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. 1997 Jun 16;137(6):1421-31.
doi: 10.1083/jcb.137.6.1421.

The small GTPases Rho and Rac are required for the establishment of cadherin-dependent cell-cell contacts

V M Braga  1 , L M Machesky, A Hall, N A Hotchin
Affiliations

The small GTPases Rho and Rac are required for the establishment of cadherin-dependent cell-cell contacts

V M Braga et al. J Cell Biol. .

Abstract

Cadherins are calcium-dependent cell-cell adhesion molecules that require the interaction of the cytoplasmic tail with the actin cytoskeleton for adhesive activity. Because of the functional relationship between cadherin receptors and actin filament organization, we investigated whether members of the Rho family of small GTPases are necessary for cadherin adhesion. In fibroblasts, the Rho family members Rho and Rac regulate actin polymerization to produce stress fibers and lamellipodia, respectively. In epithelial cells, we demonstrate that Rho and Rac are required for the establishment of cadherin-mediated cell-cell adhesion and the actin reorganization necessary to stabilize the receptors at sites of intercellular junctions. Blocking endogenous Rho or Rac selectively removed cadherin complexes from junctions induced for up to 3 h, while desmosomes were not perturbed. In addition, withdrawal of cadherins from intercellular junctions temporally precedes the removal of CD44 and integrins, other microfilament-associated receptors. Our data showed that the concerted action of Rho and Rac modulate the establishment of cadherin adhesion: a constitutively active form of Rac was not sufficient to stabilize cadherindependent cell-cell contacts when endogenous Rho was inhibited. Upon induction of calcium-dependent intercellular adhesion, there was a rapid accumulation of actin at sites of cell-cell contacts, which was prevented by blocking cadherin function, Rho or Rac activity. However, if cadherin complexes are clustered by specific antibodies attached to beads, actin recruitment to the receptors was perturbed by inhibiting Rac but not Rho. Our results provide new insights into the role of the small GTPases in the cadherin-dependent cell- cell contact formation and the remodelling of actin filaments in epithelial cells.

