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RhoD regulates endosome dynamics through Diaphanous-related Formin and Src tyrosine kinase

Nature Cell Biology volume 5, pages 195–204 (2003)Cite this article

A Corrigendum to this article was published on 01 July 2003

Abstract

Early endosomes move bidirectionally between the cell periphery and the interior through a mechanism regulated by the low molecular weight GTPase RhoD. Here, we identify a novel splice variant of human Diaphanous, hDia2C, which specifically binds to RhoD and is recruited onto early endosomes. Expression of RhoD and hDia2C induces a striking alignment of early endosomes along actin filaments and reduces their motility. This activity depends on the membrane recruitment and activation of c-Src kinase, thus uncovering a new role in endosome function. Our results define a novel signal transduction pathway, in which hDia2C and c-Src are sequentially activated by RhoD to regulate the motility of early endosomes through interactions with the actin cytoskeleton.

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Figure 1: hDia2C, a new member of the Formin-Related family, is a RhoD effector.
Figure 2: The GTPase-deficient mutant RhoDG26V targets hDia2C on endosomes.
Figure 3: RhoD and hDia2C regulate endosomes motility in vivo.
Figure 4: RhoD and hDia2C regulate endosome–cytoskeleton interactions.
Figure 5: hDia2C stimulates Src-kinase activity in HeLa cells.
Figure 6: RhoD/hDia2C regulate endosomes motility through Src kinase.

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References

  1. Mellman, I. Endocytosis and molecular sorting. Annu. Rev. Cell Dev. Biol. 12, 575–625 (1996).

    Article  CAS  PubMed  Google Scholar 

  2. Kubler, E. & Riezman, H. Actin and fimbrin are required for the internalization step of endocytosis in yeast. EMBO J. 12, 2855–2862 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Fujimoto, L.M., Roth, R., Heuser, J.E. & Schmid, S.L. Actin assembly plays a variable, but not obligatory role in receptor-mediated endocytosis in mammalian cells. Traffic 1, 161–171 (2000).

    Article  CAS  PubMed  Google Scholar 

  4. Buss, F., Arden, S.D., Lindsay, M., Luzio, J.P. & Kendrick-Jones, J. Myosin VI isoform localized to clathrin-coated vesicles with a role in clathrin-mediated endocytosis. EMBO J. 20, 3676–3684 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Durrbach, A., Louvard, D. & Coudrier, E. Actin filaments facilitate two steps of endocytosis. J. Cell Sci. 109, 457–465 (1996).

    CAS  PubMed  Google Scholar 

  6. Raposo, G. et al. Association of myosin I α with endosomes and lysosomes in mammalian cells. Mol. Biol. Cell 10, 1477–1494 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Taunton, J. Actin filament nucleation by endosomes, lysosomes and secretory vesicles. Curr. Opin. Cell Biol. 13, 85–91 (2001).

    Article  CAS  PubMed  Google Scholar 

  8. Matteoni, R. & Kreis, T.E. Translocation and clustering of endosomes and lysosomes depends on microtubules. J. Cell Biol. 105, 1253–1265 (1987).

    Article  CAS  PubMed  Google Scholar 

  9. McGraw, T.E., Dunn, K.W. & Maxfield, F.R. Isolation of a temperature-sensitive variant Chinese hamster ovary cell line with a morphologically altered endocytic recycling compartment. J. Cell Physiol. 155, 579–594 (1993).

    Article  CAS  PubMed  Google Scholar 

  10. Nielsen, E., Severin, F., Backer, J.M., Hyman, A.A. & Zerial, M. Rab5 regulates motility of early endosomes on microtubules. Nature Cell Biol. 1, 376–382 (1999).

    Article  CAS  PubMed  Google Scholar 

  11. Aniento, F., Emans, N., Griffiths, G. & Gruenberg, J. Cytoplasmic dynein-dependent vesicular transport from early to late endosomes. J. Cell Biol. 123, 1373–1387 (1993).

