Contractility Dominates Adhesive Ligand Density in Regulating Cellular De-adhesion and Retraction Kinetics

Abstract

Cells that are enzymatically detached from a solid substrate rapidly round up as the tensile prestress in the cytoskeleton is suddenly unopposed by cell–ECM adhesions. We recently showed that this retraction follows sigmoidal kinetics with time constants that correlate closely with cortical stiffness values. This raises the promising prospect that these de-adhesion measurements may be used for high-throughput screening of cell mechanical properties; however, an important limitation to doing so is the possibility that the retraction kinetics may also be influenced and potentially rate-limited by the time needed to sever matrix adhesions. In this study, we address this open question by separating contributions of contractility and adhesion to cellular de-adhesion and retraction kinetics. We first develop serum-free conditions under which U373 MG glioma cells can be cultured on substrates of fixed fibronectin density without direct matrix contributions from the medium. We show that while spreading area increases with ECM protein density, cortical stiffness and the time constants of retraction do not. Conversely, addition of lysophosphatidic acid (LPA) to stimulate cell contractility strongly speeds retraction, independent of the initial matrix protein density and LPA’s contributions to spreading area. All of these trends hold in serum-rich medium commonly used in tissue culture, with the time constants of retraction much more closely tracking cortical stiffness than adhesive ligand density or cell spreading. These results support the use of cellular de-adhesion measurements to track cellular mechanical properties.

Probing Cellular Mechanoadaptation Using Cell-Substrate De-Adhesion Dynamics: Experiments and Model

“…Specifically, for a given pre-stretch, with increase in substrate stiffness, pre-stress initially increases and then saturates on stiffer substrates. Furthermore, on a substrate of given stiffness, as contractility increases, cell de-adhere faster, as is experimentally observed in faster de-adhesion of cells treated with contractility-activating drugs like LPA and nocodazole [9], [10]. Collectively, these results demonstrate how substrate stiffness influences de-adhesion dynamics via modulation of cell contractility.…”

“…Consequently, de-adhesion timescales depend both on the initial state of the cystokeleton and the rate of bond breakage. This has been demonstrated in previous experiments where contractile stimulation via drugs including LPA and nocodazole, or faster dis-assembly of adhesions effected by using a higher concentration of trypsin, both led to faster de-adhesion [9], [10]. Such a coupled dependency of on the two factors can be appreciated from the analytical expression  = .…”

Constitutive Activation of Myosin-Dependent Contractility Sensitizes Glioma Tumor-Initiating Cells to Mechanical Inputs and Reduces Tissue Invasion

“…It has also been shown that continuous culture models of GBM require myosin II and its upstream regulators to sense and respond to ECM rigidity (4,5,37,38). However, it has remained unclear if these principles apply to primary TICs, or if manipulation of the mechanosensing machinery can influence tumor initiation and progression in vivo .…”

Platelet mechanosensing of substrate stiffness during clot formation mediates adhesion, spreading, and activation

“…or density (15)(16)(17). More specifically, one can modulate the stiffness of these materials as an isolated variable by manipulating cross-linking density in ways that do not require addition of more matrix ligand (i.e., fibrinogen), which could independently alter platelet function and confound interpretation of results.…”