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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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AChR clusters are induced by transgenic MuSK lacking the ectodomain. Single muscle fibers of MuSKloxP/− mice were injected with DNA of ΔectoMuSK–GFP and Cre. Expression of transgenes was analyzed 21 d after injection. The excised muscles were incubated with r-bgt to identify AChR clusters. Maximum projections of confocal image series are shown. (A) Expression of ΔectoMuSK–GFP (green fluorescence indicates successful expression of transgenes) and Cre. (B) AChR clusters are formed in absence of endogenous MuSK which has been inactivated by transgenic Cre (a MuSKloxP/− mouse was used for injection; a total of 22 fibers were injected; 11 fibers expressed ΔectoMuSK–GFP and 10 of these fibers had ectopic AChR clusters). (C) Overlay of green and red fluorescence images: ΔectoMuSK–GFP is not colocalized with AChR clusters.
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fig7: AChR clusters are induced by transgenic MuSK lacking the ectodomain. Single muscle fibers of MuSKloxP/− mice were injected with DNA of ΔectoMuSK–GFP and Cre. Expression of transgenes was analyzed 21 d after injection. The excised muscles were incubated with r-bgt to identify AChR clusters. Maximum projections of confocal image series are shown. (A) Expression of ΔectoMuSK–GFP (green fluorescence indicates successful expression of transgenes) and Cre. (B) AChR clusters are formed in absence of endogenous MuSK which has been inactivated by transgenic Cre (a MuSKloxP/− mouse was used for injection; a total of 22 fibers were injected; 11 fibers expressed ΔectoMuSK–GFP and 10 of these fibers had ectopic AChR clusters). (C) Overlay of green and red fluorescence images: ΔectoMuSK–GFP is not colocalized with AChR clusters.

Mentions: To exclude any contribution of endogenous MuSK, we performed the same experiments in MuSKloxP/− mice where endogenous MuSK was inactivated locally in the injected muscle fiber by transgenic Cre. The results demonstrate that ΔectoMuSK–GFP was expressed efficiently (Fig. 7 A) and induced the appearance of AChR clusters (Fig. 7 B). Since endogenous MuSK had been inactivated by Cre, this observation confirms that the ectodomain of MuSK is not an integral component of the mechanisms underlying MuSK-induced AChR expression. The overlay of green and red fluorescence images (Fig. 7 C) suggests that ΔectoMuSK–GFP is not colocalized with r-bgt–labeled AChR.


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

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

AChR clusters are induced by transgenic MuSK lacking the ectodomain. Single muscle fibers of MuSKloxP/− mice were injected with DNA of ΔectoMuSK–GFP and Cre. Expression of transgenes was analyzed 21 d after injection. The excised muscles were incubated with r-bgt to identify AChR clusters. Maximum projections of confocal image series are shown. (A) Expression of ΔectoMuSK–GFP (green fluorescence indicates successful expression of transgenes) and Cre. (B) AChR clusters are formed in absence of endogenous MuSK which has been inactivated by transgenic Cre (a MuSKloxP/− mouse was used for injection; a total of 22 fibers were injected; 11 fibers expressed ΔectoMuSK–GFP and 10 of these fibers had ectopic AChR clusters). (C) Overlay of green and red fluorescence images: ΔectoMuSK–GFP is not colocalized with AChR clusters.
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Related In: Results  -  Collection

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fig7: AChR clusters are induced by transgenic MuSK lacking the ectodomain. Single muscle fibers of MuSKloxP/− mice were injected with DNA of ΔectoMuSK–GFP and Cre. Expression of transgenes was analyzed 21 d after injection. The excised muscles were incubated with r-bgt to identify AChR clusters. Maximum projections of confocal image series are shown. (A) Expression of ΔectoMuSK–GFP (green fluorescence indicates successful expression of transgenes) and Cre. (B) AChR clusters are formed in absence of endogenous MuSK which has been inactivated by transgenic Cre (a MuSKloxP/− mouse was used for injection; a total of 22 fibers were injected; 11 fibers expressed ΔectoMuSK–GFP and 10 of these fibers had ectopic AChR clusters). (C) Overlay of green and red fluorescence images: ΔectoMuSK–GFP is not colocalized with AChR clusters.
Mentions: To exclude any contribution of endogenous MuSK, we performed the same experiments in MuSKloxP/− mice where endogenous MuSK was inactivated locally in the injected muscle fiber by transgenic Cre. The results demonstrate that ΔectoMuSK–GFP was expressed efficiently (Fig. 7 A) and induced the appearance of AChR clusters (Fig. 7 B). Since endogenous MuSK had been inactivated by Cre, this observation confirms that the ectodomain of MuSK is not an integral component of the mechanisms underlying MuSK-induced AChR expression. The overlay of green and red fluorescence images (Fig. 7 C) suggests that ΔectoMuSK–GFP is not colocalized with r-bgt–labeled 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