Rho family GTPase functions in Drosophila epithelial wound repair

Abstract

Epithelial repair in the Drosophila embryo is achieved through 2 dynamic cytoskeletal machineries: a contractile actomyosin cable and actin-based cellular protrusions. Rho family small GTPases (Rho, Rac, and Cdc42) are cytoskeletal regulators that control both of these wound repair mechanisms. Cdc42 is necessary for cellular protrusions and, when absent, wounds are slow to repair and never completely close. Rac proteins accumulate at specific regions in the wound leading edge cells and Rac-deficient embryos exhibit slower repair kinetics. Mutants for both Rho1 and its effector Rok impair the ability of wounds to close by disrupting the leading-edge actin cable. Our studies highlight the importance of these proteins in wound repair and identify a downstream effector of Rho1 signaling in this process.

Keywords: Cdc42; Drosophila; GAPs, GTPase activating proteins; GDIs, guanine nucleotide dissociation inhibitors; GEFs, guanine nucleotide exchange factors; RBD, Rho Binding Domain; Rac; Rho; Rho family GTPases; Rok; cytoskeleton; epithelial wound repair.

Figure 1.

Rho family GTPases are essential for efficient epithelial repair in the dorsal closure (stage 15) Drosophila embryo. (A-D) Time-lapse projections of embryos expressing an actin marker (sGMCA) in wildtype (A), Cdc42⁴/Cdc42⁶ (B), Rac1^J10, Rac2^Δ, Mtl^Δ (referred to as Rac*; C), and Rho1^1B (D) embryos during wound repair. Wildtype embryos display an actin cable and actin protrusions during wound repair (A). Cdc42⁴/Cdc42⁶ mutant embryos exhibit significantly less protrusions (B; adapted from and reproduced with permission from The Company of Biologists). Despite their significant delay in repair, Rac* mutant embryos do not show gross defects in actin cable formation or protrusions (C). Rho1^1B mutant embryos show incomplete actin cable formation and increased protrusions during wound repair (D). (E) Time series of size matched wildtype and Cdc42⁴/Cdc42⁶ wounds (orthogonal view) entering closure; Cdc42⁴/Cdc42⁶ mutants fail to reseal the epithelium. (F) Confocal projections of wounds in wildtype (top) and Rho1^1B (bottom) mutant embryos showing that Rho1¹ mutants fail to form a continuous actin cable along the lead edge (arrows) and do not become rounded (jagged leading edge) indicating a defective actomyosin purse string. Rho1^1B embryos have large protrusions (asterisk). (G–I) Quantification of wound area versus time in medium-size wounds showing that Rho1^1B, Cdc42⁴/Cdc42⁶, and Rac* mutant embryos exhibit delays throughout the repair process (wildtype, n = 10; Rho1^1B, n = 10; Cdc42⁴/Cdc42⁶, n = 6; Rac*, n = 6; results are given as means ± s.e.m.). (H) Quantification of wound area vs. time in small (<500 μm²), medium (500-1000 μm²), and large (1000-1500 μm²) wounds generated in wildtype or Rho1^1B mutant embryos showing that time of repair is scalar to wound size (all sizes: wildtype, n = 3; Rho1^1B, n = 3). Scale bar: 10 μm.

Figure 2.

Expression of Cdc42 and Rac1/2 fluorescent proteins and activity biosensors during epithelial repair. (A) Surface projections of wound repair in embryos expressing an actin marker (sGMCA) and fluorescent Cdc42; Cdc42 does not accumulate specifically in response to wounding. (B) Surface projections of wound repair in embryos expressing an actin marker (sChMCA) and the Cdc42 biosensor GFP-WASp^RBD showing no accumulation indicative of activated Cdc42. This biosensor is functional as there is specific accumulation in the hemocytes responding to the wound (arrow). (C–C”) Surface projections of wound repair in embryos expressing an actin marker (sChMCA) and fluorescent Rac1; Rac1 accumulates in protrusions (asterisk; C’) and at some cell junctions (arrows; C’–C’’). (D–D’) Surface projections of wound repair in embryos expressing fluorescent Rac2. Rac2 accumulates in protrusions (asterisk; D’) and at some cell junctions (arrows; D’). (E–F) Surface projections of wound repair in embryos expressing an actin marker (sGMCA or sChMCA) and the Rac proteins biosensors ChFP-PlexinB^RBD (E) or GFP-Pak3^RBD (F) The ChFP-PlexinB^RBD biosensor does not accumulate at the wound edge (E), whereas the GFP-Pak3^RBD biosensor accumulates at the wound along portions of the leading edge (F). Scale bars: 10 μm (A–C, D, E, F) and 5 μm (C’, C’’, D’).

Figure 3.

Expression of Rho1 and its downstream effectors and Rok mutant during wound repair. (A) Surface projections of wound repair in embryos expressing an actin marker (sGMCA) and mChFP-Rho1. Rho1 does not accumulate at the leading edge. (B) Confocal projection of 3 adjacent wounds in embryos expressing an actin marker (sGMCA) and stained with α-Rho1 antibody (P1D9; 1:50). Rho1 does not accumulate at the wound edge (arrows). (C–E) Surface projections of wound repair in embryos expressing an actin marker (sChMCA) and the Rho1 biosensor GFP-Pkn^RBD (C) or the full-length Rho1 downstream effectors Pkn (Pkn-GFP; D) or Rok (Rok-GFP; E). The Pkn^RBD biosensor does not accumulate at the wound edge (C), whereas the full-length Pkn and Rok effectors accumulate at the wound and co-localize with the actin cable (D–E). (F) Quantification of wound area versus time in medium-size wounds shows that Rok² mutant embryos exhibit delays throughout the repair process, albeit less severe than that observed with Rho1^1B (wildtype, n = 10; Rho1^1B, n = 10; Rok², n = 5; results are given as means ± s.e.m.). (G) Time-lapse projections of embryos expressing an actin marker (sGMCA) in wound Rok² mutant embryos during wound repair. Rok² mutant embryos show incomplete actin cable formation and increased cellular protrusions during wound repair similar to that observed with Rho1^1B.