Spatiotemporal Analyses of Cellular Tractions Describe Subcellular Effect of Substrate Stiffness and Coating

Abstract

Cells interplay with their environment through mechanical and chemical interactions. To characterize this interplay, endothelial cells were cultured on polyacrylamide hydrogels of varying stiffness, coated with either fibronectin or collagen. We developed a novel analysis technique, complementary to traction force microscopy, to characterize the spatiotemporal evolution of cellular tractions: We identified subpopulations of tractions, termed traction foci, and tracked their magnitude and lifetime. Each focus consists of tractions associated with a local single peak of maximal traction. Individual foci were spread over a larger area in cells cultured on collagen relative to those on fibronectin and exerted higher tractions on stiffer hydrogels. We found that the trends with which forces increased with increasing hydrogel stiffness were different for foci and whole-cell measurements. These differences were explained by the number of foci and their average strength. While on fibronectin multiple short-lived weak foci contributed up to 30% to the total traction on hydrogels with intermediate stiffness, short-lived foci in such a number were not observed on collagen despite the higher tractions. Our approach allows for the use of existing traction force microscopy data to gain insight at the subcellular scale without molecular probes or spatial constraining of cellular tractions. KEY TERMS cell mechanics; traction force microscopy; endothelial cell; ECM protein MATERIALS AND METHODS Polyacrylamide hydrogel preparation and functionalization Polyacrylamide (PA) hydrogels were prepared as described by Tse et al. (22). To obtain the desired stiffness values (1.4, 2.7, and 4.5 kPa), hydrogels were prepared by mixing different volumes of 40% acrylamide, 2% bis-acrylamide (Bio-Rad, Hercules, California) with distilled water. Stiffness of the gels was corroborated using Atomic Force Microscopy (AFM) (see Supplemental Materials for details). As fiducial markers for TFM, 200 nm carboxylate-modified microspheres (FluoSpheres®, ThermoFisher Scientific, Waltham, MA) were added to the mixture at a 1:60 ratio. Hydrogels were functionalized with either 5 μg/ml of human fibronectin (Sigma-Aldrich, St. Louis, MO) or 100 μg/ml rat tail collagen I (BD bioscience, San Jose, CA). Even though the ECM protein concentrations used are different, in both situations the gels are saturated with the ECM protein, meaning the entire surface of the gel is covered by the coating molecule without any gaps of the order of magnitude of the traction foci (Figure S1 in Supplemental Materials). For a more detailed description of the hydrogel preparation and functionalization processes, see Supplemental Materials. Cell culture Green fluorescence protein (GFP)-expressing human umbilical vein endothelial cells (HUVECs) (Angioproteomie, Boston, MA), were seeded at low density on polyacrylamide hydrogels with desired stiffness. Cells were allowed to attach and spread overnight before imaging. For a more detailed description of the culture setup, see Supplemental...

Cells on Hydrogels with Micron-Scaled Stiffness Patterns Demonstrate Local Stiffness Sensing

“…As a result, we found that the cells actually probe the local, micron-scaled, mechanical properties of the matrix with traction stresses that are proportional to the local rigidity, at least in the range of rigidity we explored, from 0.4 to 12.4 kPa (Figure 4F). This observation was consistent with data reported at the cell scale, on substrates with uniform stiffness [63,64]. Larger stresses were thus observed on the stiff dots, more specifically in places of large paxillin-stained focal adhesions (Figure 4D).…”

Modeling of Mechanosensing Mechanisms Reveals Distinct Cell Migration Modes to Emerge From Combinations of Substrate Stiffness and Adhesion Receptor–Ligand Affinity

“…This meant that for a certain range of k ECM values, cell traction decreased with increasing stiffness. An increase in cellular traction was observed for the k ECM range (0.25,3) kPa; this behavior was observed for the same range in Human Umbilical Vein Endothelial Cells (HUVECs) (Izquierdo-Álvarez et al, 2018). In this study, HUVECs were shown to exert a total force between 0.1 and 0.45 µN.…”

“…Cells are highly active systems that interact with their environment. Multiple studies have demonstrated that substrate stiffness affects the magnitude of tractions exerted on the substrate; in some cases, a stiffer substrate will result in higher tractions (Ghibaudo et al, 2008;Califano and Reinhart-King, 2010;Izquierdo-Álvarez et al, 2018), while in others the opposite is observed (Chan and Odde, 2008). This observation has been credited with resulting in cell migration speed also being affected by substrate stiffness (Pathak and Kumar, 2012).…”

The role of cellular traction forces in deciphering nuclear mechanics

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