Viral actin-based motility is regulated by the rate of N-WASP turnover during ARP2/3-complex-dependent actin polymerization.
ARP2/3 complex activity is crucial for the reorganization of the actin cytoskeleton at the cell cortex during events such as vesicular trafficking, pathogen infection and cell movement. The study of pathogens undergoing actin-based motility has provided insight into how this activity is regulated. Now, Michael Way and colleagues show that ARP2/3-complex-dependent motility of vaccinia virus is controlled by the dynamic interaction between neuronal Wiskott–Aldrich syndrome protein (N-WASP) and growing actin filaments.
The vaccinia virus promotes actin polymerization by locally activating a signalling cascade that consists of N-WASP — which binds to an actin monomer and to the ARP2/3 complex — and the adaptor proteins NCK, WASP-interacting protein (WIP) and growth factor receptor-bound protein 2 (GRB2). Activation of the ARP2/3 complex induces the formation of actin tails, which enhance the cell-to-cell spread of the virus.
To clarify the dynamics of this signalling network, the authors used fluorescence recovery after photobleaching (FRAP) to analyse the turnover rates of green fluorescent protein-tagged N-WASP, NCK, WIP and GRB2 recruited to the tips of virus-induced actin tails. In the absence of GRB2, the recovery rates of the three remaining proteins were significantly increased and actin tail formation was reduced. Thus, GRB2 stabilizes the actin tail-nucleating complex. N-WASP turnover was slower than that of NCK and WIP, prompting the authors to analyse which N-WASP interactions might increase its stability. An N-WASP mutant that cannot bind actin monomers has a higher turnover and induces fewer actin tails, showing that the interaction with actin stabilizes N-WASP. Furthermore, N-WASP mutants that cannot bind and activate the ARP2/3 complex are essentially stable, suggesting that N-WASP turnover relies on active actin polymerization. This finding was confirmed by treatment with an actin polymerization inhibitor.
The authors suggest that slow N-WASP turnover can be explained by its capacity to interact with growing barbed ends as well as the ARP2/3 complex. However, interaction with the barbed ends might also limit filament extension, thus limiting the rate of virus movement. Consistently, both disrupting the interaction of N-WASP with the barbed ends and reducing its stability by depleting GRB2 in cells resulted in increased virus motility.
The results presented by Michael Way and colleagues show that higher N-WASP turnover enhances ARP2/3-complex-dependent virus motility. Nevertheless, actin tails were shorter, suggesting that the interaction between N-WASP and the barbed ends does not limit filament extension. Thus, N-WASP turnover might regulate actin filament growth by antagonizing capping proteins. Further studies are required to elucidate how the balance between N-WASP stability, barbed end capping and ARP2/3-complex-dependent actin nucleation is achieved and how this might affect filament organization.
Kim Baumann
References
Weisswange, I. et al. The rate of N-WASP exchange limits the extent of ARP2/3-complex-dependent actin-based motility. Nature458, 87–91 (2009) | Article | PubMed |
Takenawa, T. & Suetsugu, S. The WASP–WAVE protein network: connecting the membrane to the cytoskeleton. Nature Rev. Mol. Cell Biol.8, 37–48 (2007) | Article | PubMed |
