Neurofilaments (NFs), a neuron-specific subtype of intermediate filament (IF), are an essential structural component of the axon. Although the sequence and secondary structure of NF subunits are established, the higher-level organization of subunits into approximately 10-nm- diameter filaments remains unknown, in part due to compositional heterogeneity and difficulties in obtaining native preparations. Squid NFs contain low- (L-) and high- (H-) molecular-weight subunits (Mr = 60 kDa and Mr = 120 kDa, respectively). The N-terminal and approximately 50-nm-long alpha-helical rod domains of the L- and H-subunits are identical, but the H-subunit has an additional C-terminal tail containing 63 potential phosphorylation sites. These highly charged phosphorylated tails are believed to stabilize the NF cytoskeleton. A novel combination of scanning transmission electron microscopy (STEM) and parallel electron energy-loss spectroscopy (PEELS) has been used to show that native NFs isolated from squid giant axon are essentially fully phosphorylated and contain only 8 plus minus 1 coiled-coil dimers per cross section.
