Clathrin packets move in slow axonal transport and deliver functional payloads to synapses

In non-neuronal cells, clathrin has established roles in endocytosis, with clathrin cages enclosing plasma membrane infoldings, followed by rapid disassembly and reuse of monomers. However, in neurons, clathrin is conveyed in slow axonal transport over days to weeks, and the underlying transport/targeting mechanisms, mobile cargo structures, and even its precise presynaptic localization and physiologic role are unclear. Combining live imaging, photobleaching/conversion, mass spectrometry, electron microscopy, and super-resolution imaging, we found that unlike in dendrites, where clathrin cages rapidly assemble and disassemble, in axons, clathrin and related proteins organize into stable “transport packets” that are unrelated to endocytosis and move intermittently on microtubules, generating an overall slow anterograde flow. At synapses, multiple clathrin packets abut synaptic vesicle (SV) clusters, and clathrin packets also exchange between synaptic boutons in a microtubule-dependent “superpool.” Within synaptic boundaries, clathrin is surprisingly dynamic, continuously exchanging between local clathrin assemblies, and its depletion impairs SV recycling. Our data provide a conceptual framework for understanding clathrin trafficking and presynaptic targeting that has functional implications.