Mechanical Properties of Individual Actin Crosslinks

Abstract

The internal organization of eukaryotic cells is provided by the cytoskeleton, a highly dynamic protein network mainly composed of actin filaments, microtubules and intermediate filaments. The mechanical and dynamical properties of this network are not simply given by the filament mechanics, but can be modulated specifically by various crosslinking and motor proteins. The characteristics of these crosslinked actin networks have been studied extensively in ensemble experiments, while detailed information on crossing-overs of single filaments is still rare. We developed a four-bead optical tweezers assay to probe mechanical properties of individual, freely suspended actin-crosslinker-actin bonds. We then compared the forced unbinding of the crosslinking proteins filamin A and alpha-actinin 2 which share a high similarity in their actin binding domains. Interestingly, our single-molecule unbinding assay reveals a significantly different behavior: An increase in force results in faster unbinding of alpha-actinin-crosslinks (‘slip bond'), however, the opposite is true for filamin (‘catch bond'). We further used genetically engineered variants of the crosslinkers to investigate the molecular mechanism underlying this significant difference in the mechanical unbinding behavior of these crosslinking proteins.