@article {Ganguly2020.02.20.958140, author = {Archan Ganguly and Florian Wernert and S{\'e}bastien Phan and Daniela Boassa and Utpal Das and Rohan Sharma and Ghislaine Caillol and Xuemei Han and John R. Yates III and Mark H. Ellisman and Christophe Leterrier and Subhojit Roy}, title = {Mechanistic Determinants of Slow Axonal Transport and Presynaptic Targeting of Clathrin Packets}, elocation-id = {2020.02.20.958140}, year = {2020}, doi = {10.1101/2020.02.20.958140}, publisher = {Cold Spring Harbor Laboratory}, abstract = {Clathrin has established roles in endocytosis, with clathrin-cages enclosing membrane infoldings, followed by rapid disassembly and reuse of monomers. However, in neurons, clathrin synthesized in cell-bodies is conveyed into axons and synapses via slow axonal transport; as shown by classic pulse-chase radiolabeling. What is the cargo-structure, and mechanisms underlying transport and presynaptic-targeting of clathrin? What is the precise organization at synapses? Combining live-imaging, mass-spectrometry (MS), Apex-labeled EM-tomography and super-resolution, we found that unlike dendrites where clathrin transiently assembles/disassembles as expected, axons contain stable {\textquoteleft}transport-packets{\textquoteright} that move intermittently with an anterograde bias; with actin/myosin-VI as putative tethers. Transport-packets are unrelated to endocytosis, and the overall kinetics generate a slow biased flow of axonal clathrin. Synapses have integer-numbers of clathrin-packets circumferentially abutting the synaptic-vesicle cluster, advocating a model where delivery of clathrin-packets by slow axonal transport generates a radial organization of clathrin at synapses. Our experiments reveal novel trafficking mechanisms, and an unexpected nanoscale organization of synaptic clathrin.}, URL = {https://www.biorxiv.org/content/early/2020/03/05/2020.02.20.958140}, eprint = {https://www.biorxiv.org/content/early/2020/03/05/2020.02.20.958140.full.pdf}, journal = {bioRxiv} }