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MuSK induces in vivo acetylcholine receptor clusters in a ligand-independent manner.

Sander A, Hesser BA, Witzemann V - J. Cell Biol. (2001)

Bottom Line: Expression of kinase-inactive MuSK did not result in the formation of acetylcholine receptor (AChR) clusters, whereas a mutant MuSK lacking the ectodomain did induce AChR clusters.Thus, the kinase activity of MuSK initiates signals that are sufficient to induce the formation of AChR clusters.This process does not require additional determinants located in the ectodomain.

View Article: PubMed Central - PubMed

Affiliation: Abteilung Zellphysiologie, Max-Planck-Institut für Medizinische Forschung, D-69120 Heidelberg, Germany.

ABSTRACT
Muscle-specific receptor tyrosine kinase (MuSK) is required for the formation of the neuromuscular junction. Using direct gene transfer into single fibers, MuSK was expressed extrasynaptically in innervated rat muscle in vivo to identify its contribution to synapse formation. Spontaneous MuSK kinase activity leads, in the absence of its putative ligand neural agrin, to the appearance of epsilon-subunit-specific transcripts, the formation of acetylcholine receptor clusters, and acetylcholinesterase aggregates. Expression of kinase-inactive MuSK did not result in the formation of acetylcholine receptor (AChR) clusters, whereas a mutant MuSK lacking the ectodomain did induce AChR clusters. The contribution of endogenous MuSK was excluded by using genetically altered mice, where the kinase domain of the MuSK gene was flanked by loxP sequences and could be deleted upon expression of Cre recombinase. This allowed the conditional inactivation of endogenous MuSK in single muscle fibers and prevented the induction of ectopic AChR clusters. Thus, the kinase activity of MuSK initiates signals that are sufficient to induce the formation of AChR clusters. This process does not require additional determinants located in the ectodomain.

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MuSK lacking the ectodomain induces AChR clusters. Rat muscle fibers were injected with DNA of rapsyn and MuSK mutants. After 21 d, muscles were excised. MuSK-induced AChR clusters were visualized by r-bgt. (A) Expression of MuSK–GFP and rapsyn, as indicated, induces AChR clusters. Several different AChR cluster patterns are presented and demonstrate that size and shape of MuSK-induced clusters may vary, probably depending on the concentration of transgenic MuSK expressed upon direct gene transfer. (B) Expression of ΔectoMuSK–GFP and rapsyn, as indicated, induces AChR clusters similar to that shown in A (4 rats were used for DNA injection; a total of 47 fibers were injected and AChR clusters were detected on 28 fibers). (C) Diagram: ΔectoMuSK–GFP induces AChR clusters as observed in A and B. In all transgene-expressing fibers, AChR clusters are formed (3 rats were used for DNA injection; a total of 48 fibers were injected; 28 fibers expressed ΔectoMuSK–GFP and AChR clusters were detected on all 28 fibers). (D) Diagram: kinase-inactive ΔectoMuSKK608A–GFP fails to induce AChR clusters (2 rats were used for DNA injection; a total of 25 fibers were injected and 14 ΔectoMuSKK608A–GFP-expressing fibers were detected; none of the fibers had ectopic AChR clusters).
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fig6: MuSK lacking the ectodomain induces AChR clusters. Rat muscle fibers were injected with DNA of rapsyn and MuSK mutants. After 21 d, muscles were excised. MuSK-induced AChR clusters were visualized by r-bgt. (A) Expression of MuSK–GFP and rapsyn, as indicated, induces AChR clusters. Several different AChR cluster patterns are presented and demonstrate that size and shape of MuSK-induced clusters may vary, probably depending on the concentration of transgenic MuSK expressed upon direct gene transfer. (B) Expression of ΔectoMuSK–GFP and rapsyn, as indicated, induces AChR clusters similar to that shown in A (4 rats were used for DNA injection; a total of 47 fibers were injected and AChR clusters were detected on 28 fibers). (C) Diagram: ΔectoMuSK–GFP induces AChR clusters as observed in A and B. In all transgene-expressing fibers, AChR clusters are formed (3 rats were used for DNA injection; a total of 48 fibers were injected; 28 fibers expressed ΔectoMuSK–GFP and AChR clusters were detected on all 28 fibers). (D) Diagram: kinase-inactive ΔectoMuSKK608A–GFP fails to induce AChR clusters (2 rats were used for DNA injection; a total of 25 fibers were injected and 14 ΔectoMuSKK608A–GFP-expressing fibers were detected; none of the fibers had ectopic AChR clusters).

Mentions: There is evidence that MuSK signaling requires the ectodomain of MuSK (Apel et al., 1997; Glass et al., 1997). To determine whether MuSK-induced AChR clustering requires the ectodomain, we constructed a mutant lacking the complete extracellular region (ΔectoMuSK; see Fig. 2). Fig. 6 A shows several representative AChR cluster patterns induced by transgenic MuSK. The number and size of the AChR clusters vary to some extent and is probably due to the variable amounts of MuSK expressed upon injection. The expression of ΔectoMuSK led to the formation of AChR clusters (Fig. 6 B), which were comparable to the clusters induced by full length MuSK. Again, to prove successful expression of the transgene in the injected muscle fibers, the ΔectoMuSK construct was fused with GFP where GFP replaced the ectodomain (ΔectoMuSK–GFP; see Fig. 2). Injection of this DNA led to efficient expression of ΔectoMuSK–GFP, which induced AChR clusters (Fig. 6 C). The kinase-inactive mutant ΔectoMuSKK608A–GFP (Fig. 2) was also expressed efficiently, but was unable to induce AChR clusters (Fig. 6 D). Thus, deletion of the ectodomain does not interfere with MuSK's ability to induce aggregation of AChR.


