Transplantation of Quail Collagen-tailed Acetylcholinesterase Molecules Onto the Frog Neuromuscular Synapse

Frogs (Rana pipiens) obtained from Hazen Co. (Alburg, VT) were housed at 20–22°C in the animal care facility at the Hebrew University Hadassah Medical School under veterinary supervision and fed twice weekly. Frozen sections of frog muscle, 30 μm thick, were cut from the anterior tibialis muscle, mounted on gelatin-coated slides, and stored frozen until used. Before extraction the slides were warmed to room temperature, and a 0.5-cm high by 1.2-cm diam plastic ring was placed around the sections and held in place with rubber cement. The plastic rings formed the wells in which the various extractions, incubations, and immunohistochemical procedures were performed.

Empty basal lamina sheaths of cutaneous pectoris muscles were prepared as described in detail elsewhere (Anglister et al., 1994a). Muscles were damaged by freezing or crushing and were denervated by excision and removal of a 1–2-cm segment of the second spinal nerve near the vertebral column. The frogs were X-irradiated once on each of the first three days following surgery to prevent regeneration of the muscle fibers from the remaining satellite cells. After allowing 5–6 wk for degeneration of the muscle fibers, the sheaths of the cutaneous pectoris muscles were dissected, pinned in small chambers, and extracted with several changes of borate extraction buffer (see below). All extractions and incubations were carried out in the 150-μl chamber volume. When necessary, the chambers were sealed by pressing a 2.5-cm diam glass coverslip onto the surface to prevent evaporation.

The globular and collagen-tailed AChE forms were isolated from tissuecultured quail myotubes by preparative sucrose gradient sedimentation as previously described (Rotundo, 1984a). The pooled fractions containing the G₄ tetramers and the G₂ dimers (globular forms) or A₁₂ collagen- tailed AChE forms were diluted with borate buffer (see below) and passed through a 1-ml immunoaffinity column made by coupling the monoclonal anti-avian AChE antibody 1A2 (Rotundo, 1984b) to CNBr activated Sepharose 2B (Pharmacia LKB Biotechnology, Piscataway, NJ) at a concentration of 1 mg IgG/ml swelled gel. After washing the column with additional buffer the enzyme was eluted with 2 mM triethanolamine, pH 11, with 1 M NaCl and 100-μl fractions collected in microfuge tubes containing 50 μl 0.5 M Tris, pH 7. The fractions containing the catalytically active AChE forms were pooled, concentrated, and stored frozen until used. The concentration of AChE protein was estimated by enzymatic assay using the turnover number for chicken AChE, 1.45 mmol/min/mg protein, determined by Vigny et al. (1978). The stock solutions of the A₁₂ and G₄/G₂ were 97 ng/ml and 23 ng/ml, respectively.

Tissue sections and empty basal lamina sheaths were pre-extracted with detergent-containing buffer before all experiments to remove muscle plasma membranes, soluble proteins, and/or cellular debris that could interfere with access to the synaptic basal lamina. All extractions were carried out in microwells using borate extraction buffer consisting of 20 mM sodium borate, pH 9.0, 5 mM EDTA, 1 M NaCl, 0.5% Triton X-100, and 5 mg/ml bovine serum albumin high salt buffer (HSB), unless otherwise indicated. In some experiments the salt concentration was reduced to 130 mM low salt buffer (LSB) and/or the Triton X-100 was eliminated. Because of the small size of the tissue sections, protease inhibitors were generally not included in the extraction buffers. However, the sections were always washed with several changes to dilute and remove the extracted proteins.

After extraction, the tissue sections or empty basal lamina sheaths were preincubated for 30 min in HSB containing 5 mg/ml each of bovine serum albumin, chicken ovalbumin, and gelatin (all from Sigma Chem. Co., St. Louis, MO) which have isoelectric points in the same range as avian AChE. The addition of this protein mixture was essential in order to reduce nonspecific binding of AChE to the tissues. The stock AChE solutions were diluted 1:50–1:20 in HSB containing the protein mix, to give 1–2 ng AChE/well final, and the NaCl concentration was adjusted to 0.5 M. This solution was placed in the wells, 100 μl/well, and the ionic strength lowered to 0.3 M stepwise over a period of 2 h to reduce the chances of the collagen-tailed AChE form aggregating which can occur at salt concentrations below 0.3 M. After adjustment to 0.3 M NaCl the sections were incubated overnight at room temperature on a rotary platform. The next day the NaCl concentration was lowered to 0.25–0.15 M NaCl to reduce detachment of bound AChE, the sections rinsed with LSB, and the samples prepared for immunofluorescence. This procedure was modified slightly for use with the empty basal lamina sheaths. For these thicker samples the amount of quail AChE was reduced by a half, the protein added directly to HSB adjusted to 0.3 M NaCl, and the incubations were performed for 2 d in the microchambers sealed with a glass coverslip.

