Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

EB1 and APC bind to mDia to stabilize microtubules downstream of Rho and promote cell migration

Abstract

Lysophosphatidic acid (LPA) stimulates Rho GTPase and its effector, the formin mDia, to capture and stabilize microtubules in fibroblasts. We investigated whether mammalian EB1 and adenomatous polyposis coli (APC) function downstream of Rho–mDia in microtubule stabilization. A carboxy-terminal APC-binding fragment of EB1 (EB1-C) functioned as a dominant-negative inhibitor of microtubule stabilization induced by LPA or active mDia. Knockdown of EB1 with small interfering RNAs also prevented microtubule stabilization. Expression of either full-length EB1 or APC, but not an APC-binding mutant of EB1, was sufficient to stabilize microtubules. Binding and localization studies showed that EB1, APC and mDia may form a complex at stable microtubule ends. Furthermore, EB1-C, but not an APC-binding mutant, inhibited fibroblast migration in an in vitro wounding assay. These results show an evolutionarily conserved pathway for microtubule capture, and suggest that mDia functions as a scaffold protein for EB1 and APC to stabilize microtubules and promote cell migration.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1: EB1-C inhibits stable microtubule formation.
Figure 2: EB1 is necessary and sufficient for stable microtubule formation.
Figure 3: EB1 functions downstream of Rho and mDia in the microtubule stabilization pathway.
Figure 4: EB1 binding to APC is important for stable microtubule formation.
Figure 5: mDia domain analysis.
Figure 6: EB1 and APC interact with mDia.
Figure 7: EB1, APC and mDia1 are localized at Glu-MT ends in TC-7 cells.
Figure 8: Inhibition of Glu-MT formation by EB1-C inhibits cell migration.

Similar content being viewed by others

Accession codes

Accessions

BINDPlus

References

  1. Gundersen, G.G. & Bulinski, J.C. Microtubule arrays in differentiated cells contain elevated levels of a post-translationally modified form of tubulin. Eur. J. Cell Biol. 42, 288–294 (1986).

    CAS  PubMed  Google Scholar 

  2. Gundersen, G.G. & Bulinski, J.C. Selective stabilization of microtubules oriented toward the direction of cell migration. Proc. Natl Acad. Sci. USA 85, 5946–5950 (1988).

    Article  CAS  Google Scholar 

  3. Gundersen, G.G., Khawaja, S. & Bulinski, J.C. Generation of a stable, posttranslationally modified microtubule array is an early event in myogenic differentiation. J. Cell. Biol. 109, 2275–2288 (1989).

    Article  CAS  Google Scholar 

  4. Cook, T.A., Nagasaki, T. & Gundersen, G.G. Rho guanosine triphosphatase mediates the selective stabilization of microtubules induced by lysophosphatidic acid. J. Cell Biol. 141, 175–185 (1998).

    Article  CAS  Google Scholar 

  5. 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  Google Scholar 

  6. Palazzo, A.F., Eng, C.H., Schlaepfer, D.D., Marcantonio, E.E. & Gundersen, G.G. Localized stabilization of miicrotubules by integrin and FAK facilitated Rho signaling. Science 303, 836–839 (2004).

    Article  CAS  Google Scholar 

  7. Webster, D.R., Gundersen, G.G., Bulinski, J.C. & Borisy, G.G. Differential turnover of tyrosinated and detyrosinated microtubules. Proc. Natl Acad. Sci. USA 84, 9040–9044 (1987).

    Article  CAS  Google Scholar 

  8. Infante, A.S., Stein, M.S., Zhai, Y., Borisy, G.G. & Gundersen, G.G. Detyrosinated (Glu) microtubules are stabilized by an ATP-sensitive plus-end cap. J. Cell Sci. 113, 3907–3919 (2000).

    CAS  PubMed  Google Scholar 

  9. Westermann, S. & Weber, K. Post-translational modifications regulate microtubule function. Nature Rev. Mol. Cell Biol. 4, 938–947 (2003).

    Article  CAS  Google Scholar 

  10. Gundersen, G.G., Kalnoski, M.H. & Bulinski, J.C. Distinct populations of microtubules: tyrosinated and nontyrosinated α-tubulin are distributed differently in vivo. Cell 38, 779–789 (1984).

    Article  CAS  Google Scholar 

  11. Liao, G. & Gundersen, G.G. Kinesin is a candidate for cross-bridging microtubules and intermediate filaments. Selective binding of kinesin to detyrosinated tubulin and vimentin. J. Biol. Chem. 273, 9797–9803 (1998).

    Article  CAS  Google Scholar 

  12. Lin, S.X., Gundersen, G.G. & Maxfield, F.R. Export from pericentriolar endocytic recycling compartment to cell surface depends on stable, detyrosinated (glu) microtubules and kinesin. Mol. Biol. Cell 13, 96–109 (2002).

