Michael Way: Projects

Intracellular pathogens have developed numerous strategies to manipulate and use the many cellular systems of their hosts to facilitate their entry, replication, survival and spread.

Investigating exactly how pathogens hijack and subvert their unwilling hosts offers a unique opportunity to obtain mechanistic insights into the regulation and function of a multitude of cellular processes. To this end, we use a combination of quantitative imaging and biochemical approaches to study vaccinia virus as a model system to interrogate the regulation and function of Src and Rho GTPase signalling networks, actin and microtubule-based transport as well as cell migration.

Outside the context of vaccinia infection, we also examine the mechanisms regulating the assembly and function of invadopodia as well as the cellular function of Tes, a tumour suppressor that regulates Mena-dependent cell migration.

Signalling networks and regulation of actin polymerisation

The correct spatial and temporal regulation of actin polymerisation by phosphotyrosine-based signalling networks plays a critical role during many cellular processes. This includes cell migration, which plays an essential role during the development and throughout the lifetime of multicellular organisms. Unfortunately, deregulation of phosphotyrosine-based signalling networks can be devastating for example if it stimulates tumour cells to undergo metastasis.

Understanding how phosphotyrosine-based signalling induces actin polymerisation involves identifying how specificity is generated and regulated within a network by a common set of domains with relatively modest binding affinities and multiple interaction partners. This task is further complicated by the dynamic and co-operative nature of the interactions within any network. Consequently, unravelling exactly how signalling cascades stimulate actin polymerisation will require a molecular understanding and detailed knowledge of the interactions, dynamics and stoichiometry of proteins in the network.

The localised and sustained nature of the phosphotyrosine-based signalling network recruited by vaccinia make the virus an ideal model to tackle these fundamental questions and understand regulation of Arp2/3 dependent actin polymerisation (Weisswange et al., 2009; Nature. 458: 87-91).

WIP links Nck to N-WASP during vaccinia induced actin polymerisation

During vaccinia infection, a proportion of cell-associated enveloped viruses (CEV) attached to the plasma membrane induce Arp2/3 complex driven actin polymerisation to enhance their spread into neighbouring cells. CEV achieve this feat by locally activating Src and Abl family kinases, which results in the phosphorylation of tyrosine 112 and 132 of A36, an integral viral membrane protein localised beneath the virus in the plasma membrane. Phosphorylation of these two tyrosine residues leads to the recruitment of a signalling network, consisting of Grb2, Nck, WIP and N-WASP, the latter of which locally activates the Arp2/3 complex, stimulating actin polymerisation beneath the virus.

Recruitment of this signalling network is not unique to vaccinia, as it is also at the heart of a number of actin dependent cellular processes, including the formation of invadopodia during tumour cell invasion. Vaccinia therefore provides an excellent model to understand exactly how a co-operative phosphotyrosine-based Nck and N-WASP signalling network stimulates actin polymerisation.

Over the years, we have identified the main players involved in vaccinia-induced actin polymerisation (Welch and Way, 2013; Cell Host Microbe. 14: 242-55) and also uncovered an unexpected role for clathrin in enhancing this process (Humphries et al., 2012; Cell Host Microbe. 12: 346-59). Nck and N-WASP are essential, as they are required to recruit the WIP:N-WASP complex to the virus and activate the Arp2/3 complex respectively. In contrast, the role of WIP in vaccinia actin tail formation has been more obscure. We have now found that WIP, or its homologue WIRE are required for N-WASP recruitment and actin-based motility of the virus (Donnelly et al., 2013; Current Biol. 23: 999-1006).

Using a combination of biochemical approaches and expression of mutants in cells lacking endogenous Nck, WIP or N-WASP we have obtained essential insights into the hierarchy and connections within this network. WIP contains two Nck binding sites and is recruited to the virus, bound to N-WASP, by interacting with the second SH3 domain of Nck. N-WASP also contains two Nck binding sites, but its recruitment is dependent on its interaction with WIP rather than Nck. The first and third SH3 domains of Nck are not required to recruit the WIP:N-WASP complex but are involved in promoting actin assembly.

Our observations have established that WIP plays an essential role in linking Nck to N-WASP (Donnelly et al., 2013). Curiously, however, the interaction between Nck and N-WASP is not required for vaccinia actin tail formation, suggesting that an additional factor regulates the ability of N-WASP to activate the Arp2/3 complex beneath the virus.

Vaccinia F11, a PDZ scaffolding protein that inhibits RhoA by binding Myosin-9A

Prior to fusing with the plasma membrane and inducing actin polymerisation, vaccinia needs to traverse the cortical actin cytoskeleton.

Figure 1

Figure 1. Schematic representation of F11 interactions and its down regulation of RhoA via Myosin-9A.

The cortical actin, which is regulated by RhoA signalling, provides the cell with mechanical resilience and represents a significant physical barrier to the spread of infection. We have previously demonstrated that vaccinia inhibits RhoA signalling using F11, a viral protein that interacts directly with RhoA using a motif that is also found in the RhoA effector ROCK (Valderrama et al., 2006; Science. 311: 377-81).

During infection, F11-mediated inhibition of RhoA signalling stimulates cell migration, increases microtubule dynamics and also enhances viral release by modulating the cortical actin beneath the plasma membrane. F11 promotes viral spread by binding RhoA, however, the mechanistic basis of this inhibition remained to be established.

We have now found that F11 contains a central PDZ-like domain that is required to down regulate RhoA signalling and enhance viral spread (Handa et al., 2013; Cell Host Microbe. 14: 51-62). The F11 PDZ-like domain interacts with the PDZ binding motif of the RhoGTPase activating protein (GAP) Myosin-9A. In the absence of Myosin-9A, RhoA signalling is not inhibited resulting in fewer CEV inducing actin polymerisation and reduced virus release concomitant with less viral spread. The loss of the GAP activity of Myosin-9A or its ability to bind F11 also reduces the number of CEV inducing actin polymerisation. Furthermore, the ability of Myosin-9A to promote viral spread also depends on the capacity of F11 to bind RhoA.

Our observations demonstrate that F11, which is the first example of a viral protein with a functional PDZ domain, acts as a scaffolding protein to inhibit RhoA signalling by binding Myosin-9A (Handa et al., 2013).

Michael Way

+44 (0)20 379 62068

  • Qualifications and History
  • 1988 PhD in Structural Studies, Cambridge University, UK
  • 1989 Postdoctoral Fellow, LMB, Cambridge, UK
  • 1992 Postdoctoral Fellow, Whitehead Institute, Massachusetts, USA
  • 1995 Group Leader, Cell Biology Programme, EMBL, Germany
  • 2001 Established lab at the Imperial Cancer Research Fund, UK (in 2002 the Imperial Cancer Research Fund became Cancer Research UK)
  • 2015 Group Leader, the Francis Crick Institute, London, UK