The fraction of the quail AChE that bound to the tissue sections was determined biochemically by treating frozen sections of frog muscle mounted on glass slides with diisopropylfluorophosphate, to irreversibly inhibit AChE activity, followed by extensive washing to remove unreacted inhibitor. The sections were then incubated overnight with HSB containing the protein mixture as described above, either with or without A₁₂ or G₄/G₂ AChE. The sections were rinsed to remove unbound enzyme, scraped into 100-μl HSB and sonicated to disperse the tissue. Aliquots of the tissue extract were assayed for AChE activity, together with samples of the AChE solution before incubation with the tissue sections, to determine the percent AChE bound. In both cases, only a very small percentage of the total AChE added actually bound to the tissue sections, 1.0% and 1.5% for the A₁₂ and G₄/G₂ AChE forms, respectively. Although small amounts of both globular and collagen-tailed AChE binding could be detected biochemically, on the order of about 10 pg each, most of this probably reflects nonspecific binding since the available surface area of the junctional regions on these frozen sections is <0.1% of the total cross-sectional area. Therefore, in practice, it is not possible to estimate the extent of specific AChE binding in these experiments and for this reason we used immunofluorescence localization of the quail AChE.

After incubation with purified AChE forms, the tissue samples were washed with several chamber volumes of 10 mM PBS, pH 7.4, containing 10% horse serum (PBS/HS), to remove unbound enzyme and saturate nonspecific binding sites on the tissue sections. The sections were incubated with PBS/HS containing 20 μg/ml 1A2 anti-avian AChE monoclonal antibody for 60 min followed by three washes of PBS/HS over a 30– 60-min period. The second antibody was fluorescein-conjugated rabbit anti–mouse IgG (Cappel Laboratories, Malvern, PA) used at a concentration of 10 μg/ml in PBS/HS, for 60 min. After washing with additional PBS/HS, samples were rinsed with PBS alone, fixed with 4% paraformaldehyde in PBS, and mounted in 90% glycerol/10% 100 mM bicarbonate buffer, pH 9.5, containing 1 mg/ml phenylenediamine (Sigma) to reduce photobleaching. In some experiments 1 μg/ml TRITC-conjugated α-bungarotoxin (αBtx) (Molecular Probes, Inc., Eugene, OR) was included together with the first antibody to label the frog nicotinic acetylcholine receptors, and the frog AChE was localized using either a cross-reacting polyclonal antibody generated against the enzyme isolated from the electric ray (antibody R80, a generous gift from Dr. P. Taylor, U.C. San Diego, La Jolla, CA) or by immunocytochemistry following the procedure of Karnovsky (1964).

The frog neuromuscular junction consists of several nerve terminal branches coursing in parallel to the long axis of the muscle fiber. On cross sections of muscle fibers these synaptic gutters appear as a series of several peripherally localized clusters of AChR and AChE molecules (see Figs. 2 and 3). To determine the extent of colocalization, all TRITC-αBtx labeled AChR clusters were counted on three randomly chosen cross sections of frog muscle per slide. The fraction of TRITC-αBtx labeled AChR clusters that were also positive for fluorescein-labeled AChE was then determined and expressed as percent of total. At least three slides per group were quantitated and the results expressed as the mean percentage ± SEM.

