structure and knowledge of how the distribution of individual components is controlled. Although the exact nature and composition of extracellular matrices produced by various tissues are still to be elucidated, the extracellular matrix is composed for the most part of collagen, proteoglycans and glycoproteins (Kefalides et al., 1979; Kleinman et al., 1981). In one case, an enzymatic activity has been associated with the extracellular matrix (McMahan et al., 1978). On the surface of muscle fibers, acetylcholinesterase (AChE) activity is highly concentrated at the neuromuscular junction, where it can be seen by histochemical methods (Couteaux, 1955). The enzyme plays an essential role in the highly phasic transmission that occurs at this synapse by rapidly removing, through hydrolysis, the acetylcholine that is released into the synaptic cleft from presynaptic nerve terminals (Eccles et al., 1942; Kuffler and Nicholls, 1979). At least some of the enzyme activity at the neuromuscular junction is associated with the basal tamina, an extracellular coat that surrounds each muscle fiber and that at the junction extends through the synaptic cleft separating nerve and muscle. Thus in damaged frog muscle, from which nerve and muscle cells have been removed, AChE activity remains at the old synaptic sites (Marshall et al., 1977; McMahan et al., 1978). Electron microscopy of these sites has established that only basal lamina remains, with no plasma membrane attached (McMahan et al., 1978). The molecular structure of AChE has been determined by the study of enzyme purified from tissues that are homologous to muscle, the electric organs of the eel and of the marine ray (Massoulie, 1980). In these species two classes of enzyme occur: the globular forms G,, G1 and Gil, and the asymmetric forms Ad, As and AIP (the subscript refers to the number of catalytic subunits; Bon et al., 1979). The catalytic subunits in all forms are thought to be the same or closely related (Vigny et al., 1978). The asymmetric forms are of particular interest because they have an unusual structure in which the catalytic subunits are attached to a long, collagen-like tail (Massoulie, 1980). Asymmetric forms of AChE with properties similar to those from the electric organ exist in mammalian muscle (Bon et al., 1979); in some species these forms are found only in extracts from regions of muscle containing endplates (Hall, 1973; Vigny et al., 1976). The association of the asymmetric or tailed forms with parts of muscle containing endplates, coupled with the histochemical demonstration that at least part of the endplate enzyme is in the basal lamina, has led to the idea that the asymmetric forms are associated with the basal lamina, and that they represent the activity that is seen by histochemistry at the endplate. The selective release of enzyme activity from the endplate by treatment of intact muscle with the enzyme collagenase (Hall and Kelly, 1971; Betz and

Cell 72

Sakmann, 1973; lnestrosa et al., 1977) is consistent with this-idea. The subcellular location of the tailed forms of AChE in muscle cells, however, has never been established. Identification of the tailed species as part of the basal lamina would allow investigation of their mode of attachment and restriction to specific areas of the muscle surface. These problems could be investigated most conveniently in vitro with a cell line. We have recently found that a substantial portion of the AChE activity produced by myotubes of a mouse muscle-cell line is a 16s species similar to that found at endplates in adult muscles of other species. Our experiments show that this enzyme has the properties of a tailed or asymmetric form, that it is associated with extracellular structures and that its distribution on the cell surface is restricted to discrete patches. Results 16s Acetylcholinesterase Has a Collagen-Like Tail Myotubes were examined for AChE forms several days after fusion by extraction into a high salt-EDTA buffer containing Triton X-l 00, a solution that solubilized over 95% of the total AChE activity. The extract was analyzed by velocity sedimentation in a sucrose gradient. A prominent peak of enzyme activity was detected at 16S, representing about 20% of the total activity, as well as major peaks at 10s and at 4-6s (Figure 1 a). In the adult mouse, a form of the enzyme with the same sedimentation properties was found exclusively in regions of muscle containing endplates (Figure 1 b). We used the criteria proposed by Bon et al. (1979) to determine whether the 16s species made by C2 cells has a collagen-like tail similar to that of the asymmetric Electrophorus enzyme. The tail of Electrophorus AChE is responsible for aggregation of the enzyme at low ionic strength. This property is lost and the sedimentation constant of the enzyme is increased after removal of the tail with collagenase (Bon et al., 1979). We examined the aggregation of the AChE forms from C2 cells by dialyzing a high ionic strength extract of myotubes into a low ionic strength medium. Both the resulting precipitate and the supernatant were analyzed by sucrose gradient sedimentation. The dialysis quantitatively precipitated the 16s form, and separated it from the 4-6s forms and the 10s form, which were recovered in the supernatant (Figure 2a). Two minor forms (presumably corresponding to As and Aq forms; Bon et al., 1979) were also precipitated. The 16s form was sensitive to collagenase. Treatment of myotube extracts with collagenase converted the 16s form to a new species that sedimented at 18s and that did not aggregate at low ionic strength (Figure 2b). From this indirect evidence we conclude, by analogy with the Electrophorus enzyme (Bon et al.,

