Nonlinear Cellular Mechanical Behavior Adaptation to Substrate Mechanics Identified by Atomic Force Microscope

Abstract

Cell–substrate interaction plays an important role in intracellular behavior and function. Adherent cell mechanics is directly regulated by the substrate mechanics. However, previous studies on the effect of substrate mechanics only focused on the stiffness relation between the substrate and the cells, and how the substrate stiffness affects the time-scale and length-scale of the cell mechanics has not yet been studied. The absence of this information directly limits the in-depth understanding of the cellular mechanotransduction process. In this study, the effect of substrate mechanics on the nonlinear biomechanical behavior of living cells was investigated using indentation-based atomic force microscopy. The mechanical properties and their nonlinearities of the cells cultured on four substrates with distinct mechanical properties were thoroughly investigated. Furthermore, the actin filament (F-actin) cytoskeleton of the cells was fluorescently stained to investigate the adaptation of F-actin cytoskeleton structure to the substrate mechanics. It was found that living cells sense and adapt to substrate mechanics: the cellular Young’s modulus, shear modulus, apparent viscosity, and their nonlinearities (mechanical property vs. measurement depth relation) were adapted to the substrates’ nonlinear mechanics. Moreover, the positive correlation between the cellular poroelasticity and the indentation remained the same regardless of the substrate stiffness nonlinearity, but was indeed more pronounced for the cells seeded on the softer substrates. Comparison of the F-actin cytoskeleton morphology confirmed that the substrate affects the cell mechanics by regulating the intracellular structure.

Effect of F-actin and Microtubules on Cellular Mechanical Behavior Studied Using Atomic Force Microscope and an Image Recognition-Based Cytoskeleton Quantification Approach

“…During the indenting process, the used AFM probe was spherical and its indenting speed at 20 µm/s was significantly faster than the intracellular fluid efflux rate [28]. Therefore, the measured cell can be treated as incompressible.…”

“…Therefore, the probe-cell interaction could be treated as a poroelastic half-space indented by a sphere indenter during the force-relaxation process. The diffusion coefficient, D, was quantified using the empirical mathematical model as follows [28,29],…”

An Image Recognition-Based Approach to Actin Cytoskeleton Quantification

“…Moreover, the changes of F-actin orientation and quantity due to different latrunculin B treatment dosages are clearly shown in the F-actin fluorescence images. Therefore, it is clear that the proposed IRAQ is quite sensitive to detecting the F-actin cytoskeleton change, and the comparison indicated that IRAQ can be reliably used in further research to study the quantitative relation between the actin cytoskeleton morphology and the cellular mechanical properties, as previous studies have shown that latrunculin B treatment tends to alter the cell mechanical properties significantly [9][10][11][12].…”

“…As one of the three main components of the cellular cytoskeleton, the actin cytoskeleton (i.e., actin filaments) controls cell mechanotransduction dynamics through directly affecting the motility, contractility, and mechanical stability of the cells [2,6,8]. Specifically, the actin cytoskeleton responds to the external stimuli, such as the change of the substrate stiffness and external forces, applied on the cells through polymerization or depolymerization in order to regulate cellular physiological properties [9][10][11][12]. Therefore, the morphology of the actin cytoskeleton is of great importance in understanding the cellular dynamic mechanotransduction responses.…”

Simultaneous Quantification of the Interplay Between Molecular Turnover and Cell Mechanics by AFM–FRAP

“…Using AFM electronic feedback loops allows the recording of nanoscale force indentations of the cell surface as a function of the applied constant mechanical force. For example, this type of approach has been applied to investigate the biological behavior and function of living cells in response to external mechanical force …”