Recreating turnover conditions in vitro - the cell cortex as a model system

The cell cortex is a contractile actomyosin network of branched and unbranched actin filaments, myosin motors, and associated proteins that lies underneath the plasma membrane of most animal cells. The cell cortex is a central player in controlling the cell mechanical properties and tension built-up which are essential for various cellular functions and for tissue development. Currently, however, many features of the actomyosin cortex remain unknown. Due to the complexity of in vivo environment, identifying the physical principles governing actomyosin cortex self-organization remains a difficult challenge. This problem can be overcome with in vitro reconstitution systems.

Here we recreate the cell cortex in vitro using purified proteins. Our experimental set-up includes a Supported Lipid Bilayer (SLB) which includes physiologically relevant lipid composition. In addition, actin nucleation factors that are known to localize to the cell cortex are confined to the SLB surface. Finally, an actin solution of purified proteins identified to localize to the cortex of cells is used to stimulate actin cortex assembly at the SLB surface. We show how the interplay between actin nucleation, branching, and filament elongation rates regulate cortical network self-organization. Furthermore, we demonstrate how cortex disassembly plays a crucial role in achieving steady state conditions and show that network turnover is comparable to the rates measured in vivo. Beyond the experimental achievement, defining the conditions for recreating fast turnover was one of the major obstacles to overcome before a synthetic motile cell could be created.