A possible mechanism for neurofilament slowing down in myelinated axon: Phosphorylation-induced variation of NF kinetics

Neurofilaments(NFs) are the most abundant intermediate filaments that make up the inner volume of axon, with possible phosphorylation on their side arms, and their slow axonal transport by molecular motors along microtubule tracks in a “stop-and-go” manner with rapid, intermittent and bidirectional motion. The kinetics of NFs and morphology of axon are dramatically different between myelinate internode and unmyelinated node of Ranvier. The NFs in the node transport as 7.6 times faster as in the internode, and the distribution of NFs population in the internode is 7.6 folds as much as in the node of Ranvier. We hypothesize that the phosphorylation of NFs could reduce the on-track rate and slow down their transport velocity in the internode. By modifying the ‘6-state’ model with (a) an extra phosphorylation kinetics to each six state and (b) construction a new ‘8-state’ model in which NFs at off-track can be phosphorylated and have smaller on-track rate, our model and simulation demonstrate that the phosphorylation-induced decrease of on-track rate could slow down the NFs average velocity and increase the axonal caliber. The degree of phosphorylation may indicate the extent of velocity reduction. The Continuity equation used in our paper predicts that the ratio of NFs population is inverse proportional to the ratios of average velocity of NFs between node of Ranvier and internode. We speculate that the myelination of axon could increase the level of phosphorylation of NF side arms, and decrease the possibility of NFs to get on-track of microtubules, therefore slow down their transport velocity. In summary, our work provides a potential mechanism for understanding the phosphorylation kinetics of NFs in regulating their transport and morphology of axon in myelinated axons, and the different kinetics of NFs between node and internode.

Bidirectional, unlike unidirectional transport, allows transporting axonal cargos against their concentration gradient

Even though most axonal cargos are synthesized in the soma, the concentration of many of these cargos is larger at the presynaptic terminal than in the soma. This requires transport of these cargos from the soma to the presynaptic terminal or other active sites in the axon. Axons utilize both bidirectional (for example, slow axonal transport) and unidirectional (for example, fast anterograde axonal transport) modes of cargo transport. Bidirectional transport seems to be less efficient because it requires more time and takes more energy to deliver cargos. In this paper, bidirectional and unidirectional axonal transport processes are investigated with respect to their ability to transport cargos against their concentration gradient. We argue that because bidirectional axonal transport includes both the anterograde and retrograde cargo populations, information about cargo concentration at the axon entrance and at the presynaptic terminal can travel in both anterograde and retrograde directions. This allows bidirectional axonal transport to account for the concentration of cargos at the presynaptic terminal. In unidirectional axonal transport, on the contrary, cargo transport occurs only in one direction, and this disallows transport of information about the cargo concentration at the opposite boundary. For the case of unidirectional anterograde transport, this means that proximal regions of the axon do not receive information about cargo concertation in the distal regions. This does not allow for the imposition of a higher concentration at the presynaptic terminal in comparison to the cargo concentration at the axon hillock. To the best of our knowledge, our paper presents the first explanation for the utilization of seemingly inefficient bidirectional transport in neurons.

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

SUMMARYClathrin 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 ‘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.

Stochastic models of polymerization based axonal actin transport

Pulse-chase and radio-labeling studies have shown that actin is transported in bulk along the axon at rates consistent with slow axonal transport. In a recent paper, using a combination of live cell imaging, super resolution microscopy and computational modeling, we proposed that biased polymerization of metastable actin fibers (actin trails) along the axon shaft forms the molecular basis of bulk actin transport. The proposed mechanism is unusual, and can be best described as molecular hitch hiking, where G-actin molecules are intermittently incorporated into actin fibers which grow preferably in anterograde direction giving rise to directed transport, released after the fibers collapse only to be incorporated into another fiber. In this paper, we use our computational model to make additional predictions that can be tested experimentally to further scrutinize our proposed mechanism for bulk actin transport. In the previous paper the caliber of our model axon, the density of the actin nucleation sites to form the metastable actin fibers, the length distribution of the actin trails and their growth rate were adapted to the biologic axons used for measurements. Here we predict how the transport rate will change with axon caliber, density of nucleation sites, nucleation rates and trail lengths. We also discuss why a simple diffusion-based transport mechanism can not explain bulk actin transport.