Rheological Characterization of the Bundling Transition in F-Actin Solutions Induced by Methylcellulose

In many in vitro experiments Brownian motion hampers quantitative data analysis. Therefore, additives are widely used to increase the solvent viscosity. For this purpose, methylcellulose (MC) has been proven highly effective as already small concentrations can significantly slow down diffusive processes. Beside this advantage, it has already been reported that high MC concentrations can alter the microstructure of polymer solutions such as filamentous actin. However, it remains to be shown to what extent the mechanical properties of a composite actin/MC gel depend on the MC concentration. In particular, significant alterations might occur even if the microstructure seems unaffected. Indeed, we find that the viscoelastic response of entangled F-actin solutions depends sensitively on the amount of MC added. At concentrations higher than 0.2% (w/v) MC, actin filaments are reorganized into bundles which drastically changes the viscoelastic response. At small MC concentrations the impact of MC is more subtle: the two constituents, actin and MC, contribute in an additive way to the mechanical response of the composite material. As a consequence, the effect of methylcellulose on actin solutions has to be considered very carefully when MC is used in biochemical experiments

Micro- and Macrorheological Properties of Actin Networks Effectively Cross-Linked by Depletion Forces

The structure and rheology of cytoskeletal networks are regulated by actin binding proteins. Aside from these specific interactions, depletion forces can also alter the properties of cytoskeletal networks. Here we demonstrate that the addition of poly(ethylene glycol) (PEG) as a depletion agent results not only in severe structural changes, but also in alterations in mechanical properties of actin solutions. In the plateau of the elastic modulus two regimes can be distinguished by micro and macrorheological methods. In the first, the elastic modulus increases only slightly with increasing depletion agent, whereas above a critical concentration c*, a strong increase of [Formula: see text] is observed in a distinct second regime. Microrheological data and electron microscopy images show a homogenous network of actin filaments in the first regime, whereas at higher PEG concentrations a network of actin bundles is observed. The concentration dependence of the plateau modulus G(0), the shift in entanglement time τ(e), and the nonlinear response indicate that below c* the network becomes effectively cross-linked, whereas above c* G(0)(c(PEG6k)) is primarily determined by the network of bundles that exhibits a linearly increasing bundle thickness

Microstructure and viscoelasticity of confined semiflexible polymer networks

The rapidly decreasing dimensions of many technological devices have spurred interest in confinement effects1. Long before, living organisms invented ingenious ways to cope with the requirement of space saving designs down to the cellular level. Typical length scales in cells range from nanometres to micrometres so that the polymeric constituents of the cytoskeleton are often geometrically confined. Hence, the mechanical response of polymers to external confinement has potential implications both for technology and for our understanding of biological systems alike. Here we report a study of in vitro polymerized filamentous actin confined to emulsion droplets. We correlate observations of the microstructure, local rheological properties and single filament fluctuations. Enforcing progressively narrower confinement is found to induce a reduction of polymer fluctuations, network stiffening, structural heterogeneities and eventually cortex formation. We argue that the structural and mechanical effects can be consistently explained by a gradual suppression of single polymer eigenmodes

A Bmp Reporter Transgene Mouse Embryonic Stem Cell Model as a Tool to Identify and Characterize Chemical Teratogens

Stem cells & developmental biolog

Biomimetic models of the actin cytoskeleton

The cytoskeleton is a complex polymer network that plays an essential role in the functionality of eukaryotic cells. It endows cells with mechanical stability, adaptability, and motility. To identify and understand the mechanisms underlying this large variety of capabilities and to possibly transfer them to engineered networks makes it necessary to have in vitro and in silico model systems of the cytoskeleton. These models must be realistic representatives of the cellular network and at the same time be controllable and reproducible. Here, an approach to design complementary experimental and numerical model systems of the actin cytoskeleton is presented and some of their properties discussed

Biomimetic models of the actin cytoskeleton

The cytoskeleton is a complex polymer network that plays an essential role in the functionality of eukaryotic cells. It endows cells with mechanical stability, adaptability, and motility. To identify and understand the mechanisms underlying this large variety of capabilities and to possibly transfer them to engineered networks makes it necessary to have in vitro and in silico model systems of the cytoskeleton. These models must be realistic representatives of the cellular network and at the same time be controllable and reproducible. Here, an approach to design complementary experimental and numerical model systems of the actin cytoskeleton is presented and some of their properties discussed

Testing of the toxicity of volatile compounds on human lung cells using the Air/Liquid Interface (ALI) culturing and exposure technique: A prevalidation study

Actually, precision cut lung slices are under extensive investigation to serve as a model for acute inhalation toxicology. Slices can be employed as an ex-vivo/in-vitro model as alternative method in the context of REACH and the 3Rs. Moreover, this in vitro model represents in vivo tissue including all naturally occurring cell types of the respiratory tract situated in their physiological environment. For the reason of these beneficial characteristics, PCLS may be an in vitro model of first choice in the future, especially with respect to studies on inflammatory changes in the lung. However, since safety control and toxicology demand studies on genotoxicity, we also started to establish a Comet assay with live lung tissue in vitro from mouse lungs to expand the experimental possibilities from investigations with PCLS on toxicity and inflammatory responses to genotoxicity. The work focused on optimization of a simple and fast cell separation method by enzymatic digestion of the lung tissue, application of the alkaline Comet assay and exposure of lung slices to culture media (negative control) and test compounds in a first series of experiments. Ethyl methanesulfonate (EMS) and formalin (FA) were tested as model substances known to induce DNA damage. These alterations can be observed in the Comet assay as, respectively, an increased rate of DNA breaks (EMS) or a reduced rate of DNA breaks by induction of cross-links (FA). Factors of concern for the reproducibility and meaningfulness of results obtained with the method established here to investigate genotoxicity induced in this in-vivo-like live complex cell system by application of the Comet assay to PCLS are discussed. These include the slice-to-slice- and individual-to-individual-reproducibility as well as the background signal and the dose-dependency of the effects induced by the model substances. In summary, the experiments showed that dose-dependent effects induced by EMS and FA could be detected in mouse PCLS, and by application of the Comet assay, effects from strand-breaking and cross-linking substances could be reproducibly discriminated