Effects of Purified Recombinant Neural and Muscle Agrin

Effects of Purified Recombinant Neural and Muscle Agrin
Effects of Purified Recombinant Neural and Muscle Agrin
on Skeletal Muscle Fibers In Vivo
Gabriela Bezakova,* Johannes P. Helm,‡ Maura Francolini,§ and Terje Lømo*
*Department of Physiology and ‡Department of Anatomy, University of Oslo, 0317 Oslo, Norway; and §C.N.R. Center for
Cellular and Molecular Pharmacology, Department of Medical Pharmacology, University of Milano, 20129 Milan, Italy
Abstract. Aggregation of acetylcholine receptors
(AChRs) in muscle fibers by nerve-derived agrin plays a
key role in the formation of neuromuscular junctions.
So far, the effects of agrin on muscle fibers have been
studied in culture systems, transgenic animals, and in
animals injected with agrin–cDNA constructs. We have
applied purified recombinant chick neural and muscle
agrin to rat soleus muscle in vivo and obtained the following results. Both neural and muscle agrin bind uniformly to the surface of innervated and denervated
muscle fibers along their entire length. Neural agrin
causes a dose-dependent appearance of AChR aggregates, which persist 7 wk after a single application.
Muscle agrin does not cluster AChRs and at 10 times the
concentration of neural agrin does not reduce binding
or AChR-aggregating activity of neural agrin. Electrical
muscle activity affects the stability of agrin binding and
the number, size, and spatial distribution of the neural
agrin–induced AChR aggregates. Injected agrin is recovered from the muscles together with laminin and
both proteins coimmunoprecipitate, indicating that
agrin binds to laminin in vivo. Thus, the present approach provides a novel, simple, and efficient method
for studying the effects of agrin on muscle under controlled conditions in vivo.
Key words: agrin • acetylcholine receptors • laminin •
electrical activity • neuromuscular junction
Signaling between nerve and muscle occurs at neuromuscular junctions (NMJs)1, which consist of specialized and precisely apposed pre- and postsynaptic structures separated
by a synaptic cleft (Sanes and Lichtman, 1999). Agrin, a
heparan sulfate proteoglycan of 400–600 kD, is essential
for the induction and organization of the postsynaptic
structures (McMahan, 1990; Gautam et al., 1996; Cohen et
al., 1997; Jones et al., 1997; Meier et al., 1997; Rimer et al.,
1997). Agrin is synthesized by motor neurons, transported
to axon terminals, and released into the synaptic cleft of
NMJs, where it binds to the basal lamina (Magill-Solc and
McMahan, 1988, 1990; Cohen and Godfrey, 1992; Reist et
al., 1992). Motor neurons express a mixture of agrin isoforms, alternatively spliced at two sites (A and B in chick, y
and z in rat) in their COOH-terminal halves (Ruegg et al.,
1992; Rupp et al., 1992; Hoch et al., 1993). The neural isoform of agrin containing inserts of four and eight amino acAddress correspondence to Gabriela Bezakova, Department of Pharmacology/Neurobiology, Biozentrum, University of Basel, Klingelbergstrasse
50, CH-4056 Basel, Switzerland. Tel.: 41-61-267-2214. Fax: 41-61-2672208. E-mail: [email protected]
Abbreviations used in this paper: AChR, acetylcholine receptor; EB,
extraction buffer; Fl-BuTx, FITC-bungarotoxin; NMJ, neuromuscular
junction; PFA, paraformaldehyde; Rh-BuTx, TRITC--bungarotoxin;
SOL, soleus.
ids at sites A and B, respectively, is most effective in clustering acetylcholine receptors (AChRs) in vitro (Gesemann
et al., 1995). Muscle cells and other nonneuronal cells also
express agrin, but as isoforms that lack inserts at sites A and
B and fail to cluster AChRs (Ruegg et al., 1992; Hoch et al.,
1993; Ma et al., 1994; Smith and O’Dowd, 1994).
The AChR-clustering activity of neural agrin has been
well characterized in vitro, where the activity is both concentration- and Ca2-dependent (Nastuk et al., 1991;
Gesemann et al., 1996; Megeath and Fallon, 1998). When
bound to extracellular matrix, neural agrin increases the
transcription of AChR -subunits in addition to clustering
AChRs (Jones et al., 1996). Biochemical and immunological studies show that agrin binds in a Ca2-dependent
manner to -dystroglycan, a component of the dystrophinassociated glycoprotein complex present on the surface of
muscle fibers (Gee et al., 1994), as well as to laminin in the
basal lamina (Denzer et al., 1997; Kammerer et al., 1999).
However, the physiological consequences of these bindings are not clear. Other proteins have been implicated to
interact with agrin in vitro but none of them has been
shown to be critical for postsynaptic differentiation in vivo
(Sanes et al., 1998).
The most compelling evidence that agrin is essential for
NMJ formation comes from loss- and gain-of-function
 The Rockefeller University Press, 0021-9525/2001/06/1441/12 $5.00
The Journal of Cell Biology, Volume 153, Number 7, June 25, 2001 1441–1452
studies. In agrin-deficient mutant mice, the postsynaptic
differentiation was profoundly impaired and the mice died
perinatally (Gautam et al., 1996). A similar phenotype was
observed in mutant mice lacking muscle specific kinase
(MuSK), a transmembrane protein tyrosine kinase selectively expressed at the NMJ in innervated skeletal muscle
(Valenzuela et al., 1995; DeChiara et al., 1996), making it a
good candidate for the agrin receptor. However, the physical interaction between agrin and MuSK has not yet been
identified. Instead, MuSK and other proteins have been
suggested to create a multisubunit agrin receptor complex
(Glass et al., 1996).
Experiments involving implantation of agrin-secreting
myoblasts or injection of agrin expression constructs into
muscles demonstrated agrin’s activity in vivo (Cohen et
al., 1997; Jones et al., 1997; Meier et al., 1997; Rimer et al.,
1997). Expression of neural agrin in transfected cells followed by its release and deposition on neighboring fibers
induced multiple AChR aggregates. When expressed in
electrically active muscles, the ectopic AChR aggregates
acquired features typical for adult NMJ, having junctional
folds and functional electrophysiological properties (Meier
et al., 1997). Thus, agrin alone appears capable of assembling a fully functional postsynaptic apparatus.