0 BOTTOM

Figure 1. Presence in Endplate Region

10

20

30

FRACTION NUMBER

of 16s Acetylcholinesterase of Mouse Skeletal Muscles

40 TOP

in C2 Myotubes

and

(a) Sucrose density gradient fractionation of different molecular forms of AChE in C2 muscle cells. Muscle cultures were grown for 5 days in fusion medium on a tissue culture plastic substrate, and then examined for AChE activity. C2 myotubes from T75 flasks were rinsed 3 times with 10 mM Tris-Cl (pH 7.41, removed from the dishes mechanically and homogenized by hand in a Duall-Kontes ground glass homogenizer in buffer A (see Experimental Procedures). Aliquots (0.2 ml) were loaded onto a 5%-20% sucrose gradient. (b) Sucrose density gradient fractionation of different molecular forms of AChE in mouse skeletal muscle. Endplate (U) and nonendplate (-1 regions were dissected from BALB/c mouse diaphragm muscle as described in Experimental Procedures. The muscle regions were cleaned and homogenized as described above for C2 myotubes. The enzyme activity shown for the two fractions has been adjusted according to the relative protein concentration in each. Arrows: positions of marker enzymes in the gradients-from left to right, Pgalactosidase (16s) and alkaline phosphatase (6.1 S).

1979; Massoulie, 1980), that the 16s AChE is an A,, form containing a collagen-like tail. The Active Site of 16s Acetylcholinesterase Is External We then attempted to localize the AI2 enzyme in intact cells. To determine if the active site of the 16s AChE in C2 cells is exposed to the extracellular medium, we examined the effects on AChE activity of enzyme inhibitors that differ in lipid solubility and hence in ability to cross cell membranes (Mclsaac and Koelle, 1959; Rotundo and Fambrough, 1980a; Taylor et al., 1981; lnestrosa et al., 1981). Incubation of cells with diisopropylfluorophosphate, which is lipid-soluble, results in complete and irreversible inactivation of all enzyme activity associated with the cells. If an imper-

Extracellular

Patches

of 16s

AChE

73

meable, reversible inhibitor, such as BW284c51, is present during the diisopropylfluorophosphate treatment, it will protect from inactivation those enzyme molecules to which it has access. C2 cultures were incubated for 30 set with 750 PM BW284c51, then treated with 25 PM diisopropylfluorophosphate. Following removal of diisopropylfluorophosphate, the cells were washed free of the reversible inhibitor and assayed. Approximately 30% of the total AChE remained. Analysis of the protected AChE by velocity sedimentation showed that at least 90% of

the 16s AChE was recovered, and that this form accounted for more than 70% of the total protected AChE activity (Figure 3). A small amount of AChE activity corresponding to other forms was also protected, indicating either that some of the BW284c51 may have penetrated to the cell interior or that other forms were external. The latter explanation appears to be the case, since a 90% reduction in BW284c51 concentration gave no change in the relative amount of the protected forms. We conclude that most of the active sites associated with the globular forms are intracellular, while that of the AI2 enzyme appears to be almost exclusively extracellular. Collagenase Specifically Releases 16s Acetylcholinesterase from Intact C2 Cells In adult muscle, collagenase treatment specifically releases part of the AChE (Hall and Kelly, 1971; lnestrosa et al., 1977; Sketelj and Brzin, 1979) at the endplate. Because the 16s form is concentrated in this region (Hall, 1973; Vigny et al., 19761, and is

