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

Related Pub­li­ca­tions

  • Hall, A. (1998). Rho GTPases and the Actin Cytoskele­ton. Sci­ence, 279, 509514. NCBI PubMed
  • 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, 690701. NCBI PubMed
  • Kave­rina, I., Krylyshk­ina, O., Small, J.V. (2002). Reg­u­la­tion of sub­strate adhe­sion dynam­ics dur­ing cell motil­ity. Int. J. Biochem. Cell Biol. 34, 74661. PDF
  • Rot­tner, K., Behrendt, B., Small, J.V., Wehland, J. (1999). VASP dynam­ics dur­ing lamel­lipo­dia pro­tru­sion. Nat. Cell. Biol. 1, 321322. PDF