    Article  CAS  PubMed  Google Scholar 

  12. Bomsel, M., Parton, R., Kuznetsov, S.A., Schroer, T. & Gruenberg, J. Microtubule- and motor-dependent fusion in vitro between apical and basolateral endocytic vesicles from MDCK cells. Cell 62, 719–731 (1990).

    Article  CAS  PubMed  Google Scholar 

  13. Zerial, M. & McBride, H. Rab proteins as membrane organizers. Nature Rev. Mol. Cell Biol. 2, 107–117 (2001).

    Article  CAS  Google Scholar 

  14. Ridley, A.J. Rho proteins: linking signaling with membrane trafficking. Traffic 2, 303–310 (2001).

    Article  CAS  PubMed  Google Scholar 

  15. Lamaze, C., Chuang, T.-H., Terlecky, L.J., Bokoch, G.M. & Schmid, S.L. Regulation of receptor-mediated endocytosis by Rho and Rac. Nature 382, 177–179 (1996).

    Article  CAS  PubMed  Google Scholar 

  16. Kroschewski, R., Hall, A. & Mellman, I. Cdc42 controls secretory and endocytic transport to the basolateral plasma membrane of MDCK cells. Nature Cell Biol. 1, 8–13 (1999).

    Article  CAS  PubMed  Google Scholar 

  17. Murphy, C. et al. Endosome dynamics regulated by a novel rho protein. Nature 384, 427–432 (1996).

    Article  CAS  PubMed  Google Scholar 

  18. Gampel, A., Parker, P.J. & Mellor, H. Regulation of epidermal growth factor receptor traffic by the small GTPase RhoB. Curr. Biol. 9, 955–958 (1999).

    Article  CAS  PubMed  Google Scholar 

  19. Bishop, A.L. & Hall, A. Rho GTPases and their effector proteins. Biochem J. 348, 241–255 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Evangelista, M. et al. Bni1p, a yeast formin linking cdc42p and the actin cytoskeleton during polarized morphogenesis. Science 276, 118–122 (1997).

    Article  CAS  PubMed  Google Scholar 

  21. Kohno, H. et al. Bni1p implicated in cytoskeletal control is a putative target of Rho1p small GTP binding protein in Saccharomyces cerevisiae. EMBO J. 15, 6060–6068 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Alberts, A.S., Bouquin, N., Johnston, L.H. & Treisman, R. Analysis of RhoA-binding proteins reveals an interaction domain conserved in heterotrimeric G protein β subunits and the yeast response regulator protein Skn7. J. Biol. Chem. 273, 8616–8622 (1998).

    Article  CAS  PubMed  Google Scholar 

  23. Watanabe, N. et al. p140mDia, a mammalian homolog of Drosophila diaphanous, is a target protein for Rho small GTPase and is a ligand for profilin. EMBO J. 16, 3044–3056 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Westendorf, J.J. The formin/diaphanous-related protein, FHOS, interacts with Rac1 and activates transcription from the serum response element. J. Biol. Chem. 276, 46453–46459 (2001).

    Article  CAS  PubMed  Google Scholar 

  25. Yayoshi-Yamamoto, S., Taniuchi, I. & Watanabe, T. FRL, a novel formin-related protein, binds to Rac and regulates cell motility and survival of macrophages. Mol. Cell Biol. 20, 6872–6881 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Wasserman, S. FH proteins as cytoskeletal organizers. Trends Cell Biol. 8, 111–115 (1998).

    Article  CAS  PubMed  Google Scholar 

  27. Tanaka, K. Formin family proteins in cytoskeletal control. Biochem. Biophys. Res. Commun. 267, 479–481 (2000).

    Article  CAS  PubMed  Google Scholar 

  28. Rutherford, K. et al. Artemis: sequence visualization and annotation. Bioinformatics 16, 944–945 (2000).

    Article  CAS  PubMed  Google Scholar 

  29. Chistoforidis, S., McBride, H.M., Burgoyne, R.D. & Zerial, M. The Rab5 effector EEA1 is a core component of endosome docking. Nature 397, 621–625 (1999).