    Article  CAS  Google Scholar 

  13. Gurland, G. & Gundersen, G.G. Stable, detyrosinated microtubules function to localize vimentin intermediate filaments in fibroblasts. J. Cell Biol. 131, 1275–1290 (1995).

    Article  CAS  Google Scholar 

  14. Kreitzer, G., Liao, G. & Gundersen, G.G. Detyrosination of tubulin regulates the interaction of intermediate filaments with microtubules in vivo via a kinesin-dependent mechanism. Mol. Biol. Cell 10, 1105–1118 (1999).

    Article  CAS  Google Scholar 

  15. Schuyler, S.C. & Pellman, D. Microtubule “plus-end-tracking proteins”: The end is just the beginning. Cell 105, 421–424 (2001).

    Article  CAS  Google Scholar 

  16. Kohno, H., Tanaka, K., Mino, A., Umikawa, M. & Takai, Y. Bni1 implicated in cytoskeletal control is a putative target of Rho1p small GTP binding protein in S. cerevisiae. EMBO J. 15, 6060–6068 (1996).

    Article  CAS  Google Scholar 

  17. Lee, L., Klee, S.K., Evangelista, M., Boone, C. & Pellman, D. Control of mitotic spindle position by the Saccharomyces cerevisiae Formin Bni1p. J. Cell Biol. 144, 947–961 (1999).

    Article  CAS  Google Scholar 

  18. Adames, N.R. & Cooper, J.A. Microtubule interactions with the cell cortex causing nuclear movements in Saccharomyces cerevisiae. J. Cell Biol. 149, 863–874 (2000).

    Article  CAS  Google Scholar 

  19. Bloom, K. It's a kar9ochore to capture microtubules. Nature Cell Biol. 2, E96–E98 (2000).

    Article  CAS  Google Scholar 

  20. Schuyler, S.C. & Pellman, D. Search, capture and signal: games microtubules and centrosomes play. J. Cell Sci. 114, 247–255 (2001).

    CAS  PubMed  Google Scholar 

  21. Kusch, J., Liakopoulos, D. & Barral, Y. Spindle asymmetry: a compass for the cell. Trends Cell Biol. 13, 562–569 (2003).

    Article  CAS  Google Scholar 

  22. Yin, H., Pruyne, D., Huffaker, T.C. & Bretscher, A. Myosin V orientates the mitotic spindle in yeast. Nature 406, 1013–1015 (2000).

    Article  CAS  Google Scholar 

  23. Beach, D.L., Thibodeaux, J., Maddox, P., Yeh, E. & Bloom, K. The role of the proteins Kar9 and Myo2 in orienting the mitotic spindle of budding yeast. Curr. Biol. 10, 1497–1506 (2000).

    Article  CAS  Google Scholar 

  24. Su, L.K. et al. APC binds to the novel protein EB1. Cancer Res. 55, 2971–2977 (1995).

    Google Scholar 

  25. Bienz, M. Spindles cotton on to junctions, APC and EB1. Nature Cell Biol. 3, E67–E68 (2001).

    Article  CAS  Google Scholar 

  26. Munemitsu, S. et al. The APC gene product associates with microtubules in vivo and promotes their assembly in vitro. Cancer Res. 54, 3676–3681 (1994).

    CAS  PubMed  Google Scholar 

  27. Berrueta, L. et al. The adenomatous polyposis coli-binding protein EB1 is associated with cytoplasmic and spindle microtubules. Proc. Natl Acad. Sci. USA 95, 10596–10601 (1998).

    Article  CAS  Google Scholar 

  28. Zumbrunn, J., Kinoshita, K., Hyman, A.A. & Nathke, I.S. Binding of the adenomatous polyposis coli protein to microtubules increases microtubule stability and is regulated by GSK3β phosphorylation. Curr. Biol. 11, 44–49 (2001).

    Article  CAS  Google Scholar 

  29. Askham, J.M., Vaughan, K.T., Goodson, H.V. & Morrison, E.E. Evidence that an interaction between EB1 and p150(Glued) is required for the formation and maintenance of a radial microtubule array anchored at the centrosome. Mol. Biol. Cell 13, 3627–3645 (2002).

    Article  CAS  Google Scholar 

  30. Ligon, L.A., Shelly, S.S., Tokito, M. & Holzbaur, E.L. The microtubule plus-end proteins EB1 and dynactin have differential effects on microtubule polymerization. Mol. Biol. Cell 14, 1405–1417 (2003).

    Article  CAS  Google Scholar 

  31. Gundersen, G.G. Evolutionary conservation of microtubule-capture mechanisms. Nature Rev. Mol. Cell Biol. 3, 296–304 (2002).

    Article  CAS  Google Scholar 

  32. Berrueta, L., Tirnauer, J.S., Schuyler, S.C., Pellman, D. & Bierer, B.E. The APC-associated protein EB1 associates with components of the dynactin complex and cytoplasmic dynein intermediate chain. Curr. Biol. 9, 425–428 (1999).

