Role of cross-linkers in yeast branched actin networks: Linking biochemistry and mechanics

The actin cytoskeleton is an assembly of organized polymer structures. In cells, actin contributes to their internal organization, their rigidity, and their ability to exert forces. The properties of the actin networks are regulated by multiple families of actin binding proteins (ABPs). In this work, we focus on Arp2/3-branched networks, which are implicated in a variety of cellular functions such as motility or endocytosis. We propose to examine the relationship between the mechanical properties of actin networks and the biochemical composition of these gels.While atomic force microscopy and micropipette techniques have been successfully used to probe the mechanics of actin gels in vitro, their main drawback for our purpose is the limited amount of measurements per experiment. To overcome this limitation, we use instead a quantitative high-throughput system of magnetic colloids (Pujol et al., 2012). Actin networks are assembled around the colloidal particles from sets of purified proteins (bottom-up approach) or from yeast protein extracts. The advantage of these extracts is that proteins can be genetically removed one-by-one, in order to test for their functions in a near-physiological environment (top-down approach) (Michelot and Drubin, 2014).In this first study, we focus on the impact of crosslinkers, which create attachment points between neighboring filaments. The absence of two crosslinkers Sac6 (fimbrin) and Scp1 (calponin) softens and modifies the long-term evolution of actin gels assembled from extracts. Indeed, networks’ structural integrity is not recovered after load. In agreement with previous data, the addition of purified fimbrin in the bottom-up approach increases the elastic modulus of actin gels in a sigmoidal dose-dependent manner. Moreover, the system seems to evolve from little plasticity and non-linear behavior under load to a more plastic and less non-linear response.