MuSK induces in vivo acetylcholine receptor clusters in a ligand-independent manner.

Sander A, Hesser BA, Witzemann V - J. Cell Biol. (2001)

MuSK lacking the ectodomain induces AChR clusters. Rat muscle fibers were injected with DNA of rapsyn and MuSK mutants. After 21 d, muscles were excised. MuSK-induced AChR clusters were visualized by r-bgt. (A) Expression of MuSK–GFP and rapsyn, as indicated, induces AChR clusters. Several different AChR cluster patterns are presented and demonstrate that size and shape of MuSK-induced clusters may vary, probably depending on the concentration of transgenic MuSK expressed upon direct gene transfer. (B) Expression of ΔectoMuSK–GFP and rapsyn, as indicated, induces AChR clusters similar to that shown in A (4 rats were used for DNA injection; a total of 47 fibers were injected and AChR clusters were detected on 28 fibers). (C) Diagram: ΔectoMuSK–GFP induces AChR clusters as observed in A and B. In all transgene-expressing fibers, AChR clusters are formed (3 rats were used for DNA injection; a total of 48 fibers were injected; 28 fibers expressed ΔectoMuSK–GFP and AChR clusters were detected on all 28 fibers). (D) Diagram: kinase-inactive ΔectoMuSKK608A–GFP fails to induce AChR clusters (2 rats were used for DNA injection; a total of 25 fibers were injected and 14 ΔectoMuSKK608A–GFP-expressing fibers were detected; none of the fibers had ectopic AChR clusters).
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fig6: MuSK lacking the ectodomain induces AChR clusters. Rat muscle fibers were injected with DNA of rapsyn and MuSK mutants. After 21 d, muscles were excised. MuSK-induced AChR clusters were visualized by r-bgt. (A) Expression of MuSK–GFP and rapsyn, as indicated, induces AChR clusters. Several different AChR cluster patterns are presented and demonstrate that size and shape of MuSK-induced clusters may vary, probably depending on the concentration of transgenic MuSK expressed upon direct gene transfer. (B) Expression of ΔectoMuSK–GFP and rapsyn, as indicated, induces AChR clusters similar to that shown in A (4 rats were used for DNA injection; a total of 47 fibers were injected and AChR clusters were detected on 28 fibers). (C) Diagram: ΔectoMuSK–GFP induces AChR clusters as observed in A and B. In all transgene-expressing fibers, AChR clusters are formed (3 rats were used for DNA injection; a total of 48 fibers were injected; 28 fibers expressed ΔectoMuSK–GFP and AChR clusters were detected on all 28 fibers). (D) Diagram: kinase-inactive ΔectoMuSKK608A–GFP fails to induce AChR clusters (2 rats were used for DNA injection; a total of 25 fibers were injected and 14 ΔectoMuSKK608A–GFP-expressing fibers were detected; none of the fibers had ectopic AChR clusters).
Mentions: There is evidence that MuSK signaling requires the ectodomain of MuSK (Apel et al., 1997; Glass et al., 1997). To determine whether MuSK-induced AChR clustering requires the ectodomain, we constructed a mutant lacking the complete extracellular region (ΔectoMuSK; see Fig. 2). Fig. 6 A shows several representative AChR cluster patterns induced by transgenic MuSK. The number and size of the AChR clusters vary to some extent and is probably due to the variable amounts of MuSK expressed upon injection. The expression of ΔectoMuSK led to the formation of AChR clusters (Fig. 6 B), which were comparable to the clusters induced by full length MuSK. Again, to prove successful expression of the transgene in the injected muscle fibers, the ΔectoMuSK construct was fused with GFP where GFP replaced the ectodomain (ΔectoMuSK–GFP; see Fig. 2). Injection of this DNA led to efficient expression of ΔectoMuSK–GFP, which induced AChR clusters (Fig. 6 C). The kinase-inactive mutant ΔectoMuSKK608A–GFP (Fig. 2) was also expressed efficiently, but was unable to induce AChR clusters (Fig. 6 D). Thus, deletion of the ectodomain does not interfere with MuSK's ability to induce aggregation of AChR.

Bottom Line: Expression of kinase-inactive MuSK did not result in the formation of acetylcholine receptor (AChR) clusters, whereas a mutant MuSK lacking the ectodomain did induce AChR clusters.Thus, the kinase activity of MuSK initiates signals that are sufficient to induce the formation of AChR clusters.This process does not require additional determinants located in the ectodomain.

View Article: PubMed Central - PubMed

Affiliation: Abteilung Zellphysiologie, Max-Planck-Institut für Medizinische Forschung, D-69120 Heidelberg, Germany.

ABSTRACT
Muscle-specific receptor tyrosine kinase (MuSK) is required for the formation of the neuromuscular junction. Using direct gene transfer into single fibers, MuSK was expressed extrasynaptically in innervated rat muscle in vivo to identify its contribution to synapse formation. Spontaneous MuSK kinase activity leads, in the absence of its putative ligand neural agrin, to the appearance of epsilon-subunit-specific transcripts, the formation of acetylcholine receptor clusters, and acetylcholinesterase aggregates. Expression of kinase-inactive MuSK did not result in the formation of acetylcholine receptor (AChR) clusters, whereas a mutant MuSK lacking the ectodomain did induce AChR clusters. The contribution of endogenous MuSK was excluded by using genetically altered mice, where the kinase domain of the MuSK gene was flanked by loxP sequences and could be deleted upon expression of Cre recombinase. This allowed the conditional inactivation of endogenous MuSK in single muscle fibers and prevented the induction of ectopic AChR clusters. Thus, the kinase activity of MuSK initiates signals that are sufficient to induce the formation of AChR clusters. This process does not require additional determinants located in the ectodomain.

Show MeSH
Related in: MedlinePlus