(Received for publication, July 12, 1995; and in revised form, October 26, 1995)
From the
Heparin is capable of solubilizing a subset of collagen-tailed
(A) acetylcholinesterase (AChE) molecules from skeletal
muscle fibers, but cannot detach AChE from the synaptic basal lamina
(Rossi, S. G., and Rotundo, R. L.(1993) J. Biol. Chem. 268,
19152-19159). In the present study, we used tissue-cultured quail
myotubes to show that, like adult fibers, neither heparin- nor high
salt-containing buffers detached AChE molecules from cell-surface
clusters. Prelabeling clustered AChE molecules with anti-AChE
monoclonal antibody 1A2 followed by incubation in heparin-containing
medium showed that there was no reduction in the number or size of
pre-existing AChE clusters. In contrast, incubation of myotubes with
culture medium containing heparin for up to 4 days reversibly blocked
the accumulation of new cell-surface AChE molecules without affecting
the rate of AChE synthesis or assembly. Newly synthesized A
AChE becomes tightly attached to the extracellular matrix
following externalization. However, in the presence of heparin,
blocking the initial interactions between A
AChE and the
extracellular matrix results in release of AChE into the medium with a t
of
3 h. Together, these results suggest
that once A
AChE is localized on the cell surface,
initially attached via electrostatic interactions, additional factors
or events are responsible for its selective and more permanent
retention on the basal lamina.
The asymmetric collagen-tailed (A)
acetylcholinesterase (AChE) (
)molecule is the predominant
oligomeric form of this enzyme at the neuromuscular junction, where it
is attached to the synaptic basal lamina (reviewed by Taylor(1991) and
Massouliéet al.(1993)). The molecular
mechanisms underlying the highly selective targeting and retention of
this synaptic component at the appropriate location on the cell surface
are still not clearly understood, but probably involve a combination of
transcriptional, post-transcriptional, and post-translational events.
In tissue-cultured skeletal muscle fibers, the AChE catalytic subunits
are locally translated and assembled around the nuclei encoding their
transcripts (Rotundo, 1990), and the newly synthesized AChE oligomers
are selectively localized to regions of the cell surface over the
nucleus of origin (Rossi and Rotundo, 1992). The levels of AChE mRNA,
studied in tissue-cultured cells, appear to be regulated
post-transcriptionally (Fuentes and Taylor, 1993). In vivo,
transcripts encoding AChE are highly concentrated at the vertebrate
neuromuscular synapse (Jasmin et al., 1993), suggesting that,
like the tissue-cultured myotubes, AChE oligomers are locally
transcribed, translated, assembled, and selectively localized to the
overlying synaptic basal lamina. The inability of conventional
extraction procedures to remove the junctional AChE molecules, such as
high ionic strength buffers, polyanions, and chaotropic agents,
suggests that the enzyme is covalently attached to the extracellular
matrix (Rossi and Rotundo, 1993).
A likely candidate molecule
involved in localizing AChE to the neuromuscular junction is a heparan
sulfate proteoglycan (HSP). A AChE binds specifically to
heparin and sulfated glycosaminoglycans (Bon et al., 1978;
Vigny et al., 1983; Brandan et al., 1985; Brandan and
Inestrosa, 1986) and can be solubilized from muscle with heparin
(Torres and Inestrosa, 1983; Barat et al., 1986) or other
polyanions (Pérez-Tur et al., 1991a).
Electron microscopy of negatively stained aggregates of A
AChE and polyanionic components of the extracellular matrix from Torpedo electric organs shows that the distal regions of the
collagen-like tail are involved in the binding (Bon et al.,
1978). Brandan and Inestrosa(1984) demonstrated that only the A
AChE form binds to heparin-agarose, indicating that binding is
dependent on the noncatalytic collagen-like tail subunit, and only
heparin is able to displace the bound AChE. In contrast, however,
heparin does not detach A
AChE from the synaptic basal
lamina (Rossi and Rotundo, 1993).
