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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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Rapsyn–GFP identifies ectopically induced AChR clusters. Single muscle fibers of MuSKloxP/− mice were injected with DNA, as indicated by asterisks, and expression of transgenic products was analyzed 21 d after injection. The excised muscles were incubated with r-bgt to identify AChR clusters. Overlays of green and red fluorescence images of a confocal image series are shown as maximum projections. (A) Expression of agrin and rapsyn–GFP as indicated. Agrin induces ectopic AChR clusters. AChR in the injected muscle fiber form complexes with rapsyn–GFP and AChR clusters appear yellow in the overlay. The r-bgt labeled AChR clusters on noninjected fibers are red. (2 MuSKloxP/− mice were used for injection; a total of 43 fibers were injected and 26 fibers expressed agrin and rapsyn–GFP. In all injected transgene-expressing fibers AChRs were colocalized with rapsyn–GFP and appeared yellow). (B) Expression of agrin, Cre, and rapsyn–GFP as indicated. Transgenic Cre inactivates endogenous MuSK. The absence of AChR/rapsyn–GFP clusters demonstrates that the formation of AChR clusters was prevented in injected muscle fibers. Agrin-induced AChR clusters are found only on neighboring noninjected fibers. 2 MuSKloxP/− mice were used for DNA injection; a total of 40 fibers were injected; 28 fibers expressed agrin and rapsyn–GFP; no AChR clusters were detected on the injected fibers. (C) Expression of agrin, Cre, rapsyn–GFP, and MuSK as indicated. AChR induced in the injected fiber form complexes with rapsyn–GFP and AChR clusters appear yellow in the overlay. The Cre-mediated inactivation of the MuSK gene was rescued by expression of transgenic MuSK (3 MuSKloxP/− mice were used for DNA injection; a total of 62 fibers were injected and 33 fibers expressed agrin and rapsyn–GFP. Endogenous MuSK inactivation was rescued in 22 fibers, which contained AChR/rapsyn–GFP complexes.) (D) Graphic summary of experiments described in A–C shows that Cre-mediated inactivation of endogenous MuSK prevents the formation of AChR clusters. The conditional MuSK gene knock out is rescued with high efficiency by transgenic MuSK: AChR clusters are found in 73% of transgene-expressing fibers.
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fig5: Rapsyn–GFP identifies ectopically induced AChR clusters. Single muscle fibers of MuSKloxP/− mice were injected with DNA, as indicated by asterisks, and expression of transgenic products was analyzed 21 d after injection. The excised muscles were incubated with r-bgt to identify AChR clusters. Overlays of green and red fluorescence images of a confocal image series are shown as maximum projections. (A) Expression of agrin and rapsyn–GFP as indicated. Agrin induces ectopic AChR clusters. AChR in the injected muscle fiber form complexes with rapsyn–GFP and AChR clusters appear yellow in the overlay. The r-bgt labeled AChR clusters on noninjected fibers are red. (2 MuSKloxP/− mice were used for injection; a total of 43 fibers were injected and 26 fibers expressed agrin and rapsyn–GFP. In all injected transgene-expressing fibers AChRs were colocalized with rapsyn–GFP and appeared yellow). (B) Expression of agrin, Cre, and rapsyn–GFP as indicated. Transgenic Cre inactivates endogenous MuSK. The absence of AChR/rapsyn–GFP clusters demonstrates that the formation of AChR clusters was prevented in injected muscle fibers. Agrin-induced AChR clusters are found only on neighboring noninjected fibers. 2 MuSKloxP/− mice were used for DNA injection; a total of 40 fibers were injected; 28 fibers expressed agrin and rapsyn–GFP; no AChR clusters were detected on the injected fibers. (C) Expression of agrin, Cre, rapsyn–GFP, and MuSK as indicated. AChR induced in the injected fiber form complexes with rapsyn–GFP and AChR clusters appear yellow in the overlay. The Cre-mediated inactivation of the MuSK gene was rescued by expression of transgenic MuSK (3 MuSKloxP/− mice were used for DNA injection; a total of 62 fibers were injected and 33 fibers expressed agrin and rapsyn–GFP. Endogenous MuSK inactivation was rescued in 22 fibers, which contained AChR/rapsyn–GFP complexes.) (D) Graphic summary of experiments described in A–C shows that Cre-mediated inactivation of endogenous MuSK prevents the formation of AChR clusters. The conditional MuSK gene knock out is rescued with high efficiency by transgenic MuSK: AChR clusters are found in 73% of transgene-expressing fibers.

Mentions: To strengthen the observation that AChR cluster formation was completely prevented in muscle fibers expressing Cre, we developed an additional bioassay using rapsyn–GFP. In cell culture experiments it has been shown previously that rapsyn–GFP forms fluorescently labeled complexes with AChR (Ramarao and Cohen, 1998). If such complexes were also formed in rapsyn–GFP-injected muscle fibers, one could directly and reliably monitor the appearance of AChR clusters. In fact, coinjecting rapsyn–GFP with agrin DNA resulted in the appearance of AChR clusters that were exactly colocalized with rapsyn–GFP (Fig. 5 A) and demonstrated that induction and aggregation of AChR can be readily detected and assigned to the injected muscle fiber. Transgenically expressed rapsyn–GFP is therefore a specific and sensitive marker to monitor the appearance of newly formed AChR clusters in vivo. We never observed rapsyn–GFP–AChR clusters in Cre-expressing fibers (Fig. 5 B), demonstrating that local, Cre-mediated DNA recombination in single muscle fibers efficiently inactivated endogenous MuSK and thus prevented agrin-induced activation of AChR expression.