Formation of ectopic NMJs through the interaction of
transplanted axons with soleus (SOL) muscles is strongly
affected by electrical muscle activity (Lømo and Slater,
1978; Brenner et al., 1987; Rotzler and Brenner, 1990; Skorpen et al., 1999). Neural agrin induces ectopic postsynaptic-like apparatuses in SOL muscles that are similarly affected by electrical activity (Mathiesen et al., 1999). Hence,
these experimental models allowed the comparison the
formation of nerve- and agrin-induced NMJs in vivo under
the most physiological conditions examined so far.
Injecting agrin–cDNA into muscles is a useful in vivo approach, which nonetheless has drawbacks because it is difficult to transfect more than a few fibers in each muscle and
to determine the dose and site of agrin release. Here, we
present the results of a different approach based on in vivo
applications of known concentrations of purified recombinant neural and muscle chick agrin. We use this approach to
study properties of neural and muscle agrin with regard to
their binding to the surface of muscle fibers, AChR-aggregating activity, and modulation by electrical muscle stimulation. We find that a single injection of neural agrin induces
ectopic AChR aggregates along muscle fibers, which are
dose dependent and persist 7 wk. After injection, neural
agrin binds to laminin and uniformly to the surface of muscle fibers along their entire length. Subsequently, the
amount of bound neural agrin and the distribution and appearance of neural agrin–induced AChR aggregates are
regulated by electrical muscle activity. Muscle agrin displays similar binding but fails to aggregate AChRs.
Materials and Methods
Purification of Recombinant Agrin
medium was then used for 5–7 d. The conditioned medium was collected,
and the protein was purified by a batch technique using mono Q Sepharose
beads (Amersham Pharmacia Biotech). After rotating at 4C overnight,
the beads were washed on the column with 20 mM Tris-HCl, pH 7.2, 0.5 M
NaCl. Bound proteins were eluted with 2 M NaCl, and 1 ml fractions were
collected. Protein-containing fractions were further analyzed by SDSPAGE (Laemmli, 1970) on a 3–12% gradient gel and visualized by Coomassie blue and silver stainings. Agrin-containing fractions were dialyzed
against PBS, pH 7.4. Protein concentration was determined by Bradford
protein assay (Bio-Rad Laboratories), using BSA as a standard.
Metabolic Labeling of Agrin
Confluent cultures, as described above, were switched to 25% DME and
75% DME without methionine and cysteine supplemented with Tran Sulfur-35 label (20 Ci/ml; ICN Biomedicals). The purified proteins were
separated by SDS-PAGE on 3–12% gradient gels. Gels were dried and
exposed to the film (Eastman Kodak Co.).
Surgical Procedures and In Vivo Stimulation
The experiments were carried out on adult male Wistar rats (250 g body
weight). All surgical procedures were done under general anesthesia by
Equithesin (0.4 ml/100 g body weight) injected i.p. SOL muscles were denervated by removing 5 mm of the sciatic nerve in the thigh. For stimulation, uninsulated ends of two wires (AS 632, Cooner) were placed across
the muscle, run under the skin through an attachment by screws, dental
cement to the skull, and a flexible plastic tube to rotating contacts 0.5 m
above the rat (Windisch et al., 1998). Stimulation started 1 h later and consisted of 60 0.4-ms bipolar square pulses at 100 Hz every 60 s for 9 d.
Identical experiments have been inspected and approved by the Norwegian Experimental Board and Ethical Committee for Animal Experiments on several occasions. The present experiments were overseen by
the veterinarian responsible for the animal house. The animals were
checked daily. The flexible tube overhead allowed free movements within
the cage. Apart from one leg being denervated and contractions being visible during stimulation, the animals did not show obvious abnormal behavior or signs of pain.
Application of Recombinant Agrin
Intramuscular Injection. SOL muscles were injected intramuscularly with
70 l of 1 M recombinant chick neural or muscle agrin. The sciatic nerve
was either cut immediately thereafter or kept intact. The muscle was excised at different time points, labeled with TRITC--bungarotoxin (RhBuTx; Molecular Probes) for 30 min, washed with PBS, and fixed with
1.5% paraformaldehyde (PFA). Fixed muscle was teased into 30 thin
bundles containing 50–100 muscle fibers. Bundles showing signs of damage were few, attributed to the injection or overlying electrodes, and excluded from analysis. We did not observe morphological changes or
mononucleated cells indicative of significant immune response.
Bathing of SOL Muscle with Agrin. To study the effects of different concentrations of agrin, SOL was exposed in situ and carefully dissected free
from surrounding tissue except at tendons and entries of nerve and blood
vessels. Under deep anesthesia, SOL was then bathed for 2 h in PBS alone
or PBS containing from 100 pM–10 M agrin. Fresh solution was repeatedly added to the bath to keep the SOL fully immersed. After 2 h, the
opening in the leg was rinsed with PBS and closed with sutures through
overlying muscles, fascias, and skin. The muscles were excised 4 or 7 d
later, labeled with Rh-BuTx, washed with PBS, and fixed with 1.5% PFA.
4 d were chosen for denervated fibers because at 4 d obvious reorganization of the AChR aggregates had not yet occurred and the aggregates were
still uniformly distributed along fibers (see Fig. 2). 7 d were chosen for innervated fibers because comparable reorganization of AChR aggregates
did not occur on innervated fibers and staining was stronger after 7 than
4 d (see Fig. 2). At 4 or 7 d, a thin layer of surface fibers, which had been in
direct contact with the agrin solution, was dissected out and examined with
an Olympus AX70 fluorescence microscope. Images were captured with a
Colour Coolview charge-coupled device camera (Photonic Science).
Quantification of AChR-aggregating Activity
Recombinant full-length chick neural and muscle agrin were purified from
the conditioned media of stably transfected HEK 293 cells (gift of Dr.
