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.

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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.

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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.).

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Correspondence to Gregg G. Gundersen.

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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

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