2 1 0

10

20

30

BOTTOM

40 TOP

FRACTION NUMBER Figure Buffer,

2. Aggregation and Collagenase

of 16s Acetylcholinesterase Sensitivity

in

Low

Salt

(a) For the aggregation experiments, gradients were run in a SW41 Ti rotor, at 40,000 rpm for 21 hr. C2 myotubes obtained after 4 days in fusion medium were homogenized in buffer A and centrifuged at 15,000 rpm for 15 min, and the final supernatant was dialyzed overnight at 4°C against buffer A without 1 M NaCI. The low-saltsoluble and -insoluble fractions were separated by centrifugation at 15,000 X g for 15 min. and the pellet thus obtained was redissolved in buffer A. Samples of the supernatant and redissolved pellet were then analyzed by velocity sedimentation on linear 5%-20% sucrose gradients. (u) Gradient of material redissolved from precipitated protein pellet, containing the A ,*, As and A4 AChE molecular forms. (0- - -0) Gradient showing forms remaining in the supernatant following dialysis, 1 OS (G4) and 4-6s (G, and G2). The peak of maximum activity in each gradient was assigned a value of 100, and the other points were normalized accordingly. Arrows: positions of marker enzymes in the gradients-from left to right, P-galactosidase and alkaline phosphatase. (b) Collaganese treatment. C2 myotubes obtained after 4 days in fusion medium were first homogenized in low salt buffer, to remove some of the nonaggregating forms of AChE. Aliquots of a second extract, in saline buffer (1 M NaCI, 50 mM MgCk and 10 mM Tris-Cl [pH 7.4]), were incubated with (w) or without (0- - -0) collagenase (150 pg of collagenase type Ill in 1 ml of extract) for 50 min at 29”C, and then dialyzed against IO mM Tris-Cl (PH 7.4). The supernatant was analyzed in low-salt sucrose gradients (containing 0.1 mg/ml bacitracin and 10 mM Tris-Cl [pH 7.41) at 36,000 rpm for 15 hr in an SW50.1 rotor.

BOTTOM

TOP

FRACTION Figure 3. Molecular Cell Surface

Forms

NUMBER

of Acetylcholinesterase

on the Muscle-

Sucrose gradient profile of AChE obtained from extracts of C2 myotubes grown for 4 days in fusion medium. The curves represent forms of AChE in control cells (0- - -0) and in cells treated for 30 set in 750 PM EW284c51 before inactivation with diisopropylfluorophosphate (w). C2 muscle cultures from 75 cm* flasks were washed 3 times with Dulbecco’s modified Eagle’s medium with 1 g/l glucose, and then preincubated for 30 set in the presence of 750 PM BW284c51 dissolved in Dulbecco’s modified Eagle’s medium and 1 g/l glucose to block the active site of surface AChE. Diisopropylfluorophosphate was then added in the continued presence of BW284c51 to a final concentration of 25 PM for 3 min at room temperature (20°C). The solution containing diisopropylfluorophosphate was removed, and the cells were washed once with 750 PM BW284c51 in Dulbecco’s modified Eagle’s medium and 1 g/l glucose, and then at least 4 times with phosphate-buffered saline. Pretreatment of the cells with 750 PM BW284c51 for 60 set gave similar results, while pretreatment with 750 pM for 90 set resulted in additional protection of the 4-6s (35% instead of 13%) and 1 OS (30% instead of 17%) forms, suggesting that the BW284c51 was able to cross the membrane after longer exposure times.

Cell 74

susceptible to collagenase (Bon et al., 1979; Rotundo and Fambrough, 19791, it has been assumed that collagenase specifically solubilizes 16s AChE from intact muscle cells. This has not been directly demonstrated, however. We thus studied the effect of collagenase on intact myotubes. C2 cells, like chick skeletal-muscle cells in primary culture (Wilson et al., 1973; Rotundo and Fambrough, 1980a, 1980b), normally released AChE into the medium, at a rate of about 2% of the total AChE per hour. When the cells were incubated with collagenase at 37’C, this rate was increased to 8% of the total AChE per hour over the first 2-3 hr of treatment (Figure 4). Because chondroitin sulfate, a glycosaminoglycan, has been shown to be important in the aggregation of the AI2 AChE from Electrophorus (Bon et al., 19781, we also incubated C2 myotubes with chondroitinase ABC. This treatment, however, caused no change in the rate of AChE release. When cells treated with collagenase for 4-5 hr were extracted, and the AChE forms were analyzed, a specific decrease in the 16s form was observed (Figure 5). In contrast, none of the globular forms were affected by collagenase treatment. Analysis of the medium showed that the enzyme activity released by collagenase sedimented at 1 OS. The susceptibility of the 16s enzyme in intact cells to enzymatic removal is further evidence that the location of the enzyme is extracellular. High Ionic Strength, but Not Detergent, Solubilizes 16s Acetylcholinesterase To determine if the A,, AChE is a membrane-bound enzyme, we performed a series of differential solubilization experiments. When C2 myotubes were ho-

