Regulation of neurofilament interactions in vitro by natural and synthetic polypeptides sharing Lys-Ser-Pro sequences with the heavy neurofilament subunit NF-H: Neurofilament crossbridging by antiparallel sidearm overlapping

Neurofilaments are organised into parallel bundles in axons through cross-bridges formed by lateral projections of neurofilament subunits. Pure neurofilaments form gels in vitro, consisting of interconnected parallel arrays of filaments regulated by the phosphorylation level of neurofilament subunits. Neurofilament-associated polypeptides sharing phosphorylated epitopes with the repetitive lysine-serine-proline (Lys-Ser-Pro) motifs of the neurofilament heavy subunit sidearm are characterised: they regulate in vitro the neurofilament gelation kenetics in a concentration-and phosphorylation-dependent manner. Studies with synthetic peptides show that interactions between neurofilaments involve both acid and base amino acid residues of neurofilament sidearms and demonstrate the opposite effects of peptides containing either one (inhibition) or two (activation) Lys-Ser-Pro motifs. Electron microscopy reveals an organised network of native neurofilament sidearms, regulated by the phosphorylation level of neurofilament subunits, suggesting a structural transition between intra- and inter-neurofilament sidearm interactions. These results favour the hypothesis of a mechanism of neurofilament crossbridging through the variable antiparallel overlapping of the phosphorylable Lys-Ser-Pro domains of neurofilament sidearms from adjacent filaments, following an equilibrium regulated by neurofilament-associated proteins, bivalent cations and the phosphorylation level of Lys-Ser-Pro motifs from both neurofilament sidearms and neurofilament-associated proteins.