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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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Induction of ectopic AChR clusters requires MuSK kinase activity. Rat muscle fibers were injected with expression vectors encoding MuSK–GFP or MuSKK608A–GFP and rapsyn. After 21 d, the muscles were excised. Maximum projections of confocal image series show expression of GFP fusion protein (green) and the formation of AChR clusters. AChR clusters are visualized by r-bgt (red). (A) Expression of MuSK–GFP and rapsyn, as indicated. (B) AChR clusters induced by MuSK–GFP. (C) overlay of A and B indicates that MuSK–GFP and AChR clusters are not colocalized. (2 rats were used for DNA injection; a total of 26 fibers were injected and AChR clusters were detected in 14 fibers.) (D) Expression of kinase-inactive MuSKK608A–GFP and rapsyn, as indicated. (E) MuSKK608A–GFP does not induce AChR clusters and coinjected rapsyn is unable to form AChR/rapsyn aggregates. 4 rats were used for DNA injection; a total of 50 fibers were injected and 38 GFP-positive fibers were detected. (F) Graph illustrates that AChR clusters are detected on all fibers expressing transgenic MuSK. (G) Fibers expressing MuSKK608A–GFP display no ectopic AChR clusters.
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fig3: Induction of ectopic AChR clusters requires MuSK kinase activity. Rat muscle fibers were injected with expression vectors encoding MuSK–GFP or MuSKK608A–GFP and rapsyn. After 21 d, the muscles were excised. Maximum projections of confocal image series show expression of GFP fusion protein (green) and the formation of AChR clusters. AChR clusters are visualized by r-bgt (red). (A) Expression of MuSK–GFP and rapsyn, as indicated. (B) AChR clusters induced by MuSK–GFP. (C) overlay of A and B indicates that MuSK–GFP and AChR clusters are not colocalized. (2 rats were used for DNA injection; a total of 26 fibers were injected and AChR clusters were detected in 14 fibers.) (D) Expression of kinase-inactive MuSKK608A–GFP and rapsyn, as indicated. (E) MuSKK608A–GFP does not induce AChR clusters and coinjected rapsyn is unable to form AChR/rapsyn aggregates. 4 rats were used for DNA injection; a total of 50 fibers were injected and 38 GFP-positive fibers were detected. (F) Graph illustrates that AChR clusters are detected on all fibers expressing transgenic MuSK. (G) Fibers expressing MuSKK608A–GFP display no ectopic AChR clusters.

Mentions: By direct gene transfer into individual muscle fibers it was shown that agrin induced the formation of ectopic AChR clusters (Cohen et al., 1997; Jones et al., 1997; Meier et al., 1997; Rimer et al., 1997). Using the same approach, it was found that injection of MuSK plasmid DNA alone results in the absence of neural agrin and also in the formation of AChR clusters (Fig. 1 A; Hesser et al., 1999; Jones et al., 1999). MuSK-induced AChR clusters differed, however, from clusters induced following overexpression of neural agrin. Agrin-induced clusters appeared larger in size and more spread out both on the injected fiber as well as on adjacent fibers (Jones et al., 1997; see Figs. 3 and 4). In the case of rapsyn, which is essential for the clustering of AChR at developing synapses (Gautam et al., 1995), we observed that ectopic injection of rapsyn plasmid DNA alone did not induce AChR clustering (unpublished data). Since rapsyn could be linked to MuSK and AChR (Apel et al., 1997), we asked whether MuSK-induced AChR clusters might be altered when expressed in the presence of transgenic rapsyn. Coinjection of MuSK and rapsyn DNA resulted in the formation of AChR clusters (Fig. 1 B) which were similar to the clusters induced by MuSK alone as shown in Fig. 1 A.


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

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

Induction of ectopic AChR clusters requires MuSK kinase activity. Rat muscle fibers were injected with expression vectors encoding MuSK–GFP or MuSKK608A–GFP and rapsyn. After 21 d, the muscles were excised. Maximum projections of confocal image series show expression of GFP fusion protein (green) and the formation of AChR clusters. AChR clusters are visualized by r-bgt (red). (A) Expression of MuSK–GFP and rapsyn, as indicated. (B) AChR clusters induced by MuSK–GFP. (C) overlay of A and B indicates that MuSK–GFP and AChR clusters are not colocalized. (2 rats were used for DNA injection; a total of 26 fibers were injected and AChR clusters were detected in 14 fibers.) (D) Expression of kinase-inactive MuSKK608A–GFP and rapsyn, as indicated. (E) MuSKK608A–GFP does not induce AChR clusters and coinjected rapsyn is unable to form AChR/rapsyn aggregates. 4 rats were used for DNA injection; a total of 50 fibers were injected and 38 GFP-positive fibers were detected. (F) Graph illustrates that AChR clusters are detected on all fibers expressing transgenic MuSK. (G) Fibers expressing MuSKK608A–GFP display no ectopic AChR clusters.
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Related In: Results  -  Collection

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fig3: Induction of ectopic AChR clusters requires MuSK kinase activity. Rat muscle fibers were injected with expression vectors encoding MuSK–GFP or MuSKK608A–GFP and rapsyn. After 21 d, the muscles were excised. Maximum projections of confocal image series show expression of GFP fusion protein (green) and the formation of AChR clusters. AChR clusters are visualized by r-bgt (red). (A) Expression of MuSK–GFP and rapsyn, as indicated. (B) AChR clusters induced by MuSK–GFP. (C) overlay of A and B indicates that MuSK–GFP and AChR clusters are not colocalized. (2 rats were used for DNA injection; a total of 26 fibers were injected and AChR clusters were detected in 14 fibers.) (D) Expression of kinase-inactive MuSKK608A–GFP and rapsyn, as indicated. (E) MuSKK608A–GFP does not induce AChR clusters and coinjected rapsyn is unable to form AChR/rapsyn aggregates. 4 rats were used for DNA injection; a total of 50 fibers were injected and 38 GFP-positive fibers were detected. (F) Graph illustrates that AChR clusters are detected on all fibers expressing transgenic MuSK. (G) Fibers expressing MuSKK608A–GFP display no ectopic AChR clusters.
Mentions: By direct gene transfer into individual muscle fibers it was shown that agrin induced the formation of ectopic AChR clusters (Cohen et al., 1997; Jones et al., 1997; Meier et al., 1997; Rimer et al., 1997). Using the same approach, it was found that injection of MuSK plasmid DNA alone results in the absence of neural agrin and also in the formation of AChR clusters (Fig. 1 A; Hesser et al., 1999; Jones et al., 1999). MuSK-induced AChR clusters differed, however, from clusters induced following overexpression of neural agrin. Agrin-induced clusters appeared larger in size and more spread out both on the injected fiber as well as on adjacent fibers (Jones et al., 1997; see Figs. 3 and 4). In the case of rapsyn, which is essential for the clustering of AChR at developing synapses (Gautam et al., 1995), we observed that ectopic injection of rapsyn plasmid DNA alone did not induce AChR clustering (unpublished data). Since rapsyn could be linked to MuSK and AChR (Apel et al., 1997), we asked whether MuSK-induced AChR clusters might be altered when expressed in the presence of transgenic rapsyn. Coinjection of MuSK and rapsyn DNA resulted in the formation of AChR clusters (Fig. 1 B) which were similar to the clusters induced by MuSK alone as shown in Fig. 1 A.

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