TIME OF INCUBATION

Figure 4. Effect linesterase

of Collagenase

mogenized in 1 M NaCl at neutral pH without detergent (Triton X-100), at least 90% of the 16s form was solubilized as well as a minor proportion of the 4-6s forms (Figure 6). Extraction of C2 cells in the presence of a low ionic strength buffer with or without detergent only released globular forms (data not shown). Thus high ionic strength alone is sufficient to solubilize the enzyme; detergent is not required. These results, taken with those above, indicate that the AI2 AChE is not an integral membrane protein, but appears to be associated with insoluble extracellular structures by ionic interactions. Focal Patches of Acetylcholinesterase Correspond to the 16s Form We have reported previously (Silberstein et al., 1982) that histochemical staining of C2 cells for AChE shows that in older cultures some myotubes have a patchy distribution of enzyme activity on their surface. These patches of activity only occur on myotubes and appear 3-5 days after fusion, shortly after the appearance of 16s AChE. The focal staining is abolished by IO ,uM BW284c51. Because of the association of 16s AChE with endplates in adult muscle, we wished’to determine if the focal staining observed in C2 myotubes was due to 16s AChE. We tested for this in two ways: by examining the sensitivity of the staining to BW284c51 protection, and by examining its sensitivity to collagenase. C2 myotubes were stained for AChE after the impermeable inhibitor BW284c51 had been used to block reversibly the active sites of the externally located enzyme during inactivation of the internal AChE by treatment with the irreversible inhibitor, diisopropylfluorophosphate, as described above. The

TOP

(h)

on the Rate of Release

FRACTION of Acetylcho-

C2 cells were grown for 4 days in fusion medium, washed to remove serum and then incubated in the presence (M) or absence (O- - -0) of 0.1 mg/ml collagenase in 0.1 M Tris-Cl (pH 7.4) and 5 mM CaCI,. Incubations were carried out at 37’C.

Figure

5. Effect

of Collagenase

NUMBER

on 16s Acetylcholinesterase

Sucrose gradient profile of AChE forms obtained from C2 cells grown for 4 days in fusion medium. incubated at 37°C for 4-5 hr in the absence (O- - -0) or presence (u) of 0.1 mg/ml collagenase in 0.1 M Tris-Cl (PH 7.4) and 5 mM CaCI, and then harvested, extracted and analyzed as described in Figure 1.

Extracellular 75.

Patches

of 16s AChE

majority of cells showed highly localized staining with strongly positive patches of AChE activity (Figure 76). Thus the patches, like 16s AChE, are on the external surface. Because this treatment appeared to increase the frequency and staining intensity of patches, we assume that in untreated C2 cultures the focal staining was somewhat obscured by a diffuse background of staining throughout the myotube. In some cases these regions of focal staining were clearly localized close

x .r_ E I E 2 t t > F 2 2 2

0

Discussion

6 4 2

0

10

20

30

40 TOP

BOTTOM

FRACTION Figure 6. Solubilization Concentration

of 16s

NUMBER

Acetylcholinesterase

by High

Salt

(O- - -0) Sucrose gradient profile of AChE obtained from C2 cells grown for 4 days in fusion medium, harvested, extracted and analyzed as described in Figure 1. (-1 Sucrose gradient profile of AChE forms extracted in buffer A without detergent.

Figure

to the sarcolemma of the myotube (Figure 76, arrow). By microscopic examination with close focusing it was possible to see at these sites specializations in which the whole deeply staining area appeared to bulge slightly from the cell surface. In a second type of experiment, C2 myotubes were pretreated with collagenase, and then stained for AChE. Almost all the patches of AChE activity disappeared from the surface of the myotubes (Figure 7D), although the diffuse staining remained. The susceptibility of the enzyme in the patches both to protection by impermeable inhibitors and to removal by collagenase indicates that most of the activity must be due to 16s AChE.