Although widely distributed throughout the extracellular matrix surrounding muscle fibers, HSP also extends into the junctional region, where its density is increased severalfold on the synaptic basal lamina (Bayne et al., 1981; Anderson and Fambrough, 1983; Sanes et al., 1986). The deposition of HSP is spatially and temporally correlated with AChR aggregation in developing myotubes and at developing neuromuscular synapses in culture (see Anderson and Fambrough(1983) and Bayne et al.(1984); reviewed by Hall and Sanes(1993)). Furthermore, there is a positive correlation between the formation of acetylcholine receptor (AChR) clusters and subsequent accumulation of extracellular matrix components, including HSPs, in cultured muscle cells (Bayne et al., 1984; Swenarchuk et al., 1990). Agrin, a protein originally isolated from basal lamina-rich extracts of Torpedo electric organs, induces the clustering of AChR, AChE, butyrylcholinesterase, the cytoplasmic AChR-associated 43-kDa protein, and HSP on chick myotubes in culture (Wallace et al., 1985; Nitkin et al., 1987; Wallace, 1989; Lieth and Fallon, 1993). However, in myotubes pretreated with inhibitors of protein synthesis, aggregates of HSP and AChE were not detected even though agrin continued to induce the formation of AChR aggregates (Wallace, 1989). These observations indicate that the formation of HSP and AChE clusters are downstream events from the initial AChR clustering and suggest that they may possibly be linked.
In quail skeletal muscle cultures, the
A AChE form is clustered on the upper surface of the
myotubes, where it can be removed using purified collagenase (Rossi and
Rotundo, 1992). However, the mechanism of attachment of the clustered
A
AChE has not been analyzed in detail. In this study, we
show that heparin does not detach A
AChE previously
clustered on the surface of myotubes. In contrast, heparin reversibly
blocks the accumulation of newly synthesized A
AChE in a
time-dependent manner. Furthermore, we show that only the newly
synthesized A
AChE molecules can be solubilized from the
cell surface. In untreated cultures, these molecules become tightly
linked to the extracellular matrix, whereas in the presence of heparin,
they are readily removed. This study suggests that HSPs are involved in
the initial targeting of AChE to specialized regions of the cell
surface, but that once localized, more permanent mechanisms of
attachment are formed.
Alternatively, AChE oligomeric forms expressed on the cell surface were analyzed by velocity sedimentation following protection with the water-soluble reversible AChE inhibitor BW284c51 and irreversible inactivation of the intracellular AChE with diisopropyl fluorophosphate (DFP) as described previously (Rotundo, 1984b). Under these conditions, >80% of the total cell-surface AChE is protected by BW284c51 (data not shown).
To quantitate newly synthesized AChE, three 35-mm cultures dishes per group were rinsed three times with HBSS, followed by a 10-min incubation with 10 µM DFP in HBSS to irreversibly inhibit all AChE activity. The cultures were then rinsed with HBSS and returned to the culture incubator for 24 h in modified defined medium with or without heparin. To identify secreted AChE forms, 200-µl aliquots of medium from each dish were analyzed by velocity sedimentation. The three cultures per group were then rinsed with HBSS and extracted in a total volume of 600 µl of borate extraction buffer containing 0.5% Triton X-100 and 1 M NaCl (HSB) to determine total cell-associated AChE forms. The pooled culture extracts were homogenized and centrifuged for 20 min in a microcentrifuge (4 °C, 14,000 rpm). The AChE oligomeric forms were resolved by velocity sedimentation on 5-20% sucrose gradients for 16 h at 32,000 rpm in an SW 50.1 rotor. Fifteen-drop fractions were collected, and AChE activity was assayed by a modification of the radiometric method of Johnson and Russell(1975) as described previously (Rotundo and Fambrough, 1979).
For quantitation
of AChE clusters, only accumulations >3 µm localized on the
upper surface of the myotubes were counted in each field. The total
number of nuclei in myotubes and the number of AChR clusters in the
same field were also determined. Ten fields (350 µm each) were sampled from each coverslip culture, and three
coverslips were quantitated for each point. The results are expressed
as clusters per myotube nuclei (mean ± S.E.).
