Members of the Rho family of small GTPases have been shown to operate in distinct pathways signaling the formation of different organisational arrays of the actin filaments in the actin cytoskeleton (illustration below). Rac and Cdc42 signal the formation of lamellipodia and filopodia, respectively and Rho signals the formation of actin stress fibre bundles (Hall, 1998). The formation of these actin arrays is accompanied by the formation of adhesion foci that associate with them (see Heasman and Ridley, 2008).
A schematic illustration of the actin cytoskeleton of a fibroblast, indicating the Rho-family members involved in signaling different subcompartment assemblies of actin filaments: Rac, lamellipodia and focal complexes; Cdc42, filopodia and focal complexes; Rho, stress fibre bundles and focal adhesions (modified from Kaverina et al., 2002). Abbreviations: FX, focal complexes; FA, focal adhesions, Lam, lamellipodium; Fil, Filopodium; SF, stress fibre bundle; CB, Concave bundle (essentially stress fibre bundle at non-motile cell edges); Arc, arc shaped bundles sometimes observed under the dorsal cell surface; LM, loose meshwork of actin filaments; Rf, ruffle (corresponding to upfolding lamellipodium).
The transition of a focal complex associated with a lamellipodium, to a focal adhesion associated with a stress fibre bundle is effected by a change in the balance of signaling, from Rac to Rho. This signaling transition can be illustrated in living cells in which the balance of Rac and Rho activities are experimentally manipulated (Rottner et al., 1999).
Rho can be specifically inhibited by ribosylation with C3 transferase. The Rho-proteins can also be mutated to produce dominant negative and constitutively active forms. By injecting a single cell with both C3 transferase and the constitutively active Rac mutant (L61Rac), Rho can be downregulated and Rac upregulated. The result of injecting such a mixture into a Swiss 3T3 fibroblast is shown here:
Focal complexes induced by Rac. Video shows a Swiss 3T3 fibroblast that was injected with C3 transferase to inactivate Rho and constitutively active L61 Rac to activate Rac. The cell was also injected with rhodamine-tagged vinculin to mark adhesion sites. Only small peripheral adhesions are formed in association with ruffling lamellipodia; these correspond to focal complexes. (From Rottner et al., 1999).
Starting with the experimental set-up as in the last figure, the activity of Rho can be up-regulated by a subsequent injection with a constitutively active Rho mutant (L63Rho). This experiment is shown in the figure below. The upregulation of Rho (during the video sequence) causes a transition of the Rac-induced focal complexes at the periphery into larger, elongated adhesions, corresponding to Rho-induced focal adhesions. This experiment also shows that focal complexes can serve as precursors of focal adhesions:
At the beginning of the experiment, this cell was injected with the same protein mixture as in the figure before, to induce “Rac focal complexes” at the cell periphery. Subsequently, the cell was injected with constitutively active L63Rho. The resulting up-regulation of Rho was followed by the transition of the individual, punctate focal complexes, into elongated focal adhesions. (From Rottner et al., 1999).
Rac and Rho antagonise each other– in consequence the expression of different actin filament sub-compartments is influenced by the balance of Rho-protein activities. This antagonism can be illustrated by monitoring changes in the reoganisatioin of cells labelled with an adhesion site marker (e.g. vinculin), in response to manipulations of the activites of Rac and Rho. Two examples are shown in the figures below:
An example of the antagonism between Rac and Rho. A Swiss 3T3 fibroblast was injected with rhodamine-tagged vinculin to mark adhesion sites. The cell was intially immotile and expressed mainly focal adhesions (elongated adhesion sites). During the experiment, Rho-kinase inhibitor (Y-27632) was added to inhibit the downstream pathway of Rho that leads to focal adhesion assembly. This caused the rapid disassembly of the focal adhesions. In addition, the cell actively initiated the formation of lamellipodia and associated focal complexes, diagnostic of the activation of Rac. (From Rottner et al., 1999).
A second example of the antagonism between Rac and Rho. In this experiment a fibroblast was first injected with rhodamine-tagged vinculin to mark adhesion foci. Mid-way through the video sequence it was then injected with dominant negative Rac (N17Rac). The resulting down-regulation of Rac activity is recognised from the arrest of protrusive and ruffling activity at the cell front. Note, that in addition, the down-regulation of Rac was accompanied by the growth of the elongated focal adhesions, diagnostic of the up-regulation of Rho. (Top) fluorescence image; (bottom) phase contrast image
- Hall, A. (1998). Rho GTPases and the Actin Cytoskeleton. Science, 279, 509–514.
- Heasman, S., J. and Ridley, A., J. (2008). Mammalian Rho GTPases: new insights into their functions from in vivo studies. Nat. Rev. Mol. Cell. Biol. 9, 690–701.
- Kaverina, I., Krylyshkina, O., Small, J.V. (2002). Regulation of substrate adhesion dynamics during cell motility. Int. J. Biochem. Cell Biol. 34, 746–61.
- Rottner, K., Behrendt, B., Small, J.V., Wehland, J. (1999). VASP dynamics during lamellipodia protrusion. Nat. Cell. Biol. 1, 321 – 322.