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N-WASP regulates the epithelial junctional actin cytoskeleton through a non-canonical post-nucleation pathway

Nature Cell Biology volume 13, pages 934–943 (2011)Cite this article

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Abstract

N-WASP is a major cytoskeletal regulator that stimulates Arp2/3-mediated actin nucleation. Here, we identify a nucleation-independent pathway by which N-WASP regulates the cytoskeleton and junctional integrity at the epithelial zonula adherens. N-WASP is a junctional protein whose depletion decreased junctional F-actin content and organization. However, N-WASP (also known as WASL) RNAi did not affect junctional actin nucleation, dominantly mediated by Arp2/3. Furthermore, the junctional effect of N-WASP RNAi was rescued by an N-WASP mutant that cannot directly activate Arp2/3. Instead, N-WASP stabilized newly formed actin filaments and facilitated their incorporation into apical rings at the zonula adherens. A major physiological effect of N-WASP at the zonula adherens thus occurs through a non-canonical pathway that is distinct from its capacity to activate Arp2/3. Indeed, the junctional impact of N-WASP was mediated by the WIP-family protein, WIRE, which binds to the N-WASP WH1 domain. We conclude that N-WASP–WIRE serves as an integrator that couples actin nucleation with the subsequent steps of filament stabilization and organization necessary for zonula adherens integrity.

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Figure 1: The epithelial junctional actin cytoskeleton contains two pools of actin filaments that are predominantly nucleated at the membrane.
Figure 2: N-WASP regulates the junctional actin cytoskeleton without affecting filament nucleation.
Figure 3: N-WASP regulates junctional filament stability and organization by a VCA-independent, WH1-dependent mechanism.
Figure 4: The N-WASP WH1 domain regulates junctional filament stability and dynamic spatiotemporal organization of F-actin.
Figure 5: N-WASP recruits WIRE to regulate the junctional actin cytoskeleton.
Figure 6: Junctional biogenesis is perturbed by N-WASP or WIRE knockdown.

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Acknowledgements

We thank our laboratory colleagues for their advice and support and other colleagues for the gifts of reagents, notably S. Thomas (Birmingham University, UK) for an early gift of N-WASP antibodies and J. Demmers (Erasmus MC, Rotterdam, The Netherlands) for proteomics analysis. This work was financially supported by the National Health and Medical Council of Australia and the Human Frontiers Science Program. A.S.Y. is a Research Fellow of the NHMRC. Confocal imaging was carried out at the IMB/ACRF Cancer Biology Imaging Facility, established with the generous support of the Australian Cancer Research Foundation.

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Authors and Affiliations

  1. Division of Molecular Cell Biology, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia

    Eva M. Kovacs, Suzie Verma, Radiya G. Ali, Aparna Ratheesh & Alpha S. Yap

  2. School of Biomedical Sciences, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia

    Radiya G. Ali

  3. Division of Genomics and Computational Biology, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia

    Nicholas A. Hamilton

  4. Cell Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

    Anna Akhmanova

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Contributions

E.M.K., R.G.A. and A.S.Y. conceived the project and designed experiments; E.M.K., S.V. and R.G.A. carried out experiments; A.R. developed reagents; A.A. carried out protein partner analysis; E.M.K., N.A.H. and A.S.Y. analysed the data; E.M.K. and A.S.Y. wrote the manuscript.

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Correspondence to Alpha S. Yap.

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Kovacs, E., Verma, S., Ali, R. et al. N-WASP regulates the epithelial junctional actin cytoskeleton through a non-canonical post-nucleation pathway. Nat Cell Biol 13, 934–943 (2011). https://doi.org/10.1038/ncb2290

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