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

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

Rapsyn–GFP identifies ectopically induced AChR clusters. Single muscle fibers of MuSKloxP/− mice were injected with DNA, as indicated by asterisks, and expression of transgenic products was analyzed 21 d after injection. The excised muscles were incubated with r-bgt to identify AChR clusters. Overlays of green and red fluorescence images of a confocal image series are shown as maximum projections. (A) Expression of agrin and rapsyn–GFP as indicated. Agrin induces ectopic AChR clusters. AChR in the injected muscle fiber form complexes with rapsyn–GFP and AChR clusters appear yellow in the overlay. The r-bgt labeled AChR clusters on noninjected fibers are red. (2 MuSKloxP/− mice were used for injection; a total of 43 fibers were injected and 26 fibers expressed agrin and rapsyn–GFP. In all injected transgene-expressing fibers AChRs were colocalized with rapsyn–GFP and appeared yellow). (B) Expression of agrin, Cre, and rapsyn–GFP as indicated. Transgenic Cre inactivates endogenous MuSK. The absence of AChR/rapsyn–GFP clusters demonstrates that the formation of AChR clusters was prevented in injected muscle fibers. Agrin-induced AChR clusters are found only on neighboring noninjected fibers. 2 MuSKloxP/− mice were used for DNA injection; a total of 40 fibers were injected; 28 fibers expressed agrin and rapsyn–GFP; no AChR clusters were detected on the injected fibers. (C) Expression of agrin, Cre, rapsyn–GFP, and MuSK as indicated. AChR induced in the injected fiber form complexes with rapsyn–GFP and AChR clusters appear yellow in the overlay. The Cre-mediated inactivation of the MuSK gene was rescued by expression of transgenic MuSK (3 MuSKloxP/− mice were used for DNA injection; a total of 62 fibers were injected and 33 fibers expressed agrin and rapsyn–GFP. Endogenous MuSK inactivation was rescued in 22 fibers, which contained AChR/rapsyn–GFP complexes.) (D) Graphic summary of experiments described in A–C shows that Cre-mediated inactivation of endogenous MuSK prevents the formation of AChR clusters. The conditional MuSK gene knock out is rescued with high efficiency by transgenic MuSK: AChR clusters are found in 73% of transgene-expressing fibers.
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Related In: Results  -  Collection

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fig5: Rapsyn–GFP identifies ectopically induced AChR clusters. Single muscle fibers of MuSKloxP/− mice were injected with DNA, as indicated by asterisks, and expression of transgenic products was analyzed 21 d after injection. The excised muscles were incubated with r-bgt to identify AChR clusters. Overlays of green and red fluorescence images of a confocal image series are shown as maximum projections. (A) Expression of agrin and rapsyn–GFP as indicated. Agrin induces ectopic AChR clusters. AChR in the injected muscle fiber form complexes with rapsyn–GFP and AChR clusters appear yellow in the overlay. The r-bgt labeled AChR clusters on noninjected fibers are red. (2 MuSKloxP/− mice were used for injection; a total of 43 fibers were injected and 26 fibers expressed agrin and rapsyn–GFP. In all injected transgene-expressing fibers AChRs were colocalized with rapsyn–GFP and appeared yellow). (B) Expression of agrin, Cre, and rapsyn–GFP as indicated. Transgenic Cre inactivates endogenous MuSK. The absence of AChR/rapsyn–GFP clusters demonstrates that the formation of AChR clusters was prevented in injected muscle fibers. Agrin-induced AChR clusters are found only on neighboring noninjected fibers. 2 MuSKloxP/− mice were used for DNA injection; a total of 40 fibers were injected; 28 fibers expressed agrin and rapsyn–GFP; no AChR clusters were detected on the injected fibers. (C) Expression of agrin, Cre, rapsyn–GFP, and MuSK as indicated. AChR induced in the injected fiber form complexes with rapsyn–GFP and AChR clusters appear yellow in the overlay. The Cre-mediated inactivation of the MuSK gene was rescued by expression of transgenic MuSK (3 MuSKloxP/− mice were used for DNA injection; a total of 62 fibers were injected and 33 fibers expressed agrin and rapsyn–GFP. Endogenous MuSK inactivation was rescued in 22 fibers, which contained AChR/rapsyn–GFP complexes.) (D) Graphic summary of experiments described in A–C shows that Cre-mediated inactivation of endogenous MuSK prevents the formation of AChR clusters. The conditional MuSK gene knock out is rescued with high efficiency by transgenic MuSK: AChR clusters are found in 73% of transgene-expressing fibers.
Mentions: To strengthen the observation that AChR cluster formation was completely prevented in muscle fibers expressing Cre, we developed an additional bioassay using rapsyn–GFP. In cell culture experiments it has been shown previously that rapsyn–GFP forms fluorescently labeled complexes with AChR (Ramarao and Cohen, 1998). If such complexes were also formed in rapsyn–GFP-injected muscle fibers, one could directly and reliably monitor the appearance of AChR clusters. In fact, coinjecting rapsyn–GFP with agrin DNA resulted in the appearance of AChR clusters that were exactly colocalized with rapsyn–GFP (Fig. 5 A) and demonstrated that induction and aggregation of AChR can be readily detected and assigned to the injected muscle fiber. Transgenically expressed rapsyn–GFP is therefore a specific and sensitive marker to monitor the appearance of newly formed AChR clusters in vivo. We never observed rapsyn–GFP–AChR clusters in Cre-expressing fibers (Fig. 5 B), demonstrating that local, Cre-mediated DNA recombination in single muscle fibers efficiently inactivated endogenous MuSK and thus prevented agrin-induced activation of AChR expression.

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