Amino acid sequence alignment of keratin assembly partners K18 (blue letters) and K8 (green letters).

<p>The order of the subdomains is as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093194#pone-0093194-g001" target="_blank">Figure 1</a> of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093194#pone.0093194-Herrmann5" target="_blank">[46]</a>. Hydrophobic amino acids in the non-α-helical head and tail domains are indicated in red <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093194#pone.0093194-Kyte1" target="_blank">[47]</a>. Significant hydrophobic motifs in these domains are underlined. In the rod domain, the a- and d-heptad positions are highlighted in yellow; these amino acids are responsible for the formation of a coiled-coil dimer from two individual α-helices. Hydrophilic domains on the surface of a coiled-coil dimer, generated by amino acids positioned in the b-, c-, e-, f-, and g-positions of the heptad pattern, are highlighted in cyan.</p

Influence of TX-100 on network structure of K8/K18 characterized by electron microscopy and MPT.

<p>Electron micrographs of K8/K18 without surfactant (A) and with 0.01% TX-100 (B). Scale bar represents 100 nm. (C) and (D) show the MSDs without surfactant and with 0.01% TX-100 at <i>c</i> = 1.0 g/l (19.7 µM) as a function of lag time <i>τ</i>. The black line illustrates the ensemble average of the MSDs. The insets show the histogram of the MSDs normalized by the averaged MSD after <i>τ</i> = 1 s.</p

Attractive Interactions among Intermediate Filaments Determine Network Mechanics In Vitro

<div><p>Mechanical and structural properties of K8/K18 and vimentin intermediate filament (IF) networks have been investigated using bulk mechanical rheometry and optical microrheology including diffusing wave spectroscopy and multiple particle tracking. A high elastic modulus <i>G</i>₀ at low protein concentration <i>c</i>, a weak concentration dependency of <i>G</i>₀ (<i>G</i>₀∼<i>c</i>^0.5±0.1) and pronounced strain stiffening are found for these systems even without external crossbridgers. Strong attractive interactions among filaments are required to maintain these characteristic mechanical features, which have also been reported for various other IF networks. Filament assembly, the persistence length of the filaments and the network mesh size remain essentially unaffected when a nonionic surfactant is added, but strain stiffening is completely suppressed, <i>G</i>₀ drops by orders of magnitude and exhibits a scaling <i>G</i>₀∼<i>c</i>^1.9±0.2 in agreement with microrheological measurements and as expected for entangled networks of semi-flexible polymers. Tailless K8Δ/K18ΔT and various other tailless filament networks do not exhibit strain stiffening, but still show high <i>G</i>₀ values. Therefore, two binding sites are proposed to exist in IF networks. A weaker one mediated by hydrophobic amino acid clusters in the central rod prevents stretched filaments between adjacent cross-links from thermal equilibration and thus provides the high <i>G</i>₀ values. Another strong one facilitating strain stiffening is located in the tail domain with its high fraction of hydrophobic amino acid sequences. Strain stiffening is less pronounced for vimentin than for K8/K18 due to electrostatic repulsion forces partly compensating the strong attraction at filament contact points.</p></div

Influence of Triton X-100 on <i>G</i>₀ at <i>ω</i> = 0.5 rad/s.

<p>*exp. errors calculated from st. dev. of at least three independent measurements.</p

Influence of protein concentration on <i>G</i>₀ and comparison of the respective apparent mesh sizes <i>ξ</i>.

<p>(A) presents <i>G</i>₀ data obtained from shear rheology and particle tracking at <i>ω</i> = 1 rad/s as a function of K8/K18 concentration. The dotted line shows the results obtained by equation (4). (B) Comparison of the mesh sizes <i>ξ</i> for different biological filament networks calculated from protein concentration using the cubic grid model in equation (1) with the mesh size calculated from the plateau modulus <i>G</i>₀ according to equation (3). The data used for K8/K18 is the same as in (A). Data for vimentin and desmin were taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093194#pone.0093194-Schopferer1" target="_blank">[7]</a>. Actin data was extracted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093194#pone-0093194-g003" target="_blank">Fig. 3</a> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093194#pone.0093194-Hinner1" target="_blank">[8]</a>. The dark grey zone illustrates the region where the simplistic model can be used to calculate the mesh size of networks from G₀ with an uncertainty of ±0.15 µm. This holds if the filaments are in thermal equilibrium. The light grey area shows that this is not the case for many IF-networks.</p

Schematic representation of the K8/K18 complex and the protein domains responsible for the network viscoelastity.

<p>Illustration adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093194#pone.0093194-Herrmann4" target="_blank">[45]</a>.</p

MC simulations of filaments with a <i>l</i>_p of 1000 nm.

<p>MC simulations of filaments with a <i>l</i>_p of 1000 nm.</p

Length distributions of vimentin intermediate filaments normalized to the persistence length.

<p>Filament length distributions were measured at three assembly times. The proportion of filaments lengths observed of the indicated lengths is shown at the specific time points.</p

Dependency of the radius of gyration and the filament length for different persistence lengths.

<p>Single filaments of various lengths were simulated. After they have been equilibrated 2*10⁹ sweeps were performed. If a maximal bond angel of 15° is allowed (black squares) we found a good agreement with the theoretical prediction for a worm-like chain with a <i>l</i>_p of 1000 nm (black line). For a bond angle of 25° (red circle) a good comparison with the theoretical prediction for a worm-like chain with a <i>l</i>_p of 333 nm (red line) was found.</p

Assembly kinetics of vimentin.

<p>Assembly kinetics of vimentin.</p