Limits...
Laminin-induced acetylcholine receptor clustering: an alternative pathway.

Sugiyama JE, Glass DJ, Yancopoulos GD, Hall ZW - J. Cell Biol. (1997)

Bottom Line: Most importantly, laminin- 1-induced clustering does not require MuSK, a receptor tyrosine kinase that is part of the receptor complex for agrin.Laminin-1 does not cause tyrosine phosphorylation of MuSK in C2 myotubes and induces AChR clustering in myotubes from MuSK-/- mice that do not respond to agrin.In contrast to agrin, laminin-1 also does not induce tyrosine phosphorylation of the AChR, demonstrating that AChR tyrosine phosphorylation is not required for clustering in myotubes.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
The induction of acetylcholine receptor (AChR) clustering by neurally released agrin is a critical, early step in the formation of the neuromuscular junction. Laminin, a component of the muscle fiber basal lamina, also induces AChR clustering. We find that induction of AChR clustering in C2 myotubes is specific for laminin-1; neither laminin-2 (merosin) nor laminin-11 (a synapse-specific isoform) are active. Moreover, laminin-1 induces AChR clustering by a pathway that is independent of that used by neural agrin. The effects of laminin-1 and agrin are strictly additive and occur with different time courses. Most importantly, laminin- 1-induced clustering does not require MuSK, a receptor tyrosine kinase that is part of the receptor complex for agrin. Laminin-1 does not cause tyrosine phosphorylation of MuSK in C2 myotubes and induces AChR clustering in myotubes from MuSK-/- mice that do not respond to agrin. In contrast to agrin, laminin-1 also does not induce tyrosine phosphorylation of the AChR, demonstrating that AChR tyrosine phosphorylation is not required for clustering in myotubes. Laminin-1 thus acts by a mechanism that is independent of that used by agrin and may provide a supplemental pathway for AChR clustering during synaptogenesis.

Show MeSH
Laminin-1 induces AChR clustering on MuSK−/− myotubes. MuSK−/− myotubes were untreated (A) or treated with  150 pM neural agrin (B) or 120 nM laminin-1 (C). Cultures were  stained with rhodamine-conjugated α-bungarotoxin and visualized under 400×. Only laminin-1 treatment induces AChR aggregation on MuSK−/− myotubes. Bar, 25 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139811&req=5

Figure 6: Laminin-1 induces AChR clustering on MuSK−/− myotubes. MuSK−/− myotubes were untreated (A) or treated with 150 pM neural agrin (B) or 120 nM laminin-1 (C). Cultures were stained with rhodamine-conjugated α-bungarotoxin and visualized under 400×. Only laminin-1 treatment induces AChR aggregation on MuSK−/− myotubes. Bar, 25 μm.

Mentions: Neural agrin requires MuSK, a receptor tyrosine kinase, to induce the formation of AChR clusters. Mice with a targeted disruption of the gene encoding MuSK do not form neuromuscular synapses (DeChiara et al., 1996), and myotubes from such mice fail to cluster AChRs in response to neural agrin (Glass et al., 1996). To determine whether MuSK is also required for laminin-1 clustering activity, we treated immortalized myotube cultures made from MuSK−/− mouse muscle with either laminin-1 or neural agrin and then stained them with rhodamine–α-bungarotoxin. When treated with laminin-1, the MuSK−/− myotubes produced AChR clusters of varying sizes, ranging from short, dense clusters on the myotube surface to long clusters along the edge of the myotube, similar to those seen on C2 myotubes (Fig. 6). Quantitation of these results by counting clusters in random fields showed that only myotubes treated with laminin-1 were able to produce AChR clusters (Fig. 7). The number of AChR clusters induced by laminin-1 on the MuSK−/− myotubes, however, was not as great as those observed on C2 myotubes (∼20-fold fewer clusters compared to C2 myotubes), even when concentrations as high as 120 nM of laminin-1 were used. In general, the MuSK−/− myotubes differed in appearance and were thinner than the C2 myotubes. Thus, the reduced number of AChR aggregates on the mutant myotubes could be the result of inherent differences between C2 myotubes and the immortalized MuSK−/− myotubes, or it could be a specific consequence of the absence of MuSK. As expected, we did not observe AChR clustering on MuSK−/− myotubes treated with neural agrin, even at concentrations up to 15 nM. In the absence of laminin-1, occasional bright spots and faint edge staining were observed on the MuSK−/− myotubes but did not resemble AChR clusters. This nonspecific staining accounted for the nominal clustering values recorded for untreated and agrin-treated MuSK−/− myotubes (Fig. 7).


