In addition to understanding the GEF GAP cycle

In addition to understanding the GEF-GAP cycle regulating Rho-family GTPases, studies have continued to address the role of the Rho-GDIs, which binds to the geranylgeranyl tail to sequester Rho-family GTPases from interacting with lipid membranes, preventing their activation. Whilst it would be tempting to speculate that Rho-GDIs act to maintain a pool of unused Rho-family GTPases, a recent study has shown that Rho-GDI serves a specific role in controlling the level of Rho-GTPase activation by coordinating GTPase activity and re-activation on a ∼10 s timescale [39]. Furthermore, a role for the lipid composition of the plasma membrane in regulating this association has been proposed. By inhibiting fatty Phenytoin sodium synthetase (FAS) in migrating inflammatory macrophages, Wei et al. were able to demonstrate a role for fatty acids in stabilising the presence of Rho-family GTPases at the plasma membrane, in the context of diabetes [] (Figure 1). Rho-GTPases have long been known to signal downstream of a variety of receptors such as receptor tyrosine kinases (RTKs), G-protein-coupled receptors (GPCRs) and integrins to name a few. Recently, a study has extended this list, identifying a novel role for non-canonical Notch signalling in driving Rac1 activity via the GEF TRIO, which in turn reinforces the formation of adherens junctions [] in endothelial cells both in vitro and in vivo. It will therefore be interesting to observe if this pathway plays a role in collective cell migration, which is co-ordinated via cadherin-based adhesions (Figure 2a). In terms of placing Rho-family GTPases within the context of a signalling network, Boolean modelling of Rac/RhoA signalling in invasive cancer cells has established a link between MAP kinase signalling downstream of RTKs, and the activation of RhoA, which we had previously shown to drive invasion into fibronectin-rich extracellular matrix [22,23]. Model simulations predicted MAPK signalling controls a negative feedback loop via the Sos1-Eps8-Abi1 complex that supresses Rac1 activity, enabling the activation of RhoA in cells migrating both in 2D plastic and 3D cell-derived matrix. Experimental inhibition of MAP kinase signalling enabled the re-activation of Rac1 at the leading edge of the cell, supressing filopodia formation and invasion into extracellular matrix and on cell-derived matrix. Critically, knockdown of Eps8 (a key component of the RacGEF complex in this system) rendered cells insensitive to MAPK inhibition, re-enabling cells to activate RhoA at the leading edge of the cell, driving invasive migration []. Such feedback loops may provide plasticity to the migrating cell, enabling it to re-programme its leading edge in response to a changes in the surrounding environment [43]. Similar approaches using more sophisticated kinetic modelling identified a role for PAK signalling in mediating a bi-stable switch [44]. Exposing MDA-MB-231 breast carcinoma cells in 2D culture to increasing amounts of PAK inhibitor had different effects on Rac and RhoA signalling depending on whether cells had been pre-incubated with the same inhibitor, demonstrating the predicted hysteresis. Interestingly this bi-stability is conserved in actin dynamics, and suggests that cytoskeletal signalling pathways encode a memory of activation status [44].
Rho GTPases in cell–matrix interactions The importance of Rho-family GTPases in cell matrix interactions has been well appreciated ever since the initial identification of RhoA as a regulator of stress fibres, which showed that focal adhesions are unable to form in the absence of RhoA signalling [6]. Since then, a number of studies have shown extensive reciprocal signalling between matrix receptors and Rho-family GTPases, however for the purpose of this review, we shall focus on a handful of recent studies that have extended our understanding of direct signalling between focal adhesions and Rho GTPases. Focal adhesions have long been known to control the activity of Rho-family GTPases via adaptor proteins that can signal to GEFs and GAPS, such as paxillin, which can signal to both activate Rac1 and suppress RhoA, and FAK which can signal to supress RhoA activity [45,46]. β-Pix is a Rac GEF recruited to adhesion complexes through interaction with Git1/2 recruitment to paxillin [47,48]. Interestingly Git1/2-β -Pix can also be recruited to adhesion complexes by RhoJ, which mediates adhesion turnover by sustaining Rac1 activity and preventing RhoA activation [49]. These types of interactions can govern the transition of nascent adhesion complex to focal complexes, but restrain the maturation to focal adhesion (which requires RhoA–driven contractility [6,50]). Interestingly, RhoU is stabilised by interaction with PAK4 in a Cdc42 and kinase-independent manner to regulate adhesion turnover [51]. This suggests that complex feedback networks exist between Rho GTPases and adhesion complexes that might determine the intricate and subtle morphological adaptations of adhering and migrating cells (Figure 2b). All these studies were performed principally in 2D cell culture and thus it remains to be understood how RhoJ and RhoU mediate crosstalk with focal complexes in 3D matrix environments.