Molecular Basis for the Dual Function of Eps8 on Actin Dynamics: Bundling and Capping

IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy.
PLoS Biology (Impact Factor: 11.77). 06/2010; 8(6):e1000387. DOI: 10.1371/journal.pbio.1000387
Source: PubMed

ABSTRACT Actin capping and cross-linking proteins regulate the dynamics and architectures of different cellular protrusions. 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, molecular, and genetic approaches with electron microscopy and image analysis to dissect the molecular mechanism responsible for the distinct activities of Eps8. We propose that bundling activity of Eps8 is mainly mediated by a compact four helix bundle, which is contacting three actin subunits along the filament. The capping activity is mainly mediated by a amphipathic helix that binds within the hydrophobic pocket at the barbed ends of actin blocking further addition of actin monomers. Single-point mutagenesis validated these modes of binding, permitting us to dissect Eps8 capping from bundling activity in vitro. We further showed that the capping and bundling activities of Eps8 can be fully dissected in vivo, demonstrating the physiological relevance of the identified Eps8 structural/functional modules. Eps8 controls actin-based motility through its capping activity, while, as a bundler, is essential for proper intestinal morphogenesis of developing Caenorhabditis elegans.

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    • "It has been previously shown that the capping activity of Eps8 is primarily mediated by the amphipathic H1 helix, while the globular H2–H5 core is responsible for bundling (Hertzog et al, 2010). We then took advantage of the Eps8 capping mutant Eps8H1, in which the hydrophobic residues in the amphipathic helix, H1, critical for actin capping, were mutated while leaving intact the actin bundling activity (Hertzog et al, 2010). A total of 10–11 DIV hippocampal cultures were transfected with constructs expressing either the Eps8 wt protein or its actin-capping mutant, Eps8H1. "
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