Structure of the peripheral domains of neurofilaments revealed by low angle rotary shadowing

The structure of the peripheral domains of neurofilaments (NFs) was revealed by rotary shadowing electron microscopy. NFs were isolated from bovine spinal cords by Sepharose CL-4B gel filtration and examined by low angle rotary shadowing. The peripheral domains appeared as thin, flexible, filamentous structures projecting from the intermediate filament core, with a constant density along their entire length. The average length of the projections was approximately 85 nm and the width about 4 nm. These projections appeared from regularly distributed sites, at 22 nm spacing, which seemed to correspond to the typical repeat of the alpha-helix-rich rod domain of the core filament. The density of the projections was found to be 4.1 (+/- 0.6) per 22 nm. We performed reconstitution experiments using purified NF polypeptides to confirm that the projection was indeed the NF peripheral domain. Individual components of the NF triplet, i.e. NF-L, NF-M and NF-H, were purified by DE-52 and Mono-Q anion exchange chromatographies in the presence of 6 M-urea and were assembled in various combinations into filaments. Reassembled filaments were somewhat more slender than the isolated NFs and exhibited a distinct 22 nm axial periodicity. While prominent projections were not observed in the filaments assembled from NF-L alone, reconstructed filaments containing NF-L plus either NF-M or NF-H revealed many projections. The average length of the projections in the filaments reconstructed from NF-L and NF-H was about 63 nm. The projections of reconstructed filaments from NF-L and NF-M were about 55 nm in length. The difference in the lengths of the projections might reflect the difference in the length of the carboxy-terminal tail domain between NF-M and NF-H. The results are interpreted to show that the carboxy-terminal tail domains of NFs project in a regular pattern from the core filament, which is consistent with a half-staggered organization of the tetrameric subunits.