cdc37 is essential for JNK pathway activation and wound closure in Drosophila

Abstract

Wound closure in the Drosophila larval epidermis mainly involves nonproliferative, endocyling epithelial cells. Consequently, it is largely mediated by cell growth and migration. We discovered that both cell growth and migration in Drosophila require the cochaperone-encoding gene cdc37. Larvae lacking cdc37 in the epidermis failed to close wounds, and the cells of the epidermis failed to change cell shape and polarize. Likewise, wound-induced cell growth was significantly reduced, and correlated with a reduction in the size of the cell nucleus. The c-Jun N-terminal kinase (JNK) pathway, which is essential for wound closure, was not typically activated in injured cdc37 knockdown larvae. In addition, JNK, