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

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Abstract
We used long-term multi-field time-lapse imaging to analyze the movement, severing and annealing of single neurofilaments along axons of cultured neurons. All filaments were capable of rapid movement. However, 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. Site-directed mutagenesis to mimic phosphorylation at known phosphorylation sites in the neurofilament protein L head domain increased the severing rate, generating shorter neurofilaments that moved more frequently. A dephospho-mimic mutant had the opposite effect. Treatment with activators of protein kinase A increased filament severing, and this effect was blocked by the dephosphomimic. Thus, neurofilament length and transport are regulated by a dynamic cycle of severing and annealing. We propose that focal destabilization of intermediate filaments by N-terminal phosphorylation may be a general enzymatic mechanism for severing these cytoskeletal polymers. SUMMARY Uchida & Brown demonstrate that neurofilament length and transport are regulated by a dynamic cycle of neurofilament polymer severing and end-to-end annealing and propose an enzymatic severing mechanism for neurofilaments involving N-terminal phosphorylation of their constituent polypeptides.

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