M.A. Ruegg, University of Basel, Basel, Switzerland) by modified method
(Denzer et al., 1997). The cells were cultured in a DME supplemented with
10% FBS (BioWhittaker) until they became confluent. FBS-free DME
Relative Area Occupied by AChR Aggregates. Agrin-induced AChR aggregates that could be viewed en face on the surface of single muscle fiber at
1,000 magnification were selected for measurements using an Open Lab
imaging software (Improvision). A rectangular region enclosing a collec-
The Journal of Cell Biology, Volume 153, 2001
tion of these aggregates was drawn, and the areas of all the individual aggregates within the region were measured and summed. The sum of such
areas was expressed as a percentage of the rectangular region and taken as
a measure of agrin AChR-aggregating activity. Collections of AChR aggregates on differently treated muscle fibers were summed within the rectangle of the same size.
Intensity of Fluorescent Labeling. The imaging system was calibrated,
using the InSpeck Microscope Image Intensity Calibration Kit (Molecular
Probes) containing microspheres coated with six different concentrations
of fluorescent dye. To obtain the specific mean labeling intensity of aggregates, the mean intensity of an adjacent aggregate-free part of the fiber
(background) was subtracted from the mean intensity of the aggregates
(Turney et al., 1996). The subtracted value was then normalized by the
value given by microspheres coated with 3% of fluorescent dye obtained
in the same session. 20 images in each of 3 similarly treated muscles were
analyzed in randomly chosen extrajunctional regions of denervated fibers
and in regions near the myotendinous junctions of innervated fibers,
where most of the aggregates were located (see Fig. 3 A).
Teased muscle bundles were labeled with Rh-BuTx for 30 min, washed
with PBS, and fixed in 1.5% PFA. The labeling of - and -subunits was
performed as described by Missias et al. (1996) and Gu et al. (1991), respectively (gift of Dr. J. Sanes, Washington University, St. Louis, MO).
The primary antibodies were visualized by FITC-conjugated anti–rabbit
secondary antibodies (Sigma-Aldrich) at dilution 1:200. The samples were
observed with a confocal laser-scanning microscope (TCS-SP; Leica)
equipped with an Ar Kr ion laser. Excitation was done at 488 and 568
nm, and the spectrometer settings (width and positions of the slits in front
of the photomultiplier tubes) were selected in order to minimize crossbleeding between the FITC and the TRITC channels.
Autoradiography of Muscle Fibers from Muscles
Injected with 35S-labeled Agrin
SOL muscles were injected with 70 l of 1 M 35S-labeled neural or muscle agrin. The muscles were excised 1 and 4 d later and fixed with 2.5%
glutaraldehyde overnight. Single muscle fibers were teased out and placed
on gelatine-coated slides. The muscle fibers were covered with a film
emulsion (Eastman Kodak Co.), as previously described (Salpeter and
Azabo, 1976). The film was developed after appropriate time and the
amount and distribution of silver grains were analyzed using a confocal laser-scanning microscope (TCS-SP; Leica) equipped with an Ar Kr ion
laser. The specimens were scanned using a Plan Apochromat 63 1.32 oil
immersion lens. The scans were done in the reflection mode. When examining the specificity of 35S-labeled agrin binding, the muscle was injected
with 70 l of 10 M unlabeled agrin isoform 6 h before injection of the
same volume of 1 M labeled agrin isoform. Similar studies were performed using combination of muscle and neural agrin. Fibers teased from
muscle injected with PBS were used to control for unspecific background.
10 fibers teased at random from each of 3 similarly treated muscles were
placed on glass slides such that each slide contained fibers for each experimental condition and control. In this way, only fibers undergoing identical
processing were compared.
Immunoprecipitation and Western Blot. SOL muscle was dissected from
rats previously injected with recombinant neural or muscle agrin and frozen in liquid nitrogen. The tissue was then extracted in 10 vol (wt/vol) of
ice-cold EB using a Polytron. The homogenate was centrifuged for 20 min
at 50,000 g. The supernatant was collected and incubated with mAb 5B1
overnight at 4C. Protein A–agarose (Sigma-Aldrich) was added for 4 h at
4C. The beads were then applied on a column and washed with 50 vol of
EB. Bound proteins were eluted with SDS sample buffer. The eluted fraction was separated on 3–12% SDS-PAGE gel. Proteins were either visualized by silver staining or transferred to nitrocellulose membrane using
standard methods (Towbin et al., 1979). After blocking, the membranes
were incubated overnight with polyclonal antibody against 2-laminin diluted 1:5,000 (gift of Dr. R. Timpl, Max-Plank Institute, Martinstried, Germany). The primary antibody was detected with appropriate secondary
antibody conjugated to HRP (Jackson ImmunoResearch Laboratories)
diluted 1:2,000. Bands were visualized by chemiluniscence (Pierce Chemical Co.) and exposed to the film (Eastman Kodak Co.).
Purification of Full-Length Chick Neural and Muscle Agrin
Full-length chick neural and muscle recombinant agrin
(Fig. 1 A) was purified from cultures of stably transfected
293 HEK cells using an ion exchange chromatography as
described in Materials and Methods. Both proteins appeared on 3–12% SDS-PAGE gradient gels stained with
Coomassie blue or silver as single smeared bands with an
apparent molecular weight of 400–600 kD (Fig. 1, B and
C). Comparable bands were observed after immunoprecipitation with mAb 5B1 (Reist et al., 1987) or a polyclonal antiserum against chick agrin polyclonal antibody
3228 (Gesemann et al., 1995), confirming the identity of
the proteins (Fig. 1 D).
Fate of AChR Aggregates after a Single
Injection of Neural Agrin
Sequential Protein Extraction. 1 d after the injection of recombinant neural or muscle 35S-labeled agrin, the muscles were dissected out and frozen
in liquid nitrogen. The tissues were then homogenized in 10 vol (wt/vol) of
ice-cold homogenization buffer containing 10 mM Na phosphate, pH 7.4,
150 mM NaCl, 5 mM EDTA plus a cocktail of protease inhibitors (aprotinin, leupeptin, benzamidin, pepstatin at 0.5 g/ml each and 2 mM PMSF)
using a polytron. The homogenate was centrifuged for 20 min at 12,000 g.
