title: Structure of the peripheral domains of neurofilaments revealed by low angle rotary shadowing.
abstract: 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.
authors.affiliation: Department of Anatomy and Cell Biology, School of Medicine, University of Tokyo, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Hisanaga
authors.forename: N
authors.initials: N
authors.lastname: Hirokawa
mesh_terms: D000818:Animals; D002417:Cattle; D003599:Cytoskeleton / Q000648:ultrastructure*; D007381:Intermediate Filament Proteins; D007382:Intermediate Filaments / Q000648:ultrastructure*; D046911:Macromolecular Substances; D008854:Microscopy, Electron / Q000379:methods
chemical_list: D007381:Intermediate Filament Proteins; D046911:Macromolecular Substances