Abstract
The measurement of local mechanical properties of living cells by nano/micro indentation relies on the foundational assumption of locally isotropic cellular deformation. As a consequence of assumed isotropy, the cell membrane and underlying cytoskeleton are expected to locally deform axisymmetrically when indented by a spherical tip. Here, we directly observe the local geometry of deformation of membrane and cytoskeleton of different living adherent cells during nanoindentation with the integrated Atomic Force (AFM) and spinning disk confocal (SDC) microscope. We show that the presence of the perinuclear actin cap (apical stress fibers), such as those encountered in cells subject to physiological forces, causes a strongly non-axisymmetric membrane deformation during indentation reflecting local mechanical anisotropy. In contrast, axisymmetric membrane deformation reflecting mechanical isotropy was found in cells without actin cap: cancerous cells MDA-MB-231, which naturally lack the actin cap, and NIH 3T3 cells in which the actin cap is disrupted by latrunculin A. Careful studies were undertaken to quantify the effect of the live cell fluorescent stains on the measured mechanical properties. Using finite element computations and the numerical analysis, we explored the capability of one of the simplest anisotropic models – transverse isotropy model with three local mechanical parameters (longitudinal and transverse modulus and planar shear modulus) – to capture the observed non-axisymmetric deformation. These results help identifying which cell types are likely to exhibit non-isotropic properties, how to measure and quantify cellular deformation during AFM indentation using live cell stains and SDC, and suggest modelling guidelines to recover quantitative estimates of the mechanical properties of living cells.
Highlights
Recent developments in fluorescent live-cell imaging and biophysical methods have significantly advanced our understanding of the dynamic biochemical and mechanical processes underlying cellular functions such as cell migration
We found a strong correlation between presence of the perinuclear actin cap fibers and cell mechanical properties; highly anisotropic indentation geometry was found in cells with actin cap
In NIH 3T3 fibroblasts, most of the F-actin was found in the form of stress fibers, whereas MDA-MB-231 cells exhibited a more irregular and disorganized F-actin network www.nature.com/scientificreports located underneath the plasma membrane (Fig. 1a), which is in agreement with previous reports for different types of cancer cells[35,36]
Summary
Recent developments in fluorescent live-cell imaging and biophysical methods have significantly advanced our understanding of the dynamic biochemical and mechanical processes underlying cellular functions such as cell migration. Without the visualization of the cell structure and geometry of deformation simultaneously during cell indentation, it is extremely difficult, if not impossible, to verify if many underlying assumptions of the model are met. Such simultaneous visualization can help assess how the inhomogeneity of the cell structure affects the indentation; how the underlying cytoskeleton behaves to produce observed cellular mechanical behaviour; and to check the presence of any effects of the indentation on cells, like distant cytoskeletal rearrangements, residual damage or induced mechanoresponse[12,13,14,15,16,17,18,19,20,21,22,23,24]. Our observations suggest a significant role of an anisotropic deformability and stiffness in the mechanics of cells
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