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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/AChR and MuSK–GFP/AChR distribution at ectopic MuSK-induced AChR clusters. Rat muscle fibers were injected with plasmid DNA. After 21 d, the muscles were excised. AChR were stained with r-bgt. Confocal image series were recorded to analyze at high resolution whether rapsyn and MuSK are colocalized with AChR clusters. (A) A single section of a confocal image series shows expression of MuSK and rapsyn–GFP (green), as indicated. (B) MuSK-induced AChR clusters (red). (C) AChR clusters and rapsyn–GFP are colocalized as shown by overlay of green and red fluorescence images. (E) A single section of a confocal image series shows expression of MuSK–GFP (green) and rapsyn, as indicated. (F) MuSK-induced AChR clusters (red). (G) AChR clusters and MuSK–GFP are not colocalized as shown by overlay of green and red fluorescence images. (D) Statistical colocalization analysis of all sections of a confocal image series of a transfected fiber was performed (see Materials and methods). The relative fluorescence intensities of rapsyn–GFP (x-axis) are plotted against the relative fluorescence intensities of r-bgt–labeled AChR clusters (y-axis). Data points are located near the diagonal, showing that rapsyn–GFP is colocalized with AChR. (H) Statistical colocalization analysis, as in D, for MuSK–GFP. Data points are scattered, showing that MuSK–GFP is not colocalized with AChR.
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fig8: Rapsyn–GFP/AChR and MuSK–GFP/AChR distribution at ectopic MuSK-induced AChR clusters. Rat muscle fibers were injected with plasmid DNA. After 21 d, the muscles were excised. AChR were stained with r-bgt. Confocal image series were recorded to analyze at high resolution whether rapsyn and MuSK are colocalized with AChR clusters. (A) A single section of a confocal image series shows expression of MuSK and rapsyn–GFP (green), as indicated. (B) MuSK-induced AChR clusters (red). (C) AChR clusters and rapsyn–GFP are colocalized as shown by overlay of green and red fluorescence images. (E) A single section of a confocal image series shows expression of MuSK–GFP (green) and rapsyn, as indicated. (F) MuSK-induced AChR clusters (red). (G) AChR clusters and MuSK–GFP are not colocalized as shown by overlay of green and red fluorescence images. (D) Statistical colocalization analysis of all sections of a confocal image series of a transfected fiber was performed (see Materials and methods). The relative fluorescence intensities of rapsyn–GFP (x-axis) are plotted against the relative fluorescence intensities of r-bgt–labeled AChR clusters (y-axis). Data points are located near the diagonal, showing that rapsyn–GFP is colocalized with AChR. (H) Statistical colocalization analysis, as in D, for MuSK–GFP. Data points are scattered, showing that MuSK–GFP is not colocalized with AChR.

Mentions: When comparing the distribution of r-bgt–labeled AChR clusters and MuSK–GFP (Fig. 3 C) or ΔectoMuSK–GFP (Fig. 7 C), it appeared that the two proteins were not strictly colocalized on the surface of the muscle fibers and MuSK–GFP fusion proteins were observed outside of AChR clusters and vice versa. This was surprising in view of the current model, in which the ectodomain of MuSK has to be structurally linked to AChR. Therefore, one would expect that a significant portion of the AChR clusters overlaps with the GFP-labeled MuSK molecules. Rapsyn is thought to interconnect AChR and MuSK via the hypothetical linker RATL (Apel et al., 1997). Therefore, we analyzed at higher resolution whether rapsyn was distributed like MuSK or was associated more strictly with AChR (as indicated already in Fig. 5). MuSK and rapsyn–GFP were injected ectopically to induce AChR clusters and the distribution of rapsyn–GFP relative to AChR clusters was analyzed by confocal microscopy. Single sections revealed that rapsyn–GFP was strictly colocalized with AChR stained by r-bgt (Fig. 8, A–C). In contrast, when MuSK–GFP and rapsyn were injected, MuSK–GFP displayed a different distribution and was spatially distinct from AChR clusters (Fig. 8, E–G).


