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: 9.34). 06/2010; 8(6):e1000387. DOI: 10.1371/journal.pbio.1000387
Source: PubMed


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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    • "Eps8 actin capping activity limits leader bleb size by decreasing actin density and mechanical properties in the cell body cortex, and antagonizing actin bundling at the leader bleb tip We next sought to determine whether the actin filament capping activity of Eps8 is required for leader bleb formation, cytoskeletal organization, and cellular mechanical properties. We made aspartic acid substitutions within the C-terminus of Emerald-tagged mouse Eps8 (V689D/L693D, referred to as Emerald-mEps8Δcap) which have been previously shown to specifically block Eps8 capping activity (Figure 4A) (Hertzog et al., 2010) and regulate blebbing during cell division (Werner et al., 2013). Strikingly, when confined under agar, Eps8-KD cells expressing Emerald- mEps8Δcap formed significantly larger leader blebs and the proportion of cells migrating nearly doubled compared to either untransfected controls or to Eps8-KD cells expressing Emerald- mEps8 (Figure 5A,B,D, Supplementary file 1 and Video 5). "
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    ABSTRACT: Within the confines of tissues, cancer cells can use blebs to migrate. Eps8 is an actin bundling and capping protein whose capping activity is inhibited by Erk, a key MAP kinase that is activated by oncogenic signaling. We tested the hypothesis that Eps8 acts as an Erk effector to modulate actin cortex mechanics and thereby mediate bleb-based migration of cancer cells. Cells confined in a non-adhesive environment migrate in the direction of a very large 'leader bleb.' Eps8 bundling activity promotes cortex tension and intracellular pressure to drive leader bleb formation. Eps8 capping and bundling activities act antagonistically to organize actin within leader blebs, and Erk mediates this effect. An Erk biosensor reveals concentrated kinase activity within leader blebs. Bleb contents are trapped by the narrow neck that separates the leader bleb from the cell body. Thus, Erk activity promotes actin bundling by Eps8 to enhance cortex tension and drive the bleb-based migration of cancer cells under non-adhesive confinement.
    eLife Sciences 07/2015; 4. DOI:10.7554/eLife.08314 · 9.32 Impact Factor
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    • "This identifies Eps8 as a mediator of integrin effector signaling and FAK-dependent downstream phenotypes in SCC cancer cells, likely by controlling the actin organization in the proximity of Src–Eps8 or FAK–Eps8 complexes. DISCUSSION Eps8 is a molecular scaffold that regulates actin by assembling the complexes that control filament capping and bundling (Croce et al., 2004; Disanza et al., 2004; Frittoli et al., 2011; Hertzog et al., 2010). Through multi-protein complexes and signaling to the Rho GTPases Rac and Cdc42, Eps8 also controls the generation of membrane ruffles and filopodia, respectively (Disanza et al., 2006; Goicoechea et al., 2006; Innocenti et al., 2003). "
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    ABSTRACT: Eps8 is an actin regulatory scaffold protein increased in Squamous Cell Carcinoma (SCC) cells. It forms a complex with both Focal Adhesion Kinase (FAK) and c-Src in SCC cells derived from the DMBA/TPA model of skin carcinogenesis. Here, we describe two new roles for Eps8. Firstly, it controls the spatial distribution of active c-Src in a FAK-dependent manner. Specifically, Eps8 participates in, and regulates, a biochemical complex with c-Src and drives c-Src's trafficking to autophagic structures that SCC cells use to cope with high levels of active c-Src when FAK is absent. Secondly, when FAK is expressed in SCC cells, so tethering active c-Src at focal adhesion complexes, Eps8 is also recruited to focal adhesions and is required for FAK-dependent polarization and invasion. Therefore, Eps8 is a critical mediator of Src/FAK-regulated processes; it participates in specific biochemical complexes and promotes actin re-arrangements that determine c-Src's spatial localization and Src/FAK functions in invasive migration.
    Journal of Cell Science 10/2014; DOI:10.1242/jcs.157560 · 5.43 Impact Factor
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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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    ABSTRACT: Actin-based remodelling underlies spine structural changes occurring during synaptic plasticity, the process that constantly reshapes the circuitry of the adult brain in response to external stimuli, leading to learning and memory formation. A positive correlation exists between spine shape and synaptic strength and, consistently, abnormalities in spine number and morphology have been described in a number of neurological disorders. In the present study, we demonstrate that the actin-regulating protein, Eps8, is recruited to the spine head during chemically induced long-term potentiation in culture and that inhibition of its actin-capping activity impairs spine enlargement and plasticity. Accordingly, mice lacking Eps8 display immature spines, which are unable to undergo potentiation, and are impaired in cognitive functions. Additionally, we found that reduction in the levels of Eps8 occurs in brains of patients affected by autism compared to controls. Our data reveal the key role of Eps8 actin-capping activity in spine morphogenesis and plasticity and indicate that reductions in actin-capping proteins may characterize forms of intellectual disabilities associated with spine defects.
    The EMBO Journal 05/2013; 32(12). DOI:10.1038/emboj.2013.107 · 10.43 Impact Factor
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