Figure 1: Immunofluorescence localization of cell-surface AChE clusters following extraction with high salt- or heparin-containing buffers. Seven-day-old quail muscle cultures were extracted for 1 h with one of the different detergent-containing extraction buffers prior to localization of cell-surface AChE by indirect immunofluorescence using anti-AChE mAb 1A2. A, myotubes extracted with LSB; B, myotubes extracted with HSB; C, myotubes extracted with HSB containing 0.5 mg/ml heparin. Neither high salt buffers nor heparin released AChE from cell-surface clusters. Bar = 25 µm.
To quantitate the amount of cell-surface AChE remaining after the different extraction procedures, three cultures per group were incubated for 1 h with PBS alone, PBS plus 1.0 mg/ml heparin, PBS with 1.0 M NaCl, or PBS with 1 M NaCl and 1.0 mg/ml heparin; and the remaining cell-surface AChE activity was assayed. Control cultures were incubated with PBS alone. The results show that neither heparin nor high concentrations of NaCl removed catalytically active AChE molecules from the cell surface (Table 1). In addition, other polyanion-containing buffers have also been shown to solubilize at least some asymmetric AChE from adult chicken muscle (Pérez-Tur et al., 1991a). To determine whether any of these buffers could detach AChE clusters on quail myotubes, the number of clusters per nucleus was quantitated following solubilization. The results, shown in Table 2, indicate that AChE was not detached even after 1 h of extraction using high salt- or polyanion-containing buffers.
Figure 2:
Heparin prevents the accumulation of
cell-surface AChE on tissue-cultured myotubes. Muscle cells were grown
in complete medium with or without 1 mg/ml heparin beginning at the
time of myoblast fusion on day 3 of culture. At the indicated times,
three cultures per group were rinsed with PBS and incubated in the
buffer/substrate mixture to assay cell-surface AChE activity. ,
normal medium;
, medium plus heparin (H) for 1 h;
, medium plus heparin, days 5-7;
, medium plus
heparin, days 3-7. A, linearity of the assay for each
experimental group on day 7; B, long-term effect of heparin in
culture on cell-surface AChE activity. There was no effect between days
3 and 5, whereas accumulation was blocked between days 5 and 7
following the onset of asymmetric AChE expression around day 5 in
culture. Prolonged exposure to heparin reduced the accumulation of
catalytically active AChE molecules on the cell
surface.
Figure 7:
Newly synthesized asymmetric AChE is
secreted into the medium in heparin-treated muscle cultures.
Six-day-old muscle cultures were treated with DFP to irreversibly
inhibit all AChE molecules and allowed to recover for 24 h in defined
medium containing 20 mg/ml bovine serum albumin. The AChE forms
associated with the cells and secreted into the medium were analyzed by
velocity sedimentation as described under ``Experimental
Procedures.'' A, AChE forms secreted into the medium in
the presence () or absence (
) of heparin; B, AChE
oligomeric forms synthesized by myotubes incubated in the presence
(
) or absence (
) of heparin. Although the total amount of
AChE synthesized during the 24-h period was not significantly different
between the two groups, the accumulation of the asymmetric
collagen-tailed form was attenuated in the presence of heparin. More
important, in the presence of heparin, the collagen-tailed form of the
enzyme was secreted into the medium rather than retained on the cell
surface.
Figure 3: Heparin concentration effects on formation of cell-surface AChE clusters. Tissue-cultured myotubes were incubated with complete medium in the absence or presence of heparin at concentrations ranging from 0.15 to 1,500 µg/ml (10 nM to 100 µM) from days 5 to 7. On day 7, the cultures were rinsed with HBSS, and the numbers of cell-surface AChE clusters were determined by immunofluorescence after labeling the nuclei with Hoechst 33342. Maximal inhibition of cluster formation was observed at heparin concentrations in the 500-1,500 µg/ml (10-100 µM) range.