7. Patches

of Acetylcholinesterase

Are Protected

by BW284c51

This report on the subcellular distribution of the 16s AChE form in C2, a mouse cell line, is the first clear expertmental demonstration of an extracellular localization for the muscle AI2 AChE form. Although it may be attached to membrane elements, it is not an intrinsic membrane protein and appears to be associated with the extracellular matrix. Two lines of evidence lead to the conclusion that the AI2 form of AChE is on the external surface of the cell: the accessibility of the active site to the membrane-impermeable inhibitor, BW284c51, and the susceptibility of the enzyme to collagenase. As the enzyme is quantitatively extracted by a high ionic strength buffer containing no detergent, we conclude that it is bound at the cell surface

and Digested

by Collagenase

C2 muscle cells were first submitted to either the BW284c51 protection or the collagenase digestion procedures, and then processed for AChE histochemistry. BW284c51 protection. Muscle cultures were washed 3 times with Dulbecco’s modified Eagle’s medium and 1 g/l glucose, and then preincubated for 30 set in the presence of 750 pM BW284c51. In control cells (A) the BW284c51 was subsequently removed, and the cells were washed with Dulbecco’s modified Eagle’s medium. In treated cells(B) diisopropylfluorophosphate was added in the continued presence of BW284c51 to a final concentration of 25 PM for 3-5 min. Diisopropylfluorophosphate was removed, and the cells were washed once with 750 pM BW284c51 and then several times with Dulbecco’s modified Eagle’s medium. Pretreatment of the cells with 75 PM BW284c51 for 1 min gave similar results. Collagenase treatment. Muscle cultures were washed to remove serum with 10 mM Tris-Cl (pH 7.4), and then incubated at 37°C in the absence (C) or presence (D) of 0.1 mg/ml collagenase in 0.1 M Tris-Cl (pH 7.4) and 5 mM CaCb. After 4- 5 hr of incubation, the cells were prepared for AChE histochemistry as described in Experimental Procedures. 500X.

Cdl 76

by ionic interactions. C2 ceils in culture produce an extensive extracellular matrix (Silberstein et al., 1982) and the AI2 AChE is presumably part of that matrix. Another major finding of our study is that in C2 myotubes the 16s or AI2 form of AChE is segregated in focal patches on the external cell surface. This restricted distribution is also found in adult rat muscle, in which the 16s form is localized in the basal lamina at the synapse (Hall and Kelly, 1971; H. L. Fernandez and N. C. Inestrosa, manuscript in preparation). Two well characterized components of the basal lamina, laminin and fibronectin, have also been reported to be focally localized in differentiated myotubes (Chen, 1977; Foidart et al., 1980; but see Furcht et al., 1978; Walsh et al., 1981). In contrast to 16s AChE, however, these components are present in adult muscle in both synaptic and extrasynaptic basement membranes (Sanes, 1982). Our experiments with BW284c51 suggest that in addition to the AI2 form, small amounts of other forms of AChE may also be on the cell surface. These could be components of the surface membrane. Several neuronal (Lazar and Vigny, 1980; Taylor et al., 1981; lnestrosa et al., 1981) and muscle (Rotundo and Fambrough, 1980a) cells in culture have a Gq form on their surface that is associated with the membrane; globular forms of the enzyme are also found in Torpedo membranes (Bon and Massoulie, 1980; Viratelle and Bernhard, 1980). Alternatively, small amounts of the Aq or As enzyme could be attached to the matrix. Our experiments show that almost all of the 16s AChE in C2 myotubes has an extracellular location, where it can be protected from diisopropylfluorophosphate by BW284c51. About 5%-l 0% was not protected; similarly, 7%-10% of the 16s activity was resistant to 4-5 hr of collagenase digestion (Figure 5). Because longer times of pretreatment with BW284c51 gave increasing protection of the 1 OS and 4-6s forms (see legend to Figure 3), it is likely that some BW284c51 crosses the membrane and protects internal AChE. It is thus possible that there is a small internal pool of 16s AChE. Similar experiments in adult rat muscle show that about 15% of the 16s AChE has an intracellular location by these criteria (H. L. Fernandez and N. C. Inestrosa, manuscript in preparation). We have recently obtained clear evidence for an internal pool that comprises about 50% of the total 16s AChE in a mutant strain of PC12 cells (N. C. Inestrosa, W. D. Matthew, C. G. Reiness, 2. W. Hall and L. F. Reichardt, manuscript in preparation). The production of small amounts of A,2 AChE has been reported for other cultured muscle cells (Sugiyama, 1977; Koenig and Vigny, 1978) but it has never previously been established that the enzyme is present on the external cell surface. A novel finding in these experiments is that the A,* enzyme occurs in focal patches on the surface. Focal accumulations of

