Presynapses contain distinct actin nanostructures

Abstract

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.

Myosin VI controls localization of Golgi satellites at active presynaptic boutons

“…The F-actin cytoskeleton is known to regulate the transport and localization of dendritic secretory organelles, such as lysosomes and the spine apparatus (van Bommel et al, 2019; Konietzny et al, 2022). It is also highly enriched in presynaptic boutons (Bingham et al, 2023). We therefore asked if presynaptic F-actin can regulate organelle positioning in distal axons.…”

“…Guedes-Dias et al have reported that dynamic MT ends at en passant synaptic boutons cause synaptic vesicles transported by the kinesin-3 motor protein to hop off the MT track (Guedes-Dias et al, 2019). However, presynaptic sites are also enriched in F-actin (Bingham et al, 2023), and we hypothesized that an additional mechanism, namely F-actin acting as a mesh to stop passing organelles, might be at play. A similar process has also been described in dendrites, where F-actin hot spots found along the dendrite at the base of spines or excitatory shaft synapses stop and anchor passing lysosomes (van Bommel et al, 2019).…”

The actin-spectrin submembrane scaffold restricts endocytosis along proximal axons

“…To confirm the specific stability of CCPs at the AIS of neurons, and avoid potential stabilizing effects of overexpressed clathrin or spectrin, we next used a modified AAV-mediated HiUGE strategy (Ogawa et al, 2023; Bingham et al, 2023) to endogenously tag clathrin light chain with EGFP in cultured hippocampal neurons (CLC-GFP KI), and imaged clathrin dynamics using super-resolution spinning disk microscopy (Figure 6, E; Supporting Movie 2). The enhanced temporal resolution (one frame every 5s for 10 min) allowed us to discern scission events for both static pits at the AIS (appearing as interruptions in the vertical traces on kymographs) and transient pits in dendrites (appearing as J-shaped traces in kymographs; Figure 6, F).…”

Single-Molecule-Resolution Approaches in Synaptic Biology

“…, MAP2). A recent study used single-molecule localization microscopy to resolve the actin cytoskeleton at the presynaptic compartment and found three distinct types of local actin nanostructures (mesh, rails, and corrals) . However, this study is still in fixed cells.…”

“…A recent study used single-molecule localization microscopy to resolve the actin cytoskeleton at the presynaptic compartment and found three distinct types of local actin nanostructures (mesh, rails, and corrals). 58 However, this study is still in fixed cells. To observe the cytoskeleton dynamics in live cells, MINFLUX, a singlemolecule-resolution microscopy that combines STED and SMLM, has recently showcased its potential.…”