Since the inhibition of
cell-surface AChE accumulation and lack of cell-surface AChE cluster
formation could also result from an inhibition of AChE biosynthesis, we
measured the effect of heparin on newly synthesized AChE. Six-day-old
muscle cultures were preincubated in complete medium with or without 1
mg/ml heparin for 24 h. The cultures were then treated with DFP in HBSS
to inhibit all AChE, followed by washing and recovery for 2 h in
complete medium with or without heparin. Under these conditions, the
rate of appearance of AChE activity is linear with time and cell
numbers and reflects the rate of de novo AChE synthesis
(Rotundo and Fambrough, 1980). After a 2-h recovery, the total AChE
activity was (4.89 ± 0.08) 10
cpm in control
cultures versus (4.70 ± 0.09)
10
cpm in cultures incubated with heparin. We conclude that there
are no long-term effects of heparin on AChE synthesis.
Figure 4:
Heparin specifically blocks the
accumulation of cell-surface AChE, but does not affect AChR clustering
during myotube development in culture. Tissue-cultured myotubes were
incubated in either complete medium (controls) or medium supplemented
with 1 mg/ml heparin (H) for the indicated times. For each
time point, cultures were labeled with anti-AChE mAb 1A2, TRITC
-BTX to localize AChR clusters, and Hoechst 33342 to visualize the
nuclei. The density of cell-surface AChE and AChR clusters was
determined by counting myotube nuclei and clusters in 10 fields per
culture. Each point is the mean of three cultures, and the standard
deviations are <10%. The AChE clusters formed after the period of
cell fusion and differentiation from days 1 to 3, with the largest
increase occurring after the myotubes became spontaneously contractile
at approximately day 5. The presence of heparin blocked the formation
of new AChE clusters (A) without affecting the accumulation of
AChR clusters (B).
To determine whether heparin could disperse or release previously clustered AChE molecules, 6-day-old myotubes were incubated with anti-AChE mAb 1A2 for 2 h, washed in complete medium, and incubated for 24 h in the presence or absence of complete medium containing 1 mg/ml heparin. The cultures were rinsed and incubated with fluorescein isothiocyanate-conjugated second antibody on day 7, and the number of AChE clusters per nucleus was quantitated. Our results show that heparin did not remove AChE once localized to clusters since the number of clusters per nucleus in cultures incubated in heparin-containing medium from days 6 to 7 following mAb 1A2 addition on day 6 is virtually identical to that in the day 6 controls (Fig. 5).
Figure 5: Heparin does not disrupt cell-surface AChE clusters once they have formed. Six-day-old muscle cultures were incubated for 2 h with 20 µg/ml anti-AChE mAb 1A2 in complete medium to label all cell-surface AChE clusters, washed three times with complete medium to remove unbound antibody, and incubated in complete medium with or without 1 mg/ml heparin (H). Twenty-four hours later, the cultures were rinsed with PBS/horse serum, and the remaining AChE clusters were localized by incubation with fluorescein isothiocyanate-conjugated second antibody. Positive control (CONT) cultures were labeled on day 7 with both anti-AChE mAb 1A2 and fluorescein isothiocyanate second antibody. The number of AChE clusters per nucleus was quantitated as described under ``Experimental Procedures.'' The presence of heparin in the medium did not detach AChE molecules previously clustered on the cell surface.
Figure 6: Effects of heparin on cell-surface AChE clusters are reversible. AChE molecules were clustered on the upper surface of myotubes after 5 days in culture using EMEM 210. AChE clusters were visualized by indirect immunofluorescence on day 7 (A). After long-term heparin treatment (days 3-6), clusters of AChE did not form (B). To determine the reversibility of long-term heparin treatment, 1 mg/ml heparin was added to the medium from days 3 to 6 and removed on day 6, and the cells were fed with normal medium from days 6 to 7 (C). Heparin blocked the accumulation of AChE molecules; however, the effect was reversible since returning the cells to normal medium partially restored the appearance of AChE clusters. Bar = 25 µm.
