Experimental estimation of preexisting tension in single actin stress fiber of vascular cells

Actin stress fibers (SFs) in vascular smooth muscle cells and endothelial cells play a critical role in transmitting intracellular forces between separate focal adhesion sites. However, quantitative studies on the berable tension level for single SFs have not yet appeared. Here, we estimated magnitude of preexisting tension in SFs based on measurements of their preexisting stretching strain and tensile properties. Cultured cells expressing fluorescentlylabeled actin were treated with detergents to extract the acin bundles. One end of an individual SF was then dislodged from the substrate by using a microneedle, resulting in a shortening of the SF due to a release of preexisting tension. Tensile tests of the isolated single SFs were conducted with a pair of cantilevers to measure the force required for stretching it up to the original length that corresponds to preexisting tension. The magnitude of the preexisting tension, ~10 nN on average, was comparable in magnitude to previously reported data on the cell traction force generated by living adherent cells at focal adhesion sites to keep cell integrity. The Young’s modulus of the isolated SFs was estimated to be ~300-1500 kPa from the tensile tests. These data will be fundamental in considering the intracellular force transmission mechanism in vascular cells.