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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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Cre-mediated inactivation of the MuSK gene in single muscle fibers. Single muscle fibers of MuSKloxP/− mice were injected with DNA and expression of transgenic products was analyzed 21 d after injection. Excised muscles were incubated with r-bgt to identify AChR clusters. Overlays of green and red fluorescence images are shown. (A) Maximum projection of a confocal image series shows ectopic AChR clusters (red) induced by agrin. The nGFP-expressing fiber (green) on the right has been injected with agrin and nGFP DNA as indicated. Expression of nGFP and the appearance of AChR clusters shows successful expression of the transgenic proteins. The clusters are apparently located on the injected as well as on noninjected fibers. (B) Cross-sections of the confocal images allow to assign AChR clusters to individual muscle fibers. Reconstructed xz projection of the confocal image series (A) over the range (y) marked by white arrowheads. AChR clusters, located in the plasma membrane, surround the nGFP, transgene-expressing muscle fiber (marked by asterisk). AChR clusters are also located on neighboring noninjected fibers. (Bottom) Location of muscle fibers is indicated schematically and AChR cluster–expressing fibers are labeled red. Transgene-expressing fiber is marked by asterisk. (C) Maximum projection of a confocal image series. Two nGFP-expressing fibers (green) had been injected with agrin and nGFP DNA plus Cre DNA to conditionally inactivate endogenous MuSK. Agrin-induced AChR clusters (red) are apparently distributed as in A. Expression of nGFP and AChR clusters shows successful expression of the transgenic proteins. (D) Reconstructed xz projection of the confocal image series (C) over the range (y) marked by white arrowheads. AChR clusters are not found on the nGFP, Cre-expressing muscle fibers (marked by asterisks), but only on neighboring noninjected fibers. Cre-mediated inactivation of endogenous MuSK prevents formation of AChR clusters. (Bottom) Location of muscle fibers is indicated schematically. AChR cluster-expressing fibers are labeled red. Transgene-expressing fibers are marked by asterisks. 7 mice were used for DNA injection; a total of 39 GFP-expressing fibers were analyzed but contained no ectopic AChR clusters.
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fig4: Cre-mediated inactivation of the MuSK gene in single muscle fibers. Single muscle fibers of MuSKloxP/− mice were injected with DNA and expression of transgenic products was analyzed 21 d after injection. Excised muscles were incubated with r-bgt to identify AChR clusters. Overlays of green and red fluorescence images are shown. (A) Maximum projection of a confocal image series shows ectopic AChR clusters (red) induced by agrin. The nGFP-expressing fiber (green) on the right has been injected with agrin and nGFP DNA as indicated. Expression of nGFP and the appearance of AChR clusters shows successful expression of the transgenic proteins. The clusters are apparently located on the injected as well as on noninjected fibers. (B) Cross-sections of the confocal images allow to assign AChR clusters to individual muscle fibers. Reconstructed xz projection of the confocal image series (A) over the range (y) marked by white arrowheads. AChR clusters, located in the plasma membrane, surround the nGFP, transgene-expressing muscle fiber (marked by asterisk). AChR clusters are also located on neighboring noninjected fibers. (Bottom) Location of muscle fibers is indicated schematically and AChR cluster–expressing fibers are labeled red. Transgene-expressing fiber is marked by asterisk. (C) Maximum projection of a confocal image series. Two nGFP-expressing fibers (green) had been injected with agrin and nGFP DNA plus Cre DNA to conditionally inactivate endogenous MuSK. Agrin-induced AChR clusters (red) are apparently distributed as in A. Expression of nGFP and AChR clusters shows successful expression of the transgenic proteins. (D) Reconstructed xz projection of the confocal image series (C) over the range (y) marked by white arrowheads. AChR clusters are not found on the nGFP, Cre-expressing muscle fibers (marked by asterisks), but only on neighboring noninjected fibers. Cre-mediated inactivation of endogenous MuSK prevents formation of AChR clusters. (Bottom) Location of muscle fibers is indicated schematically. AChR cluster-expressing fibers are labeled red. Transgene-expressing fibers are marked by asterisks. 7 mice were used for DNA injection; a total of 39 GFP-expressing fibers were analyzed but contained 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)

