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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 fashion (1-5). 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 virus (6-9), 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., During infection, vaccinia virus enhances its cell-to-cell spread by stimulating ARP2/3-complex-dependent actin polymerization beneath the extracellular enveloped virus by locally activating Src-and Abl-dependent signalling cascadesb. (6,10-12) consisting of GRB2, NCK, [...]

Phosphorylation of tyrosine 112 in the viral protein A36R by Src-family kinases is required for the actin-based motility of vaccinia. Tyrosine phosphorylation of A36R leads to direct interaction with Nck, the adaptor protein, and recruitment of N-WASP. Nck and N-WASP are required for actin-based motility of vaccinia virus.

Cell movement is essential for the development and survival of multicellular organisms, although it can be detrimental as when cancer cells move to distant parts of the body. The polymerization of actin monomers into filaments close to the plasma membrane is thought to be responsible for cell movement. Mullins and colleagues have shown that a seven-protein complex is central to the organization of actin-related proteins Arp2 and Arp3. A model has been proposed for the mechanism of actin polymerization at the leading edge of the cell.