Membrane-Bound Vimentin Filaments Reorganize and Elongate under Strain.

Within a cell, intermediate filaments interact with other cytoskeletal components, altogether providing the cell's mechanical stability. However, little attention has been drawn to intermediate filaments close to the plasma membrane. In this cortex configuration, the filaments are coupled and arranged in parallel to the membrane, and the question arises of how they react to the mechanical stretching of the membrane. To address this question, we set out to establish an in vitro system composed of a polydimethylsiloxane-supported lipid bilayer. With a uniaxial stretching device, the supported membrane was stretched up to 34% in the presence of a lipid reservoir that was provided by adding small unilamellar vesicles in the solution. After vimentin attachment to the membrane, we observed structural changes of the vimentin filaments in networks of different densities by fluorescence microscopy and atomic force microscopy. We found that individual filaments respond to the membrane stretching with a reorganization along the stretching direction as well as an intrinsic elongation, while in a dense network, mainly filament reorganization was observed.

Epithelial cells sacrifice excess area to preserve fluidity in response to external mechanical stress.

Viscoelastic properties of epithelial cells subject to shape changes were monitored by indentation-retraction/relaxation experiments. MDCK II cells cultured on extensible polydimethylsiloxane substrates were laterally stretched and, in response, displayed increased cortex contractility and loss of excess surface area. Thereby, the cells preserve their fluidity but inevitably become stiffer. We found similar behavior in demixed cell monolayers of ZO-1/2 double knock down (dKD) cells, cells exposed to different temperatures and after removal of cholesterol from the plasma membrane. Conversely, the mechanical response of single cells adhered onto differently sized patches displays no visible rheological change. Sacrificing excess surface area allows the cells to respond to mechanical challenges without losing their ability to flow. They gain a new degree of freedom that permits resolving the interdependence of fluidity ß on stiffness [Formula: see text]. We also propose a model that permits to tell apart contributions from excess membrane area and excess cell surface area.

Epithelial cells fluidize upon adhesion but display mechanical homeostasis in the adherent state.

Atomic force microscopy is used to study the viscoelastic properties of epithelial cells in three different states. Force relaxation data are acquired from cells in suspension, adhered but single cells, and polarized cells in a confluent monolayer using different indenter geometries comprising flat bars, pyramidal cones, and spheres. We found that the fluidity of cells increased substantially from the suspended to the adherent state. Along this line, the prestress of suspended cells generated by cortical contractility is also greater than that of cells adhering to a surface. Polarized cells that are part of a confluent monolayer form an apical cap that is soft and fluid enough to respond rapidly to mechanical challenges from wounding, changes in the extracellular matrix, osmotic stress, and external deformation. In contrast to adherent cells, cells in the suspended state show a pronounced dependence of fluidity on the external areal strain. With increasing areal strain, the suspended cells become softer and more fluid. We interpret the results in terms of cytoskeletal remodeling that softens cells in the adherent state to facilitate adhesion and spreading by relieving internal active stress. However, once the cells spread on the surface they maintain their mechanical phenotype displaying viscoelastic homeostasis.

Non-negative blind deconvolution for signal processing in a CRISPR-edited iPSC-cardiomyocyte model of dilated cardiomyopathy.

We developed an integrated platform for analysis of parameterized data from human disease models. We report a non-negative blind deconvolution (NNBD) approach to quantify calcium (Ca2+ ) handling, beating force and contractility in human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) at the single-cell level. We employed CRISPR/Cas gene editing to introduce a dilated cardiomyopathy (DCM)-causing mutation in troponin T (TnT), TnT-R141W, into wild-type control iPSCs (MUT). The NNDB-based method enabled data parametrization, fitting and analysis in wild-type controls versus isogenic MUT iPSC-CMs. Of note, Cas9-edited TnT-R141W iPSC-CMs revealed significantly reduced beating force and prolonged contractile event duration. The NNBD-based platform provides an alternative framework for improved quantitation of molecular disease phenotypes and may contribute to the development of novel diagnostic tools.

Prestress and Area Compressibility of Actin Cortices Determine the Viscoelastic Response of Living Cells.

Shape, dynamics, and viscoelastic properties of eukaryotic cells are primarily governed by a thin, reversibly cross-linked actomyosin cortex located directly beneath the plasma membrane. We obtain time-dependent rheological responses of fibroblasts and MDCK II cells from deformation-relaxation curves using an atomic force microscope to access the dependence of cortex fluidity on prestress. We introduce a viscoelastic model that treats the cell as a composite shell and assumes that relaxation of the cortex follows a power law giving access to cortical prestress, area-compressibility modulus, and the power law exponent (fluidity). Cortex fluidity is modulated by interfering with myosin activity. We find that the power law exponent of the cell cortex decreases with increasing intrinsic prestress and area-compressibility modulus, in accordance with previous finding for isolated actin networks subject to external stress. Extrapolation to zero tension returns the theoretically predicted power law exponent for transiently cross-linked polymer networks. In contrast to the widely used Hertzian mechanics, our model provides viscoelastic parameters independent of indenter geometry and compression velocity.