Lifetime of Membrane-Cytoskeleton Bonds is Mediated by RHO-GTPases in a Cancer Cell

Abstract

Cells especially cancer cells are capable of rapid cytoskeleton remodeling and often membrane reorganization in response to environmental cues. The propensity to remodel is reflected in part by the lifetime τ of the membrane-cytoskeleton bonds at the edges of cells; τ increases as their number density and on-rate increases. We measure τ by monitoring the time-course of the membrane peeling force Fp at the edge of a cancer cell. Experiments are conducted at the near-equilibrium region (cf. irreversible), where an optical tweezers is used to apply a load with a handle which is bound to a slowly moving cell, and the handle displacement is detected at a resolution of 500 μs after averaging.Under constant load, Fp increases monotonically with time; at force fR and time tR this slope abruptly changes indicating membrane-cytoskeleton bond rupture. Repeating for many cells we find fR is not constant but increases with tR. We propose it represents the rupture of a cluster of bonds with larger clusters demonstrating greater rupture forces and lifetimes. This is in agreement with theory that calculates the lifetime of a cluster of bonds between two bodies (J.Chem.Phys.121:8997). Comparing experimental data with this theory, we find that molecular parameters are within expected ranges reported for biomolecular bonds in vitro withevidence that the bonds can re-bind. Cells treated with Rho-GTPase inhibitors possess membrane-cytoskeleton bonds with lower stiffness that show no rebinding on the timescale of the measurements (zero on-rate). This is predictable since active Rho-GTPases form linkages between the membrane and actin-effector proteins of the cytoskeleton. This measurable change in bond properties provides a quantitative method for evaluating the role of Rho-GTPases in dynamic cytoskeleton remodeling. This is relevant as Rho-GTPases are upregulated in many human cancers including the HN-31 cell line used in this study.