Correspondence to: Gabriela Bezakova, Department of Pharmacology/Neurobiology, Biozentrum, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland. Tel:41-61-267-2214 Fax:41-61-267-2208 E-mail:gabriela.bezakova{at}unibas.ch.
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Abstract |
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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 agrincDNA 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 agrininduced 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
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Introduction |
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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 (400600 kD, is essential for the induction and organization of the postsynaptic structures (
The AChR-clustering activity of neural agrin has been well characterized in vitro, where the activity is both concentration- and Ca2+-dependent (-subunits in addition to clustering AChRs (
-dystroglycan, a component of the dystrophin-associated glycoprotein complex present on the surface of muscle fibers (
The most compelling evidence that agrin is essential for NMJ formation comes from loss- and gain-of-function studies. In agrin-deficient mutant mice, the postsynaptic differentiation was profoundly impaired and the mice died perinatally (
Experiments involving implantation of agrin-secreting myoblasts or injection of agrin expression constructs into muscles demonstrated agrin's activity in vivo (
Formation of ectopic NMJs through the interaction of transplanted axons with soleus (SOL) muscles is strongly affected by electrical muscle activity (
Injecting agrincDNA 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 agrininduced AChR aggregates are regulated by electrical muscle activity. Muscle agrin displays similar binding but fails to aggregate AChRs.
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Materials and Methods |
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Purification of Recombinant Agrin
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 (
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 µCi/ml; ICN Biomedicals). The purified proteins were separated by SDS-PAGE on 312% 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 (
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 µl of 1 µM 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 (Rh-BuTx; Molecular Probes) for 30 min, washed with PBS, and fixed with 1.5% paraformaldehyde (PFA). Fixed muscle was teased into
30 thin bundles containing 50100 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 pM10 µM 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).
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Quantification of AChR-aggregating Activity
Relative Area Occupied by AChR Aggregates.
Agrin-induced AChR aggregates that could be viewed en face on the surface of single muscle fiber at 1,000x magnification were selected for measurements using an Open Lab imaging software (Improvision). A rectangular region enclosing a collection 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 (
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Immunocytochemistry
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
Autoradiography of Muscle Fibers from Muscles Injected with 35S-labeled Agrin
SOL muscles were injected with 70 µl of 1 µM 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 (
Identification of Agrin Binding to Laminin
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 µg/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 312% 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.
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 4°C. Protein Aagarose (Sigma-Aldrich) was added for 4 h at 4°C. 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 312% SDS-PAGE gel. Proteins were either visualized by silver staining or transferred to nitrocellulose membrane using standard methods (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.).
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Results |
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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 312% SDS-PAGE gradient gels stained with Coomassie blue or silver as single smeared bands with an apparent molecular weight of 400600 kD (Fig 1B and Fig C). Comparable bands were observed after immunoprecipitation with mAb 5B1 (
Fate of AChR Aggregates after a Single Injection of Neural Agrin
A single injection of purified recombinant neural agrin (1 µM, 70 µl) 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 µm), punctate, and uniformly distributed along the fibers. During the next 12 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 FITC-bungarotoxin (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 0.50.8-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 µm), structurally 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
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).
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These results are shown quantitatively in Fig 5 and Table 1. 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 predenervated muscles. On the other hand, the EC50 for the response of innervated muscles to neural agrin was 10 times higher.
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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 (Fig 4 and Fig 5 B, Table 1). Furthermore, there was no significant difference between EC50 for half maximal labeling intensity in predenervated, denervated, and innervated muscles (Table 1).
The size and organization of AChR aggregates depended strongly on the concentration of neural agrin (Fig 4 and Fig 5). To examine if these differences could be related to the switch from - to
-subunitcontaining 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.
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Muscle agrin did not aggregate AChRs at any concentration (Fig 4 and Fig 5), even though muscle agrin bound well to the muscle surface (see below). Immunoprecipitation of injected neural and muscle chick agrin by species-specific 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
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 agrininduced AChR aggregates.
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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 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, cf). 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).
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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).
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 µM in 70 µl) 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 µM EDTA and protease inhibitors in PBS, which is known to extract efficiently laminin (
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Recombinant neural and muscle agrin were immunoprecipitated from injected muscles and separated on 5% or 312% SDS-PAGE gradient gels. Silver staining revealed double bands of Mr 200 and 400 kD, corresponding to the sizes of
, ß, 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.
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Discussion |
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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 AChRaggregating 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 (
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 (
-subunits in nonsynaptic regions (
-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 (
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 (-subunit and innervated muscles containing mainly
-subunit of AChR behaved similarly.
A single injection of neural agrin (1 µM) 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). Second, 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 (
Binding of Injected Agrin to the Surface of Muscle Fibers
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, 0.50.8-mm long, on either side of the original NMJs. This region, as opposed to the rest of the fiber, is also refractory to ectopic NMJ formation by transplanted axons (
-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 active (
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 (
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 (
7ß1 integrin (
7 subunit of integrin. The integrin receptor associates with the cytoskeleton (
7ß1 integrin is particularly pronounced (
In vitro, agrin binds to both laminin and, in a Ca2+-dependent manner, to -dystroglycan (
-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 (
-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.
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Footnotes |
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1 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.
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Acknowledgements |
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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.
Submitted: 27 February 2001
Revised: 11 May 2001
Accepted: 15 May 2001
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References |
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