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Figures

Figure 1
Figure 1
Establishment of cadherin-dependent cell–cell contacts requires Rho and Rac activities. Confluent patches of keratinocytes grown in low calcium medium were microinjected with different recombinant proteins and immediately transferred to standard medium for 55 min to induce calcium-dependent adhesion. Injected proteins were: C3 transferase to block endogenous Rho (A and B), dominant negative N17Rac (C and D), constitutively active L63Rho (E and F), or constitutively active L61Rac (G and H). Microinjected cells were identified by coinjection of Dextran–Texas red (A, C, E, and G). Cadherin-mediated adhesiveness was evaluated by the presence of immunopositive staining for E-cadherin at intercellular junctions (B, D, F, and H, arrows). Arrowheads (B and D) point to the absence of cadherin staining in the microinjected cells when Rho or Rac function was blocked. Arrowheads show the presence of cadherin-mediated adhesion between cells microinjected with activated Rho (F) or activated Rac (H). Bar, 50 μm.
Figure 2
Figure 2
Inhibition of endogenous Rho and Rac perturbs stable cadherin-mediated adhesion but not desmosomes. Keratinocytes grown in low calcium medium were transferred to standard medium for up to 3 h before microinjection of C3 transferase (A–C), N17Rac (D–F), or buffer alone (G–I). Cells were incubated for 25 min in the same medium before fixation. A, D, and G show the microinjected cells, visualized by coinjection of Dextran–Texas red. Keratinocytes were double labeled for E-cadherin followed by FITC-conjugated anti–rat IgG (B, E, and H) and desmoplakin followed by Cy5conjugated anti–mouse IgG (C, F, and I). Arrows (C, F, and I) show immunopositive staining for desmoplakin in the microinjected cells; arrows in B and E show that cadherin staining is not detected at the intercellular junctions. The arrow in H indicates that cadherin immunostaining is not perturbed by microinjection of buffer. Bar, 50 μm.
Figure 3
Figure 3
E-cadherin is selectively removed from stable cell–cell contacts. Keratinocytes grown in low calcium medium were transferred to standard medium for up to 3 h before microinjection of C3 transferase (A–C and G–I) or N17Rac (D–F). After incubation for 25 min, cells were fixed and injected cells identified by coinjection of Dextran–Texas red (A, D, and G). Keratinocytes were double labeled for E-cadherin followed by FITC-conjugated anti–rat IgG (B, E, and H) and CD44 (C and F) or β1-integrins (I) followed by Cy5-conjugated anti–mouse IgG. Arrows show immunopositive staining in the microinjected cells for CD44 (C and F) or integrins (I); arrows (B, E, and H) indicate the absence of cadherin staining at intercellular junctions of injected cells. Bar, 50 μm.
Figure 4
Figure 4
Both Rho and Rac are required for the establishment of cadherin-dependent cell–cell adhesion. Keratinocytes grown in low calcium medium were microinjected with C3 transferase alone (A and B) or coinjected with an activated form of Rac (L61Rac; C and D ). Alternatively, C3 was injected with a Rho chimera insensitive to C3 ADP ribosylation, V12Rac74Rho (RacRho; E and F). Cells were immediately transferred to standard medium and incubated for 55 min. Injected cells were visualized by coinjected Dextran–Texas red (A, C, and E). Arrows in B and D indicate that cadherin-dependent cell–cell contacts were absent in the microinjected cells. The arrow in F shows that the exogenous Rho chimera was able to restore cadherin immunostaining at the intercellular junctions. Bar, 50 μm.
Figure 5
Figure 5
Cy3-actin is rapidly concentrated at sites of cadherinmediated adhesion upon induction of stable cell–cell contacts. Keratinocytes grown in low calcium medium were microinjected with Cy3-actin and either maintained in the same medium (A and B) or transferred to standard medium for 20 min (C–J). Control IgG (G and H) or blocking-functional antibodies against cadherins (E and F) were incubated with the cells before the microinjection of labeled actin. In other experiments, Cy3-actin was coinjected with C3 (I and J) before induction of cell–cell contacts for 20 min. The same results were obtained by coinjection of Cy3actin with N17Rac (data not shown). Microinjected cells were identified (A, C, E, G, and I), and immunostaining for E-cadherin was performed (B, D, F, H, and J). Bar, 50 μm.
Figure 6
Figure 6
Recruitment of actin and β-catenin, but not talin, to latex beads coated with anti-cadherin antibodies. Keratinocytes were incubated for 1 h with a suspension of latex beads coated with BSA (A and B), anti–E-cadherin mAb (C, D, G, and H), or anti-integrin α3β1 mAb (E and F). Cells were double labeled with phalloidin (A, C, E, and G) and anti–β-catenin (B, D, and F) or anti-talin (H) rabbit polyclonal antibodies. A less confluent area is shown in G and H to demonstrate talin staining at focal contact sites. Arrows point to attached beads. Bar, 50 μm.
Figure 7
Figure 7
Actin recruitment to beads coated with anti-cadherin antibodies is perturbed by N17Rac. Keratinocytes grown in low calcium medium were microinjected with C3 transferase (A and B), N17Rac (C and D), or buffer alone (E and F). After 15 min, a suspension of latex beads coated with anti-cadherin antibodies was incubated with the cells for an additional 45 min. Injected cells were identified by coinjection of Dextran–FITC (A, C, and E); keratinocytes were labeled with phalloidin–Texas red (B, D, and F, actin). Arrows in B and F show beads on the microinjected cells with clustered actin around them. Arrows in D show beads on N17Rac-injected cells; top arrow shows a bead negative for actin recruitment. Bar, 50 μm.
Figure 8
Figure 8
Quantitation of actin recruitment to beads coated with anti-cadherin antibodies (HECD-1 beads) or anti-α3β1 integrins (VM-2 beads). The percentage of beads with recruited actin on the microinjected cells and on control noninjected cells was quantified from experiments similar to the one shown in Fig. 7 and expressed as percentage of total attached beads (see Materials and Methods). Around 20% of the few BSA-coated beads able to attach to keratinocytes showed some weak phalloidin staining, and this was considered our baseline (negative or nonspecific binding, dashed line). Error bars represent standard deviation. *, statistically significant (Student's t test, P < 0.001).
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