    Article  CAS  Google Scholar 

  33. Tirnauer, J.S., O'Toole, E., Berrueta, L., Bierer, B.E. & Pellman, D. Yeast Bim1p promotes the G1-specific dynamics of microtubules. J. Cell Biol. 145, 993–1007 (1999).

    Article  CAS  Google Scholar 

  34. Rogers, S.L., Rogers, G.C., Sharp, D.J. & Vale, R.D. Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle. J. Cell Biol. 158, 873–884 (2002).

    Article  CAS  Google Scholar 

  35. Mimori-Kiyosue, Y., Shiina, N. & Tsukita, S. The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules. Curr. Biol. 10, 865–868 (2000).

    Article  CAS  Google Scholar 

  36. Alberts, A.S. Identification of a carboxyl-terminal diaphanous-related formin homology protein autoregulatory domain. J. Biol. Chem. 276, 2824–2830 (2001).

    Article  CAS  Google Scholar 

  37. Palazzo, A.F. et al. Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-regulated microtubule stabilization. Curr. Biol. 11, 1536–1541 (2001).

    Article  CAS  Google Scholar 

  38. Askham, J.M., Moncur, P., Markham, A.F. & Morrison, E.E. Regulation and function of the interaction between the APC tumour suppressor protein and EB1. Oncogene 19, 1950–1958 (2000).

    Article  CAS  Google Scholar 

  39. Fukata, M. et al. Rac1 and Cdc42 capture microtubules through IQGAP1 and CLIP-170. Cell 109, 873–885 (2002).

    Article  CAS  Google Scholar 

  40. 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  Google Scholar 

  41. Wallar, B.J. & Alberts, A.S. The formins: active scaffolds that remodel the cytoskeleton. Trends Cell Biol. 13, 435–446 (2003).

    Article  CAS  Google Scholar 

  42. Yasuda, S. et al. Cdc42 and mDia3 regulate microtubule attachment to kinetochores. Nature 428, 767–771 (2004).

    Article  CAS  Google Scholar 

  43. Nakamura, M., Zhou, X.Z., Kishi, S. & Lu, K.P. Involvement of the telomeric protein Pin2/TRF1 in the regulation of the mitotic spindle. FEBS Lett. 514, 193–198 (2002).

    Article  CAS  Google Scholar 

  44. Subramanian, A. et al. Shortstop recruits EB1/APC1 and promotes microtubule assembly at the muscle-tendon junction. Curr. Biol. 13, 1086–1095 (2003).

    Article  CAS  Google Scholar 

  45. Leung, C.L., Sun, D., Zheng, M., Knowles, D.R. & Liem, R.K. Microtubule actin cross-linking factor (MACF): a hybrid of dystonin and dystrophin that can interact with the actin and microtubule cytoskeletons. J. Cell Biol. 147, 1275–1286 (1999).

    Article  CAS  Google Scholar 

  46. Karakesisoglou, I., Yang, Y. & Fuchs, E. An epidermal plakin that integrates actin and microtubule networks at cellular junctions. J. Cell Biol. 149, 195–208 (2000).

    Article  CAS  Google Scholar 

  47. Sun, D., Leung, C.L. & Liem, R.K. Characterization of the microtubule binding domain of microtubule actin crosslinking factor (MACF): identification of a novel group of microtubule associated proteins. J. Cell Sci. 114, 161–172 (2001).

    CAS  PubMed  Google Scholar 

  48. Kodama, A., Karakesisoglou, I., Wong, E., Vaezi, A. & Fuchs, E. ACF7. An essential integrator of microtubule dynamics. Cell 115, 343–354 (2003).

    Article  CAS  Google Scholar 

  49. Evangelista, M., Zigmond, S. & Boone, C. Formins: signaling effectors for assembly and polarization of actin filaments. J. Cell Sci. 116, 2903–2911 (2003).

    Article  Google Scholar 

  50. Elbashir, S.M., Harborth, J., Weber, K. & Tuschl, T. Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 26, 199–213 (2002).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Tsukita, Y. Mimori-Kiyosue, K. Kinzler, B. Voglestein, S. Narumiya, R. Vallee and K. Vaughan for DNA constructs; E. Keohane for assistance preparing the GST–EB1-C construct and protein; and R. Liem for comments on the manuscript. C.H.E. was supported by a Howard Hughes Predoctoral Fellowship. This work was supported by National Institutes of Health grant GM62939 and a grant from the Steward Trust (to G.G.G.) and DOD grant DAMD17-00-1-0190 (to A.S.A.).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Gregg G. Gundersen.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wen, Y., Eng, C., Schmoranzer, J. et al. EB1 and APC bind to mDia to stabilize microtubules downstream of Rho and promote cell migration. Nat Cell Biol 6, 820–830 (2004). https://doi.org/10.1038/ncb1160

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb1160

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing