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Effects of purified recombinant neural and muscle agrin on skeletal muscle fibers in vivo.

Bezakova G, Helm JP, Francolini M, Lømo T - J. Cell Biol. (2001)

Bottom Line: We have applied purified recombinant chick neural and muscle agrin to rat soleus muscle in vivo and obtained the following results.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.Thus, the present approach provides a novel, simple, and efficient method for studying the effects of agrin on muscle under controlled conditions in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Oslo, 0317 Oslo, Norway. gabriela.bezakova@unibas.ch

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 > or = 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.

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Ectopic AChR aggregates induced by purified recombinant chick neural agrin. (A) A single injection of 1 μM 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 Rh-BuTx and Fl-BuTx indicates the structural stability of aggregates.
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Figure 2: Ectopic AChR aggregates induced by purified recombinant chick neural agrin. (A) A single injection of 1 μM 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 Rh-BuTx and Fl-BuTx indicates the structural stability of aggregates.

Mentions: 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 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).


Effects of purified recombinant neural and muscle agrin on skeletal muscle fibers in vivo.

Bezakova G, Helm JP, Francolini M, Lømo T - J. Cell Biol. (2001)

Ectopic AChR aggregates induced by purified recombinant chick neural agrin. (A) A single injection of 1 μM 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 Rh-BuTx and Fl-BuTx indicates the structural stability of aggregates.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2150725&req=5

Figure 2: Ectopic AChR aggregates induced by purified recombinant chick neural agrin. (A) A single injection of 1 μM 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 Rh-BuTx and Fl-BuTx indicates the structural stability of aggregates.
Mentions: 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 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).

Bottom Line: We have applied purified recombinant chick neural and muscle agrin to rat soleus muscle in vivo and obtained the following results.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.Thus, the present approach provides a novel, simple, and efficient method for studying the effects of agrin on muscle under controlled conditions in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Oslo, 0317 Oslo, Norway. gabriela.bezakova@unibas.ch

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 > or = 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.

Show MeSH
Related in: MedlinePlus