In both mammals and birds there is an accumulation of nuclei in the innervated regions of the muscle fibers that express higher levels of AChR (Merlie and Sanes, 1985; Klarsfeld et al., 1991; Simon et al., 1992) and AChE (Jasmin et al., 1993; Michel et al., 1995; Legay et al., 1995) transcripts. To determine the numbers and distribution of myonuclei at frog neuromuscular junctions, teased fibers from the cutaneous pectoris muscles were stained histochemically for AChE (Karnovsky, 1964) and the nuclei labeled with HOECHST 33342 (Fig. 1). The average neuromuscular junction in the cutaneous pectoris muscle is located along a 340 ± 50 μm (mean ± SEM, n = 24) long central segment of the myofiber and has about 3–4 nuclei distributed along its length. These myonuclei are typically elongated, sausage-shaped, organelles that can be easily distinguished from the rounded nuclei in the Schwann cells, fibroblasts, and satellite cells. The nuclei within the innervated region of the fibers were counted and compared to the distribution of nuclei within the same length of non-innervated regions in the same fibers. In these fibers the ratio of junctional to extrajunctional nuclei was 1.09 ± 0.04 (mean ± SEM; n = 24). Thus, in contrast to higher vertebrates where a dozen or more disc-shaped nuclei can be distributed immediately beneath the sites of nerve-muscle contact, at the frog neuromuscular junction there does not appear to be such an accumulation of myonuclei. These observations indicate that, in frog, the AChE molecules that are destined to become incorporated at the neuromuscular junction must move long distances following translation before their extracellular attachment at precise and orderly locations on the synaptic basal lamina of the elongated junction.

This initial study was performed to determine the extractability of frog AChE and the extent to which the AChR and AChE remained colocalized following incubation with extraction buffers. Frog muscles were pretreated so that synaptic sites would retain the putative “association sites” for AChE while releasing at least some of the frog AChE from those sites. We have previously shown that AChE is poorly extracted from quail and rat neuromuscular junctions even using high ionic strength buffers, with or without detergents (Rossi and Rotundo, 1993). Similar extraction procedures were applied to frog muscles, and the extent to which the AChR and AChE clusters remained colocalized under the different conditions was examined as follows. Frozen sections of anterior tibialis muscle were incubated with frog Ringer's solution, LSB, or HSB, with or without detergent, for either 90 min or overnight. The distribution of AChR and AChE were examined by incubating the sections with TRITC-αBtx to label AChR and by indirect immunofluorescence to label AChE (Fig. 2). The different extractions had only a small effect on the intensity of AChR and AChE staining, indicating that under the conditions of this study most of these molecules remained attached to the frog muscle frozen sections throughout the incubation period. These results, presented in Table I, indicate that the distribution of AChR clusters accurately matches the distribution of AChE molecules even after extensive extraction with high salt and detergent containing buffers. Similar results were obtained using whole mounts of cutaneous pectoris muscle (data not shown).

Newly synthesized A₁₂ AChE molecules undergo transient electrostatic interactions with components of the extracellular matrix before becoming more permanently attached (Rossi and Rotundo, 1996). The initial interactions are blocked in the presence of heparin, whereas once the A₁₂ molecules are attached, heparin cannot remove them. These observations suggest that specific attachment sites for the A₁₂ AChE form exist on the extracellular matrix and that the availability of such sites at the neuromuscular junctions could determine the distribution of AChE on the synaptic basal lamina.

To determine whether exogenous quail AChE could attach to specific sites on frog muscle, purified globular G₄/G₂ or collagen-tailed A₁₂ forms, prepared from tissue-cultured quail myotubes as described in Materials and Methods, were incubated with high salt-detergent extracted sections of frog muscle under conditions where the ionic strength of the medium could be gradually reduced. After washing the chambers to remove unbound enzyme, the sections were incubated with TRITC-αBtx to localize AChRs and mAb 1A2 to localize quail AChE (Fig. 3). The monoclonal anti-AChE antibody used in these experiments is species-specific and does not recognize the frog enzyme (Fig. 3, A and B). When frog muscle was incubated with the globular G₄/G₂ AChE forms there was little or no binding of the enzyme molecules to sites of nervemuscle contact represented by the clusters of AChR accumulation (Fig. 3, C and D). In contrast, when the frog muscle sections were incubated with purified A₁₂ AChE most sites of nerve-muscle contact also had quail AChE attached (Fig. 3, E and F). This attachment was specific in that inclusion of heparin in the incubation medium prevented attachment of the A₁₂ (Fig. 3, G and H), just as it prevented attachment of the newly synthesized AChE to cell surface clusters on tissue-cultured myotubes (Rossi and Rotundo, 1996). These results are presented quantitatively in Fig. 4 showing that more than 85% of the sites of frog nerve-muscle contact were also positive for quail AChE, while inclusion of heparin reduced binding to <15%.

