Severing and end-to-end annealing of neurofilaments in neurons

We have shown previously that neurofilaments and vimentin filaments expressed in nonneuronal cell lines can lengthen by joining ends in a process known as end-to-end annealing. To test if this also occurs for neurofilaments in neurons, we transfected cultured rat cortical neurons with fluorescent neurofilament fusion proteins and then used photoconversion or photoactivation strategies to create distinct populations of red and green fluorescent filaments. Within several hours we observed the appearance of chimeric filaments consisting of alternating red and green segments, which is indicative of end-to-end annealing of red and green filaments. However, the appearance of these chimeric filaments was accompanied by a gradual fragmentation of the red and green filament segments, which is indicative of severing. Over time we observed a progressive increase in the number of red-green junctions along the filaments accompanied by a progressive decrease in the average length of the alternating red and green fluorescent segments that comprised those filaments, suggesting a dynamic cycle of severing and end-to-end-annealing. Time-lapse imaging of the axonal transport of chimeric filaments demonstrated that the red and green segments moved together, confirming that they were indeed part of the same filament. Moreover, in several instances, we also were able to capture annealing and severing events live in time-lapse movies. We propose that the length of intermediate filaments in cells is regulated by the opposing actions of severing and end-to-end annealing, and we speculate that this regulatory mechanism may influence neurofilament transport within axons.