The architecture of the actin cytoskeleton that concentrates at presynapses remains poorly known, hindering our understanding of its roles in synaptic physiology. In this work, we measure and visualize presynaptic actin by diffraction-limited and super-resolution microscopy, thanks to a validated model of bead-induced presynapses in cultured neurons. We identify a major population of actin-enriched presynapses that concentrates more presynaptic components and shows higher synaptic vesicle cycling than their non-enriched counterparts. Pharmacological perturbations point to an optimal actin amount and the presence of distinct actin structures within presynapses. We directly visualize these nanostructures using Single Molecule Localization Microscopy (SMLM), defining three distinct types: an actin mesh at the active zone, actin rails between the active zone and deeper reserve pools, and actin corrals around the whole presynaptic compartment. Finally, CRISPR-tagging of endogenous actin allows us to validate our results in natural synapses between cultured neurons, confirming the role of actin enrichment and the presence of three types of presynaptic actin nanostructures.

Synapses are the nexus of signal transmission and processing in the nervous system. Despite decades of work, the architecture of the actin cytoskeleton that concentrates at presynapses remain poorly known, hindering our comprehensive understanding of its roles in presynaptic physiology. In this work, we take advantage of a validated model of bead-induced presynapses to measure and visualize isolated presynaptic actin by diffraction-limited and super-resolution microscopy. We first identify a major population of actin-enriched presynapses that concentrates more presynaptic components, and shows higher synaptic vesicle cycling than their non-enriched counterparts. Using pharmacological perturbations, we determine that an optimal amount of actin is necessary for this effect of actin enrichment. Contrasted modulation of this effect by actin nucleation inhibitors indicates its dependance on distinct presynaptic actin assemblies. Using Single Molecule Localization Microscopy (SMLM), we directly visualize these nano-structures in isolated presynapses, defining an actin mesh at the active zone, actin rails between the active zone and deeper reserve pools, and actin corrals around the whole presynaptic compartment. We finally show that these three types of presynaptic actin nano-structures are differentially affected by actin nucleation inhibitors, consistent with their effect on presynaptic component concentration and on synaptic vesicle cycling.

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