* Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, Florida, and Department of
Anatomy and Cell Biology, Hebrew University-Hadassah Medical School, Jerusalem, Israel
The highly organized pattern of acetylcholinesterase (AChE) molecules attached to the basal
lamina of the neuromuscular junction (NMJ) suggests
the existence of specific binding sites for their precise
localization. To test this hypothesis we immunoaffinity purified quail globular and collagen-tailed AChE forms
and determined their ability to attach to frog NMJs
which had been pretreated with high-salt detergent
buffers. The NMJs were visualized by labeling acetylcholine receptors (AChRs) with TRITC--bungarotoxin and AChE by indirect immunofluorescence; there
was excellent correspondence (>97%) between the distribution of frog AChRs and AChE. Binding of the
exogenous quail AChE was determined using a speciesspecific monoclonal antibody. When frog neuromuscular junctions were incubated with the globular G4/G2
quail AChE forms, there was no detectable binding
above background levels, whereas when similar preparations were incubated with the collagen-tailed A12
AChE form >80% of the frog synaptic sites were also
immunolabeled for quail AChE attached. Binding of
the A12 quail AChE was blocked by heparin, yet could
not be removed with high salt buffer containing detergent once attached. Similar results were obtained using
empty myofiber basal lamina sheaths produced by mechanical or freeze-thaw damage. These experiments
show that specific binding sites exist for collagen-tailed AChE molecules on the synaptic basal lamina of the
vertebrate NMJ and suggest that these binding sites
comprise a "molecular parking lot" in which the AChE
molecules can be released, retained, and turned over.
Acetylcholinesterase (AChE),1 the enzyme responsible for hydrolyzing acetylcholine released
at the neuromuscular junction, is localized in the
synaptic cleft interposed between the nerve terminals and
the postsynaptic membrane, where a substantial fraction of the enzyme is attached to the synaptic basal lamina
(McMahan et al., 1978 The junctional AChE molecules can be contributed either by the muscle fibers (Anglister and McMahan, 1985 In birds and mammals the predominant, if not exclusive,
form of AChE attached to the synaptic basal lamina is the
asymmetric A12 form of the enzyme consisting of three tetramers covalently linked to a three-stranded collagen-like
tail (reviewed in Massoulié et al., 1993 Tissue-cultured muscle cells express the same oligomeric forms of AChE as adult muscle including the globular monomeric (G), dimeric (G2), and tetrameric (G4) forms
and the asymmetric collagen-tailed form (A12). When the
myotubes differentiate into actively contracting myofibers
they assemble the A12 collagen-tailed form which in turn
becomes organized into clusters on the extracellular matrix overlying individual myonuclei (Rossi and Rotundo,
1992 In the present study we sought to determine whether
specific attachment sites for the collagen-tailed AChE
form were situated on the synaptic basal lamina of adult
muscle. Using purified quail globular and collagen-tailed
AChE forms, we demonstrate that only the collagen-tailed
form of the enzyme can bind to the frog neuromuscular junction where it attaches to the extracellular matrix.
These experiments show that specific sites exist for localizing AChE at the neuromuscular synapse and that the
properties of these sites are shared across species as divergent as amphibians and birds. Furthermore, since AChE
molecules can be turned over at the neuromuscular junction while maintaining their appropriate distributions (Kasprzak and Salpeter, 1985 Preparation of Tissues and Empty Basal
Lamina Sheaths
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 Isolation and Purification of Avian AChE Forms
The globular and collagen-tailed AChE forms were isolated from tissuecultured quail myotubes by preparative sucrose gradient sedimentation as
previously described (Rotundo, 1984a Extraction of AChE from Tissue Sections and Transfer
of Quail Enzyme
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.
Immunofluorescence Localization of AChE and
Labeling of AChR
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 Quantitation of AChR and AChE Colocalization
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-
Distribution of Nuclei at the Frog
Neuromuscular Junction
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
Attachment of Acetylcholinesterase to the Frog
Neuromuscular 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 Table I.
Colocalization of AChR and AChE after Different
Extraction Procedures
; for review see Massoulié et al.,
1993
). The distribution of AChE molecules on the synaptic basal lamina closely matches the distribution of nicotinic acetylcholine receptors (AChRs), as well as other
molecules on the pre- and postsynaptic membranes, indicating a high degree of organization of the molecular components at the neuromuscular junction (reviewed in Hall
and Sanes, 1993
). Although most of the AChE expressed
in muscle can be solubilized, the AChE molecules attached to the synaptic basal lamina in birds and mammals
are not removed by high ionic strength buffers, detergents, or chaotropic agents (Rossi and Rotundo, 1993
). On the
other hand, the basal lamina-associated AChE can be detached using mixtures of collagenase and other proteases
(Hall and Kelly, 1971
; Betz and Sakmann, 1973
) or highly
purified collagenase (Rossi and Rotundo, 1993
).
;
De La Porte et al., 1986) or by the motoneurons (Anglister, 1991
), with the former most likely contributing most of
the basal lamina-associated enzyme. After denervation,
there is a large decrease in the density of AChE molecules
at the neuromuscular synapse which can be restored by
electrical stimulation of the denervated muscles or by their reinnervation either at the original (Lømo and Slater, 1980
)
or at ectopic sites (Weinberg and Hall, 1979
). Furthermore, regenerating myofibers re-accumulate basal lamina
AChE at original synaptic sites in the absence of innervation (Anglister and McMahan, 1985
). Altogether, these
studies show that muscle fibers can produce synaptic
AChE and, moreover, indicate that the information necessary for organizing AChE molecules on the synaptic basal
lamina is associated either with the muscle or contained
within the synaptic matrix itself.
