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Regulation of cell shape by Cdc42 is mediated by the synergic actin-bundling activity of the Eps8–IRSp53 complex

Nature Cell Biology volume 8, pages 1337–1347 (2006)Cite this article

Abstract

Actin-crosslinking proteins organize actin into highly dynamic and architecturally diverse subcellular scaffolds that orchestrate a variety of mechanical processes, including lamellipodial and filopodial protrusions in motile cells. How signalling pathways control and coordinate the activity of these crosslinkers is poorly defined. IRSp53, a multi-domain protein that can associate with the Rho-GTPases Rac and Cdc42, participates in these processes mainly through its amino-terminal IMD (IRSp53 and MIM domain). The isolated IMD has actin-bundling activity in vitro and is sufficient to induce filopodia in vivo. However, the manner of regulation of this activity in the full-length protein remains largely unknown. Eps8 is involved in actin dynamics through its actin barbed-ends capping activity and its ability to modulate Rac activity. Moreover, Eps8 binds to IRSp53. Here, we describe a novel actin crosslinking activity of Eps8. Additionally, Eps8 activates and synergizes with IRSp53 in mediating actin bundling in vitro, enhancing IRSp53-dependent membrane extensions in vivo. Cdc42 binds to and controls the cellular distribution of the IRSp53–Eps8 complex, supporting the existence of a Cdc42–IRSp53–Eps8 signalling pathway. Consistently, Cdc42-induced filopodia are inhibited following individual removal of either IRSp53 or Eps8. Collectively, these results support a model whereby the synergic bundling activity of the IRSp53–Eps8 complex, regulated by Cdc42, contributes to the generation of actin bundles, thus promoting filopodial protrusions.

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Figure 1: IRSp53 binds Eps8 through a multi-surfaces interaction.
Figure 2: IRSp53 and Eps8 cooperate in bundling actin filaments in vitro.
Figure 3: Eps8 enhances IRSp53-mediated membrane protrusive activity in vivo.
Figure 4: IRSp53-mediated membrane protrusions require Eps8, but not WAVE complex components or N-WASP.
Figure 5: IRSp53 modulates Eps8 localization downstream Cdc42.
Figure 6: The IRSp53–Eps8 complex is required for Cdc42-induced filopodia formation.

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Acknowledgements

This work was supported by grants: from AIRC (Associazione Italiana Ricerca sul Cancro) to G.S.; from the Human Science Frontier Program to G.S. (grant # RGP0072/2003-C); from the Italian Ministry of Health (grant R.F. 02/184) to G.S.; from European Community (VI Framework) to G.S.; from the European Molecular Biology Organization (EMBO) to M.H.; from Deutsche Forschungsgemeinschaft (DFG) to T.E.B.S. and H.J.K. (SPP 1150 and Ri192/24-1, respectively); from Associazione Italiana per la Ricerca sul Cancro European Community (VI Framework), Ministero della Salute, Ministero dell' Università e della Ricerca (MIUR), Fondazione Monzino to P.P.D.F. We would like to thank M. Garre, M. Faretta and D. Dominique (from M.-F. Carlier's laboratory) for technical help and S. Confalonieri for assistance with bioinformatic analysis. We are especially thankful to: M.-F. Carlier for continuous support and advice, and for critically reading the manuscript; A. Hall, T. Takenawa and S. Ahmed for providing IRSp53 constructs; and C. Brakebusch for cdc42 null cells.

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Author notes

  1. Andrea Disanza, Sara Mantoani, Maud Hertzog and Pier Paolo Di Fiore: These authors contributed equally to this work.

Authors and Affiliations

  1. Istituto FIRC di Oncologia Molecolare (IFOM), Via Adamello 16, Milan, 20139, Italy

    Andrea Disanza, Sara Mantoani, Maud Hertzog, Silke Gerboth, Emanuela Frittoli, Anika Steffen, Francesca Milanesi, Pier Paolo Di Fiore, Andrea Ciliberto & Giorgio Scita

  2. Department of Experimental Oncology, Istituto Europeo di Oncologia (IEO), Via Ripamonti 435, Milan, 20141, Italy

    Andrea Disanza, Sara Mantoani, Maud Hertzog, Silke Gerboth, Emanuela Frittoli, Anika Steffen, Francesca Milanesi, Pier Paolo Di Fiore, Andrea Ciliberto & Giorgio Scita

  3. Signalling and Motility Group, German Research Centre for Biotechnology (GBF), Mascheroder Weg 1, Braunschweig, D-38124, Germany

    Anika Steffen & Theresia E. B. Stradal

  4. Institute for Human Genetics, University Hospital Eppendorf, Hamburg Martinistrasse 52, Hamburg, D-20246, Germany

    Kerstin Berhoerster & Hans-Juergen Kreienkamp

  5. Dipartimento di Medicina, Chirurgia ed Odonoiatria, Università degli Studi di Milano, Milan, 20122, Italy

    Andrea Disanza & Pier Paolo Di Fiore

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Contributions

A.D., S.M and M.E. contributed to experimental work, project planning and data analysis. S.G., E.F., A.S., F.M. and K.B. contributed to experimental work. A.C., H..K., P.P.D.F. and T.E.B.S contributed to project planning and data analysis.

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Correspondence to Giorgio Scita.

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Disanza, A., Mantoani, S., Hertzog, M. et al. Regulation of cell shape by Cdc42 is mediated by the synergic actin-bundling activity of the Eps8–IRSp53 complex. Nat Cell Biol 8, 1337–1347 (2006). https://doi.org/10.1038/ncb1502

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