AChE have been observed at sites of nerve-muscle contact in culture, under conditions in which the cultured cells are known to contain A,* AChE (Koenig and Vigny, 1978; Rubin et al., 1980) but have in general not been seen in aneural cultures, even those that produce the AI2 form (Rieger et al., 1980). Patches of cholinesterase associated with basal lamina have recently been reported in aneural cultures of Xenopus myotomal cells (Moody-Corbett and Cohen, 1981; Weldon et al., 1981); it will be of interest to determine whether these cells contain a tailed species of AChE. In C2 myotubes we do not know what proportion of the AIP form is in patches, as opposed to having a diffuse distribution. Our experiments establish, however, that the patches are composed almost exclusively of the AI2 form, since collagenase treatment, which removes only the AI2 enzyme, completely abolishes focal staining. We have shown that the 16s enzyme in C2 cells has the properties that are associated in Electrophorus and Torpedo enzymes (Bon et al., 1979; Massoulie, 1980) with a long, collagen-like tail. Perhaps the tail, which distinguishes this form from globular forms, is necessary for the accumulation of enzyme seen in the patches on myotubes. Complex association with other matrix components, such as glycosaminoglycans and fibronectin, both of which have been shown to interact with the isolated tailed form of AChE (Bon et al., 1978; Emmerling et al., 1981) may serve to anchor the enzyme in the extracellular matrix. This possibility is consistent with studies showing that multiple interactions of fibronectin, collagen and glycosaminoglycans could play a role in the deposition of these and other substances as an insoluble extracellular matrix (Perkins et al., 1979; Ruoslahti and Engvall, 1980; Yamada et al., 1980). Only a few of the components of the extracellular matrix at the neuromuscular junction have been identified (Sanes and Hall, 1979; Bayne et al., 1981; Sanes, 1982); thus far, AChE is the only enzyme that has been identified as part of an extracellular matrix. The demonstration that only a particular form of the enzyme is found here raises questions about how molecules are incorporated into the extracellular matrix and how they are localized to particular sites within it. Experimental

Procedures

Tissue Culture The C2 cell line was established and initially described by Yaffe and Saxel (1977). The cells were maintained as exponentially growing myoblasts in a medium consisting of Dulbecco’s modified Eagle’s medium with 1 g/l glucose, supplemented with 20% fetal calf serum and 0.5% chick embryo extract (all obtained from the University of California at San Francisco Cell Culture Facility). The cells were grown on Falcon tissue culture plastic flasks, under a humidified 90% air, 10% CO* atmosphere at 37°C. Cell stocks were maintained at less than 75% confluence by passage every 2-3 days. For studies involving differentiated cells, the myoblasts were harvested with trypsin and replated at a density of 5,000-i 0,000 cells/cm*. The cells

Extracellular 77

Patches

of 16s

AChE

were maintained in growth medium for 24-48 hr, until they reached approximately 709/o-80% confluency, at which time the growth medium was removed and replaced with 0.2 ml/cm’ of Dulbecco’s modified Eagle’s medium with 1 g/l glucose supplemented with 10% horse serum (fusion medium). Each day thereafter, the cells were fed with 0.2 ml/cm’ of fresh fusion medium, increasing to 0.4 ml/cm’ on the 4th and 5th days in C2 fusion medium. Within 24 hr. myoblasts began to fuse into multinucleated myotubes that increased in size and branching complexity during the next 5-6 days. Mouse Diaphragm-Muscle Dissection Mouse diaphragm muscles together with attached ribs were removed, washed in ice-cold saline, rapidly cleaned of most of the connective tissue and separated from the ribs. We divided diaphragms into regions containing endplates (the middle third of the muscle; Hebb et al., 1964) and those without endplates, by transversely cutting 2-3 mm strips of muscle, under a dissecting microscope with illumination from below.

for the distribution of reaction product. With much longer fixation times (15-30 min) the amount of background staining of C2 myotubes increased. Chemicals and Special Reagents The AChE inhibitor BW284c51 (1,5-bis[4-allyldimethylammoniumphenyllpentane-3-one dibromide) was a gift from Burroughs Wellcome. Diisopropylfluorophosphate was obtained from Sigma. Sucrose (enzyme grade) was purchased from Schwarz Mann Biochemicals. All other chemicals were of reagent grade. Escherichia coli alkaline phosphatase and b-galactosidase were obtained from Worthington Biochemicals. Chondroitinase ABC (lot E9803,5 U) was a Seikagaku product. Collagenase (form Ill), chromatographically purified and free of detectable nonspecific proteases, was obtained from Advance Biofactures.

