Morphological analysis of glutaraldehyde-fixed vimentin intermediate filaments and assembly-intermediates by atomic force microscopy

Abstract

Atomic force microscopy (AFM) was used to study the morphology of vimentin intermediate filaments (IFs) and their assembly intermediates. At each time after initiation of IF assembly in vitro of recombinant mouse vimentin, the sample was fixed with 0.1% glutaraldehyde and then applied to AFM analysis. When mature vimentin IFs were imaged in air on mica, they appeared to have a width of ∼28 nm, a height of ∼4 nm and a length of several micrometers. Taking into account the probe tip's distortion effect, the exact width was evaluated to be ∼25 nm, suggesting that the filaments flatten on the substrate rather than be cylindrical with a diameter of ∼10 nm. Vimentin IFs in air clearly demonstrated ∼21-nm repeating patterns along the filament axis. The three-dimensional profiles of vimentin IFs indicated that the characteristic patterns were presented by repeating segments with a convex surface. The repeating patterns close to 21 nm were also observed by AFM analysis in a physiological solution condition, suggesting that the segments along the filaments are an intrinsic substructure of vimentin IFs. In the course of IF assembly, assembly intermediates were analyzed in air. Many short filaments with a full-width and an apparent length of ∼78 nm (evaluated length ∼69 nm) were observed immediately after initiation of the assembly reaction. Interestingly, the short full-width filaments appeared to be composed of the four segments. Further incubation enabled the short full-width filaments to anneal longitudinally into longer filaments with a distinct elongation step of ∼40 nm, which corresponds to the length of the two segments. To explain these observations, we propose a vimentin IF formation model in which vimentin dimers are supercoiling around the filament axis.