Rho-family GTPases, including Cdc42, Rac1 and RhoA, mediate the interaction of cytoskeleton proteins during cell adhesion and migration. These GTPases have been implicated in several different cell types: fibroblasts, T cells, macrophages, astrocytes, epithelial cells and neuronal cells (Fukata et al., 2003). Recent intensive analyses have started to clarify how Rho-family GTPases regulate cell migration: basically they re- organise the cytoskeleton by producing a gradient of signalling molecules (Fukata et al., 2003; Raftopoulou and Hall, 2004; Scales and Parsons, 2011).
The Rho GTPase switch
Rho-family GTPases are ubiquitously expressed and 20 members have been identified in mammals. The Rho-family GTPases cycle between a GDP-bound inactive form and a GTP-bound active form (Fukata et al., 2003). This process is regulated by three factors: guanine nucleotide exchange factors (GEF), Rho GDP
dissociation inhibitor (GDIs), and GTPase-activating proteins (GAPs). Their best- characterised function is to regulate actin dynamics in the lamellipodium. The in vitro analyses focused mainly on fibroblasts have clarified that Rho regulates the assembly of actin and myosin filaments, while Rac1 and Cdc42 organize polymerization of actin to form peripheral lamellipodia and filopodia protrusions, respectively (Raftopoulou and Hall, 2004). Moreover, these three GTPases are all involved in the assembly of integrin-based adhesions (Nobes and Hall, 1995).
Regulation of Rho GTPase during cell migration
High resolution fluorescence images have shown that Rac is found at the front of lamellipodia and regulates actin polymerization during cell migration (Chen et al., 2008). Rho, on the other hand, is thought to regulate the contraction and retraction at the rear of cell body, and would be expected to follow an inverse distribution to Rac. For example, by using FLAIR technology, Rac can be visualized in migrating fibroblasts with a high concentration at leading edge and Rho at the rear (Kraynov et al., 2000; Stuart et al., 2008). Cdc42, unlike Rac and Rho, is thought to establish cell polarity in order to initiate migration as live fluorescence imaging showed that Cdc42 was located in the Golgi apparatus where it mediated secretion in endocytic transport (Kroschewski et al., 1999). In the 3T3 fibroblast scratch wound model, a gradient of Rac1 activity exists across the flank of the wound with highest concentration at the leading edge of the wound. A comparison of localization between Rac1 and Cdc42 showed that the highest concentration of Rac is closer to the leading edge than that of Cdc42 (Itoh et al., 2002).
PI3-Kinase and PIP3 in regulation of Rho GTPases activity
PI3K and its product PIP3 have been widely implicated in a pivotal role in regulating cell polarity and migration in physiological and pathological conditions (Stephens et al., 2002). During chemotaxis, PI3K is required for lamellipodium extension and migration via signalling from G-coupled receptors (Li et al., 2000). Fluorescence imaging of GFP-tagged PIP3 has shown that extracellular stimuli induce their redistribution to the leading edge of migrating cells and co-localize with centres of actin polymerisation (Haugh et al., 2000; Servant et al., 2000).
But how does the PI3K signalling pathway interact with Rho-family GTPase activation? It has been found that both regulatory and catalytic subunits (p85 and p110, respectively) of PI3k are present in the eluates of GTPγS-bound affinity column chromatographs (Kobayashi et al., 1998; Raftopoulou and Hall, 2004). In addition GTP-bound Rac1 and Cdc42 can enhance PI3K activity and work as the upstream signals of PI3K (van Leeuwen et al., 1999). Due to the evidence that Rac1 GEFs are activated by PIP3, a positive feedback loop for cell migration is proposed for the PI3K signalling pathway: PIP3—GEFs—Rho GTPases—PI3K—PIP3 (Raftopoulou and Hall, 2004). Recently, PTEN, a lipid phosphatase and a negative regulator of cell survival mediated by the PI3k-Akt pathway, exhibits reciprocal localization to PI3K in Dictyostelium (Funamoto et al., 2002).
Downstream effects of Rho family GTPases during cell migration
Rac and Cdc42 are both required for cell migration, with Rac needed for the generation of protrusion force and Cdc42 for polymerisation of actin in filopodia. Recent studies on Drosophila have shown that Cdc42 loss of function mutations do not affect the migration speed of peripheral glial cells but instead reduce the directedness of migration (Sepp and Auld, 2003). The interactions between Rac/Cdc42 and actin dynamics have been intensely investigated. The Ser/Thr kinase PAK is commonly activated upon Rac or Cdc42 activation and is believed to play a role in regulating actin dynamics and adhesions. In addition, PAK phosphorylates and activates LIM kinase, which in turn inactivates cofilin. Cofilin is crucial for actin treadmilling at the front of migrating cells by dissociating actin subunits from pointed-end actin filaments (Arber et al., 1998) (Figure 1-12).
The WASP/SCAR/WAVE family of scaffold proteins are the essential regulators of actin dynamics. They are able to stimulate the Arp2/3 complex, which can initiate actin polymerisation either de novo or at the barbed end or sides of pre-existing filaments (Amann and Pollard, 2001). Cdc42 activates WASP/N-WASP directly and PIP3 is an important cofactor (Rohatgi et al., 2001), whereas Rac1 activates SCAR/WAVE family members indirectly (Eden et al., 2002). Rho activity is responsible for focal adhesion assembly and cell contractility. In addition, Rho activity is associated with actin and myosin interaction and therefore induces the contraction of the rear of the cell body via ROCK (Alblas et al., 2001) (Figure 1-12).
Clearly, if Rho activity is at the front of a migrating cell this will block membrane protrusion, hence mechanisms must be in place to inhibit its activity at the leading edge. Rac may have this function because expression activated Rac has been shown to inhibit Rho activity in many cell types, ranging from fibroblast to neurons (Raftopoulou and Hall, 2004).
Rho-family GTPase and polarity
Although Rho-family GTPases have received attention mainly for their actin modification effects, it is now clear they also possess functions for the regulation of microtubules and cell polarity (Wittmann and Waterman-Storer, 2001). While it seems that microtubules are not involved in lamellipodium dynamics and chemotaxis over a short distance, long distance migration and the persistence of cell polarity are achieved by the microtubule cytoskeleton. Rho was shown to promote the stabilization of microtubules by mDia, which is involved with microtubule capping (Ishizaki et al., 2001). Rac, on the other hand, may elongate microtubules through PAK and the inactivation of the microtubule destabilizing protein, stathmin (Daub et al., 2001).
Cdc42 plays an essential role in defining cell polarity in response to external environmental stimuli. Inhibition of Cdc42 blocked macrophage chemotaxis to CSF without impairing their motility (Ridley, 2001). The polarized migration in many cells is reflected in the reorganization of the microtubule skeleton and centrosome, which usually face the direction of migration (Raftopoulou and Hall, 2004). Cdc42 has been shown to regulate reorientation of the microtubule skeleton and centrosome in migrating astrocytes and fibroblasts. The mechanism involves GTP-bound Cdc42 activating Par6 and PKCζ at the leading edge of cells, which is essential for determining the orientation of cell migration (Etienne-Manneville and Hall, 2001).
Figure 1-12 Rho-family GTPases regulated pathways affect actin filaments organization
Rho promotes contractile actin:myosin filament assembly through two effectors, mDia and ROCK. Rac and Cdc42 both regulate actin polymerization through the WASP/SCAR/WAVE family of proteins acting on Arp2/3 complex (Alblas et al., 2001).