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Scientific Unit
The mechanical properties of cells are important for many biological processes, including wound healing, cancers, and embryogenesis. Currently, our understanding of cell mechanical properties remains incomplete. Different techniques have been used to probe different aspects of the mechanical properties of cells, among them microplate rheology, optical tweezers, micropipette aspiration, and magnetic twisting cytometry. These techniques have given rise to different theoretical descriptions, reaching from simple Kelvin-Voigt or Maxwell models to fractional such as power law models, and their combinations. Atomic force microscopy (AFM) is a flexible technique that enables global and local probing of adherent cells. Here, using an AFM, we indented single retinal pigmented epithelium cells adhering to the bottom of a culture dish. The indentation was performed at two locations: above the nucleus, and towards the periphery of the cell. We applied creep compliance, stress relaxation, and oscillatory rheological tests to wild type and drug modified cells. Considering known fractional and semi-fractional descriptions, we found the extracted parameters to correlate. Moreover, the Young’s modulus as obtained from the initial indentation strongly correlated with all of the parameters from the applied power-law descriptions. Our study shows that the results from different rheological tests are directly comparable. This can be used in the future, for example, to reduce the number of measurements in planned experiments. Apparently, under these experimental conditions, the cells possess a limited number of degrees of freedom as their rheological properties change.
The cellular cortex is an approximately 200-nm-thick actin network that lies just beneath the cell membrane. It is responsible for the mechanical properties of cells, and as such, it is involved in many cellular processes, including cell migration and cellular interactions with the environment. To develop a clear view of this dense structure, high-resolution imaging is essential. As one such technique, electron microscopy, involves complex sample preparation procedures. The final drying of these samples has significant influence on potential artifacts, like cell shrinkage and the formation of artifactual holes in the actin cortex. In this study, we compared the three most used final sample drying procedures: critical-point drying (CPD), CPD with lens tissue (CPD-LT), and hexamethyldisilazane drying. We show that both hexamethyldisilazane and CPD-LT lead to fewer artifactual mesh holes within the actin cortex than CPD. Moreover, CPD-LT leads to significant reduction in cell height compared to hexamethyldisilazane and CPD. We conclude that the final drying procedure should be chosen according to the reduction in cell height, and so CPD-LT, or according to the spatial separation of the single layers of the actin cortex, and so hexamethyldisilazane.
The rapid development of advanced microscopy techniques over recent decades has significantly increased the quality of imaging and our understanding of subcellular structures, such as the organization of the filaments of the cytoskeleton using fluorescence and electron microscopy. However, these recent improvements in imaging techniques have not been matched by similar development of techniques for computational analysis of the images of filament networks that can now be obtained. Hence, for a wide range of applications, reliable computational analysis of such two-dimensional methods remains challenging. Here, we present a new algorithm for tracing of filament networks. This software can extract many important parameters from grayscale images of filament networks, including the mesh hole size, and filament length and connectivity (also known as Coordination Number). In addition, the method allows sub-networks to be distinguished in two-dimensional images using intensity thresholding. We show that the algorithm can be used to analyze images of cytoskeleton networks obtained using different advanced microscopy methods. We have thus developed a new improved method for computational analysis of two-dimensional images of filamentous networks that has wide applications for existing imaging techniques. The algorithm is available as open-source software.
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 obtaintime-dependent rheological responses of fibroblasts and MDCK II cells from deformation-relaxationcurves using an atomic force microscope to access the dependence of cortex fluidity on pre-stress. We introduce a viscoelastic model that treats the cell as a composite shell and assumes thatrelaxation of the cortex follows a power law giving access to cortical pre-stress, area compressibilitymodulus, and the power law (fluidity) exponent. Cortex fluidity is modulated by interferingwith myosin activity. We find that the power law exponent of the cell cortex decreases withincreasing intrinsic pre-stress and area compressibility modulus, in accordance with previousfinding for isolated actin networks subject to external stress. Extrapolation to zero tension returnsthe theoretically predicted power law exponent for transiently cross-linked polymer networks. In contrast to the widely used Hertzian mechanics, our model provides viscoelastic parametersindependent of indenter geometry and compression velocity.
The cellular cortex is an approximately 200-nm-thick actin network that lies just beneath the cell membrane. It is responsible for the mechanical properties of cells, and as such, it is involved in many cellular processes, including cell migration and cellular interactions with the environment. To develop a clear view of this dense structure, high-resolution imaging is essential. As one such technique, electron microscopy, involves complex sample preparation procedures. The final drying of these samples has significant influence on potential artifacts, like cell shrinkage and the formation of artifactual holes in the actin cortex. In this study, we compared the three most used final sample drying procedures: critical-point drying (CPD), CPD with lens tissue (CPD-LT), and hexamethyldisilazane drying. We show that both hexamethyldisilazane and CPD-LT lead to fewer artifactual mesh holes within the actin cortex than CPD. Moreover, CPD-LT leads to significant reduction in cell height compared to hexamethyldisilazane and CPD. We conclude that the final drying procedure should be chosen according to the reduction in cell height, and so CPD-LT, or according to the spatial separation of the single layers of the actin cortex, and so hexamethyldisilazane.Competing Interest StatementThe authors have declared no competing interest.
