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

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1

    Pollard, T. D. The cytoskeleton, cellular motility and the reductionist agenda. Nature 422, 741–745 (2003).

  2. 2

    Pantaloni, D., Le Clainche, C. & Carlier, M. F. Mechanism of actin-based motility. Science 292, 1502–1506 (2001).

  3. 3

    Tilney, L. G., Connelly, P. S., Vranich, K. A., Shaw, M. K. & Guild, G. M. Why are two different cross-linkers necessary for actin bundle formation in vivo and what does each cross-link contribute? J. Cell Biol. 143, 121–133 (1998).

  4. 4

    Tseng, Y. et al. How actin crosslinking and bundling proteins cooperate to generate an enhanced cell mechanical response. Biochem. Biophys. Res. Commun. 334, 183–192 (2005).

  5. 5

    Tseng, Y., Fedorov, E., McCaffery, J. M., Almo, S. C. & Wirtz, D. Micromechanics and ultrastructure of actin filament networks crosslinked by human fascin: a comparison with α-actinin. J. Mol. Biol. 310, 351–366 (2001).

  6. 6

    Revenu, C., Athman, R., Robine, S. & Louvard, D. The co-workers of actin filaments: from cell structures to signals. Nature Rev. Mol. Cell Biol. 5, 635–646 (2004).

  7. 7

    Kreis, T. & Vale, R. D. Guidebook to the Cytoskeletal and Motor Proteins 2nd edn (Oxford University Press, Oxford, 1999).

  8. 8

    Faix, J. & Rottner, K. The making of filopodia. Curr. Opin. Cell Biol. 18, 18–25 (2006).

  9. 9

    Koleske, A. J. Do filopodia enable the growth cone to find its way? Sci. STKE pe20 (2003).

  10. 10

    Svitkina, T. M. et al. Mechanism of filopodia initiation by reorganization of a dendritic network. J. Cell Biol. 160, 409–421 (2003).

  11. 11

    Bear, J. E. et al. Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility. Cell 109, 509–521 (2002).

  12. 12

    Schirenbeck, A. et al. The bundling activity of vasodilator-stimulated phosphoprotein is required for filopodium formation. Proc. Natl Acad. Sc.i USA 103, 7694–7699 (2006).

  13. 13

    Schirenbeck, A., Arasada, R., Bretschneider, T., Schleicher, M. & Faix, J. Formins and VASPs may co-operate in the formation of filopodia. Biochem. Soc. Trans 33, 1256–1259 (2005).

  14. 14

    Romero, S. et al. Formin is a processive motor that requires profilin to accelerate actin assembly and associated ATP hydrolysis. Cell 119, 419–429 (2004).

  15. 15

    Kovar, D. R., Harris, E. S., Mahaffy, R., Higgs, H. N. & Pollard, T. D. Control of the assembly of ATP- and ADP-actin by formins and profilin. Cell 124, 423–435 (2006).

  16. 16

    Pollard, T. D. Formins coming into focus. Dev. Cell 6, 312–314 (2004).

  17. 17

    Mogilner, A. & Rubinstein, B. The physics of filopodial protrusion. Biophys. J. 89, 782–795 (2005).

  18. 18

    Yeh, T. C., Ogawa, W., Danielsen, A. G. & Roth, R. A. Characterization and cloning of a 58/53-kDa substrate of the insulin receptor tyrosine kinase. J. Biol. Chem. 271, 2921–2928 (1996).

  19. 19

    Krugmann, S. et al. Cdc42 induces filopodia by promoting the formation of an IRSp53:Mena complex. Curr. Biol. 11, 1645–1655 (2001).

  20. 20

    Yamagishi, A., Masuda, M., Ohki, T., Onishi, H. & Mochizuki, N. A novel actin bundling/filopodium-forming domain conserved in insulin receptor tyrosine kinase substrate p53 and missing in metastasis protein. J. Biol. Chem. 279, 14929–14936 (2004).

  21. 21

    Govind, S., Kozma, R., Monfries, C., Lim, L. & Ahmed, S. Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing the 58-kD insulin receptor substrate to filamentous actin. J. Cell Biol. 152, 579–594 (2001).

  22. 22

    Nakagawa, H. et al. IRSp53 is colocalised with WAVE2 at the tips of protruding lamellipodia and filopodia independently of Mena. J. Cell Sci. 116, 2577–2583 (2003).

  23. 23

    Connolly, B. A., Rice, J., Feig, L. A. & Buchsbaum, R. J. Tiam1–IRSp53 complex formation directs specificity of rac-mediated actin cytoskeleton regulation. Mol. Cell Biol. 25, 4602–4614 (2005).

  24. 24

    Choi, J. et al. Regulation of dendritic spine morphogenesis by insulin receptor substrate 53, a downstream effector of Rac1 and Cdc42 small GTPases. J. Neurosci. 25, 869–879 (2005).

  25. 25

    Miki, H., Yamaguchi, H., Suetsugu, S. & Takenawa, T. IRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling. Nature 408, 732–735 (2000).

