| Abstract: | ![]()
Actin capping and cross-linking proteins regulate the dynamics and architecturesof different cellular protrusions. Eps8 is the founding member of a uniquefamily of capping proteins capable of side-binding and bundling actin filaments.However, the structural basis through which Eps8 exerts these functions remainselusive. Here, we combined biochemical, molecular, and genetic approaches withelectron microscopy and image analysis to dissect the molecular mechanismresponsible for the distinct activities of Eps8. We propose that bundlingactivity of Eps8 is mainly mediated by a compact four helix bundle, which iscontacting three actin subunits along the filament. The capping activity ismainly mediated by a amphipathic helix that binds within the hydrophobic pocketat the barbed ends of actin blocking further addition of actin monomers.Single-point mutagenesis validated these modes of binding, permitting us todissect Eps8 capping from bundling activity in vitro. We further showed that thecapping and bundling activities of Eps8 can be fully dissected in vivo,demonstrating the physiological relevance of the identified Eps8structural/functional modules. Eps8 controls actin-based motility through itscapping activity, while, as a bundler, is essential for proper intestinalmorphogenesis of developing Caenorhabditis elegans. |
