A dynamic formin-dependent deep F-actin network in axons

Although actin at neuronal growth cones is well-studied, much less is known about actin organization and dynamics along axon shafts and presynaptic boutons. Using probes that selectively label filamentous-actin (F-actin), we found focal actin hotspots along axons-spaced similar to 3-4 mu m apart-where actin undergoes continuous assembly/disassembly. These foci are a nidus for vigorous actin polymerization, generating long filaments spurting bidirectionally along axons a phenomenon we call actin trails. Super-resolution microscopy reveals intra-axonal deep actin filaments in addition to the subplasmalemmal actin rings described recently. F-actin hotspots colocalize with stationary axonal endosomes, and blocking vesicle transport diminishes the actin trails, suggesting mechanistic links between vesicles and F-actin kinetics. Actin trails are formin-but not Arp2/3-dependent and help enrich actin at presynaptic boutons. Finally, formin inhibition dramatically disrupts synaptic recycling. Collectively, available data suggest a two-tier F-actin organization in axons, with stable actin rings providing mechanical support to the plasma membrane and dynamic actin trails generating a flexible cytoskeletal network with putative physiological roles.