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Regulation of acetylcholine receptor clustering by ADF/cofilin-directed vesicular trafficking

Abstract

Postsynaptic receptor localization is crucial for synapse development and function, but the underlying cytoskeletal mechanisms remain elusive. Using Xenopus neuromuscular junctions as a model, we found that actin depolymerizing factor (ADF)/cofilin regulated actin-dependent vesicular trafficking of acetylcholine receptors (AChRs) to the postsynaptic membrane. Active ADF/cofilin was concentrated in small puncta adjacent to AChR clusters and was spatiotemporally correlated with the formation and maintenance of surface AChR clusters. Notably, increased actin dynamics, vesicular markers and intracellular AChRs were all enriched at the sites of ADF/cofilin localization. Furthermore, a substantial amount of new AChRs was detected at these ADF/cofilin-enriched sites. Manipulation of either ADF/cofilin activity through its serine-3 phosphorylation or ADF/cofilin localization via 14-3-3 proteins markedly attenuated AChR insertion and clustering. These results suggest that spatiotemporally restricted ADF/cofilin-mediated actin dynamics regulate AChR trafficking during the development of neuromuscular synapses.

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Figure 1: Localization of ADF/cofilin in spontaneous and synaptic AChR clusters.
Figure 2: Dynamics of ADF/cofilin in spontaneous and agrin-induced redistribution of AChRs.
Figure 3: Regulation of actin dynamics by ADF/cofilin in spontaneous and synaptic AChR clusters.
Figure 4: Local enrichment of vesicular trafficking machinery and intracellular AChRs in spontaneous and agrin-induced AChR clusters.
Figure 5: Time-dependent incorporation of newly inserted AChRs into the existing surface AChR clusters.
Figure 6: Regulation of agrin- and nerve-induced AChR clustering by ADF/cofilin activity.
Figure 7: Involvement of 14-3-3ζ in AChR clustering and ADF/cofilin localization.
Figure 8: Regulation of surface targeting of new AChRs by ADF/cofilin activity and 14-3-3ζ.

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Acknowledgements

We would like to thank B. Lu (National Institute of Child Health and Human Development) for his help in our electrophysiological experiments. This work is supported by grants from National Institutes of Health to J.Q.Z. (GM083889 and AG029596) and J.R.B. (NS40371). C.W.L. was supported by a postdoctoral fellowship from The Croucher Foundation.

Author information

Authors and Affiliations

Authors

Contributions

C.W.L. designed and performed most of the experiments, data analyses and manuscript writing. J.H. did the electrophysiological recording. J.R.B. provided insightful advice to the experiments and critical input to the manuscript and contributed the reagents for ADF/cofilin and 14-3-3ζ. L.H. and R.L. performed the molecular subcloning of some of the DNA constructs that were used. J.Q.Z. formulated and oversaw the research project and directed the experiments, analyses and writing.

Corresponding author

Correspondence to James Q Zheng.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 (PDF 1074 kb)

Supplementary Video 1

Time-lapse movie of paGFP-actin in the spontaneous AChR clusters. A time-lapse series of fluorescence images was taken for 18 min at 1-min intervals using the same imaging settings. The movie was made at a playback rate of 2 frames per s (120×). (AVI 574 kb)

Supplementary Video 2

Time-lapse movie of paGFP-actin in the agrin bead–induced AChR clusters. A time-lapse series of fluorescence images was taken for 18 min at 1-min intervals using the same imaging settings. The movie was made at a playback rate of 2 frames per s (120×). (AVI 487 kb)

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Lee, C., Han, J., Bamburg, J. et al. Regulation of acetylcholine receptor clustering by ADF/cofilin-directed vesicular trafficking. Nat Neurosci 12, 848–856 (2009). https://doi.org/10.1038/nn.2322

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