Intermediate filament (IF) networks are a major contributor to cell rigidity and thus serve as vital elements to preserve the integrity of entire cell layers. Keratin K8 and K18 IFs are the basic constituents of the cytoskeleton of epithelial cells. The mechanical properties of K8/K18 networks depend on the structural arrangements of individual filaments within the network. This paper investigates the architecture of these networks in vitro under the influence of the monovalent cation potassium and that of the cytolinker protein plectin. Whereas increasing amounts of potassium ions lead to filament bundling, plectin interlinks filaments at filament intersection points but does not lead to bundle formation. The mechanics of the resulting networks are investigated by microrheology with assembled K8/K18 networks. It is shown that bundling induced by potassium ions significantly stiffens the network. Furthermore, our measurements reveal an increase in plectin-mediated keratin network rigidity as soon as an amount corresponding to more than 20% of the plectin present in cells is added to the keratin IF networks. In parallel, we investigated the influence of plectin on cell rigidity in detergentextracted epithelial vulva carcinoma derived A431 cells in situ. These cytoskeletons, containing mostly IFs, actin filaments and associated proteins, exhibit a significantly decreased stiffness, when plectin is downregulated to E10% of the normal value. Therefore, we assume that plectin, via the formation of IF-IF connections and crosslinking of IFs to actin filaments, is an important contributor to cell stiffness.
SummaryActive microrheology is a valuable tool to determine viscoelastic properties of polymer networks. Observing the response of the beads to the excitation of a reference leads to dynamic and morphological information of the material. In this work we present an expansion of the well-known active two-point microrheology. By measuring the response of multiple particles in a viscoelastic medium in response to the excitation of a reference particle, we are able to determine the force propagation in the polymer network. For this purpose a lock-in technique is established that allows for extraction of the periodical motion of embedded beads. To exert a sinusoidal motion onto the reference bead an optical tweezers setup in combination with a microscope is used to investigate the motion of the response beads. From the lock-in data the so called transfer tensor can be calculated, which is a direct measure for the ability of the network to transmit mechanical forces. We also take a closer look at the influence of noise on lock-in measurements and state some simple rules for improving the signal-to-noise ratio.
The cytoskeleton of epithelial cells consists of three types of filament systems: microtubules, actin filaments and intermediate filaments (IFs). Here, we took a closer look at type I and type II IF proteins, i.e. keratins. They are hallmark constituents of epithelial cells and are responsible for the generation of stiffness, the cellular response to mechanical stimuli and the integrity of entire cell layers. Thereby, keratin networks constitute an important instrument for cells to adapt to their environment. In particular, we applied models to characterize the assembly of keratin K8 and K18 into elongated filaments as a means for network formation. For this purpose, we measured the length of in vitro assembled keratin K8/K18 filaments by transmission electron microscopy at different time points. We evaluated the experimental data of the longitudinal annealing reaction using two models from polymer chemistry: the Schulz–Zimm model and the condensation polymerization model. In both scenarios one has to make assumptions about the reaction process. We compare how well the models fit the measured data and thus determine which assumptions fit best. Based on mathematical modelling of experimental filament assembly data we define basic mechanistic properties of the elongation reaction process.
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