Cortical cell stiffness is independent of substrate mechanics

Abstract

Cortical stiffness is an important cellular property that changes during migration, adhesion, and growth. Previous atomic force microscopy (AFM) indentation measurements of cells cultured on deformable substrates suggested that cells adapt their stiffness to that of their surroundings. Here we show that the force applied by AFM onto cells results in a significant deformation of the underlying substrate if it is softer than the cells. This 'soft substrate effect' leads to an underestimation of a cell's elastic modulus when analyzing data using a standard Hertz model, as confirmed by finite element modelling (FEM) and AFM measurements of calibrated polyacrylamide beads, microglial cells, and fibroblasts. To account for this substrate deformation, we developed the 'composite cell-substrate model' (CoCS model). Correcting for the substrate indentation revealed that cortical cell stiffness is largely independent of substrate mechanics, which has significant implications for our interpretation of many physiological and pathological processes.

Atomic force microscopy reveals the mechanical properties of breast cancer bone metastases

“…Last but not least, a recent study has reported that AFM indentation can push cells into soft substrates. 40 Consequently, the resultant moduli are influenced by the properties of the soft components under the cells, and thus are significantly lower than the moduli of the cells themselves. This is likely happening in tissues, where cells sit in soft extracellular matrices.…”

Effective cell membrane tension is independent of polyacrylamide substrate stiffness

“…Another possibility is that traction forces transmitted to the cortex might dissipate quickly so that overall cortical tension would not scale with local traction forces 22 . If cortical and membrane tension are coupled, changes in membrane tension on the different hydrogels would happen only locally 29 near focal adhesions and cannot be seen when probed further away.…”

“…In contrast, actomyosin-based traction forces scale with substrate stiffnesses for both neurons and fibroblasts 22 . Also in our culture conditions, cellular traction forces significantly increased on stiffer substrates in both cell types within the investigated stiffness range (Fig.…”

“…Tether forces on hydrogels but not SSFs on glass are independent of actomyosin contractility Cellular cortical tension is mediated by actomyosin contractility 20 . In order to further examine the interplay between cortical tension and membrane tension, we measured tether forces in 6 fibroblasts cultured on glass and 10 kPa hydrogels, where we would expect the highest cortical tension 22 , following treatment with the myosin inhibitor blebbistatin.…”

Measuring the elastic modulus of soft culture surfaces and three-dimensional hydrogels using atomic force microscopy

“…Here, it should be noted that AFM, in general, is a surface characterization technique. However, on materials like hydrogels, indentations with a spherical probe will result in deformation many microns deep into the hydrogel 60 . Thus, the resulting mechanical response will be that of a combination of the overlying hydrogel, the cells or organoids themselves, and their surrounding matrix 59 .…”