Neurofilaments are transported rapidly but intermittently in axons: Implications for slow axonal transport

Slow axonal transport conveys cytoskeletal proteins from cell body to axon tip. This transport provides the axon with the architectural elements that are required to generate and maintain its elongate shape and also generates forces within the axon that are necessary for axon growth and navigation. The mechanisms of cytoskeletal transport in axons are unknown. One hypothes is states that cytoskeletal proteins are transported within the axon as pol ymers. We tested this hypothesis by visualizing individual cytoskeletal pol ymers in living axons and determining whether they undergo vectorial moveme nt. We focused on neurofilaments in axons of cultured sympathetic neurons b ecause individual neurofilaments in these axons can be visualized by optica l microscopy. Cultured sympathetic neurons were infected with recombinant a denovirus containing a construct encoding a fusion protein combining green fluorescent protein (GFP) with the heavy neurofilament protein subunit (NFH ). The chimeric GFP-NFH coassembled with endogenous neurofilaments. Time la pse imaging revealed that individual GFP-NFH-labeled neurofilaments undergo vigorous vectorial transport in the axon in both anterograde and retrograd e directions but with a strong anterograde bias. NF transport in both direc tions exhibited a broad spectrum of rates with averages of approximate to 0 .6-0.7 mm/sec. However, movement was intermittent, with individual neurofil aments pausing during their transit within the axon. Some NFs either moved or paused for the most of the time they were observed, whereas others were intermediate in behavior. On average, neurofilaments spend at most 20% of t he time moving and rest of the time paused. These results establish that th e slow axonal transport machinery conveys neurofilaments.