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Targeting of the F-actin-binding protein drebrin by the microtubule plus-tip protein EB3 is required for neuritogenesis

Abstract

Interactions between dynamic microtubules and actin filaments (F-actin) underlie a range of cellular processes including cell polarity and motility. In growth cones, dynamic microtubules are continually extending into selected filopodia, aligning alongside the proximal ends of the F-actin bundles. This interaction is essential for neuritogenesis and growth-cone pathfinding. However, the molecular components mediating the interaction between microtubules and filopodial F-actin have yet to be determined. Here we show that drebrin, an F-actin-associated protein, binds directly to the microtubule-binding protein EB3. In growth cones, this interaction occurs specifically when drebrin is located on F-actin in the proximal region of filopodia and when EB3 is located at the tips of microtubules invading filopodia. When this interaction is disrupted, the formation of growth cones and the extension of neurites are impaired. We conclude that drebrin targets EB3 to coordinate F-actin–microtubule interactions that underlie neuritogenesis.

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Figure 1: Biochemical analysis of the interaction between drebrin and EB1/EB3.
Figure 2: Drebrin co-localizes with F-actin and microtubules in filopodia.
Figure 3: Drebrin interacts directly and specifically with EB3 in growth-cone filopodia.
Figure 4: Interaction between drebrin and EB3 induces the formation of microtubule-containing filopodia.
Figure 5: Inhibition of EB3–drebrin interaction and knockdown of drebrin perturb neuritogenesis.

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Acknowledgements

We thank Britta Eickholt and her laboratory and the P.R.G.-W. laboratory for helpful discussions. We are grateful to Brigitte Keon for the gift of human drebrin E cDNA, Ewan Morrison for EB1 cDNA, Roger Tsien for CherryRFP cDNA, Britta Eickholt for drebrin–RFP, and Niels Galjart for polyclonal antibodies against CLASP 1, CLASP 2 and CLIP-170 and EB3 cDNA, and Matthias Krause for help with kymography and comments on the manuscript. This work was supported by grants from the Medical Research Council, the Royal Society and the Wellcome Trust. Sara Geraldo's PhD studentship is funded by Fundação para a Ciência e Tecnologia, Portugal.

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U.K.K. performed the biochemical experiments shown in Fig. 1, P.R.G.-W. conducted the immunofluorescence experiments shown in Fig. 2, S.G. performed the live-cell imaging experiments shown in Figs 2 and 5 and the transfection experiments in Fig. 4, M.P. and S.G. conducted the FLIM experiments shown in Fig. 3, J.K.C. made EB1–RFP and EB3–RFP and conducted preliminary transfection experiments in neurons, P.R.G.-W. coordinated the whole project and wrote the manuscript, and all authors read and edited it.

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Correspondence to Phillip R. Gordon-Weeks.

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Geraldo, S., Khanzada, U., Parsons, M. et al. Targeting of the F-actin-binding protein drebrin by the microtubule plus-tip protein EB3 is required for neuritogenesis. Nat Cell Biol 10, 1181–1189 (2008). https://doi.org/10.1038/ncb1778

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