    Article  Google Scholar 

  30. Watanabe, N., Kato, T., Fujita, A., Ishizaki, T. & Narumiya, S. Cooperation between mDia1 and ROCK in Rho-induced actin reorganization. Nature Cell Biol. 1, 136–143 (1999).

    Article  CAS  PubMed  Google Scholar 

  31. Murphy, C. et al. Dual function of RhoD in vesicular movement and cell motility. Eur. J. Cell Biol. 80, 391–398 (2001).

    Article  CAS  PubMed  Google Scholar 

  32. Sonnichsen, B., De Renzis, S., Nielsen, E., Rietdorf, J. & Zerial, M. Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11. J. Cell Biol. 149, 901–914 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Elbashir, S.M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).

    Article  CAS  PubMed  Google Scholar 

  34. Ishizaki, T. et al. Coordination of microtubules and the actin cytoskeleton by the Rho effector mDia1. Nature Cell Biol. 3, 8–14 (2001).

    Article  CAS  PubMed  Google Scholar 

  35. Tominaga, T. et al. Diaphanous-related formins bridge Rho GTPase and Src tyrosine kinase signaling. Mol. Cell 5, 13–25 (2000).

    Article  CAS  PubMed  Google Scholar 

  36. Kaplan, K.B., Swedlow, J.R., Varmus, H.E. & Morgan, D.O. Association of p60c-src with endosomal membranes in mammalian fibroblasts. J. Cell Biol. 118, 321–333 (1992).

    Article  CAS  PubMed  Google Scholar 

  37. Klinghoffer, R.A., Sachsenmaier, C., Cooper, J.A. & Soriano, P. Src family kinases are required for integrin but not PDGFR signal transduction. EMBO J. 18, 2459–2471 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Riveline, D. et al. Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK- independent mechanism. J. Cell Biol. 153, 1175–1186 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Volberg, T., Romer, L., Zamir, E. & Geiger, B. pp60(c-src) and related tyrosine kinases: a role in the assembly and reorganization of matrix adhesions. J. Cell Sci. 114, 2279–2289 (2001).

    CAS  PubMed  Google Scholar 

  40. Bione, S. et al. A human homologue of the Drosophila melanogaster diaphanous gene is disrupted in a patient with premature ovarian failure: evidence for conserved function in oogenesis and implications for human sterility. Am. J. Hum. Genet. 62, 533–541 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Krebs, A., Rothkegel, M., Klar, M. & Jockusch, B.M. Characterization of functional domains of mDia1, a link between the small GTPase Rho and the actin cytoskeleton. J. Cell Sci. 114, 3663–3672 (2001).

    CAS  PubMed  Google Scholar 

  42. Evangelista, M., Pruyne, D., Amberg, D.C., Boone, C. & Bretscher, A. Formins direct Arp2/3-independent actin filament assembly to polarize cell growth in yeast. Nature Cell Biol. 4, 32–41 (2002).

    Article  CAS  PubMed  Google Scholar 

  43. Pruyne, D. et al. Role of formins in actin assembly: nucleation and barbed-end association. Science 297, 612–615 (2002).

    Article  CAS  PubMed  Google Scholar 

  44. Palazzo, A.F., Cook, T.A., Alberts, A.S. & Gundersen, G.G. mDia mediates Rho-regulated formation and orientation of stable microtubules. Nature Cell Biol. 3, 723–729 (2001).

    Article  CAS  PubMed  Google Scholar 

  45. Uetz, P., Fumagalli, S., James, D. & Zeller, R. Molecular interaction between limb deformity proteins (formins) and Src family kinases. J. Biol. Chem. 271, 33525–33530 (1996).

    Article  CAS  PubMed  Google Scholar 

  46. Wu, H., Reynolds, A.B., Kanner, S.B., Vines, R.R. & Parsons, J.T. Identification and characterization of a novel cytoskeleton-associated pp60src substrate. Mol. Cell Biol. 11, 5113–5124 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Uruno, T. et al. Activation of Arp2/3 complex-mediated actin polymerization by cortactin. Nature Cell Biol. 3, 259–266 (2001).

