Mechanical and morphological response of confluent epithelial cell layers to reinforcement and dissolution of the F-actin cytoskeleton.

The F-actin cytoskeleton and its connection to the plasma membrane provide structure and shape of epithelial cells. In this study we focus on the impact of the F-actin cytoskeleton on the morphology and mechanical behaviour of confluent epithelial cells. F-actin depolymerisation was fostered by Latrunculin A, while depolymerisation was allayed by Jasplakinolide. The impact of drug treatment on cellular mechanics was measured using atomic force microscopy based active microrheology and force-indentation curves, while morphology was monitored by AFM imaging, electric cell-substrate impedance sensing (ECIS) experiments and fluorescence microscopy. A softening and fluidisation of the cells upon dissolution of F-actin was observed, accompanied by reduction of cell-substrate and cell-cell contacts and an altered topography. The strengthening of actin filaments upon Jasplakinolide treatment was mirrored in several mechanical properties. The largest impact was on the cellular viscosity. The cells were, however, capable of restoring their initial phenotypes, e.g., amount of actin, intercellular and cell-substrate interactions.

Importance of integrity of cell-cell junctions for the mechanics of confluent MDCK II cells.

Intercellular junctions are important mechanical couplers between cells in epithelial layers providing adhesion and intercellular communication. Regulation of the junctions occurs in cellular processes such as layer formation, epithelial-to-mesenchymal transition, embryogenesis, and cancer progression. Many studies addressed the role of force generation in cells for establishing lateral cell-cell junctions and the role of cellular force transmission in tissue formation and maintenance. Our atomic force microscopy- (AFM) based study shed light on the role of both, tight junctions and adherens junctions for the mechanical properties of individual epithelial cells that are part of a confluent monolayer. We found that tight junctions are important for the establishment of a functional barrier-forming layer but impairing them does not reduce the mechanical integrity of cells. Depletion of ZO-1 results in a weak increase in cortical tension. An opposite effect was observed for disruption of E-cadherin-mediated adherens junctions using DTT. Opening of adherens junctions leads to substantial alterations of cellular mechanics such as reduced overall stiffness, but these changes turned out to be reversible after re-establishing disulfide bridges in E-cadherin by removal of DTT. We found that regulatory mechanisms exist that preserve mechanical integrity during recovery of disrupted adherens junctions.

Cell-Substrate Dynamics of the Epithelial-to-Mesenchymal Transition.

The biological process of the epithelial-to-mesenchymal transition (EMT) allows epithelial cells to enhance their migratory and invasive behavior and plays a key role in embryogenesis, fibrosis, wound healing, and metastasis. Among the multiple biochemical changes from an epithelial to a mesenchymal phenotype, the alteration of cellular dynamics in cell-cell as well as cell-substrate contacts is crucial. To determine these variations over the whole time scale of the EMT, we measure the cell-substrate distance of epithelial NMuMG cells during EMT using our newly established metal-induced energy transfer (MIET) microscopy, which allows one to achieve nanometer axial resolution. We show that, in the very first hours of the transition, the cell-substrate distance increases substantially, but later in the process after reaching the mesenchymal state, this distance is reduced again to the level of untreated cells. These findings relate to a change in the number of adhesion points and will help to better understand remodeling processes associated with wound healing, embryonic development, cancer progression, or tissue regeneration.

Viscoelastic Properties of Confluent MDCK II Cells Obtained from Force Cycle Experiments.

The local mechanical properties of cells are frequently probed by force indentation experiments carried out with an atomic force microscope. Application of common contact models provides a single parameter, the Young's modulus, to describe the elastic properties of cells. The viscoelastic response of cells, however, is generally measured in separate microrheological experiments that provide complex shear moduli as a function of time or frequency. Here, we present a straightforward way to obtain rheological properties of cells from regular force distance curves collected in typical force indentation measurements. The method allows us to record the stress-strain relationship as well as changes in the weak power law of the viscoelastic moduli. We derive an analytical function based on the elastic-viscoelastic correspondence principle applied to Hertzian contact mechanics to model both indentation and retraction curves. Rheological properties are described by standard viscoelastic models and the paradigmatic weak power law found to interpret the viscoelastic properties of living cells best. We compare our method with atomic force microscopy-based active oscillatory microrheology and show that the method to determine the power law coefficient is robust against drift and largely independent of the indentation depth and indenter geometry. Cells were subject to Cytochalasin D treatment to provoke a drastic change in the power law coefficient and to demonstrate the feasibility of the approach to capture rheological changes extremely fast and precisely. The method is easily adaptable to different indenter geometries and acquires viscoelastic data with high spatiotemporal resolution.

Mechanical properties of MDCK II cells exposed to gold nanorods.

BACKGROUND: The impact of gold nanoparticles on cell viability has been extensively studied in the past. Size, shape and surface functionalization including opsonization of gold particles ranging from a few nanometers to hundreds of nanometers are among the most crucial parameters that have been focussed on. Cytoxicity of nanomaterial has been assessed by common cytotoxicity assays targeting enzymatic activity such as LDH, MTT and ECIS. So far, however, less attention has been paid to the mechanical parameters of cells exposed to gold particles, which is an important reporter on the cellular response to external stimuli. RESULTS: Mechanical properties of confluent MDCK II cells exposed to gold nanorods as a function of surface functionalization and concentration have been explored by atomic force microscopy and quartz crystal microbalance measurements in combination with fluorescence and dark-field microscopy. CONCLUSION: We found that cells exposed to CTAB coated gold nanorods display a concentration-dependent stiffening that cannot be explained by the presence of CTAB alone. The stiffening results presumably from endocytosis of particles removing excess membrane area from the cell's surface. Another aspect could be the collapse of the plasma membrane on the actin cortex. Particles coated with PEG do not show a significant change in elastic properties. This observation is consistent with QCM measurements that show a considerable drop in frequency upon administration of CTAB coated rods suggesting an increase in acoustic load corresponding to a larger stiffness (storage modulus).