The supernatant was collected (PBS-EDTA fraction), and the pellet was
further extracted with homogenization buffer plus 1% Triton X-100 (extraction buffer [EB]) using Dounce homogenizer. The extract was centrifuged 20 min at 50,000 g. The supernatant was separated (Triton X-100
fraction), and the pellet was boiled for 10 min with gel denaturing or nondenaturing loading buffers (pellet fraction). Individual fractions were separated on 3–12% gradient gels using denaturing and reducing or nondenaturing nonreducing conditions. The gels were dried and exposed to the
film (Eastman Kodak Co.). The position and the size of radioactively labeled bands were analyzed.
A single injection of purified recombinant neural agrin (1
M, 70 l) into innervated or denervated SOL muscles in
vivo caused aggregation of AChRs on the surface of muscle fibers outside the original NMJs (Fig. 2 A). Without
such injections, AChR aggregates were not observed. In
both muscles, AChR aggregates had already formed on
day 3, the earliest time point examined. In muscles denervated at the time of injection, the aggregates were initially
numerous, small (4 m), punctate, and uniformly distributed along the fibers. During the next 1–2 wk, they became larger and surrounded by regions with reduced number of aggregates. These larger aggregates persisted 7 wk
after the injection, the latest time point examined. They
were formed, most probably, by coalescence of smaller
clusters since aggregates labeled by injection of Rh-BuTx
on day 7 had become similarly reorganized when examined 14 d later (Fig. 2 B). In addition, the colocalization of
aggregates labeled by Rh-BuTx on day 7 and by FITCbungarotoxin (Fl-BuTx) on day 21 shows that the aggregates became structurally stable with newly synthesized
AChRs (labeled on day 21) inserted at aggregates containing old AChRs (labeled on day 7). No AChR aggregates
appeared in a 0.5–0.8-mm-long region on each side of the
NMJ, except for a few punctate clusters in the immediate
vicinity of the junction (Fig. 3 B).
The AChR aggregates induced on innervated muscles
were different. They were much fewer in number, larger,
more uniform in size (mean length 130 m), structurally
Bezakova et al. AChR Clustering by Purified Recombinant Agrin In Vivo
Identification of Agrin Binding to Laminin
Figure 1. Characterization of full-length chick neural and muscle agrin purified from stably transfected 293 HEK cells. (A) Location of alternatively spliced sites characteristic for neural
(7.4.8.) and nonneural or muscle (0.0.0.) agrin isoforms together
with their amino acid sequences. The fractions of purified agrin
collected by ion exchange chromatography contained high concentrations of agrin that were easily detectable by Comassie blue
staining (B) and were 90% pure as shown by Silver staining
(C). Immunoprecipitation (D) using the mAb against agrin
(mAb 5B1) or antiagrin antiserum (polyclonal antibody 3228)
confirmed that the purified protein is chick agrin. Proteins were
separated by 3–12% SDS-PAGE. Molecular masses of standard
proteins are indicated in kD in B. Arrows point to the smeared
bands of purified neural and muscle agrin with the apparent molecular of 400–600 kD.
stable, and preferentially located near the myotendinous
junction (Fig. 3 A), where the aggregates illustrated in Fig.
2 A were formed. Also in this case, the perisynaptic region
on each side of the original NMJs was devoid of AChR aggregates, except for those in the immediate vicinity of the
junction (Fig. 3 C).
The phenomena just described were neither species nor
muscle type specific since SOL and EDL in both rat and
mouse responded similarly (data not shown).
AChR-aggregating Activity of Neural Agrin:
Dependence on Dose and Innervation
Figure 2. Ectopic AChR aggregates induced by purified recombinant chick neural agrin. (A) A single injection of 1 M agrin
into acutely denervated (left) or innervated (right) SOL muscles
caused the appearance of numerous AChR aggregates at the indicated times after injection. Note differences in number, size, and
distribution of aggregates labeled with Rh-BuTx in denervated
and innervated muscles. In innervated muscles, the aggregates appeared predominantly near myotendinous junctions (see also Fig.
3). (B) SOL muscle was injected with agrin and denervated on day
0. On day 7, it was injected with Rh-BuTx, removed, and labeled
with Fl-BuTx on day 21. Presence of Rh-BuTx–labeled aggregates
on day 21 shows their metabolic stability; colocalization of RhBuTx and Fl-BuTx indicates the structural stability of aggregates.
To examine the influence of innervation on neural agrin’s
AChR-aggregating activity, we compared the effects of
different concentrations of neural agrin on predenervated
(7 d), acutely denervated, and innervated SOL muscles. In
these experiments, we applied neural agrin at different
concentrations to the exposed surface of SOL muscles for
2 h and examined the surface fibers for AChR aggregates
after 4 (denervated muscles) or 7 (innervated muscles) d
(see Materials and Methods). Predenervated and acutely
denervated muscles responded similarly (Fig. 4). With increasing agrin concentration, individual AChR aggregates
became first larger and then smaller in size. In innervated
fibers, similar profile of responses was observed but the
threshold concentration for induction of AChR aggregates
was 100-fold higher (Fig. 4).
These results are shown quantitatively in Fig. 5 and Table
I. There was no major difference in EC50 (agrin concentration necessary to induce half-maximal response) between
predenervated and acutely denervated muscles, except that
the area occupied by AChR aggregates for a given area of
fiber surface became moderately larger in the predener-
The Journal of Cell Biology, Volume 153, 2001
Figure 3. Distribution of AChR aggregates induced by injection of 1 M neural agrin into innervated (A and C) or denervated (B)
muscles. In innervated muscles, most of the aggregates appeared near myotendinous junction (to the left in A). In denervated muscles,
AChR aggregates were essentially absent near the original NMJs (B) but abundant elsewhere (see also Fig. 2). Small punctate aggregates appeared in the area immediately adjacent to the original NMJs in both innervated and denervated muscles (B and C). The large
majority of agrin-induced aggregates were distinguished from the original NMJs by their different appearance, location outside the
characteristic band of NMJs across the middle of the muscle, and weaker intensity of Rh-BuTx staining. The few punctate aggregates
close to the NMJs were distinguished by their appearance since nothing like them was observed near innervated or denervated NMJs
without injecting neural agrin. Double arrow points to agrin-induced ectopic AChR aggregates; single arrow points to original NMJ.
vated muscles. On the other hand, the EC50 for the response
of innervated muscles to neural agrin was 10 times higher.
