The Dynamics of the Rapsyn Scaffolding Protein at Individual Acetylcholine Receptor Clusters

Emile Bruneau,Mohammed Akaaboune

doi:10.1074/jbc.m608714200

Emile Bruneau, Mohammed Akaaboune

Open Access

https://doi.org/10.1074/jbc.m608714200

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Abstract

Rapsyn, a cytoplasmic receptor-associated protein, is required for the clustering of acetylcholine receptors (AChRs). Although AChR dynamics have been extensively studied, little is known about the dynamics of rapsyn. Here, we used a rapsyn-green fluorescent protein (GFP) fusion protein and quantitative fluorescent imaging to study the dynamics of rapsyn in transfected C2C12 myotubes. First, we found that rapsyn-GFP expression at clusters did not alter AChR aggregation, function, or turnover. Quantification of rapsyn immunofluorescence indicated that the expression of rapsyn-GFP proteins at clusters does not increase the overall rapsyn density compared with untransfected myotube clusters. Using time lapse imaging and fluorescence recovery after photobleaching, we demonstrated that the recovery of rapsyn-GFP fluorescence at clusters was very fast, with a halftime of about approximately 1.5 h (approximately 3 times faster than AChRs). Inhibition of protein kinase C significantly altered receptor insertion, but it had no effect on rapsyn insertion. When cells were treated with the broad spectrum kinase inhibitor staurosporine, receptor insertion was decreased even further. However, inhibition of protein kinase A had no effect on insertion of either rapsyn or receptors. Finally, when cells were treated with neural agrin, rapsyn and AChRs were both directed away from preexisting clusters and accumulated together in new small clusters. These results demonstrate the remarkable dynamism of rapsyn, which may underlie the stability and maintenance of the postsynaptic scaffold and suggest that the insertion of different postsynaptic proteins may be operating independently.

Highlights

The formation of the postsynaptic receptor density is best studied at the neuromuscular junction
By monitoring the fluorescence of rapsyn-GFP fusion proteins clustered on the surface of transfected C2C12 myotubes, we were able to determine that rapsyn is markedly more dynamic than acetylcholine receptors (AChRs) and is not affected by pharmacological manipulation that alters AChR dynamics, both rapsyn and AChRs are modulated by the nerve clustering factor, agrin
Rapsyn-GFP Does Not Affect Acetylcholine Receptor Clustering, Function, or Removal Rate—First, in order to verify that rapsyn-GFP was able to localize to laminin-associated AChR clusters on our cultured myotubes, we transiently transfected C2C12 myoblasts with a rapsyn-GFP fusion protein [27]

Summary

EXPERIMENTAL PROCEDURES

GFP Fusion Construct—The rapsyn-GFP fusion construct was kindly provided by Dr Jonathan Cohen (Harvard Medical School). Agrin Treatment—To determine the effect of agrin on rapsyn-GFP expression at clusters, myotubes grown on laminin-coated dishes transfected with rapsyn-GFP were incubated with BTX-594 to label all AChRs. After initial images were taken, 100 ng/ml C-terminal agrin (R & D Systems, Minneapolis, MN) was added to the culture dish. At 4 and 8 h, images were taken to determine the location of rapsyn-GFP and AChRs. Kinase Inhibition—To determine the effect of various kinase inhibitors on rapsyn-GFP insertion over time, the GFP signal from clusters on myotubes transfected with rapsyn-GFP was carefully removed with an argon laser, and one of the following drugs was added to the medium: 100 nM H-89 (to block PKA), 20 ␮M H-89 (to inhibit both PKA and PKC), 10 nM calphostin C (to inhibit PKC), or 20 nM staurosporine (to block a broad spectrum of kinases, including PKA and PKC) (drug specificity obtained from the manufacturer, Sigma). The same clusters were reimaged 4 h later, and their fluorescence intensity was measured

RESULTS

DISCUSSION

Methods