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Filopodia are required for cortical neurite initiation

Nature Cell Biology volume 9, pages 1347–1359 (2007)Cite this article

Abstract

Extension of neurites from a cell body is essential to form a functional nervous system; however, the mechanisms underlying neuritogenesis are poorly understood. Ena/VASP proteins regulate actin dynamics and modulate elaboration of cellular protrusions. We recently reported that cortical axon-tract formation is lost in Ena/VASP-null mice and Ena/VASP-null cortical neurons lack filopodia and fail to elaborate neurites. Here, we report that neuritogenesis in Ena/VASP-null neurons can be rescued by restoring filopodia formation through ectopic expression of the actin nucleating protein mDia2. Conversely, wild-type neurons in which filopodia formation is blocked fail to elaborate neurites. We also report that laminin, which promotes the formation of filopodia-like actin-rich protrusions, rescues neuritogenesis in Ena/VASP-deficient neurons. Therefore, filopodia formation is a key prerequisite for neuritogenesis in cortical neurons. Neurite initiation also requires microtubule extension into filopodia, suggesting that interactions between actin-filament bundles and dynamic microtubules within filopodia are crucial for neuritogenesis.

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Figure 1: Filopodia can dilate to form neurites.
Figure 2: Capping actin filaments in control neurons with cytochalasin D or capping protein inhibits filopodia formation and neuritogenesis.
Figure 3: mDia2 and myosin X expression rescue filopodia and neuritogenesis in mmvvee neurons.
Figure 4: Filopodia are necessary but not sufficient for neuritogenesis.
Figure 5: Filopodia are targeted by dynamic microtubules.
Figure 6: Laminin rescues neurite initiation in mmvvee mutants.
Figure 7: Laminin rescues neuritogenesis through formation of actin-rich filopodia-like extensions.
Figure 8: Myosin II inhibition rescues neuritogenesis through formation of actin-rich filopodium-like extensions.

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Acknowledgements

We thank: R. Cheney (UNC Chapel Hill) for EGFP–MyoX and MyoX antibodies; R. Tsien (UC San Diego) for mCherry; R. Makar for generous assistance with the mouse colony and advice; M. Wold and N. Enzer; and E. Pinheiro and K. O'Brien for help during early phases of the work. We appreciate the helpful advice, comments and discussion from all the Gertler lab members. E.W.D. was supported by a National Institutes of Health (NIH) grant (F32-NS45366). A.V.K. was supported by an Anna Fuller Predoctoral Fellowship. S.G. was supported by a Jane Coffin Childs fellowship. U.P. was supported by funds from ICBP (Integrative Cancer Biology Program). D.A.R. was supported by a Ludwig Fellowship. C.F. was supported by NIH grant F32-GM071156. This work was supported by NIH grant GM68678 and funds from the Stanley Medical Research Institute (F.B.G).

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Author notes

  1. Erik W. Dent

    Present address: Current address: Department of Anatomy, University of Wisconsin, Madison, WI 53706, USA.,

  2. Adam V. Kwiatkowski

    Present address: Current address: Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA.,

Authors and Affiliations

  1. Department of Biology, Massachusetts Institute of Technology, Cambridge, 02139, MA, USA

    Erik W. Dent, Adam V. Kwiatkowski, Leslie M. Mebane, Ulrike Philippar, Melanie Barzik, Douglas A. Rubinson, Stephanie Gupton, J. Edward Van Veen, Craig Furman & Frank B. Gertler

  2. Department of Radiology, Division of Nuclear Magnetic Resonance Research, Johns Hopkins University School of Medicine, Baltimore, 21205, MD, USA

    Jiangyang Zhang & Susumu Mori

  3. Laboratory of Cell Structure and Signal Integration, Van Andel Research Institute, Grand Rapids, 49503, MI, USA

    Arthur S. Alberts

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Contributions

E.W.D. and F.B.G. conceived the project. E.W.D. performed all of the cell biological experiments and wrote the manuscript with F.B.G. A.V.K. generated the EVL−/− mice and contributed substantively to revising the manuscript. A.V.K and D.A.R. established the mmvvee colony, helped with initial experiments and provided advice and guidance. L.M. executed the platinum-replica electron microscopy. M.B. generated mDia2 subclones and helped in planning all mDia2 experiments. U.P. cloned Mena2+, Mena3+ and Mena3+2+ isoforms and performed RT–PCR on prenatal cortex. J.E.V provided much help with the mouse colony, and J.E.V. and S.G. performed experiments resulting in Supplementary Fig. S7. C.F. generated the capping-protein constructs and provided much advice during the project. A.A. provided mDia2 reagents and advice. J.Z and S.M. performed all μDTI on mmvvee embryos. F.B.G provided advice, overall direction and supervised the execution of the project. All authors read and edited the manuscript.

Corresponding author

Correspondence to Frank B. Gertler.

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Dent, E., Kwiatkowski, A., Mebane, L. et al. Filopodia are required for cortical neurite initiation. Nat Cell Biol 9, 1347–1359 (2007). https://doi.org/10.1038/ncb1654

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