    Article  CAS  PubMed  Google Scholar 

  48. Tsubakimoto, K. et al. Small GTPase RhoD suppresses cell migration and cytokinesis. Oncogene 18, 2431–2440 (1999).

    Article  CAS  PubMed  Google Scholar 

  49. Warren, G. Membrane partitioning during cell division. Annu. Rev. Biochem. 62, 323–348 (1993).

    Article  CAS  PubMed  Google Scholar 

  50. Kato, T. et al. Localization of a mammalian homolog of diaphanous, mDia1, to the mitotic spindle in HeLa cells. J. Cell Sci. 114, 775–784 (2001).

    CAS  PubMed  Google Scholar 

  51. Bergeland, T., Widerberg, J., Bakke, O. & Nordeng, T.W. Mitotic partitioning of endosomes and lysosomes. Curr. Biol. 11, 644–651 (2001).

    Article  CAS  PubMed  Google Scholar 

  52. Christoforidis, S. & Zerial, M. Purification and identification of novel Rab effectors using affinity chromatography. Methods 20, 403–410 (2000).

    Article  CAS  PubMed  Google Scholar 

  53. Kalaidzidis, Y.L., Gavrilov, A.V., Zaitsev, P.V., Kalaidzidis, A.L. & Korolev, E.V. Pluk a software development environment programming. Programmirovanie 4, 38–46 (1997).

    Google Scholar 

  54. Chetverikov, D. & Verestói, J. Feature point tracking for incomplete trajectories. Computing 62, 321–338 (1999).

    Article  Google Scholar 

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Acknowledgements

We are grateful to S. Narumiya for providing pFL-mDia1 and pFL-mDia1ΔN3, H. McBride for pLexA-RhoDG26V (−CAAX) and D. Toniolo for pCMV-HA-hDia2B. We acknowledge the generosity of P. Soriano and G. Superti-Furga for kindly providing Src+/+ and Src−/− cell lines, as well as pEGFP-SrcWT and pEGFP-SrcY527F. Special thanks A. Desay, J. Howard, H. Mellor, C. Murphy and J. Rink for valuable discussions and comments on the manuscript, and to S. Chasserot-Golaz for help with confocal microscopy. We would like also to thank A. Giner for technical assistance, B. Habermann for biocomputing analysis and D. Bagnard for sharing technical resources. This work was supported by a Human Frontier Science Program (HFSP) long-term fellowship to S.G. (LT0493/1999-M) and by a HFSP grant to M.Z. (RG0260/1999-M)

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Authors and Affiliations

  1. Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, D-01307, Germany

    Stéphane Gasman & Marino Zerial

  2. CNRS UPR-2356 Neurotransmission et Sécrétion Neuroendocrine, 5 rue Blaise Pascal, Strasbourg, 67084, France

    Stéphane Gasman

  3. A.N. Belozersky Institute of Physico-chemical Biology, Build A, Moscow State University, Moscow, 119899, Russia

    Yannis Kalaidzidis

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Correspondence to Marino Zerial.

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Supplementary information

Supplementary Movie

All videos were acquired and processed as described previously in Live Cell Imaging and Image Processing. They comprise 100 frames, animated at 20 frames/s (around 7 × acquisition time). (MOV 2390 kb)

Movie 1 HeLa cells co-overexpressing hRab5-GFP and empty pCDNA3.1.

Supplementary Movie

Movie 2 HeLa cells co-overexpressing hRab5-GFP and RhoDV26. (MOV 2326 kb)

Supplementary Movie

Movie 3 HeLa cells co-overexpressing hRab5-GFP and ΔGBD-hDia2C. (MOV 1898 kb)

Supplementary Figure

Figure S1 mDia1 do not regulate endosome-cytoskeleton interactions (PDF 176 kb)

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Gasman, S., Kalaidzidis, Y. & Zerial, M. RhoD regulates endosome dynamics through Diaphanous-related Formin and Src tyrosine kinase. Nat Cell Biol 5, 195–204 (2003). https://doi.org/10.1038/ncb935

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