  26. 26

    Millard, T. H. et al. Structural basis of filopodia formation induced by the IRSp53/MIM homology domain of human IRSp53. EMBO J. 24, 240–250 (2005).

  27. 27

    Funato, Y. et al. IRSp53/Eps8 complex is important for positive regulation of Rac and cancer cell motility/invasiveness. Cancer Res. 64, 5237–5244 (2004).

  28. 28

    Innocenti, M. et al. Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8, Abi1, and Sos-1. J. Cell Biol. 160, 17–23 (2003).

  29. 29

    Innocenti, M. et al. Mechanisms through which Sos-1 coordinates the activation of Ras and Rac. J. Cell Biol. 156, 125–136 (2002).

  30. 30

    Scita, G. et al. EPS8 and E3B1 transduce signals from Ras to Rac. Nature 401, 290–293 (1999).

  31. 31

    Disanza, A. et al. Eps8 controls actin-based motility by capping the barbed ends of actin filaments. Nature Cell Biol. 6, 1180–1188 (2004).

  32. 32

    Disanza, A. et al. Actin polymerization machinery: the finish line of signaling networks, the starting point of cellular movement. Cell. Mol. Life Sci. 62, 955–970 (2005).

  33. 33

    Mongiovi, A. M. et al. A novel peptide-SH3 interaction. EMBO J. 18, 5300–5309 (1999).

  34. 34

    Offenhauser, N. et al. The eps8 family of proteins links growth factor stimulation to actin reorganization generating functional redundancy in the Ras/Rac pathway. Mol. Biol. Cell 15, 91–98 (2004).

  35. 35

    Fujiwara, T., Mammoto, A., Kim, Y. & Takai, Y. Rho small G-protein-dependent binding of mDia to an Src homology 3 domain-containing IRSp53/BAIAP2. Biochem. Biophys. Res. Commun. 271, 626–629 (2000).

  36. 36

    Stradal, T. et al. The Abl interactor proteins localize to sites of actin polymerization at the tips of lamellipodia and filopodia. Curr. Biol. 11, 891–895 (2001).

  37. 37

    Rottner, K., Behrendt, B., Small, J. V. & Wehland, J. VASP dynamics during lamellipodia protrusion. Nature Cell Biol. 1, 321–322 (1999).

  38. 38

    Samarin, S. et al. How VASP enhances actin-based motility. J. Cell Biol. 163, 131–142 (2003).

  39. 39

    Krause, M., Bear, J. E., Loureiro, J. J. & Gertler, F. B. The Ena/VASP enigma. J. Cell Sci. 115, 4721–4726 (2002).

  40. 40

    Auerbuch, V., Loureiro, J. J., Gertler, F. B., Theriot, J. A. & Portnoy, D. A. Ena/VASP proteins contribute to Listeria monocytogenes pathogenesis by controlling temporal and spatial persistence of bacterial actin-based motility. Mol. Microbiol. 49, 1361–1375 (2003).

  41. 41

    Bear, J. E. et al. Negative regulation of fibroblast motility by Ena/VASP proteins. Cell 101, 717–728 (2000).

  42. 42

    Miki, H. & Takenawa, T. WAVE2 serves a functional partner of IRSp53 by regulating its interaction with Rac. Biochem. Biophys. Res. Commun. 293, 93–99 (2002).

  43. 43

    Czuchra, A. et al. Cdc42 is not essential for filopodium formation, directed migration, cell polarization, and mitosis in fibroblastoid cells. Mol. Biol. Cell 16, 4473–4484 (2005).

  44. 44

    Tilney, L. G., Connelly, P. S., Vranich, K. A., Shaw, M. K. & Guild, G. M. Actin filaments and microtubules play different roles during bristle elongation in Drosophila. J. Cell Sci. 113, 1255–1265 (2000).

  45. 45

    Tilney, L. G., Tilney, M. S. & Guild, G. M. F actin bundles in Drosophila bristles. I. Two filament cross-links are involved in bundling. J. Cell Biol. 130, 629–638 (1995).

  46. 46

    Tseng, Y., Schafer, B. W., Almo, S. C. & Wirtz, D. Functional synergy of actin filament cross-linking proteins. J. Biol. Chem. 277, 25609–25616 (2002).

  47. 47

    Mangan, S. & Alon, U. Structure and function of the feed-forward loop network motif. Proc. Natl Acad. Sci. USA 100, 11980–11985 (2003).

  48. 48

    Innocenti, M. et al. Abi1 is essential for the formation and activation of a WAVE2 signalling complex. Nature Cell Biol. 6, 319–327 (2004).

  49. 49

    Innocenti, M. et al. Abi1 regulates the activity of N-WASP and WAVE in distinct actin-based processes. Nature Cell Biol. 7, 969–976 (2005).

Download references

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.

Author information

Affiliations

Authors

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.

Corresponding author

Correspondence to Giorgio Scita.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures S1, S2, S3, S4, S5, S5, S6, S7 and S8 (PDF 1285 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading