Rho-dependent formation of epithelial "leader" cells during wound healing

Abstract

The motile behavior of epithelial cells located at the edge of a large wound in a monolayer of cultured cells was analyzed. The initial cellular response is alignment of the edge with an accompanying formation of tangential marginal actin bundles within individual cells positioned along the wound edge. Later, coherent out-growths of cell masses occur by the formation of special "leader" cells at the tops of outgrowths and "follower" cells along the sides. Leader cells exhibit profound cytoskeletal reorganization, including disassembly of marginal bundles, the realignment of actin filament bundles, and penetration of microtubules into highly active lamellae. Additionally, cell-cell contacts acquire radial geometry indicative of increased contractile tension. Interestingly, leader cells acquire a cytoskeletal organization and motility typical of fibroblasts. IAR-2 cultures stably transfected with a dominant-negative mutant of RhoA or treated with Rho-kinase inhibitor Y-27632 transformed most edge cells into leader-like cells. Alternatively, transfection of cells with constitutively active RhoA suppressed formation of leaders. Thus, expansion of the epithelial sheet involves functional differentiation into two distinct types of edge cells. The transition between these two patterns is controlled by Rho activity, which in turn controls the dynamic distribution and activity of actin filament bundles, myosin II, and microtubules.

Fig. 1.

Movement of the epithelial sheet of IAR-2 culture into the wound. Phase contrast micrographs demonstrate nonaligned sheet (A), aligned cells at the edge (B), and a “leader” (arrowheads in C–E) initiated progressively growing protrusion. (F) Leaders forming along the lateral edge of outgrowths. (G) Wide lamellipodium formed by leader (arrowhead) and small lamelipodia (arrow) formed by its “follower” documented by differential interference contrast microscopy. (H) E-cadherin staining showing altered radial cell–cell contacts at the sides of leader. Arrow indicates the active edge of the leader. (Bars = 20 μm in A–F and 10 μm in G and H.)

Fig. 2.

Localization of actin, microtubules and paxillin in edge cells. Cells were double-stained for actin (red) and microtubules (green in A–C) or paxillin (green in D). (A and B) Irregular distribution of actin bundles and microtubules at the edge immediately after wounding (A); in aligned cells (B), marginal bundle formed and microtubules did not cross this bundle. (C) Marginal bundles were present in the followers but absent in the leader. (D) In leader cells the marginal bundles became decomposed and triangular focal contacts were seen at the ends of fragments of the bundle. (E) Actin fluorescence of the same field as in D. (Bars = 10 μm.)

Fig. 3.

Movement of microtubules in live edge cells expressing EGFP-tubulin. (A) Microtubules grew into lamella (arrowheads) of leader cells (right) but not into that of follower (left). (B) Individual microtubule of a leader cell penetrated to the active edge (arrow) and a newly formed microtubule (arrowhead) grew into the lamelipodium. (Bars = 10 μm.)

Fig. 4.

Effect of transfection of IAR-2 cells with dominant-negative RhoA (A–C) or incubation with Y-27632 inhibitor of Rho kinase (D–F). (A) Phase contrast micrograph of epithelial sheet expressing dominant-negative RhoA demonstrates several leader-type cells with large lamellas at the edge. (B) Several wide lamellipodia formed by central cell and by its neighbor; note the hole between the cells. (C) E-cadherin staining revealed fragmented contacts. (D) Many cells with lamellas and processes were seen at the edge of 10 μM Y-27632-treated cells imaged by phase contrast microscopy. (E) The inhibitor caused disruption of the center of marginal bundle (red) and microtubules (green) penetrated through the opening in the bundle to ruffling edge. (F) Movements of microtubules near the edge of the extended lamellar process observed in live cell expressing EGFP-labeled tubulin and treated with 10 μM Y-27632 for 1 h. (Bars = 20 μmin A and D and 10 μm in B, C, E, and F.)

Fig. 5.

Effect of transfection of IAR-2 cells with constitutively active RhoA. (A) Phase contrast micrograph shows that cells after 4 h of wounding had poorly aligned edge and no protrusions. (B) After 48 h, cell alignment was increased and numerous cell islands, at the free substrate, were formed. (C) Blebs (arrowheads) and small lamellipodia at the edge were observed by differential interference contrast microscopy. (D) Transfected cells stained for actin had marginal and central actin bundles. (E) Well developed tangential contacts revealed by β-catenin staining. (Bar = 20 μm in B and 10 μm in C–E.)