Regulation of neurofilament length and transport by a dynamic cycle of polymer severing and annealing

Abstract

Neurofilaments are space-filling cytoskeletal polymers that are transported into axons where they accumulate during development to expand axon caliber. We previously described novel severing and end-to-end annealing mechanisms in neurons that alter neurofilament length. To explore the functional significance of neurofilament length, we developed a long-term multi-field time-lapse method to track the movement of fluorescently tagged neurofilaments in axons of cultured neurons for up to 30 minutes. All filaments moved rapidly, but long filaments paused and reversed more often, resulting in little net movement, whereas short filaments moved persistently for long distances, pausing and reversing less often. Long filaments severed more frequently, generating shorter filaments, and short filaments annealed more frequently, generating longer filaments. Thus, neurofilament length is regulated by a dynamic cycle of severing and annealing and this influences neurofilament transport. Site-directed mutagenesis to mimic phosphorylation at four known phosphorylation sites in the head domain of neurofilament protein L generated shorter neurofilaments that moved more frequently. A non-phosphorylatable mutant had the opposite effect. Treatment of cultured neurons with activators of protein kinase A, which phosphorylates three of these sites, increased neurofilament severing. This effect was blocked by the non-phosphorylatable mutant. We propose that focal destabilization of intermediate filaments by N-terminal phosphorylation of their constituent polypeptides at specific locations along their length may be a general enzymatic mechanism for severing this class of cytoskeletal polymers. Our data suggest a novel mechanism for the control of neurofilament transport and accumulation in axons based on regulation of neurofilament polymer length. SUMMARYNeurofilaments are space-filling cytoskeletal polymers that are transported into axons where they accumulate to expand axon caliber, which is an important determinant of axonal conduction velocity. We reported previously that neurofilaments can lengthen and shorten by novel end-to-end annealing and severing mechanisms. Here, we show that neurofilament annealing and severing are robust phenomena in cultured neurons that act antagonistically to dynamically regulate neurofilament length, which in turn regulates their transport. In addition, we present evidence for a novel enzymatic mechanism of intermediate filament severing based on site-directed phosphorylation of the neurofilament subunit proteins. We propose that modulation of neurofilament length by annealing and severing may be a mechanism for the regulation of neurofilament transport and accumulation in axons.