Acknowledgments

Extraction of Acetylcholinesterase and Assay of Enzyme Activity Muscle cultures were rinsed 3 times with 10 mM Tris-Cl (PH 7.41, removed from the dishes mechanically and homogenized by hand in a Duall-Kontes ground glass homogenizer in 20 mM Tris buffer (pH 7.41, 1 M NaCI, 0.5% Triton X-i 00, 10 mM EDTA, 20 U/ml Aprotinin, 20 gg/ml pepstatin, 1 mM benzamidine, 1 mM N-ethylmaleimide and 0.1 mg/ml bacitracin (buffer A). AChE activity was assayed according to the method of Ellman et al. (1961), with 0.75 mM acetylthiocholine iodide as the substrate. Hydrolysis of acetylthiocholine by the samples was reduced by over 95% by the addition of 10 PM BW284c51 (EC. 3.1 .1.7), a selective inhibitor of AChE. The inhibitor was routinely included in parallel assays. Thus most of the enzymatic activity that we have observed represents specific AChE. The AChE enzyme assays described were linear with respect to both time and enzyme concentration under the experimental conditions used. Multiplication by 0.0735 converts the U,,,/min to micromoles of substrate hydrolyzed per minute.

We thank Dr. David Yaffe for the C2 cell line; Dr. Helen Blau for advice concerning techniques in myoblast culture; and Drs. Louis Giguere, Ralph Greenspan, Regis Kelly, Louis Reichardt and Joshua Sanes for their comments on an early draft of the manuscript. N. C. I. is a fellow of the Muscular Dystrophy Association of America. L. S. is a fellow of the National Institutes of Health. This research was supported in part by grants from the National Institutes of Health, the Muscular Dystrophy Association of America and the National Science Foundation to 2. W. H. 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.

Sedimentation Analysis of Acetylcholinesterase The molecular forms of AChE were separated by sedimentation on sucrose gradients as described previously (Hall. 1973; lnestrosa et al., 1979, 1981). The high salt-detergent extract was prepared and clarified by centrifugation at 15,000 x g for 15 min. Aliquots (200 (~1) of the resulting supernatants were loaded onto a 5 ml 5%-20% linear sucrose gradient made up in Buffer A. Sedimentation ultracentrifugation was performed in a Beckman L5-65 ultracentrifuge in an SW50.1 rotor. Centrifugation was carried out so that the square of the speed of centrifugation (in rpm) multiplied by the time (in hours) was approximately equal to 1.8 x 10”. In general, samples were centrifuged at 37,000 rpm for 12 hr at 4°C and approximately 40 fractions were collected from the bottom of each gradient. The relative proportion of different AChE molecular forms in relation to total AChE recovered from the gradient (85%-90% recovery) was evaluated by graphical integration of the area under each peak and under the total sedimentation profile. Sedimentation coefficients for AChE forms were determined by comparison with those for /3-galactosidase (16s) and alkaline phosphatase (8.1 S). P-Galactosidase was measured according to the method of Craven et al. (1965), and alkaline phosphatase was measured according to the method of Schlesinger and Barret (1985).

Bader, D. (1981). Density and distribution of a-bungarotoxin-binding sites in post-synaptic structure of regenerated rat skeletal muscle. Cell Biol. 88, 338-345.

Histochemical Reaction for Acetylcholinesterase Muscle cultures were rinsed with Dulbecco’s modified Eagle’s medium with 1 g/l glucose, and fixed for 3 min at room temperature with 1% paraformaldehyde and 0.8% glutaraldehyde in 90 mM phosphate buffer containing 130 mM sucrose. Cultures were then rinsed extensively with phosphate buffer and stained for AChE by the technique of Karnovsky and Roots (1964). The reaction was continued for 3 hr at 37°C. Staining was completely inhibited by 10 pM BW284c51, a specific inhibitor of AGhE. As has been observed in chick primary cultures (Rubin et al., 1979), short fixation times seemed important

Bon, S., Vigny. M. and Massoulie, J. (1979). Asymmetric and globular forms of acetylcholinesterase in mammals and birds. Proc. Nat. Acad. Sci. USA 76, 2546-2550.

Received

December

9. 1981

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