Mechanistic principles underlying regulation of the actin cytoskeleton by phosphoinositides

Abstract

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)P], 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)P along the lipid bilayer. Together, these findings demonstrate that membrane-interaction mechanisms of ABPs evolved to precisely fulfill their specific functions in cytoskeletal dynamics.

Molecular mechanism for inhibition of twinfilin by phosphoinositides

“…However, the isolated N-terminal ADF-H domain of twinfilin does not bind lipids with detectable affinity, and also the C-terminal ADF-H domain displays only relatively modest binding to lipids in vitro. These results are consistent with a recent study demonstrating that ADF/cofilins display only transient, low affinity interactions with phosphoinositide-rich membranes (21). Importantly, our experiments revealed that full-length twinfilin binds membranes more strongly compared to ADF/cofilins, and that the C-terminal tail of twinfilin is critical for this high affinity lipidbinding.…”

“…Our co-sedimentation and DPH anisotropy experiments as well as atomistic simulations demonstrated that, similarly to ADF/cofilins (19,21), twinfilin interacts with negatively-charged phospholipids through electrostatic interactions without penetrating into the hydrophobic acyl chain region of the bilayer. However, the isolated N-terminal ADF-H domain of twinfilin does not bind lipids with detectable affinity, and also the C-terminal ADF-H domain displays only relatively modest binding to lipids in vitro.…”

“…In support to this hypothesis, ADF/cofilins, which are entirely composed of a structurally similar ADF-H domain, bind PI(4,5)P 2 (12,19,45), although with relatively low affinity (21). We thus expressed and purified different domains of twinfilin (Fig.…”

“…7). Whereas the other actin-binding proteins examined so far, including ADF/cofilins and capping protein (19,21,50), directly associate with membranes through their actin-binding sites, twinfilin does not employ its actin-binding site for initial interactions with the membrane. The high-affinity actin-binding site is located in the C-terminal ADF-H domain of twinfilin (34).…”

Computer simulations of protein–membrane systems

“…Senju et al 380 performed unbiased AA-MD simulations, initiated by placing the protein 0.5 nm above the membrane surface. During the simulations, both proteins interact with the PI(4,5)P 2 -enriched membrane via their large positively charged surface (Fig.…”