The intensity of Rh-BuTx labeling (see Materials and
Methods) increased with increasing agrin concentrations,
also at the highest concentrations when individual aggregates became smaller and occupied a smaller area of the
muscle fiber surface (Figs. 4 and 5 B, Table I). Furthermore, there was no significant difference between EC50 for
Figure 4. AChR-aggregating activity of neural agrin is dose dependent. 7-d denervated (predenervated), acutely denervated, or innervated SOL muscles were bathed in the solution of neural or muscle agrin at concentrations as indicated in vivo for 2 h. The muscles
were dissected out after 4 (predenervated and acutely denervated) or 7 (innervated) d and labeled with Rh-BuTx. Only neural agrin induced AChR aggregates whose appearance was dependent on the dose applied.
Bezakova et al. AChR Clustering by Purified Recombinant Agrin In Vivo
Table I. Comparison of Agrin AChR-aggregating Activities
after Different Muscle Treatments
Muscle treatment
EC50 AChR clustering
EC50 AChR intensity
Acutely denervated
Acutely denervated
2.6 0.28 nM
3.2 0.35 nM
33.1 2.9 nM
32.1 2.7 nM
31.3 2.9 nM
33.1 2.4 nM
Agrin AChR-clustering activity and the intensity of AChR aggregates were determined
from fluorescence micrographs as described in Materials and Methods. Values are
means SEM of three independent experiments (20 images each).
(Figs. 4 and 5). To examine if these differences could be
related to the switch from - to -subunit–containing
AChRs that normally occurs at developing NMJs, we labeled the aggregates with antibodies specific for - and
-subunits. In denervated muscles, little or no -subunit
expression could be detected at any concentration. In contrast, in innervated muscles, the aggregates contained
-subunits and little or no detectable -subunits (Fig. 6).
Thus, the decline in size and the increase in labeling intensity observed at aggregates induced by the highest concentrations of agrin were not related to the content of - or
-subunits in the aggregates.
Muscle agrin did not aggregate AChRs at any concentration (Figs. 4 and 5), even though muscle agrin bound
well to the muscle surface (see below). Immunoprecipitation of injected neural and muscle chick agrin by speciesspecific mAbs followed by SDS-PAGE gels revealed
bands of appropriate size and similar intensities (Fig. 5 C).
Electrical Muscle Stimulation Alters the Number,
Size, and Distribution of AChR Aggregates
Induced by Neural Agrin
half maximal labeling intensity in predenervated, denervated, and innervated muscles (Table I).
The size and organization of AChR aggregates depended strongly on the concentration of neural agrin
The distribution and appearance of AChR aggregates induced by injected recombinant neural agrin were different
in innervated and denervated muscles (Fig. 2 A). To examine if lack of electrical muscle activity could account for
these differences, we started muscle stimulation 7 d after the
muscle had been denervated and injected with neural agrin.
Stimulation for 7 d removed most of the aggregates and
caused those that survived to become similar in appearance
and distribution to those observed in innervated muscles
(Fig. 7, a and b). Accordingly, electrical muscle activity appears to be a major factor in controlling the distribution and
organization of neural agrin–induced AChR aggregates.
To examine whether the number of AChR aggregates
induced in the innervated muscle after a single injection
could be increased by delayed denervation, we injected
neural agrin into innervated muscles and then denervated
the muscles for 7 d at different times afterwards. In muscles denervated 3 d after the injection (Fig. 7 f), multiple
small AChR aggregates appeared that were indistinguishable from those induced by injecting neural agrin into denervated muscles (Fig. 7 d). A few larger aggregates were
also observed, primarily near myotendinous junctions,
which probably represented the aggregates induced in the
muscle before the denervation. In muscles denervated 28 d
after the injection, additional AChR aggregates still appeared but in smaller number (Fig. 7 j, compare with nondenervated fibers in c, e, g, and i). These results suggest
that the amount of agrin initially bound along the muscle
The Journal of Cell Biology, Volume 153, 2001
Figure 5. Dose-response curves from experiments illustrated in
Fig. 4. The area of AChR aggregates as percentage of a given
area of muscle fiber surface (A) and the relative fluorescence of
the Rh-BuTx that labeled the aggregates (B) are plotted against
concentration of applied agrin (see Materials and Methods).
Plots represent mean values SEM of 20 collections of aggregates in each of three experiments for each concentration.
Amount of neural and muscle agrin bound to the muscle was determined by immunoprecipitation 12 h after application (C).
Figure 6. Presence of - and - AChR subunits in AChR aggregates induced by neural agrin. SOL muscles that were 7-d denervated
(a), acutely denervated (b and c), or innervated (d) were bathed in PBS containing 10 M neural agrin in vivo for 2 h. 4 (a and b) and 7
(c and d) d later, the muscles were excised and examined for AChR aggregates on surface fibers after labeling with Rh-BuTx and antibodies against - and -subunits, as indicated. Note that AChR aggregates on denervated fibers contained -subunits (a2, b2, and c2) and
no detectable -subunits (a4, b4, and c4), whereas innervated fibers contained -subunits (d4) but little or no detectable -subunits (d2).
fiber of the innervated muscles was gradually decreasing. 4
wk after application, agrin was, however, still detectable
by immunocytochemistry at the ectopic aggregates in the
innervated muscles (not shown).
