Lateral Subunit Coupling Determines Intermediate Filament Mechanics.

The cytoskeleton is a composite network of three types of protein filaments, among which intermediate filaments (IFs) are the most extensible ones. Two very important IFs are keratin and vimentin, which have similar molecular architectures but different mechanical behaviors. Here we compare the mechanical response of single keratin and vimentin filaments using optical tweezers.

Mutation-induced alterations of intra-filament subunit organization in vimentin filaments revealed by SAXS.

Vimentin intermediate filaments constitute a distinct filament system in mesenchymal cells that is instrumental for cellular mechanics and migration. In vitro, the rod-like monomers assemble in a multi-step, salt-dependent manner into micrometer long biopolymers. To disclose the underlying mechanisms further, we employed small angle X-ray scattering on two recombinant vimentin variants, whose assembly departs at strategic points from the normal assembly route: (i) vimentin with a tyrosine to leucine change at position 117; (ii) vimentin missing the non-α-helical carboxyl-terminal domain.

Impact of ion valency on the assembly of vimentin studied by quantitative small angle X-ray scattering.

The assembly kinetics of intermediate filament (IF) proteins from tetrameric complexes to single filaments and networks depends on the protein concentration, temperature and the ionic composition of their environment. We systematically investigate how changes in the concentration of monovalent potassium and divalent magnesium ions affect the internal organization of the resulting filaments. Small angle X-ray scattering (SAXS) is very sensitive to changes in the filament cross-section such as diameter or compactness.

Dynamics of intermediate filament assembly followed in micro-flow by small angle X-ray scattering.

The assembly of intermediate filaments (IFs) is a complex process that can be recapitulated through a series of distinct steps in vitro. The combination of microfluidics and small angle X-ray scattering (SAXS) provides a powerful tool to investigate the kinetics of this process on the relevant timescales. Microfluidic mixers based on the principle of hydrodynamic focusing allow for precise control of the mixing of proteins and smaller reagents like ions.