Interestingly, re-extraction of the frog muscle sections incubated with A₁₂ AChE using high salt and detergent containing buffers did not remove the quail AChE (Fig. 4). This result is similar to observations made both on adult neuromuscular junctions and on tissue-cultured myotubes, indicating that once the avian A₁₂ AChE molecules are attached, high ionic strength buffers cannot dissociate them from the extracellular matrix (Rossi and Rotundo, 1993, 1996).

The attachment of quail AChE to the frog neuromuscular junction could occur by direct interactions with components of the extracellular matrix or with molecules localized on the postsynaptic membrane or on the nerve terminal (or some combination). To distinguish between these possibilities, empty basal lamina sheaths were prepared by denervating the frog cutaneous pectoris muscles and destroying the muscle fibers, followed by X-irradiation to prevent fiber regeneration (Anglister et al., 1994a). After allowing 5–6 wk for the damaged fibers and nerve terminals to completely degenerate and be removed by phagocytosis, the empty basal lamina sheaths were dissected, pinned out in Silgard microwells, and extracted with HSB. The empty basal lamina sheaths were incubated in buffers containing either the globular G₄/G₂ or collagen-tailed A₁₂ AChE forms for 48 h, washed, and stained for quail AChE using mAb 1A2 and frog AChE using enzyme histochemistry (Fig. 5). Only the A₁₂ AChE bound to the empty basal lamina sheaths where it was localized along portions of the synaptic gutters in a pattern identical to the native frog enzyme molecules. These results indicate that the A₁₂ AChE molecules are binding to specific components of the synaptic basal lamina, and moreover, that this component(s) is highly organized along the region of the basal lamina contacted by the nerve terminals. Furthermore, all the components of the reaction responsible for binding the collagen-tailed AChE to the extracellular matrix must belong to the extracellular matrix itself or to the purified enzyme, since it was possible to transplant avian A₁₂ AChE to the empty basal lamina sheaths.

The multiple oligomeric forms of AChE expressed in nerves and muscle have been extensively studied and much is known about their structure, synthesis, assembly, and regulation at the cellular and molecular level. However, the mechanisms involved in localizing the newly synthesized AChE molecules at cholinergic synapses are still not well understood. In particular, the unique association of the collagen-tailed A₁₂ AChE form with the specialized extracellular matrix interposed between the motor nerve terminals and the skeletal muscle fibers, requires that a precise targeting mechanism exist to insure the appropriate distribution and density of enzyme molecules at the neuromuscular junction as well as providing a means for their periodic replacement.

The major events during the synthesis and assembly of the oligomeric AChE forms in skeletal muscle are well documented (for review see Massoulié et al., 1993). The catalytic subunits consist of single polypeptide chains that are cotranslationally glycosylated in the rough endoplasmic reticulum where they are assembled into dimers and tetramers. After vesicular transport into the Golgi apparatus a subset of the oligomers are attached to the noncatalytic subunit, the three-stranded collagen-like tail, via disulfide bonds to complete the hetero-oligomeric form consisting of twelve catalytic subunits of ∼65–100 kD each and three noncatalytic polypeptide chains of ∼55 kD each for a total M_r of ∼1.1–1.4 million and a length of ∼50 nm depending upon the species. The newly assembled A₁₂ forms are then transported to the cell surface and externalized via fusion of the transport vesicles. In tissue cultured skeletal muscle, the A₁₂ form remains associated with the cell surface, whereas in tissue-cultured neurons the A₁₂ form appears to be mostly, if not entirely, secreted. Thus, the fate of the newly synthesized A₁₂ AChE, i.e., whether it is released vs retained, depends upon the cell type in which it was synthesized and the molecular composition of its cell surface components. In tissue-cultured myotubes, the A₁₂ AChE molecules preferentially accumulate in cell surface clusters in the vicinity of the myonuclei around which they were synthesized (Rossi and Rotundo, 1992).

