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Inhibition of Arp2/3-mediated actin polymerization by PICK1 regulates neuronal morphology and AMPA receptor endocytosis

Abstract

The dynamic regulation of actin polymerization plays crucial roles in cell morphology and endocytosis. The mechanistic details of these processes and the proteins involved are not fully understood, especially in neurons. PICK1 is a PDZ–BAR-domain protein involved in regulated AMPA receptor (AMPAR) endocytosis in neurons. Here, we demonstrate that PICK1 binds filamentous (F)-actin and the actin-nucleating Arp2/3 complex, and potently inhibits Arp2/3-mediated actin polymerization. RNA interference (RNAi) knockdown of PICK1 in neurons induces a reorganization of the actin cytoskeleton resulting in aberrant cell morphology. Wild-type PICK1 rescues this phenotype, but a mutant PICK1, PICK1W413A, that does not bind or inhibit Arp2/3 has no effect. Furthermore, this mutant also blocks NMDA-induced AMPAR internalization. This study identifies PICK1 as a negative regulator of Arp2/3-mediated actin polymerization that is critical for a specific form of vesicle trafficking, and also for the development of neuronal architecture.

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Figure 1: PICK1 binds F-actin and Arp2/3 complex.
Figure 2: PICK1 inhibits VCA and Arp2/3-mediated actin polymerization.
Figure 3: Identification of functional Arp2/3 and actin binding sites on PICK1.
Figure 4: Inhibition of Arp2/3 activity by PICK1 is required for appropriate neuronal morphology in developing neurons.
Figure 5: The PICK1–Arp2/3 complex interaction is required for NMDA-induced AMPAR endocytosis in neurons.
Figure 6: Inhibition of Arp2/3-mediated actin polymerization by PICK1 is required for NMDA-induced AMPAR endocytosis in neurons.
Figure 7: PICK1-mediated inhibition of actin polymerization is enhanced by GluR2.
Figure 8: A schematic representation of a proposed model for the regulation of actin polymerization by PICK1.

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Acknowledgements

We thank G. Cory for invaluable discussions and advice on actin biology. We also thank A. Clarke and E. Compton-Daw for the use of and assistance with the fluorimeter, E. Ziff for the KK251,252EE construct, L. J. King, J. Mellor, J. Henley for critical reading of the manuscript. J.G.H is a fellow of the Wellcome Trust, D.R. is funded by an MRC studentship. This work was supported by ENI-NET.

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D.L.R. planned and performed the biochemistry and some imaging experiments. S.M. supervised generation of shRNA, planned and performed some imaging experiments. E.L.J. generated shRNA constructs. J.G.H. planned and performed imaging experiments, mutagenesis and cloning, supervised the project and wrote the paper.

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Correspondence to Jonathan G. Hanley.

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Supplementary figures S1, S2, S3, S4, S5, S6 and S7 (PDF 1677 kb)

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Rocca, D., Martin, S., Jenkins, E. et al. Inhibition of Arp2/3-mediated actin polymerization by PICK1 regulates neuronal morphology and AMPA receptor endocytosis. Nat Cell Biol 10, 259–271 (2008). https://doi.org/10.1038/ncb1688

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