Laminin-induced acetylcholine receptor clustering: an alternative pathway.

Sugiyama JE, Glass DJ, Yancopoulos GD, Hall ZW - J. Cell Biol. (1997)

Laminin-1 induces AChR clustering on MuSK−/− myotubes. MuSK−/− myotubes were untreated (A) or treated with  150 pM neural agrin (B) or 120 nM laminin-1 (C). Cultures were  stained with rhodamine-conjugated α-bungarotoxin and visualized under 400×. Only laminin-1 treatment induces AChR aggregation on MuSK−/− myotubes. Bar, 25 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139811&req=5

Figure 6: Laminin-1 induces AChR clustering on MuSK−/− myotubes. MuSK−/− myotubes were untreated (A) or treated with 150 pM neural agrin (B) or 120 nM laminin-1 (C). Cultures were stained with rhodamine-conjugated α-bungarotoxin and visualized under 400×. Only laminin-1 treatment induces AChR aggregation on MuSK−/− myotubes. Bar, 25 μm.
Mentions: Neural agrin requires MuSK, a receptor tyrosine kinase, to induce the formation of AChR clusters. Mice with a targeted disruption of the gene encoding MuSK do not form neuromuscular synapses (DeChiara et al., 1996), and myotubes from such mice fail to cluster AChRs in response to neural agrin (Glass et al., 1996). To determine whether MuSK is also required for laminin-1 clustering activity, we treated immortalized myotube cultures made from MuSK−/− mouse muscle with either laminin-1 or neural agrin and then stained them with rhodamine–α-bungarotoxin. When treated with laminin-1, the MuSK−/− myotubes produced AChR clusters of varying sizes, ranging from short, dense clusters on the myotube surface to long clusters along the edge of the myotube, similar to those seen on C2 myotubes (Fig. 6). Quantitation of these results by counting clusters in random fields showed that only myotubes treated with laminin-1 were able to produce AChR clusters (Fig. 7). The number of AChR clusters induced by laminin-1 on the MuSK−/− myotubes, however, was not as great as those observed on C2 myotubes (∼20-fold fewer clusters compared to C2 myotubes), even when concentrations as high as 120 nM of laminin-1 were used. In general, the MuSK−/− myotubes differed in appearance and were thinner than the C2 myotubes. Thus, the reduced number of AChR aggregates on the mutant myotubes could be the result of inherent differences between C2 myotubes and the immortalized MuSK−/− myotubes, or it could be a specific consequence of the absence of MuSK. As expected, we did not observe AChR clustering on MuSK−/− myotubes treated with neural agrin, even at concentrations up to 15 nM. In the absence of laminin-1, occasional bright spots and faint edge staining were observed on the MuSK−/− myotubes but did not resemble AChR clusters. This nonspecific staining accounted for the nominal clustering values recorded for untreated and agrin-treated MuSK−/− myotubes (Fig. 7).

Bottom Line: Most importantly, laminin- 1-induced clustering does not require MuSK, a receptor tyrosine kinase that is part of the receptor complex for agrin.Laminin-1 does not cause tyrosine phosphorylation of MuSK in C2 myotubes and induces AChR clustering in myotubes from MuSK-/- mice that do not respond to agrin.In contrast to agrin, laminin-1 also does not induce tyrosine phosphorylation of the AChR, demonstrating that AChR tyrosine phosphorylation is not required for clustering in myotubes.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
The induction of acetylcholine receptor (AChR) clustering by neurally released agrin is a critical, early step in the formation of the neuromuscular junction. Laminin, a component of the muscle fiber basal lamina, also induces AChR clustering. We find that induction of AChR clustering in C2 myotubes is specific for laminin-1; neither laminin-2 (merosin) nor laminin-11 (a synapse-specific isoform) are active. Moreover, laminin-1 induces AChR clustering by a pathway that is independent of that used by neural agrin. The effects of laminin-1 and agrin are strictly additive and occur with different time courses. Most importantly, laminin- 1-induced clustering does not require MuSK, a receptor tyrosine kinase that is part of the receptor complex for agrin. Laminin-1 does not cause tyrosine phosphorylation of MuSK in C2 myotubes and induces AChR clustering in myotubes from MuSK-/- mice that do not respond to agrin. In contrast to agrin, laminin-1 also does not induce tyrosine phosphorylation of the AChR, demonstrating that AChR tyrosine phosphorylation is not required for clustering in myotubes. Laminin-1 thus acts by a mechanism that is independent of that used by agrin and may provide a supplemental pathway for AChR clustering during synaptogenesis.

Show MeSH