Properties of Intermediate Filament Networks Assembled from Keratin 8 and 18 in the Presence of Mg2+

Abstract

The mechanical properties of epithelial cells are modulated by structural changes in keratin intermediate filament networks. To investigate the relationship between network architecture and viscoelasticity, we assembled keratin filaments from recombinant keratin proteins 8 (K8) and 18 (K18) in the presence of divalent ions (Mg(2+)). We probed the viscoelastic modulus of the network by tracking the movement of microspheres embedded in the network during assembly, and studied the network architecture using scanning electron microscopy. Addition of Mg(2+) at physiological concentrations (<1 mM) resulted in networks whose structure was similar to that of keratin networks in epithelial cells. Moreover, the elastic moduli of networks assembled in vitro were found to be within the same magnitude as those measured in keratin networks of detergent-extracted epithelial cells. These findings suggest that Mg(2+)-induced filament cross-linking represents a valid model for studying the cytoskeletal mechanics of keratin networks.

Investigating Cellular Nanoscale with X-rays

“…Similar effect can be expected for keratin and structural differences might play a role, that can be accessed with X-rays. Furthermore, it has been shown that Mg 2+ ions are cross-linking keratin filaments [103,190,191] or other IFs [187,233]. Very similar conclusion can be drawn from the micro-diffraction measurements.…”

“…Here, the following assumptions have been applied: i) the Gaussian chains are located at the rod surface, ii) the number of Gaussian chains corresponds to 16 monomers per ULF length (about 43 nm), thus λ = 2.69 nm, iii) the standard deviation of the filament thickness distribution is estimated to 17.02% of the average radius [114] and iv) the persistence length of about 500 nm for keratin [103] is much larger than the length scale accessible by SAXS, which corresponds to about 60 nm. …”

“…The microtubules are all converging to the same point, close to the nucleus, and form stiff rods with a persistence length of about 1 mm [101]. Finally, IFs form a dense network structure and are semi-flexible with a persistence length of 0.1 to 2 µm, depending on the actual IF: 100 to 200 nm for neurofilaments [102], 500 nm for keratin [103] and 2 µm for vimentin [104]. Both, microfilaments and microtubules are polar filaments, as shown in Fig.…”

“…In cells, keratin proteins form bundles and networks. The mechanical and structural properties of keratin bundles have been studied by microscopy techniques [103,117,[183][184][185][186][187][188][189][190][191], but the internal structure could not be accessed. One way to access the internal architecture of these bundles is by the use of X-rays.…”

Both monovalent cations and plectin are potent modulators of mechanical properties of keratin K8/K18 networks

“…2) already show an influence of 10 mM KCl on network mechanics, which is below the bundling onset concentration. The same gradual change in network mechanics was reported by Leitner et al 29 for MgCl 2 . Here also concentrations clearly below the onset concentration were sufficient to alter network mechanics.…”