Mechanical Properties of Branched Actin Networks Assembled from Yeast Extracts

Abstract

The actin cytoskeleton is essential for cell mechanics and motility. Its dynamic nature and the host of actin binding protein (ABP) that modifies its architecture and growth make its description challenging to achieve. Here we propose to study actin branched networks through their mechanical properties. We previously developed a powerful experimental method to measure the mechanics of in vitro dense branched actin networks assembled from a minimal number of regulatory proteins (Pujol et al. PNAS 2012). This method is based on magnetic beads and allows for the rapid acquisition of orders of magnitude more experiments than previous techniques. We now focus on a recent in vitro system that reconstitutes actin branched gels in a near-physiological environment, from yeast cell extracts. These reconstituted actin networks are analogous to endocytic actin patches assembled in vivo, with more than 80 regulatory proteins present (Michelot et al., Curr. Biol, 2010). Compared to gels assembled from purified proteins, these gels were notably stiffer and more prone to plastic deformations, exhibiting failure and long-time relaxation. Because of the large number of experiments that can be carried out with the magnetic technique and the ease of yeast mutant generation, we are able to test the effects of the absence of numerous ABPs on the actin network mechanics. The lack of two cross-linkers Sac6 (fimbrin) and Scp1 (calponin) softens the gels and modifies their long-term evolution. The absence of Aip1, a protein involved in actin filament severing and network disassembly, changes the plastic reorganization properties of the actin networks. In the future, the combination of the mechanical magnetic probing technique and the yeast actin reconstituted system will provide an avenue to precise the role of many other actin protein partners implicated in the mechanics of the cytoskeleton.