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

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

Rapsyn–GFP/AChR and MuSK–GFP/AChR distribution at ectopic MuSK-induced AChR clusters. Rat muscle fibers were injected with plasmid DNA. After 21 d, the muscles were excised. AChR were stained with r-bgt. Confocal image series were recorded to analyze at high resolution whether rapsyn and MuSK are colocalized with AChR clusters. (A) A single section of a confocal image series shows expression of MuSK and rapsyn–GFP (green), as indicated. (B) MuSK-induced AChR clusters (red). (C) AChR clusters and rapsyn–GFP are colocalized as shown by overlay of green and red fluorescence images. (E) A single section of a confocal image series shows expression of MuSK–GFP (green) and rapsyn, as indicated. (F) MuSK-induced AChR clusters (red). (G) AChR clusters and MuSK–GFP are not colocalized as shown by overlay of green and red fluorescence images. (D) Statistical colocalization analysis of all sections of a confocal image series of a transfected fiber was performed (see Materials and methods). The relative fluorescence intensities of rapsyn–GFP (x-axis) are plotted against the relative fluorescence intensities of r-bgt–labeled AChR clusters (y-axis). Data points are located near the diagonal, showing that rapsyn–GFP is colocalized with AChR. (H) Statistical colocalization analysis, as in D, for MuSK–GFP. Data points are scattered, showing that MuSK–GFP is not colocalized with AChR.
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fig8: Rapsyn–GFP/AChR and MuSK–GFP/AChR distribution at ectopic MuSK-induced AChR clusters. Rat muscle fibers were injected with plasmid DNA. After 21 d, the muscles were excised. AChR were stained with r-bgt. Confocal image series were recorded to analyze at high resolution whether rapsyn and MuSK are colocalized with AChR clusters. (A) A single section of a confocal image series shows expression of MuSK and rapsyn–GFP (green), as indicated. (B) MuSK-induced AChR clusters (red). (C) AChR clusters and rapsyn–GFP are colocalized as shown by overlay of green and red fluorescence images. (E) A single section of a confocal image series shows expression of MuSK–GFP (green) and rapsyn, as indicated. (F) MuSK-induced AChR clusters (red). (G) AChR clusters and MuSK–GFP are not colocalized as shown by overlay of green and red fluorescence images. (D) Statistical colocalization analysis of all sections of a confocal image series of a transfected fiber was performed (see Materials and methods). The relative fluorescence intensities of rapsyn–GFP (x-axis) are plotted against the relative fluorescence intensities of r-bgt–labeled AChR clusters (y-axis). Data points are located near the diagonal, showing that rapsyn–GFP is colocalized with AChR. (H) Statistical colocalization analysis, as in D, for MuSK–GFP. Data points are scattered, showing that MuSK–GFP is not colocalized with AChR.
Mentions: When comparing the distribution of r-bgt–labeled AChR clusters and MuSK–GFP (Fig. 3 C) or ΔectoMuSK–GFP (Fig. 7 C), it appeared that the two proteins were not strictly colocalized on the surface of the muscle fibers and MuSK–GFP fusion proteins were observed outside of AChR clusters and vice versa. This was surprising in view of the current model, in which the ectodomain of MuSK has to be structurally linked to AChR. Therefore, one would expect that a significant portion of the AChR clusters overlaps with the GFP-labeled MuSK molecules. Rapsyn is thought to interconnect AChR and MuSK via the hypothetical linker RATL (Apel et al., 1997). Therefore, we analyzed at higher resolution whether rapsyn was distributed like MuSK or was associated more strictly with AChR (as indicated already in Fig. 5). MuSK and rapsyn–GFP were injected ectopically to induce AChR clusters and the distribution of rapsyn–GFP relative to AChR clusters was analyzed by confocal microscopy. Single sections revealed that rapsyn–GFP was strictly colocalized with AChR stained by r-bgt (Fig. 8, A–C). In contrast, when MuSK–GFP and rapsyn were injected, MuSK–GFP displayed a different distribution and was spatially distinct from AChR clusters (Fig. 8, E–G).

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