Measuring the on-Rate of Membrane-Cytoskeleton Bonds at the Near-Equilibrium Region

Abstract

Cells especially cancer cells exhibit cytoskeleton remodeling. 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 equilibrium constant, K 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. Results: We find for most cells (80 %) that Fp is zero (0) for a finite time, tdelay upon application of a constant loading rate. Subsequently, Fp increases monotonically for time, Δt reaching a value that ruptures bonds at force, f. Comparing events from all cells we find tdelay exhibits an exponential distribution with a constant (15 s−1) and Δt increases monotonically with f. We estimate the on (15 s−1) and off (1 s−1) rates by comparing data with theory that calculates the lifetime of a cluster of bonds. The rates are similar when cells are treated with a microtubule-disrupting drug, but are altered by Rho-GTPase inhibitors. This is predictable as active Rho-GTPases form bridges between the membrane and actin-effector proteins of the cytoskeleton. Conclusion: The frequent detection of tdelay enabled by monitoring the force and its time course reveals an approach to experimentally estimate the intractable kon in cells. Further experiments at slower loading rates (≫60 pN/s) will confirm whether this is close to the intrinsic value, koon.