To quantitate the reversibility of long-term heparin treatment, muscle cultures maintained in heparin-containing medium for 2-3 days were rinsed and returned to normal medium for either 1 or 2 days. The cultures were then incubated with mAb 1A2 to localize and quantitate AChE clusters. As a control, AChE was immunolocalized immediately after heparin treatment (Table 3). The rapid reappearance of AChE clusters following heparin removal suggests that the AChE attachment sites remained localized in the absence of AChE deposition while heparin was in the medium.
Figure 8:
Specific binding of quail asymmetric AChE
to heparin. Individual oligomeric forms of AChE were isolated from
tissue-cultured quail muscle cultures by velocity sedimentation, and
the peak fractions corresponding to each form were pooled for analysis
and adjusted for sucrose concentration and similar levels of enzyme
activity. Aliquots of the pooled fractions were incubated overnight
with avidin-conjugated agarose beads previously saturated with
biotinylated heparin. Each incubation mixture was prepared in
quadruplicate, and NaCl was lowered to 200 mM. The beads were
then washed with PBS containing 0.5% Triton X-100 and assayed for bound
AChE. In these experiments, 5% of the total A
AChE
was bound per 10 µl of biotin-heparin-agarose beads. The results
show that the asymmetric AChE form synthesized by the tissue-cultured
myotubes preferentially bound to the immobilized heparin. The small
amount of binding observed for the G
tetramer probably
reflects some contamination from the asymmetric A
form,
which is present in the cultures and cosediments with the G
form.
Figure 9: Newly synthesized cell-surface AChE on heparin-treated myotubes is extractable. Tissue-cultured myotubes were incubated in complete medium with or without heparin from days 5-7, and 10 µg/ml puromycin was added to the medium for an additional 9 h. At the end of the puromycin chase period, half the cultures in each group were extracted with HSB to remove electrostatically bound surface AChE, and all cultures were assayed for cell-surface AChE as described under ``Experimental Procedures.'' Results are expressed as percentage untreated controls (mean ± S.E.). In untreated myotubes, essentially all of the cell-surface enzyme was tightly attached to the extracellular matrix, and even the most recently synthesized molecules were unextractable as shown by the lack of reduced surface AChE in the presence of puromycin. In contrast, most of the cell-surface enzyme that had accumulated in the presence of heparin was extractable with HSB. The significant decrease in cell-surface AChE activity in the presence of puromycin and its solubility in high ionic strength buffer indicate that a large fraction of the externalized enzyme molecules are transiently associated with the extracellular matrix via electrostatic interactions.
A
second prediction based on our results would be that the small pool of
``extractable'' cell-surface A AChE molecules,
those that have not yet formed tight associations with other molecules,
would appear less stable in the presence of heparin, where they would
be prevented from short-term interactions with the extracellular
matrix. As can be seen in Fig. 9, this pool of extractable AChE
molecules constitutes only a very small percentage of the total surface
AChE activity. To estimate the relative association time of
cell-surface A
AChE molecules in the presence or absence
of heparin, three myotube cultures per group were incubated in the same
medium with or without puromycin for 3, 5, 7, or 9 h. Following
protection of the cell-surface AChE with BW284c51, the
``soluble'' surface AChE forms were extracted with HSB and
analyzed on sucrose gradients, and A
AChE was quantitated (Fig. 10). In untreated controls, there was a small but
detectable decrease in the amount of extractable A
AChE
during the puromycin chase period, suggesting that a portion of the
matrix-associated molecules could maintain electrostatic interactions
with the cell surface. In the presence of heparin, however, this small
pool was unstable and could rapidly dissociate or diffuse away from the
matrix. In the presence of puromycin, there was a rapid decrease in the
amount of extractable A
AChE with time in puromycin to
levels <5% of control. This experiment indicates that most of the
newly synthesized A
AChE molecules could not maintain
their association with the extracellular matrix in the presence of
heparin.