In vivo, the problem of targeting the A₁₂ AChE to the mature neuromuscular junction is more complex due to the intricate architecture of the synaptic region and the requirement that the AChE molecules lie precisely between the neurotransmitter release sites on the nerve terminals and the array of nicotinic acetylcholine receptors on the postsynaptic membrane. Although there is a higher level of AChE mRNA, as well as protein expression in innervated regions of skeletal muscle fibers (Jasmin et al., 1993), which in turn can increase localized secretion of the enzyme, this alone is not sufficient to insure correct organization of the AChE molecules on the synaptic basal lamina since many of the areas of high AChE density can be tens of micrometers from the nearest nucleus (Fig. 1). Thus, secretion followed by limited diffusion of the protein must account, at least in part, for the distribution of AChE molecules along the synaptic basal lamina.

While limited diffusion can account for the dispersion of A₁₂ AChE molecules from sites of secretion on the synaptic sarcolemma, it cannot account for the highly organized pattern of matrix-associated AChE interposed between the nerve terminals and the postsynaptic membrane. Within a species, the density of AChE molecules at the neuromuscular junctions is remarkably constant from one area of nerve contact to another (Anglister et al., 1994b, and references therein). Together, these observations imply that some molecular component(s) of the synaptic basal lamina is already organized and available for the attachment of the A₁₂ AChE. That this is indeed the case is suggested by the experiments presented in Figs. 3–5 showing that only the A₁₂ AChE form can bind to the neuromuscular junctions where it colocalizes with AChRs.

Although the molecule(s) to which the A₁₂ AChE bind have not yet been identified, there is strong indirect evidence to suggest that it may be a heparan sulfate-like proteoglycan localized in the extracellular matrix. Early studies on the structure and membrane association of AChE from Torpedo electric organs showed that the collagentailed, but not the globular forms, could associate with fragments of the extracellular matrix isolated by sucrose gradient centrifugation (Lwebuga-Mukasa et al., 1976). The collagenic tail of AChE contains a heparin-binding domain (Deprez and Inestrosa, 1995) and can specifically associate with heparan sulfate glycosaminoglycans (Bon et al., 1978; Vigny et al., 1983; Brandan et al., 1985). A specific heparan sulfate proteoglycan is highly concentrated at the vertebrate neuromuscular junction (Anderson and Fambrough, 1983; Bayne et al., 1984; Sanes et al., 1986; Swenarchuk et al., 1990), where its distribution correlates very closely with AChE. Whether AChE attaches only to this sulfated proteoglycan or to other molecules with similar sulfated carbohydrates remains to be determined.

Based upon the above observations and our present studies, a plausible mechanism for localizing AChE on the synaptic basal lamina would be that the newly synthesized A₁₂ AChE molecules, preferentially expressed in innervated regions of the fibers, would be secreted into the space between the sarcolemma and the overlying synaptic basal lamina. The space would afford limited diffusion of these large molecules until they could interact electrostatically with the appropriate heparan sulfate proteoglycans. This interaction would be transient (Rossi and Rotundo, 1996) until additional events resulted in a more permanent form of attachment, such as formation of covalent bonds with neighboring molecules or additional intrachain disulfide bonds within the collagen-like tail subunits (Krejci et al., 1991). The A₁₂ AChE would then remain attached to the synaptic basal lamina until it was detached via some as yet unknown, but possibly enzymatic, mechanism. To date, the only available quantitative data on turnover of AChE at the neuromuscular junction indicates that it has a relatively long half life of ∼20 d and is removed by a process that exhibits first order decay kinetics (Kasprzak and Salpeter, 1985). A specific attachment site on the synaptic basal lamina would therefore be the molecular equivalent of a space in a “parking lot,” whereby the newly synthesized collagen-tailed AChE molecules could be inserted and removed in the region between the nerve terminals and the acetylcholine receptors, as necessary. Furthermore, these studies also emphasize the fact that, despite differences in the underlying cellular and molecular mechanisms regulating AChE biogenesis between amphibians and aves, similar mechanisms determine the ultimate numbers and patterns of distribution of AChE molecules at the neuromuscular synapse.

We would like to thank Rachel Cohen for excellent technical assistance, Drs. Kenneth J. Muller and Israel Silman for their critical reviews of the manuscript, and Dr. Palmer Taylor for the anti-AChE antibody R80.

AChE

acetylcholinesterase

AChR

acetylcholine receptors

Btx

α-bungarotoxin

HSB

high salt buffer

LSB

low salt buffer