Buckling of actin stress fibers: A new wrinkle in the cytoskeletal tapestry

Intracellular tension is considered an important determinant of cytoskeletal architecture and cell function. However, many details about cytoskeletal tension remain poorly understood because these forces cannot be directly measured in living cells. Therefore, we have developed a method to characterize the magnitude and distribution of pre-extension of actin stress fibers (SFs) due to resting tension in the cytoskeleton. Using a custom apparatus, human aortic endothelial cells (HAECs) were cultured on a pre-stretched silicone substrate coated with a fibronectin-like polymer. Release of the substrate caused SFs aligned in the shortening direction in adhered cells to buckle when compressed rapidly (5% shortening per second or greater) beyond their unloaded slack length. Subsequently, the actin cytoskeleton completely disassembled in 5 sec and reassembled within 60 sec. Quantification of buckling in digital fluorescent micrographs of cells fixed and stained with rhodamine phalloidin indicated a nonuniform distribution of 0-26% pre-extension of SFs in non-locomoting HAECs. Local variability suggests heterogeneity of cytoskeletal tension and/or stiffness within individual cells. These findings provide new information about the magnitude and distribution of cytoskeletal tension and the dynamics of actin stress fibers, and the approach offers a novel method to elucidate the role of specific cytoskeletal elements and crosslinking proteins in the force generating apparatus of non-muscle cells.