Figure 10:
Analysis of extractable cell-surface
asymmetric AChE in the presence or absence of heparin. Tissue-cultured
myotubes were incubated in the presence or absence of heparin as
described for Fig. 8, followed by incubation with 10 µg/ml
puromycin for 3, 5, 7, or 9 h. At the end of the puromycin incubation,
all cultures were treated with BW284c51 followed by DFP to inactivate
intracellular AChE. Three dishes per group were extracted using HSB;
the soluble cell-surface AChE forms were analyzed by velocity
sedimentation; and A AChE was quantitated. Comparison of
soluble cell-surface A
AChE forms obtained from control
and heparin cultures suggests that the rate of dissociation of the
enzyme from the extracellular matrix is very slow (4%/h or less), if at
all, in untreated cultures, whereas in the presence of heparin,
dissociation is rapid, at least 15%/h.
The synaptic basal lamina of skeletal muscle fibers contains
higher concentrations of several identified molecules, including AChE
and heparan sulfate proteoglycan(s), compared with non-innervated
regions (reviewed by Massouliéet
al.(1993) and Hall and Sanes(1993)). Of the several AChE
oligomeric forms expressed in muscle, the asymmetric collagen-tailed,
or A, form appears to be the most abundant at the
vertebrate neuromuscular junction. Its mechanism of attachment to the
synaptic basal lamina is generally thought to be through electrostatic
interactions with glycosaminoglycans such as heparan sulfate
proteoglycan or dermatan sulfate proteoglycan (Bon et al.,
1978; Brandan and Inestrosa, 1987; Pérez-Tur et al., 1991b; Melo and Brandan, 1993). Heparin is capable of
solubilizing A
AChE molecules from vertebrate muscle
(Torres and Inestrosa, 1983; Brandan and Inestrosa, 1984, 1986; Barat et al., 1986; Pérez-Tur et al.,
1991a) as well as from some neural preparations (Torres et
al., 1983). This selective solubilization, together with the
strong evidence for direct interactions between A
AChE and
proteoglycans, argues that they may play a role in the anchorage of
asymmetric forms to the synaptic basal lamina.
On the other hand, we
have recently shown that heparin does not detach AChE from the
neuromuscular junctions of adult fast and slow quail muscles or from
rat muscles (Rossi and Rotundo, 1993). Furthermore, we demonstrated
that essentially all of the immunohistochemically detectable enzyme
localized on the synaptic basal lamina was tightly attached and could
not be removed by high ionic strength buffers, detergents, or
chaotropic agents such as guanidine HCl or urea. Only collagenase was
able to detach the enzyme (Hall and Kelly, 1971; Betz and Sakmann,
1973; Rossi and Rotundo, 1993), indicating that the basal
lamina-associated AChE is most likely covalently linked to one or more
molecular components of the extracellular matrix. In this study, we
show that, like the adult neuromuscular junction, short-term treatment
(1 h) of tissue-cultured myotubes with heparin did not detach the
AChE molecules associated with cell-surface clusters (Fig. 1),
nor did it remove catalytically active enzyme molecules from the cell
surface ( Fig. 2and Table 1). Similar results were
obtained using the standard high salt- or polyanion-containing buffers,
which also were unable to solubilize the matrix-bound AChE ( Table 1and Table 2), whereas incubation of the cultures
with purified collagenase removed the surface AChE (Rossi and Rotundo,
1992). These results support the idea that clustered AChE molecules on
the surfaces of myotubes are attached in the same manner as at sites of
nerve-muscle contact in vivo.
In contrast with the
inability of heparin to extract previously clustered AChE molecules,
long-term exposure to heparin in culture blocked the accumulation of
catalytically active AChE on the myotube surface (Fig. 2) as
well as the formation of new surface AChE clusters ( Fig. 4and Fig. 5). This effect was detectable only after days 3-4 in
culture, coincident with the onset of A AChE expression
and its deposition in clusters on the muscle cell surface. The normal
increase in number of AChE clusters per nucleus was prevented when
incubations were extended over periods of several days in
heparin-containing medium. However, heparin did not prevent the
spontaneous formation of AChR clusters, indicating that the effects are
specific for AChE (Fig. 4). This heparin block was reversible ( Fig. 6and Table 3), and newly synthesized AChE molecules
continued to accumulate once heparin was removed from the medium,
suggesting that heparin was interacting directly with the A
AChE molecule, as shown in Fig. 8, rather than disrupting
the binding sites for AChE on the extracellular matrix.