Distribution of Injected Agrin along Muscle Fibers
The AChR aggregates induced by neural agrin were discontinuous along the fibers. To determine whether this distribution corresponds to the distribution of injected agrin after
binding onto the muscle fiber, we metabolically labeled neural and muscle agrin by [35S]methionine and [35S]cysteine using stably transformed 293 HEK cells (Fig. 8 A; see Materials and Methods). 1 d after injection, 35S-labeled neural or
muscle agrin, respectively, were detected by autoradiography at similar densities along the fibers (Fig. 8 B, a and b) of
innervated muscle. 4 d after the injection, however, the density was much lower in innervated than in denervated fibers
(Fig. 8 B, c–f). Moreover, many fibers contained in their midregion a site of higher grain density, which presumably corresponded to the original NMJs (Fig. 8, a and b, arrow). In
agreement with this finding, we also detected recombinant
chick agrin by immunocytochemistry at NMJs (not shown).
We did not detect any gaps in the distribution of bound
agrin in 1-d innervated or 4-d denervated muscles that could
correspond to the absence of AChR aggregates on each side
of the original NMJs (see above).
The binding of 35S-labeled agrin to the surface of the
muscle fiber was specific. The injection of 10 times higher
concentrations of unlabeled neural or muscle agrin 6 h before application of 35S-labeled agrin markedly reduced the
binding of 35S-labeled neural or muscle agrin, respectively
(Fig. 8 B, g and h). On the other hand, injection of 10 times
higher concentrations of unlabeled neural agrin 6 h before
application of 35S-labeled muscle agrin did not reduce the
binding of muscle agrin (Fig. 8 B, k).
Bezakova et al. AChR Clustering by Purified Recombinant Agrin In Vivo
Neural and Muscle Agrin Bind to Laminin
To examine whether the agrin binding to laminin could be
responsible for homogenous distribution of recombinant
agrin after application, muscles were injected with radioactive neural or muscle agrin (1 M in 70 l) and, 1 d later
subjected to sequential extraction or immunoprecipitation, were followed by Western blot analysis. Muscle extracts and purified 35S agrin were loaded onto 3-12% gradient gels under denaturing, reducing or non-denaturing,
non-reducing conditions. Almost all 35S agrin appeared in
the fraction extracted with 5 M EDTA and protease inhibitors in PBS, which is known to extract efficiently laminin (Paulsson et al., 1987). Under denaturing and reducing
conditions, extracted and purified 35S-labeled agrin migrated similarly but under nondenaturing, nonreducing
conditions, extracted agrin failed to penetrate into the gel,
suggesting that it was bound to other protein(s) (Fig. 9 A).
Recombinant neural and muscle agrin were immunoprecipitated from injected muscles and separated on 5%
or 3–12% SDS-PAGE gradient gels. Silver staining revealed double bands of Mr 200 and 400 kD, corresponding to the sizes of , , and chains of laminin, and agrin
(Fig. 9 B). Western blot analysis of the immunoprecipitated complex using polyclonal antibody against 2-laminin detected a positive band at 400 kD (Fig. 9 C). Together, these data indicate that both neural and muscle
agrin bind to laminin in vivo.
AChR Aggregation Induced by Recombinant Neural Agrin
In this work, we have applied purified recombinant agrin
to skeletal muscles to study its effects in vivo in a more
controlled way than done until now. We show that agrin
AChR–aggregating activities (EC50) are 3 nM in denervated and 30 nM in innervated muscle fibers, which are
100 and 1,000 times higher than reported for cultured myotubes (Gesemann et al., 1995) using the same chick agrin
isoform. We bathed the SOL in solutions of neural agrin.
When necessary, we added fresh solution of appropriate
concentration to keep the muscle fully immersed during
the 2 h of incubation and afterwards examined only surface fibers that had been in direct contact with the solu-
tion. Therefore, we think that agrin concentration was
roughly maintained at stated values during treatment and
that the higher EC50 in vivo may be related in part to differences in the composition of extracellular matrix, particularly to the amount of laminin known to bind agrin (Denzer et al., 1995, 1997; Kammerer et al., 1999), and in part to
differences in responsiveness of embryonic and adult muscle cells to neural agrin.
The threshold concentration and EC50 for AChR aggregation by neural agrin was 100 and 10 times higher in
innervated than in denervated muscles, respectively. Electrical muscle activity downregulates the expression of
MuSK, which is a part of an agrin receptor and essential
for AChR aggregation by neural agrin (DeChiara et al.,
1996). The higher threshold in innervated muscles may
therefore be related to a lower amount of MuSK along the
innervated fibers. Muscles denervated at the time of injection displayed similar threshold concentration and EC50 as
muscles denervated 7 d before the injection, consistent
with the notion that denervation causes rapid upregulation
of MuSK (Valenzuela et al., 1995), which can be then recruited into an agrin-induced scaffold. In innervated muscle fibers that normally do not express AChR -subunits in
nonsynaptic regions (Witzemann et al., 1991, 1990), the
AChR aggregates induced by neural agrin contained primarily -subunits. In contrast, in denervated muscle fibers,
the aggregates were composed mainly of -subunit. Thus,
in innervated fibers, AChR aggregation may depend on
agrin-induced synthesis of synapse-specific proteins involving upregulation of MuSK (Meier et al., 1997; Jones et
al., 1999), whereas in denervated fibers, aggregation of
proteins already upregulated by denervation may be initially sufficient.
Individual AChR aggregates induced by high concentrations of neural agrin became smaller in size but more intensely labeled with Rh-BuTx. Weak Rh-BuTx staining
often surrounded these aggregates, suggesting that AChRs
translocated from larger to smaller aggregates. This reorganization was observed in predenervated, acutely denervated and innervated muscles, and may be related to
agrin-induced organization of cytoskeletal proteins and
colocalized AChRs (Bezakova and Lømo, 2001, page
1453, this issue). It did not depend on AChR subunit composition since denervated muscles containing primarily
-subunit and innervated muscles containing mainly -subunit of AChR behaved similarly.
