Characterizing the biophysical properties of individual actin stress fibers

Abstract

Actin stress fibers (SFs) play essential roles in cellular contractile force generation and resulting adhesion regulation. Although contractile properties of whole cells have been extensively studied, those intrinsic to individual SFs remain unknown. Here we isolate functional SFs from cells and directly measure the relationship between the shortening velocity and contractile forces generated by individual SFs. These isolated SFs are associated with major structural proteins typical for SFs present within intact cells, which include phosphorylated myosin regulatory light chain (MRLC) required for actin-myosin crossbridge cycling. Thus, contractile shortening of the structurally and functionally non-impaired SFs can be induced in vitro upon application of adenosine triphosphate. We then characterize their contractile properties using functionalized microneedles for physical manipulation and force measurement. SFs shorten quickly in the absence of external loading, whereas, interestingly, the shortening velocity is dramatically decreased by more than two orders of magnitude upon application of a small tensile load. These results show a highly steep shortening velocity-load relationship that is distinct from the hyperbolic Hill relation of skeletal myofibrils, which has been assumed in other studies to be valid for SFs as well because these two have similar sarcomeric patterns along their lengths.