The dynamic regulation of actin polymerisation 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 AMPAR endocytosis in neurons. Here, we demonstrate that PICK1 binds F-actin and the actinnucleating Arp2/3 complex, and potently inhibits Arp2/3-mediated actin polymerisation. RNAi knockdown of PICK1 in neurons induces a reorganisation of the actin cytoskeleton resulting in aberrant cell morphology. Wild-type PICK1 rescues this phenotype, but a mutant PICK1 (W413A) that does not bind or inhibit Arp2/3 has no effect. Furthermore, this mutant also blocks NMDA-induced AMPAR internalisation. This study identifies PICK1 as a new negative regulator of Arp2/3-mediated actin polymerisation that is critical for a specific form of vesicle trafficking and also for the development of neuronal architecture.The dynamic actin cytoskeleton plays multiple roles in eukaryotic cells to regulate cell morphology, cell motility and vesicle trafficking by exerting mechanical forces that alter the shape of the plasma membrane. The Arp2/3 complex is the major catalyst for the formation of branched actin networks that mediate changes in membrane geometry. Proteins such as N-WASP, WAVE and cortactin bind and activate the Arp2/3 complex so that changes in cell morphology or vesicle trafficking occur at appropriate times and subcellular locations1-3.Regulation of the actin cytoskeleton is crucial for neuronal morphogenesis1,4,5. Recent reports have suggested a role for the Arp2/3 activator N-WASP in neurite elongation and branching in neuronal development6,7. However, negative regulators of the Arp2/3 complex in these processes have not yet been identified. A role for Arp2/3-mediated actin dynamics in the regulation of endocytosis in mammalian cell lines is becoming increasingly well established. Localised alterations in actin turnover are proposed to provide mechanical forces that contribute to plasma membrane curvature, vesicle scission, and propulsion of nascent vesicles away from the membrane2,8-10. The specific molecular mechanisms for regulating actin to control endocytosis in neurons are unknown. Dendritic spines, the sites of excitatory synapses in neurons, are particularly enriched in filamentous (F-) actin11-13, which is extremely dynamic, rapidly cycling between globular (G-) actin and F-actin14. Since designated endocytic sites are found in dendritic spines15,16, it seems likely that the densely packed, dynamic actin filaments influence endocytosis. PICK1 is a PDZ and BAR-* correspondence: e-mail: jon.hanley@bristol.ac.uk, tel: +44 (0)117 331 1944, fax: +44 (0)117 929 1687. AUTHOR CONTRIBUTIONS 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/cl...

Regulation of AMPA receptor (AMPAR) trafficking results in changes in receptor number at the postsynaptic membrane, and hence modifications in synaptic strength, which are proposed to underlie learning and memory. NMDA receptor-mediated postsynaptic Ca2+ influx enhances AMPAR internalisation, but the molecular mechanisms that trigger such trafficking are not well understood. We investigated whether AMPAR-associated protein–protein interactions known to regulate receptor surface expression may be directly regulated by Ca2+. PICK1 binds the AMPAR GluR2 subunit and is involved in AMPAR internalisation and LTD. We show that PICK1 is a Ca2+-binding protein, and that PICK1–GluR2 interactions are enhanced by the presence of 15 μM Ca2+. Deletion of an N-terminal acidic domain in PICK1 reduces its ability to bind Ca2+, and renders the GluR2–PICK1 interaction insensitive to Ca2+. Overexpression of this Ca2+-insensitive mutant occludes NMDA-induced AMPAR internalisation in hippocampal neurons. This work reveals a novel postsynaptic Ca2+-binding protein that provides a direct mechanistic link between NMDAR-mediated Ca2+ influx and AMPAR endocytosis

AMPA receptor (AMPAR) trafficking is crucial for synaptic plasticity that may be important for learning and memory. NSF and PICK1 bind the AMPAR GluR2 subunit and are involved in trafficking of AMPARs. Here, we show that GluR2, PICK1, NSF, and alpha-/beta-SNAPs form a complex in the presence of ATPgammaS. Similar to SNARE complex disassembly, NSF ATPase activity disrupts PICK1-GluR2 interactions in this complex. Alpha- and beta-SNAP have differential effects on this reaction. SNAP overexpression in hippocampal neurons leads to corresponding changes in AMPAR trafficking by acting on GluR2-PICK1 complexes. This demonstrates that the previously reported synaptic stabilization of AMPARs by NSF involves disruption of GluR2-PICK1 interactions. Furthermore, we are reporting a non-SNARE substrate for NSF disassembly activity.

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