A single injection of neural agrin (1 M) induced AChR
aggregates that persisted 7 wk. During this period, a single injection of Rh-BuTx into denervated muscle labeled
AChR aggregates that subsequently underwent changes
in size and distribution along the fibers. Despite these
changes, aggregates labeled by Rh-BuTx at a time when
the AChRs contained - rather than -subunits, were still
clearly visible 2 wk later. Furthermore, at this late time,
newly inserted AChRs labeled by Fl-BuTx precisely colocalized with those labeled 2 wk earlier by Rh-BuTx (Fig. 2
B). Two conclusions may be drawn from these results.
First, -subunit containing AChRs can be metabolically
stabilized by neural agrin in agreement with our earlier
finding that neural agrin alone can fully stabilize AChRs
in a dose-dependent manner (Bezakova, G., I. Rabben, G.
Fumagalli, and T. Lømo, submitted for publication). Sec-
The Journal of Cell Biology, Volume 153, 2001
Figure 7. Electrical muscle activity affects number, size, and distribution of neural agrin–induced AChR aggregates. 1 M neural
agrin was injected into SOL muscles that were immediately denervated (a, b, and d) or kept innervated (c and e–j). Muscles denervated for 7 d (a) were then electrically stimulated for additional
7 d (b). Some innervated muscles were denervated 0 (d), 3 (f), 7
(h), or 28 (j) d after the injection. At the indicated days after
these treatments (3–35 d), the muscles were excised and treated
with Rh-BuTx to label AChR aggregates as shown. Note the
changes in number, size, and distribution of AChR aggregates
that were caused by electrical stimulation of denervated muscles
(compare a with b) and the similarity between aggregates in denervated stimulated (b) and innervated (c, e, g, and i) muscles.
Also, note the appearance of a declining number of additional
small AChR aggregates after denervating the innervated muscles
28 d after the injection of neural agrin (d, f, h, and j).
Figure 8. Binding of neural and muscle agrin to the surface of SOL muscle fibers. Radioactive ( 35S) neural (isoform 7.4.8) and muscle
(isoform 0.0.0) chick agrin were purified from stably transfected 293 HEK cells (see Materials and Methods) and separated by SDSPAGE 3–12% gradient gel electrophoresis (A). 1 M 35S-labeled neural or muscle agrin was injected into SOL muscles, which were removed 1 or 4 d later for isolation of single fibers and subsequent autoradiography (B). Arrows in a and b, respectively, point to high
density of bound neural and muscle agrin at a site in the middle of the fiber that probably corresponds to original NMJ. Note similar
densities of neural and muscle agrin 4 d after injection in innervated (compare c with d) and denervated (compare e with f) fibers and
lower densities in innervated (c and d) compared with denervated (e and f) fibers. Injection of 10 M unlabeled agrin 6 h before injection of 1 M radioactive agrin markedly reduced binding of the corresponding radioactive agrin (g and h). Fibers teased from muscles
injected with PBS displayed essentially no grains (i and j, autoradiography and phase contrast, respectively). Injection of 10 M unlabeled neural agrin did not reduce the binding of radioactive muscle agrin injected at 1 M concentration 6 h later (k). The images show
representative distribution of agrin after different treatments. Three muscles for each condition, and 10 randomly teased fibers from
each muscle were examined.
ond, although, the aggregates labeled by Rh-BuTx underwent changes in size and distribution, they become stable
in the sense that they determined the site of insertion of
new AChRs. Presumably, this sort of stability is related to
the agrin-induced organization of cytoskeletal proteins
and colocalized AChR aggregates described elsewhere
(Bezakova and Lømo, 2001, page 1453, this issue).
Neural Agrin. Neural agrin bound uniformly along the entire length of the muscle fibers, in contrast to the AChR
aggregates it induced, which were nonuniformly distributed. In denervated muscles, large numbers of aggregates
appeared along the fibers except in a region, 0.5–0.8-mm
long, on either side of the original NMJs. This region, as
opposed to the rest of the fiber, is also refractory to ec-
topic NMJ formation by transplanted axons (Lømo, 1980).
Since neural agrin bound as well in this region as elsewhere, the refractoriness cannot be attributed to surface
changes that compromise agrin binding. Nor can it be attributed to lack of AChRs as their density is higher close
to denervated NMJs than further away (Salpeter et al.,
1988). Kues et al. (1995) provide evidence that nerve terminals at NMJs release a trophic signal, which in the continued presence of the nerve but independently of impulse
activity down regulates the expression of certain genes
(AChR -subunit, myogenin) in perisynaptic regions. The
refractoriness addressed here, however, does not require
the continued presence of the nerve since it persisted
around the original NMJs after denervation. But it does
require electrical muscle activity since it appears around
developing ectopic NMJs only if the muscle is electrically
Bezakova et al. AChR Clustering by Purified Recombinant Agrin In Vivo
Binding of Injected Agrin to the Surface of Muscle Fibers
Figure 9. Agrin binds to laminin. SOL muscles were injected with 1 M radioactively labeled neural or muscle agrin. 1 d later the muscles were excised, sequentially extracted, and analyzed by 3–12% SDS-PAGE electrophoresis under denaturing and reducing conditions (A, lanes 1–8) or nondenaturing and nonreducing conditions (A, lanes 1–8) followed by autoradiography. Lanes 1, 8, 1, and 8
represent the migration of purified radioactive neural and muscle agrin, respectively. Injected recombinant agrin was extracted from the
muscle by PBS containing EDTA (lanes 2, 5, 2, and 5). The migration of extracted agrin was dependent on the conditions used to run
the gel, indicating its binding to other protein(s). (B) SOL extracts were also immunoprecipitated with mAb 5B1 against agrin. Silver
staining revealed the presence of bands having molecular mass 400 and 200 kD. Western blots of the immunoprecipitated complex
and use of polyclonal antibody against 2-laminin confirmed the presence of 2-laminin (C). The molecular masses of standard proteins
are indicated in kD.
active (Skorpen et al., 1999). In agreement with this observation, the AChR aggregates induced by neural agrin became very few and far apart in innervated electrically active
as well as in denervated and electrically stimulated muscles.
Together, these findings strengthen earlier indications that
electrical muscle activity, as normally imposed by the
nerve, plays an important role in regulating the number,
size, and spatial distribution of the postsynaptic specializations induced by neural agrin (Mathiesen et al., 1999).
