Mechanistic Determinants of Slow Axonal Transport and Presynaptic Targeting of Clathrin Packets

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 pulsechase radiolabeling. What is the cargo-structure, and mechanisms underlying transport and presynaptic-targeting of clathrin? What is the precise organization at synapses? Combining liveimaging, 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 'transport-packets' 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.

Stimulation-Induced Differential Redistributions of Clathrin and Clathrin-Coated Vesicles in Axons Compared to Soma/Dendrites

“…These larger CCVs in axons may not be involved in SV recycling, but serve other functions that are more prominent during development. One possibility is that they may be involved in axon transport of clathrin [26].…”

Motors on track:

“…Axonal clathrin is assembled in "packets" that are not associated to the plasma membrane, but localized in close vicinity to MTs (Ganguly et al, 2020). These assemblies are mostly static; but when motile, they move at velocities of ~0.6 -0.7 μm/s with an anterograde bias (Ganguly et al, 2020). Such velocities are typical for kinesin-1 motor-driven cargo.…”

“…These phases of fast movement were dependent on the presence of microtubules (MTs), which would suggest that these movements are motor dependent. This view is supported by velocity measurements of synapsin, CamKIIα and clathrin as these were observed to be in the range of kinesin-and/or dynein-dependent motility parameters (Ganguly et al, 2020;Scott et al, 2011;Tang et al, 2013). Normally, cytosolic clathrin consists of triskelia containing three copies of both clathrin heavy chain (CHC) and light chain (LC), with a total molecular weight of ~650 kDa.…”

“…Early studies that used pulse-chase radiolabeling have established that axonal transport includes two populations of cargoes, fast transport generally represented by membranous vesicles and slow transport responsible for axonal distribution of cytoplasmic proteins and protein as-MAP7D2 contains a conserved motif that can tether clathrin to microtubules semblies (Black, 2016;Roy, 2020). More recent studies involving neurofilaments and clathrin have revealed that slow axonal transport comprises stationary phases alternated with phases of fast movement (Ganguly et al, 2020;Roy et al, 2000;Wang et al, 2000). These phases of fast movement were dependent on the presence of microtubules (MTs), which would suggest that these movements are motor dependent.…”

“…Normally, cytosolic clathrin consists of triskelia containing three copies of both clathrin heavy chain (CHC) and light chain (LC), with a total molecular weight of ~650 kDa. However, in axons clathrin forms larger protein assemblies named "clathrin-packets", which are associated with the plasma membrane but are rather found in close proximity to MTs and are presumably important for axonal transport of clathrin to synapses (Ganguly et al, 2020). This would suggest the presence of cytoskeletal linkers that may directly recruit clathrin to the MT network.…”