). The collagen-like
tail is generally thought to be required for attachment of
AChE to the extracellular matrix. The observation that
only proteolysis using collagenase can remove the junctional AChE is consistent with this view and suggests that
the AChE molecules become covalently attached to components of the specialized extracellular matrix at the neuromuscular synapse.
). These clustered AChE molecules behave very much
like the AChE attached to the synaptic basal lamina in
that they cannot be removed using high ionic strength
buffers or detergents, yet can be detached using purified
collagenase. When these forms are externalized at the surface plasma membrane they appear to transiently interact with heparan sulfate-like proteoglycans before attaching
more permanently to the extracellular matrix (Rossi and
Rotundo, 1996
). Although these studies show that the A12
AChE form is selectively retained on the extracellular matrix in the vicinity of the myonuclei, they do not distinguish
between several possible mechanisms of targeting including association with the extracellular matrix at the sites of
externalization, or, more interestingly, preferential attachment to specific sites organized on the basal lamina.
), our observations suggest that a
"molecular parking lot" exists for the insertion and removal of this enzyme on the synaptic basal lamina.
Materials and Methods
). 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 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.
-bungarotoxin (
Btx) (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)
.
Btx labeled AChR
clusters were counted on three randomly chosen cross sections of frog
muscle per slide. The fraction of TRITC-
Btx 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.
Fig. 2.
Localization of AChR and frog AChE at the frog neuromuscular junction; effects of extraction procedures. Frozen sections of anterior tibialis muscle were extracted for 90 min at room temperature in either frog Ringer's solution, LSB, or HSB before localizing sites of AChR accumulation using TRITC-Btx and frog AChE using the polyclonal antibody R80. Upper panels show the distribution of AChR and the lower panels show the corresponding distribution of frog AChE in the same fields. (A-B) Muscle sections incubated in
frog Ringer's solution; (C-D) sections extracted with LSB; and (E-F) sections extracted with HSB. In all cases there was a high degree
of correspondence between the sites of AChR and AChE localization, regardless of whether the sections were extracted with high ionic
strength buffers and/or detergents, indicating that the sites of AChE attachment are preserved during extraction and can still be identified using AChR as a marker.
[View Larger Version of this Image (26K GIF file)]
Fig. 3.
Binding of collagen-tailed quail AChE molecules to frog muscle and colocalization with AChR at the neuromuscular junction. Frozen sections of frog anterior tibialis muscle were incubated overnight with HSB containing either no exogenous quail enzyme (control, panels A-B), avian G4/G2 AChE (C-D), avian A12 AChE (E-F), or avian A12 AChE plus 1 mg/ml heparin (G-H) as described in
the text. After incubation the sections were rinsed with PBS and the quail AChE molecules localized by indirect immunofluorescence. Sites of nerve muscle contact were visualized using TRITC-Btx. Left panels, distribution of AChR at sites of nerve-muscle contact. Right panels, corresponding fields showing the distribution of quail AChE molecules. Only the A12 AChE form bound to the tissue sections, where it colocalized with frog AChR, indicating that this form of the enzyme binds specifically to the neuromuscular junction. In
contrast, there is little or no binding of the globular G4/G2 forms under the same conditions.
[View Larger Version of this Image (69K GIF file)]
Results
;
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.
Fig. 1.
Distribution of muscle nuclei at the frog neuromuscular
junction. Frog cutaneous pectoris muscles were pinned at resting
length in Silgard-coated dishes and the neuromuscular junctions
visualized by histochemically staining for AChE followed by 60 min incubation in frog Ringer's containing 100 U/ml of collagenase (Sigma type IV) to facilitate teasing the fibers free of fibroblasts and Schwann cells. After fixation with 4% paraformaldehyde in PBS and staining of nuclei with HOECHST 33342, the
fibers were teased and mounted in glycerol/phenylenediamine.
The myonuclei in frog muscle have the typical elongated shape
characteristic of myonuclei in other species, and can be easily distinguished from the rounded nuclei in the Schwann cells and fibroblasts. In contrast to mammals and birds, there is no accumulation of nuclei at the frog neuromuscular junction. Bar, 50 µm.
[View Larger Version of this Image (97K GIF file)]
). 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-
Btx 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).
Binding of Quail Collagen-tailed AChE Molecules to the Frog Neuromuscular Junction
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-Btx 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 Collagen-tailed AChE Molecules Attach to Specific Sites on the Synaptic Basal Lamina
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 Mr 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.
Received for publication 5 August 1996 and in revised form 4 October 1996.
This research was supported by research grants from the National Institutes of Health to R.L. Rotundo and from the Israel Academy of ScienceCharles H. Revson Foundation to L. Anglister. These experiments were carried out at the Hebrew University Hadassah Medical School while R.L. Rotundo was Hebrew University Visiting Professor of Medicine and Science.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, -bungarotoxin;
HSB, high salt buffer;
LSB, low salt buffer.