Compliant Or Stiff: Two Differing Mechanisms of Actin Network Stabilization by Calponin and Tropomyosin

Abstract

The cellular actin cytoskeleton plays a central role in the ability of cells to properly sense, propagate, and respond to external stresses and other mechanical stimuli. Calponin, an actin-binding protein found both in muscle and non-muscle cells, has been implicated in actin cytoskeletal organization and regulation. While currently thought to stabilize actin in the cell, the mechanisms of this stabilization are poorly understood due to the complicated nature of the living cell. Here we use a simplified in vitro model system to dissect the specific roles of individual actin binding proteins within a complex network environment.We studied the mechanical properties of actin networks in the presence of basic calponin and smooth muscle aortic tropomyosin. These two actin-binding proteins have distinct effects on single actin filaments; while calponin binds over subdomain 2 of actin and makes actin more flexible, tropomyosin lies along the long-pitch helix of actin and buttresses the filament. We constructed in vitro crosslinked actin networks and studied their strain behavior both macro- and microscopically using bulk rheology and active microrheology. Actin networks decorated with either calponin or tropomyosin exhibited increased tensile strength despite the very different effects these proteins have on individual actin filaments. While calponin-decorated actin networks exhibited delayed strain stiffening, tropomyosin-decorated actin networks underwent stress relaxation through local network failure. When the two actin-binding proteins were present together, each of these effects were apparent, and the network tensile strength were further increased.This work demonstrates two very different mechanisms by which calponin and tropomyosin increase the tensile strength of actin networks through purely mechanical interactions.Project funded by the NIH (HL086655), and the Harvard Materials Research Science and Engineering Center (DMR-0820484).