Bound agrin disappeared faster from extrajunctional regions of innervated electrically active fibers than from denervated electrically inactive fibers. Initially, the density of
agrin binding was similar in innervated and denervated fibers. Thus, electrical activity apparently does not affect agrin
binding as such but causes a more rapid removal of already
bound agrin. However, also in innervated fibers, the effect of
a single injection of neural agrin was long lasting since
AChR aggregates persisted 7 wk (Fig. 2 A). In addition,
new AChR aggregates appeared even when the muscle was
denervated as long as 4 wk after agrin injection (Fig. 7 j).
Electrical muscle stimulation of denervated muscle removed not only most of the agrin-induced AChR aggregates (the losers) but also reduced agrin at the sites of losers and elsewhere, except at the few aggregates that
survived (the winners). The mechanism underlying this activity-dependent removal of agrin from nonjunctional regions is unclear. Synaptic and extrasynaptic basal laminas
are immunologically distinguishable and differentially regulated by electrical muscle activity (Sanes and Lawrence,
1983). The faster removal of bound agrin from nonsynaptic
regions in electrically active fibers may therefore be secondary to an effect of activity on the basal lamina. Recently, it has been shown that matrix metalloproteinase-3
(MMP3) localized at the NMJ but also in extrajunctional
regions can selectively remove agrin from synaptic basal
lamina (VanSaun and Werle, 2000). Furthermore, agrin
contains nine folistatin domains that are related to Kazal
protease inhibitors (Rupp et al., 1991). Depending on
agrin binding to other proteins, these modules may acquire
specific structural organization (Bork et al., 1996). It is
therefore possible that the follistatin domains protect agrin
from the degradation only at sites where agrin is part of a
receptor complex that survives electrical muscle activity.
Muscle Agrin. Muscle agrin bound to the surface of innervated and denervated muscle fibers essentially as neural agrin with regard to density and distribution along the
fibers. Muscle agrin, however, did not cause aggregation of
AChRs. As was the case for neural agrin, nonradioactive
muscle agrin significantly decreased the binding of subsequently applied radioactive muscle agrin, indicating that
the binding in both cases was specific. In contrast, an excess of nonradioactive muscle agrin did not decrease subsequent binding of neural agrin nor did an excess of neural
agrin decrease subsequent binding by muscle agrin, suggesting that muscle and neural agrin bind to different receptors or different parts of the same receptor.
Laminin 2. We provide evidence here that both muscle
and neural agrin bind to laminin containing 2-chain in
vivo. Agrin binding to laminin has been well characterized
in vitro (Denzer et al., 1995, 1997; Kammerer et al., 1999).
The Journal of Cell Biology, Volume 153, 2001
Laminin isoforms 2 and 4 are predominant laminin isoforms of basal lamina and are expressed in developing
muscle fibers (Chiu and Sanes, 1984; Sanes et al., 1990).
Moreover, laminin-like immunoreactivity colocalizes with
agrin-like immunoreactivity in chick embryo hind limb
muscle in vivo and in vitro, and laminin as well as agrin are
enriched in AChR clusters (Godfrey et al., 1988; Nitkin
and Rothschild, 1990). In skeletal muscle, laminin also
binds to integrin, particularly 71 integrin (von der Mark
et al., 1991; Song et al., 1992) and induces colocalization of
integrin and AChRs (Burkin et al., 1998). The recruitment
of integrin into the AChR aggregates is further enhanced
by the presence of agrin, and the response induced by both
laminin and agrin can be inhibited by mAbs against 7 subunit of integrin. The integrin receptor associates with the
cytoskeleton (Song et al., 1993) and is capable of signal
transduction from the extracellular matrix (Clark and
Brugge, 1995; Kwon et al., 2000). It is, thus, possible that
integrin could be a component of the multisubunit receptor
involved in agrin-induced synapse formation. Interestingly,
in this work neural agrin induced the appearance of AChR
aggregates predominantly near the myotendinous junction
of innervated muscles, where the expression of 71 integrin is particularly pronounced (Mayer et al., 1997).
In vitro, agrin binds to both laminin and, in a Ca2dependent manner, to -dystroglycan (Gee et al., 1994;
Denzer et al., 1997, Kammerer et al., 1999). We did not examine whether agrin also binds to -dystroglycan in vivo
because such binding is difficult to resolve in the presence
of large amounts of laminin whose binding to -dystroglycan is also Ca2 dependent. Laminin may play a role in stabilizing and maintaining the postsynaptic apparatus (Denzer et al., 1997). Binding of neural agrin to laminin, and
possibly to -dystroglycan as well, may therefore have
contributed to the long-lasting effect of a single injection
of neural agrin that we observed.
Conclusion. We show that injected recombinant neural
and muscle agrin bind to the surface of muscle fibers in
vivo. Agrin binding involves laminin 2, is uniform along
the fibers, and is regulated by muscle activity. Aggregation
of AChRs by neural agrin is also muscle activity and dose
dependent and characterized by higher EC50 in comparison to cultured myotubes. AChR aggregation does not appear in perisynaptic regions, although neural agrin initially
binds as well in this region as elsewhere. A single application of neural agrin induces AChR aggregates that persist
in the innervated and denervated muscles 7 wk. Finally,
we show that a single application of purified recombinant
agrin is a suitable method for studying important aspects
of NMJ formation in vivo.
Submitted: 27 February 2001
Revised: 11 May 2001
Accepted: 15 May 2001
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We thank Professor Guido Fumagalli for critically discussing the results
and for supporting M. Francolini with a contribution from Telethon Italy.
We also thank Dr. Markus A. Rüegg for providing the stably transfected
293 HEK cell lines used in our experiments, Dr. Joshua R. Sanes for antibodies against - or -subunits, and Dr. Rudolf Timpl for antibodies
against 2-laminin.
This work was supported by grants from the EU Biotechnology Program (BIO4 CT96 0216 and BIO CT96 0433), Telethon Italy (grant 764),
and the Norwegian Research Council.
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The Journal of Cell Biology, Volume 153, 2001
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