The rate of N-WASP exchange limits the extent of ARP2/3-complex-dependent actin-based motility

Abstract

Understanding cell motility will require detailed knowledge not only of the localization of signalling networks regulating actin polymerization, but also of their dynamics. Unfortunately, many signalling networks are not amenable to such analysis, as they are frequently transient and dispersed. By contrast, the signalling pathways used by pathogens undergoing actin-based motility are highly localized and operate in a constitutive fashion1,2,3,4,5. Taking advantage of this, we have analysed the dynamics of neuronal Wiskott–Aldrich syndrome protein (N-WASP), WASP-interacting protein (WIP), GRB2 and NCK, which are required to stimulate actin-related protein (ARP)2/3-complex-dependent actin-based motility of vaccinia virus6,7,8,9, using fluorescence recovery after photobleaching. Here we show that all four proteins are rapidly exchanging, albeit at different rates, and that the turnover of N-WASP depends on its ability to stimulate ARP2/3-complex-mediated actin polymerization. Conversely, disruption of the interaction of N-WASP with GRB2 and/or the barbed ends of actin filaments increases its exchange rate and results in a faster rate of virus movement. We suggest that the exchange rate of N-WASP controls the rate of ARP2/3-complex-dependent actin-based motility by regulating the extent of actin polymerization by antagonizing filament capping.

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Figure 1: Components of the vaccinia actin-nucleating complex are highly dynamic.
Figure 2: GRB2 stabilizes the vaccinia actin-nucleating complex.
Figure 3: WH2:actin filament interactions contribute to N-WASP stability.
Figure 4: Virus movement and N-WASP turnover is dependent on actin polymerization.

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Acknowledgements

We would like to thank T. Pawson and S. Snapper for NCK- and N-WASP-null cell lines and the FACS laboratory (Cancer Research UK, London) for help sorting stable cell lines. We are also very grateful to the members of the Way laboratory, J. Beaudouin, M.-F. Carlier, H. Gerhardt, B. Mayer, K. Rottner, M. Rosen and M. Welch for discussions and comments on the manuscript. T.P.N. was funded in part by a fellowship from the Human Frontier Science Program (HFSP). This work was funded by Cancer Research UK.

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Correspondence to Michael Way.

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Weisswange, I., Newsome, T., Schleich, S. et al. The rate of N-WASP exchange limits the extent of ARP2/3-complex-dependent actin-based motility. Nature 458, 87–91 (2009). https://doi.org/10.1038/nature07773

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