Cre-mediated inactivation of the MuSK gene in single muscle fibers. Single muscle fibers of MuSKloxP/− mice were injected with DNA and expression of transgenic products was analyzed 21 d after injection. Excised muscles were incubated with r-bgt to identify AChR clusters. Overlays of green and red fluorescence images are shown. (A) Maximum projection of a confocal image series shows ectopic AChR clusters (red) induced by agrin. The nGFP-expressing fiber (green) on the right has been injected with agrin and nGFP DNA as indicated. Expression of nGFP and the appearance of AChR clusters shows successful expression of the transgenic proteins. The clusters are apparently located on the injected as well as on noninjected fibers. (B) Cross-sections of the confocal images allow to assign AChR clusters to individual muscle fibers. Reconstructed xz projection of the confocal image series (A) over the range (y) marked by white arrowheads. AChR clusters, located in the plasma membrane, surround the nGFP, transgene-expressing muscle fiber (marked by asterisk). AChR clusters are also located on neighboring noninjected fibers. (Bottom) Location of muscle fibers is indicated schematically and AChR cluster–expressing fibers are labeled red. Transgene-expressing fiber is marked by asterisk. (C) Maximum projection of a confocal image series. Two nGFP-expressing fibers (green) had been injected with agrin and nGFP DNA plus Cre DNA to conditionally inactivate endogenous MuSK. Agrin-induced AChR clusters (red) are apparently distributed as in A. Expression of nGFP and AChR clusters shows successful expression of the transgenic proteins. (D) Reconstructed xz projection of the confocal image series (C) over the range (y) marked by white arrowheads. AChR clusters are not found on the nGFP, Cre-expressing muscle fibers (marked by asterisks), but only on neighboring noninjected fibers. Cre-mediated inactivation of endogenous MuSK prevents formation of AChR clusters. (Bottom) Location of muscle fibers is indicated schematically. AChR cluster-expressing fibers are labeled red. Transgene-expressing fibers are marked by asterisks. 7 mice were used for DNA injection; a total of 39 GFP-expressing fibers were analyzed but contained no ectopic AChR clusters.
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fig4: Cre-mediated inactivation of the MuSK gene in single muscle fibers. Single muscle fibers of MuSKloxP/− mice were injected with DNA and expression of transgenic products was analyzed 21 d after injection. Excised muscles were incubated with r-bgt to identify AChR clusters. Overlays of green and red fluorescence images are shown. (A) Maximum projection of a confocal image series shows ectopic AChR clusters (red) induced by agrin. The nGFP-expressing fiber (green) on the right has been injected with agrin and nGFP DNA as indicated. Expression of nGFP and the appearance of AChR clusters shows successful expression of the transgenic proteins. The clusters are apparently located on the injected as well as on noninjected fibers. (B) Cross-sections of the confocal images allow to assign AChR clusters to individual muscle fibers. Reconstructed xz projection of the confocal image series (A) over the range (y) marked by white arrowheads. AChR clusters, located in the plasma membrane, surround the nGFP, transgene-expressing muscle fiber (marked by asterisk). AChR clusters are also located on neighboring noninjected fibers. (Bottom) Location of muscle fibers is indicated schematically and AChR cluster–expressing fibers are labeled red. Transgene-expressing fiber is marked by asterisk. (C) Maximum projection of a confocal image series. Two nGFP-expressing fibers (green) had been injected with agrin and nGFP DNA plus Cre DNA to conditionally inactivate endogenous MuSK. Agrin-induced AChR clusters (red) are apparently distributed as in A. Expression of nGFP and AChR clusters shows successful expression of the transgenic proteins. (D) Reconstructed xz projection of the confocal image series (C) over the range (y) marked by white arrowheads. AChR clusters are not found on the nGFP, Cre-expressing muscle fibers (marked by asterisks), but only on neighboring noninjected fibers. Cre-mediated inactivation of endogenous MuSK prevents formation of AChR clusters. (Bottom) Location of muscle fibers is indicated schematically. AChR cluster-expressing fibers are labeled red. Transgene-expressing fibers are marked by asterisks. 7 mice were used for DNA injection; a total of 39 GFP-expressing fibers were analyzed but contained 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