Clathrin mediated endocytosis (CME) at pre-synaptic terminals is functionally coupled to neuro-transmitter release. Like other cytosolic proteins, clathrin is synthesized in cell-bodies and conveyed into axons and synapses via slow axonal transport. Although the ‘triskelial’ structural conformation and endocytic function of clathrin at synapses is well-established, the cargo-structure or transport behavior of clathrin molecules moving in slow axonal transport is virtually unknown. We defined the cargo-composition and transport-kinetics of clathrin assemblies in axons using a combination of live-cell imaging, mass-spectrometry and correlative electron microscopy in primary hippocampal cultures and neurons in-vivo. We found that axonal clathrin is transported as a unique, stable cargo-complex that is distinct from the known ephemeral triskelial structures that exist at synapses and dendrites. While its transport-kinetics resembles other slow-component cargoes, surprisingly, the transport-complex contains both clathrin assembly and disassembly proteins and is ultra structurally distinct from any known clathrin assemblage. Further, we demonstrate that the stable transported clathrin cargo complex is deposited at en passant boutons along axons. Our studies thus uncover a previously-unknown form of clathrin and clarify mechanistic details of its slow transport into axons.
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