ABSTRACT
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. We show that the mechanics of vimentin strongly depends on the ionic strength of the buffer and that its force-strain curve suggests a high degree of cooperativity between subunits. Indeed, a computational model indicates that in contrast to keratin, vimentin is characterized by strong lateral subunit coupling of its charged monomers during unfolding of α helices. We conclude that cells can tune their mechanics by differential use of keratin versus vimentin.
Subject(s)
Cytoskeleton/chemistry , Keratins/chemistry , Models, Biological , Vimentin/chemistry , Biomechanical Phenomena , Buffers , Cytoskeleton/metabolism , Keratins/metabolism , Microscopy, Atomic Force , Optical Tweezers , Osmolar Concentration , Protein Conformation, alpha-Helical , Vimentin/metabolismABSTRACT
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. Y117L vimentin assembles into unit-length filaments (ULFs) only, whereas ΔT vimentin assembles into filaments containing a higher number of tetramers per cross section than normal vimentin filaments. We show that the shape and inner structure of these mutant filaments is significantly altered. ULFs assembled from Y117L vimentin contain more, less tightly bundled vimentin tetramers, and ΔT vimentin filaments preserve the number density despite the higher number of tetramers per filament cross-section.
Subject(s)
Intermediate Filaments/metabolism , Mutation , Protein Subunits/chemistry , Protein Subunits/genetics , Vimentin/chemistry , Vimentin/genetics , Humans , Scattering, Small Angle , X-Ray DiffractionABSTRACT
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. Our measurements reveal that filaments formed in the presence of magnesium chloride differ distinctly from filaments formed in the presence of potassium chloride. The principle multi-step assembly mechanism from tetramers via unit-length filaments (ULF) to elongated filaments is not changed by the valency of ions. However, the observed differences indicate that the magnesium ions free the head domains of tetramers from unproductive interactions to allow assembly but at the same time mediate strong inter-tetrameric interactions that impede longitudinal annealing of unit-length filaments considerably, thus slowing down filament growth.
Subject(s)
Intermediate Filament Proteins/chemistry , Intermediate Filaments/ultrastructure , Scattering, Small Angle , Vimentin/chemistry , Cytoskeleton/chemistry , Cytoskeleton/ultrastructure , Intermediate Filament Proteins/ultrastructure , Intermediate Filaments/chemistry , Ions/chemistry , Kinetics , Vimentin/ultrastructure , X-Ray DiffractionABSTRACT
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. Here, we present a multi-layer device that prevents proteins from adsorbing to the channel walls by engulfing the protein jet with a fluid layer of buffer. To ensure compatibility with SAXS, the device is fabricated from UV-curable adhesive (NOA 81). To demonstrate the successful prevention of contact between the protein jet and the channel walls we measure the distribution of a fluorescent dye in the device by confocal microscopy at various flow speeds and compare the results to finite element method (FEM) simulations. The prevention of contact enables the investigation of the assembly of IFs in flow by gradually increasing the salt concentration in the protein jet. The diffusion of salt into the jet can be determined by FEM simulations. SAXS data are collected at different positions in the jet, corresponding to different salt concentrations, and they reveal distinct differences between the earliest assembly states. We find that the mean square radius of gyration perpendicular to the filament axis increases from 13 nm(2) to 58 nm(2) upon assembly. Thereby we provide dynamic structural data of a complex assembly process that was amenable up to now only by microscopic techniques.
