Mechanistic principles underlying regulation of the actin cytoskeleton by phosphoinositides

Membrane phosphoinositides have emerged as key regulators of the actin cytoskeleton in cell migration, morphogenesis, cytokinesis, and endocytosis. However, the molecular mechanisms by which actin-binding proteins (ABPs) interact with phosphoinositide-rich membranes remain remarkably poorly understood. By applying a combination of biochemical, biophysical, and atomistic molecular dynamics simulation approaches on six central ABPs, we discovered that they employ multivalent electrostatic interactions for membrane binding. Strikingly, our experiments revealed that these proteins display enormous differences in their membrane interaction dynamics and in the ranges of phosphoinositide densities that they sense. These differences precisely correlate with the specific functions of these proteins in cytoskeletal dynamics. These findings uncover molecular principles by which membrane phosphoinositides regulate dynamics and architecture of the actin cytoskeleton in cells.

The actin cytoskeleton powers membrane deformation during many cellular processes, such as migration, morphogenesis, and endocytosis. Membrane phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], regulate the activities of many actin-binding proteins (ABPs), including profilin, cofilin, Dia2, N-WASP, ezrin, and moesin, but the underlying molecular mechanisms have remained elusive. Moreover, because of a lack of available methodology, the dynamics of membrane interactions have not been experimentally determined for any ABP. Here, we applied a combination of biochemical assays, photobleaching/activation approaches, and atomistic molecular dynamics simulations to uncover the molecular principles by which ABPs interact with phosphoinositide-rich membranes. We show that, despite using different domains for lipid binding, these proteins associate with membranes through similar multivalent electrostatic interactions, without specific binding pockets or penetration into the lipid bilayer. Strikingly, our experiments reveal that these proteins display enormous differences in the dynamics of membrane interactions and in the ranges of phosphoinositide densities that they sense. Profilin and cofilin display transient, low-affinity interactions with phosphoinositide-rich membranes, whereas F-actin assembly factors Dia2 and N-WASP reside on phosphoinositide-rich membranes for longer periods to perform their functions. Ezrin and moesin, which link the actin cytoskeleton to the plasma membrane, bind membranes with very high affinity and slow dissociation dynamics. Unlike profilin, cofilin, Dia2, and N-WASP, they do not require high “stimulus-responsive” phosphoinositide density for membrane binding. Moreover, ezrin can limit the lateral diffusion of PI(4,5)P2 along the lipid bilayer. Together, these findings demonstrate that membrane-interaction mechanisms of ABPs evolved to precisely fulfill their specific functions in cytoskeletal dynamics.

Read more: