MOLECULAR BASIS FOR THE DUAL FUNCTION OF EPS8 IN CONTROLLING ACTIN CYTOSKELETON DYNAMICS: BUNDLING AND CAPPING

Actin capping and cross-linking proteins regulate the dynamics and architectures of different cellular protrusions by controlling the number of free actin growing ends and organizing filaments into higher order structures, respectively. Eps8 is the founding member of a unique family of capping proteins capable of side-binding and bundling actin filaments. However, the structural basis through which Eps8 exerts these functions remains elusive. Here, we combined biochemical, structural and genetic approaches to dissect the molecular mechanism responsible for the distinct Eps8 activities. We show how diverse portions within the C-terminal region of Eps8 contribute to actin binding and mediate Eps8 functions. An N-terminal amphipathic H1 helix, homologous to WH2 domains, is responsible for high affinity interactions with the barbed end unit of filamentous actin and is crucial for the capping activity of the protein. A four helix bundle represents the second interaction surface which mediates contact with the filament side and is crucial for the F-actin bundling activity. Within this context, the two actin binding surfaces are spatially coordinated by a flexible linker that contributes to the high affinity interaction of Eps8 to filaments ends, ensuring a bimodal topological arrangement of Eps8 wrapped around actin filaments. This bimodal way of association to the terminal ends of actin filaments also accounts for the dual modes of organizing F-actin, via capping and cross-linking, respectively. Single-point mutagenesis validated this mode of binding, further permitting us to dissect Eps8 capping from bundling activity in vitro and in vivo. Thus, Eps8 controls actin-based motility through its capping activity, while, as a bundler, is essential for proper intestinal morphogenesis of developing Caernorhabditis elegans strains. In summary, we provided molecular and structural details of how Eps8 uniquely associates with barbed ends and sides of actin filaments through distinct structural modules. We further showed that the capping and the bundling activities of Eps8 can be fully dissected in vivo, demonstrating the physiological relevance of the identified Eps8 structural/functional module.