Although our
present observations on tissue-cultured myotubes, as well as those in vivo (Rossi and Rotundo, 1993), may appear contradictory to
published studies from other laboratories, they are complementary
rather than mutually exclusive. The A AChE form is
assembled intracellularly in the Golgi apparatus (Rotundo, 1984a) and
must then be transported to the cell surface and secreted prior to
attachment to the extracellular matrix. For this reason, a significant
pool of intracellular A
AChE molecules is usually found in
mature muscle fibers (Younkin et al., 1982) as well as in
cultured cells (Rotundo, 1984a; Brandan and Inestrosa, 1984). Once
externalized, most of the A
AChE molecules appear to
accumulate either in clusters on tissue-cultured myotubes or on
specialized regions of the muscle fiber surface in vivo.
Therefore, the fraction of A
molecules solubilized by
heparin most likely corresponds to those molecules in the intracellular
pool residing in the lumen of the Golgi apparatus as well as those that
have been externalized but have not yet been strongly attached to the
basal lamina. However, once attached, a large fraction of the A
AChE molecules can no longer be removed by high salt buffers or
polyanions, indicating that the association is through more than simply
electrostatic interactions. These are the A
AChE molecules
that accumulate in cell-surface AChE clusters in culture or at the
neuromuscular junction in vivo.
In addition to the tight
association between the A AChE molecules and the
extracellular matrix, we also find that the A
AChE
molecules undergo transient electrostatic interactions as well. In the
presence of heparin, the newly synthesized A
AChE forms
were secreted into the culture medium (Fig. 7) rather than
accumulated on the extracellular matrix. Under normal culture
conditions, only a small fraction of the total surface AChE appears to
be electrostatically associated with the extracellular matrix and
extractable with HSB (Fig. 9). When heparin is present to
interact with the collagen-like tail over long periods, the
accumulation of cell-surface AChE is prevented since most of the enzyme
(86% of control) either has diffused away (50% of the total
cell-surface AChE is not bound to the extracellular matrix in the
presence of heparin) or is weakly associated with the cell surface and
easily extractable with HSB (30% of control). In these experiments,
only the most recently synthesized A
enzyme appears
electrostatically attached to the extracellular matrix ( Fig. 9and 10). The relatively small pool of these molecules on
the cell surface of untreated cells would suggest that the more
permanent attachment of A
AChE occurs fairly rapidly, even
in tissue culture.
In summary, our results are consistent with
earlier observations and suggest that a sulfated proteoglycan(s) is the
target for localizing newly synthesized A AChE molecules
on the synaptic basal lamina. In the current model, HSP molecules would
initially form cell-surface clusters, either spontaneously in
nerve-free muscle cultures or induced at sites of nerve-muscle contact.
These localized accumulations would then serve as attachment sites for
the A
AChE molecules. This mechanism for localizing AChE
at sites of nerve-muscle contact may explain why the appearance of AChE
at ectopic synapses in vivo is such a late event compared with
the appearance of AChRs (Lömo and Slater, 1980) and
how AChE can reaccumulate at the original synaptic sites following
muscle regeneration in the absence of nerves (Anglister and McMahan,
1985), reinnervation of empty basal lamina sheaths (Anglister, 1991),
and restoration of muscle activity by reinnervation through ectopic
synapses (Weinberg and Hall, 1979). Furthermore, this model provides a
mechanism for regulating the numbers and distribution of AChE molecules
on the synaptic basal lamina, where the accumulation of HSP molecules
at sites of nerve-muscle contact could act as a molecular
``parking lot'' for the subsequent insertion and removal of
the A
AChE molecules at the vertebrate neuromuscular
junction.