Assembly and exchange of intermediate filament proteins of neurons: neurofilaments are dynamic structures.

Abstract

Abstract. We have explored the dynamics of intermediate filament assembly and subunit exchange using fluorescently labeled neurofilament proteins and a fluorescence resonance energy transfer assay. Neurofilaments (NFs) are assembled from three highly phosphorylated proteins with molecular masses of 180 (NF-H), 130 (NF-M), and 66 kD (NF-L) of which NF-L forms the structural core. The core component, NF-L, was stoichiometrically labeled at cysteine 321 with fluorescein, coumarin, or biotin-maleimide to produce assembly-competent fluorescent or biotinylated derivatives, respectively. Using coumarinlabeled NF-L as fluorescence donor and fluoresceinlabeled NF-L as the fluorescence acceptor, assembly of NF filaments was induced by rapidly raising the NaCI concentration to 170 mM, and the kinetics was followed by the decrease in the donor fluorescence. Assembly of NF-L subunits into filaments does not require nucleotide binding or hydrolysis but is strongly dependent on ionic strength, pH, and temperature. The critical concentration of NF-L, that concentration that remains unassembled at equilibrium with fully formed filaments, is 38/zg/ml or 0.6/~M. Under physiological salt conditions NF-L filaments also undergo extensive subunit exchange. Kinetic analysis and evaluation of several possible mechanisms indicate that subunit exchange is preceded by dissociation of subunits from the filament and generation of a kinetically active pool of soluble subunits. Given the concentration of NF-L found in nerve cells and the possibility of regulating this pool, these results provide the first information that intermediate filaments are dynamic structures and that NF-L within the NF complex is in dynamic equilibrium with a small but kinetically active pool of unassembled NF-L units.

Phosphorylation of native and reassembled neurofilaments composed of NF-L, NF-M, and NF-H by the catalytic subunit of cAMP-dependent protein kinase.

“…Otherwise, it might play a role in axonal transport of NF. NF have been shown in in vitro studies to be more dynamic structures than previously thought (Angelides et al, 1989). Further recent fluorescent photobleach-recovery and biotin immunocytochemistry studies indicated directly that NF in the axons turned over within small area of the axoplasm, possibly by the mechanism of lateral and segmental incorporation of new subunits.…”

Effects of phosphorylation of the neurofilament L protein on filamentous structures.

“…However, it also seemed certain that only 1 mol of phosphorylation could disrupt the integrity of the filaments. Angelides et al (1989) described in their recent paper that phosphorylated NF-L could not assemble but that phosphorylation of NF-L filaments appeared to stabilize them. The second of their observation is in contradiction with our results.…”

Regional modulation of neurofilament organization by myelination in normal axons

“…Thus, by increasing phosphorylation locally, myelination might encourage local retention of neurofilaments as part of the stationary cytoskeleton. A final possibility is that myelination encourages local assembly of neurofilaments from subunits and precursors (Angelides et al, 1989;Okabe et al, 1993). In any event, although demyelination reverses the phosphorylation of neurofilaments in the underlying axons, it has a lesser effect on neurofilament number (de Waegh et al, 1992).…”