Nanoscale Spatial Organization of ARC High‐ and Low‐Order Assemblies at Excitatory Synapses

ABSTRACT The Activity‐Regulated Cytoskeleton‐Associated protein (ARC) plays a pivotal role in mediating synaptic plasticity in neuronal cells. In vitro studies suggest that ARC can form both high‐ and low‐order oligomers. Despite their potentially important functions, direct nanoscale observation of ARC assemblies in cells has been lacking due to its tightly regulated spatiotemporal expression, the small size of the structures, the absence of suitable labelling strategies, and background signals from freely diffusing cytosolic proteins. Here, we combine super‐resolution microscopy and time‐resolved fluorescence anisotropy measurements with precisely designed ARC‐tagging strategies to reveal the nanoscale spatial organization of ARC in neuronal environments, with a particular focus on excitatory synapses. We identify low‐order assemblies of ARC at synapses, that colocalize with surface AMPA receptors (AMPARs), semi‐circular organizations of ARC at the endocytic zone, consistent with a role in mediating AMPAR endocytosis, and particle‐like assemblies sized 60–80 nm predominantly localized in dendritic spines. Finally, using a combination of experiments and simulations, we show that ARC can directly induce membrane bending and lipid bilayer tubulation in the absence of other protein partners.