Browsing by Author "Herrmann, Harald"

The intermediate filament proteins keratin K8 and K18 constitute an essential part of the cytoskeleton in simple epithelial cell layers, structurally enforcing their mechanical resistance. K8/K18 heterodimers form extended filaments and higher-order structures including bundles and networks that bind to cell junctions. We study the assembly of these proteins in the presence of monovalent or divalent ions by small-angle X-ray scattering. We find that both ion species cause an increase of the filament diameter when their concentration is increased; albeit, much higher values are needed for the monovalent compared to the divalent ions for the same effect. Bundling occurs also for monovalent ions and at comparatively low concentrations of divalent ions, very different from vimentin intermediate filaments, a fibroblast-specific cytoskeleton component. We explain these differences by variations in charge and hydrophobicity patterns of the proteins. These differences may reflect the respective physiological situation in stationary cell layers versus single migrating fibroblasts.

Aggregation is a commonly observed phenomenon when mixing charged biopolymers and counterions. However, it remains a challenge to find a general description for the involved mechanisms. Here, we investigate vimentin intermediate filaments, which are overall highly negatively charged, as a prime example of such biological polyelectrolytes. We experimentally determine the onset cation concentration for aggregation, and set it in relation to the calculated relative cation concentrations in the vicinity of the filament surface. Our analysis shows that to achieve aggregation, about 50% of the monovalent cations originally present have to be replaced by di- or multivalent cations. We are thus able to predict the bulk cation concentration, which mediates aggregation of highly negatively charged vimentin filaments.

Actin filaments, microtubules, and intermediate filaments (IFs) are central elements of the metazoan cytoskeleton. At the molecular level, the assembly mechanism for actin filaments and microtubules is fundamentally different from that of IFs. The former two types of filaments assemble from globular proteins. By contrast, IFs assemble from tetrameric complexes of extended, half-staggered, and antiparallel oriented coiled-coils. These tetramers laterally associate into unit-length filaments; subsequent longitudinal annealing of unit-length filaments yields mature IFs. In vitro, IFs form open structures without a fixed number of tetramers per cross-section along the filament. Therefore, a central question for the structural biology of IFs is whether individual subunits can dissociate from assembled filaments and rebind at other sites. Using the fluorescently labeled IF-protein vimentin for assembly, we directly observe and quantitatively determine subunit exchange events between filaments as well as with soluble vimentin pools. Thereby we demonstrate that the cross-sectional polymorphism of donor and acceptor filaments plays an important role. We propose that in segments of donor filaments with more than the standard 32 molecules per cross-section, subunits are not as tightly bound and are predisposed to be released from the filament.

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.

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.

Intermediate filament proteins form filaments, fibers and networks both in the cytoplasm and the nucleus of metazoan cells. Their general structural building plan accommodates highly varying amino acid sequences to yield extended dimeric alpha-helical coiled coils of highly conserved design. These 'rod' particles are the basic building blocks of intrinsically flexible, filamentous structures that are able to resist high mechanical stresses, that is, bending and stretching to a considerable degree, both in vitro and in the cell. Biophysical and computer modeling studies are beginning to unfold detailed structural and mechanical insights into these major supramolecular assemblies of cell architecture, not only in the 'test tube' but also in the cellular and tissue context.

Vimentin intermediate filaments, together with actin filaments and microtubules, constitute the cytoskeleton in cells of mesenchymal origin. The mechanical properties of the filaments themselves are encoded in their molecular architecture and depend on their ionic environment. It is thus of great interest to disentangle the influence of both the ion type and their concentration on vimentin assembly. We combine small angle X-ray scattering and fluorescence microscopy and show that vimentin in the presence of the monovalent ions, K+ and Na+, assembles into ‘‘standard filaments’’ with a radius of about 6 nm and 32 monomers per crosssection. In contrast, di- and multivalent ions, independent of type and valency, lead to the formation of thicker filaments associating over time into higher order structures. Hence, our results may indeed be of relevance for living cells, as local ion concentrations in the cytoplasm during certain physiological activities may differ considerably from average intracellular concentrations.

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.

Intermediate filaments are one of three classes of fibrous proteins in the cytoskeleton of eukaryotes, the others being actin filaments and microtubules. The dense filamentous networks and bundles provide important mechanical stability for the cell. Here we directly measure both the structure and mechanical properties of an in vitro model system for intermediate filaments reconstituted from purified vimentin protein at 1 mg mL(-1). We show that the mesh size is on the order of 1 mm, a value that is preserved upon addition of divalent ions. These ions act as effective cross-linkers, further stiffening the network.

The structural organization of metazoan cells and their shape are established through the coordinated interaction of a composite network consisting of three individual filament systems, collectively termed the cytoskeleton. Specifically, microtubules and actin filaments, which assemble from monomeric globular proteins, provide polar structures that serve motor proteins as tracks. In contrast, intermediate filaments (IFs) assemble from highly charged, extended coiled coils in a hierarchical assembly mechanism of lateral and longitudinal interaction steps into non-polar structures. IF proteins are expressed in a distinctly tissue-specific way and thereby serve to generate the precise plasticity of the respective cells and tissues. Accordingly, in the cell, numerous parameters such as pH and salt concentration are adjusted such that the generation of functional networks is ensured. Here, we transfer the problem for the mesenchymal IF protein vimentin to an in vitro setting and combine small angle x-ray scattering with microfluidics and finite element method simulations. Our approach is adapted to resolve the early assembly steps, which take place in the sub-second to second range. In particular, we reveal the influence of ion species and concentrations on the assembly. By tuning the flow rates and thus concentration profiles, we find a minimal critical salt concentration for the initiation of the assembly. Furthermore, our analysis of the surface sensitive Porod regime in the x-ray data reveals that the formation of first assembly intermediates, so-called unit length filaments, is not a one-step reaction but consists of distinct consecutive lateral association steps followed by radial compaction as well as smoothening of the surface of the full-width filament.