Distinct nano-structures support a multifunctional role of actin at presynapses

Abstract

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.

Membrane compression by synaptic vesicle exocytosis triggers ultrafast endocytosis

“…These results differ from those of a recent study using single-molecule localization microscopy of presynaptic terminals formed on polystyrene microbeads. In that system, three pools of F-actin were apparent: actin rails between the active zone and vesicles, actin corrals around the terminal 20 , and in contrast to our results, an actin mesh at the active zone. The presence of F-actin in such artificially-formed active zones may reflect cytoskeletal response due to changes in cortical tension, since a bead is much stiffer than a postsynaptic terminal 70 .…”

“…1a, Extended data Fig. 1a for more example micrographs) 19,20 and surrounds an active zone like a corral. However, it is largely excluded from the active zone (Fig.…”

pHusion: A robust and versatile toolset for automated detection and analysis of exocytosis

“…Neuronal culture Primary neuronal cell culture was obtained in a similar procedure as previously described (Bingham et al, 2022). Briefly, hippocampi were extracted from E18 rat pups from pregnant female Wistar rats (Janvier labs), dissected, and homogenized by trypsin treatment followed by mechanical trituration and seeded on 18-mm diameter round, #1.5H coverslips at a density of 30,000 cells/cm2 for 3 hours in serum-containing plating medium (MEM with 10% fetal bovine serum, 0.6% added glucose, 0.08 mg/mL sodium pyruvate, 100 UI/mL penicillin-streptomycin).…”