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

Abstract

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 redgreen 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.T he assembly dynamics of actin filaments and microtubules are well understood, but relatively little is known about the assembly dynamics of intermediate filaments. Several studies over the past two decades have suggested that intermediate filaments lengthen by fusion of preexisting filaments (1, 2), known as "endto-end annealing," and this idea is supported by mathematical modeling and electron microscopy of intermediate filament assembly in vitro (3-6). Several other studies also have suggested that the addition and loss of intermediate filament subunits is not confined to the filament ends and can occur along the entire length of intermediate filaments (7-9), which we refer to as "intercalary subunit exchange." Collectively, these studies suggest that intermediate filaments exhibit novel dynamic properties that are distinct from those of microtubules and actin filaments.In a previous study on neurofilaments and vimentin filaments in adrenal carcinoma and immortalized fibroblast cell lines, we reported experimental evidence for end-to-end annealing and intercalary subunit exchange in vivo (10). However, those cell lines are heterologous systems for studying neurofilament assembly because they do not naturally express neurofilament proteins. Moreover, the neurofilaments formed in those cells were simple heteropolymers composed of neurofilament L (NFL) and either neurofi...

Vimentin filament precursors exchange subunits in an ATP-dependent manner

“…This reorganization consists of active transport of filaments and filament precursors and also the assembly and disassembly of filaments. Photoconversion approaches were used to demonstrate that although vimentin filaments are rapidly transported along microtubules, the most dominant form of mature filament dynamics occurs by slow severing and reannealing (15,16). These experiments are consistent with in vitro data demonstrating that vimentin filaments elongate by end-to-end annealing of ULFs and short filaments (7).…”

“…However, live-cell imaging demonstrates that IF networks are in fact quite dynamic and undergo rapid rearrangement in cells (12,15,16,(28)(29)(30)(31). This reorganization consists of active transport of filaments and filament precursors and also the assembly and disassembly of filaments.…”

“…Recent studies using photoactivation and photoconversion demonstrate that reorganization of VIFs and neurofilaments mainly occur via a different mechanism. Instead of releasing soluble subunits, these filaments reorganize through a process of severing and end-to-end reannealing (15,16). In general, assembly and disassembly of IFs is believed to be regulated by phosphorylation or other posttranslational modifications (see ref.…”

Live-cell imaging of neurofilament transport in cultured neurons

“…The dynamics of intermediate filament assembly in cells is not well understood, but it is likely that the mechanism of fusion protein incorporation into neurofilaments involves de novo filament assembly, subunit exchange along the length of pre-existing filaments (which we have termed intercalary subunit exchange (Colakoglu and Brown, 2009)), and also cyclical annealing and severing mechanisms (Uchida et al, 2013). Regardless of the imaging strategy to be used, it is preferable to allow time for expression and incorporation of the fluorescent fusion protein throughout the axonal neurofilament array before imaging.…”

“…Using immunostaining to compare the distribution of the fusion protein to endogenous neurofilament proteins, we have found that this takes a minimum of several days. For SCG neurons transfected by nuclear injection 2 days after plating, we usually image 3–5 days later (Wang and Brown, 2001; Wang et al, 2000), whereas for cortical neurons transfected by electroporation we usually image 7–14 days later (Wang et al, 2010; Uchida et al, 2013). With enough time, the fusion protein should incorporate along the entire length of all neurofilaments throughout the axonal neurofilament array.…”