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.
Subscribe to Journal
Get full journal access for 1 year
only $17.42 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
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).
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).
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).
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).
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).
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).
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).
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).
Westermann, S. & Weber, K. Post-translational modifications regulate microtubule function. Nature Rev. Mol. Cell Biol. 4, 938–947 (2003).
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).
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).
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).
Gurland, G. & Gundersen, G.G. Stable, detyrosinated microtubules function to localize vimentin intermediate filaments in fibroblasts. J. Cell Biol. 131, 1275–1290 (1995).
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).
Schuyler, S.C. & Pellman, D. Microtubule “plus-end-tracking proteins”: The end is just the beginning. Cell 105, 421–424 (2001).
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).
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).
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).
Bloom, K. It's a kar9ochore to capture microtubules. Nature Cell Biol. 2, E96–E98 (2000).
Schuyler, S.C. & Pellman, D. Search, capture and signal: games microtubules and centrosomes play. J. Cell Sci. 114, 247–255 (2001).
Kusch, J., Liakopoulos, D. & Barral, Y. Spindle asymmetry: a compass for the cell. Trends Cell Biol. 13, 562–569 (2003).
Yin, H., Pruyne, D., Huffaker, T.C. & Bretscher, A. Myosin V orientates the mitotic spindle in yeast. Nature 406, 1013–1015 (2000).
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).
Su, L.K. et al. APC binds to the novel protein EB1. Cancer Res. 55, 2971–2977 (1995).
Bienz, M. Spindles cotton on to junctions, APC and EB1. Nature Cell Biol. 3, E67–E68 (2001).
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).
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).
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).
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).
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).
Gundersen, G.G. Evolutionary conservation of microtubule-capture mechanisms. Nature Rev. Mol. Cell Biol. 3, 296–304 (2002).
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).
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).
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).
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).
Alberts, A.S. Identification of a carboxyl-terminal diaphanous-related formin homology protein autoregulatory domain. J. Biol. Chem. 276, 2824–2830 (2001).
Palazzo, A.F. et al. Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-regulated microtubule stabilization. Curr. Biol. 11, 1536–1541 (2001).
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).
Fukata, M. et al. Rac1 and Cdc42 capture microtubules through IQGAP1 and CLIP-170. Cell 109, 873–885 (2002).
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).
Wallar, B.J. & Alberts, A.S. The formins: active scaffolds that remodel the cytoskeleton. Trends Cell Biol. 13, 435–446 (2003).
Yasuda, S. et al. Cdc42 and mDia3 regulate microtubule attachment to kinetochores. Nature 428, 767–771 (2004).
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).
Subramanian, A. et al. Shortstop recruits EB1/APC1 and promotes microtubule assembly at the muscle-tendon junction. Curr. Biol. 13, 1086–1095 (2003).
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).
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).
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).
Kodama, A., Karakesisoglou, I., Wong, E., Vaezi, A. & Fuchs, E. ACF7. An essential integrator of microtubule dynamics. Cell 115, 343–354 (2003).
Evangelista, M., Zigmond, S. & Boone, C. Formins: signaling effectors for assembly and polarization of actin filaments. J. Cell Sci. 116, 2903–2911 (2003).
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).
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.).
The authors declare no competing financial interests.
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
The actin polymerization factor Diaphanous and the actin severing protein Flightless I collaborate to regulate sarcomere size
Developmental Biology (2021)
Current Opinion in Cell Biology (2021)
Actin/microtubule crosstalk during platelet biogenesis in mice is critically regulated by Twinfilin1 and Cofilin1
Blood Advances (2020)
Gastric polyposis and desmoid tumours as a new familial adenomatous polyposis clinical variant associated with APC mutation at the extreme 3′-end
Journal of Medical Genetics (2020)