Regulation of Rho and Rac signaling to the actin cytoskeleton by paxillin during Drosophila development

Abstract

Paxillin is a prominent focal adhesion docking protein that regulates cell adhesion and migration. Although numerous paxillin-binding proteins have been identified and paxillin is required for normal embryogenesis, the precise mechanism by which paxillin functions in vivo has not yet been determined. We identified an ortholog of mammalian paxillin in Drosophila (Dpax) and have undertaken a genetic analysis of paxillin function during development. Overexpression of Dpax disrupted leg and wing development, suggesting a role for paxillin in imaginal disc morphogenesis. These defects may reflect a function for paxillin in regulation of Rho family GTPase signaling as paxillin interacts genetically with Rac and Rho in the developing eye. Moreover, a gain-of-function suppressor screen identified a genetic interaction between Dpax and cdi in wing development. cdi belongs to the cofilin kinase family, which includes the downstream Rho target, LIM kinase (LIMK). Significantly, strong genetic interactions were detected between Dpax and Dlimk, as well as downstream effectors of Dlimk. Supporting these genetic data, biochemical studies indicate that paxillin regulates Rac and Rho activity, positively regulating Rac and negatively regulating Rho. Taken together, these data indicate the importance of paxillin modulation of Rho family GTPases during development and identify the LIMK pathway as a critical target of paxillin-mediated Rho regulation.

FIG. 1.

Molecular characterization and expression of Drosophila paxillin. (A) Alignment of mammalian paxillin, Hic-5, and the two Dpax isoforms, DpaxA and DpaxB. The DpaxA sequence is similar to that reported by Wheeler et al. (42). Asterisks denote known tyrosine phosphorylation sites in avian and mammalian paxillin. The dotted line represents the region of sequence divergence between DpaxA and -B. LD repeats are underlined. (B) Lysates from the embryonic stage (Emb) (0 to 24 h), larval stages (L1-2, first and second instar; L3, third instar), and pupal stages (EP, early pupae; LP, late pupae) and from male (M) and female (YF, newly enclosed; F, 24-h-old) adult flies were subjected to immunoblotting with anti-Dpax antibody. (C and D) Dpax is expressed in follicle cells in the developing ovary (C) and in border cells (arrowheads) (D). Ovaries were costained with Dpax antibody and phalloidin. In panel D, the lower panel shows higher magnification. Merge, merged images of the left and center panels.

FIG. 2.

Overexpression of Dpax results in malformed wing and leg phenotypes. (A and B) Overexpressing Dpax results in mostly pupa lethal phenotypes, but a few escapers carry malformed-wing (A) and -leg (B) phenotypes. The hsGAL4/+;UAS-Dpax/+ larvae were heat shocked at 96 h after egg deposition (AED) daily for 40 min at 37°C through eclosion. Extra wing veins (arrowhead) and bent and/or twisted legs (arrow) are observed in flies expressing Dpax. (C) Overexpression of UAS-Dpax driven by 32B-GAL4 induces wing blisters.

FIG. 3.

Dpax genetically interacts with Rho and Rac in eye morphogenesis. Scanning electron micrographs of the compound eye of wild-type (A), GMR-Rho1¹Rho1³/+ (B), GMR-Rac1/+ (C), GMR-Dpax/+ (D), GMR-Rho1¹Rho1³/GMR-Dpax (E), and GMR-Rac1/GMR-Dpax (F) flies are shown. Dpax expression alleviates Rho1 but enhances Rac1-induced rough-eye phenotypes (E and F).

FIG. 4.

Dpax-induced wing blisters can be rescued by components in the Rho pathway, including Dlimk and its downstream targets. (A and D) Overexpression of UAS-Dpax driven by en-GAL4 or 32B-GAL4 results in blistered wings (arrows). (B) Overexpression of UAS-Dlimk driven by en-GAL4 results in multiple defects in the posterior part of the wing (6). (C and F) Overexpression of Dlimk (C) or DSRF (F) rescues the Dpax-induced blistered-wing phenotype. (E) A mutation in cofilin (tsr^ko5633), the downstream target of LIMK, rescues the wing-blistering defect. Flies were maintained at 18°C.

FIG. 5.

Dpax rescues Dlimk-induced F-actin accumulation. Third-instar wing imaginal disks were dissected from en >UAS-Dlimk/+ (A to C), enGAL4/+;UAS-Dpax/+ (D to F), or en >UAS-Dlimk/+;UAS-Dpax/+ (G to I) genotypes and stained with anti-Dpax antibody (A, D, and G) and tetramethyl rhodamine isocyanate-labeled phalloidin (B, E, and H). Merged images of the left and center panels are shown in panels C, F, and I.

FIG. 6.

Paxillin antagonizes the Rho-LIMK-mediated signaling pathway. (A) Paxillin suppresses LIMK1-induced SRF transcriptional activity in Cos7 cells. (B) Paxillin^−/− cells exhibit approximately twofold-increased SRF transcriptional activity compared with rescued cells. (C) Paxillin^−/− cells exhibit decreased Rac activity, as measured in pulldown assays with GST-PAK (PBD). The lower panel shows levels of rac in total cell lysate (TCL). (D) Paxillin^−/− cells have increased Rho activity. The lower panel shows that levels of rho in different lysates are similar.

FIG. 7.

Paxillin interacts with and regulates LIMK. (A and B) Lysates were prepared from randomly growing or Fn-plated Cos7 cells that had been transfected with LIMK1 or LIMK2 (see Materials and Methods). Immunoblots were probed with antibody to paxillin, LIMK1, or HA. (C) Rescued or Pxl^−/− MEFs were subjected to a fibronectin-plating assay as described above. Filters were probed with antibody to phosphocofilin (upper blot) or reprobed with antibody to cofilin (lower blot). Quantitation (shown below the phosphocofilin blot) was done on a Li-Cor infrared imaging system.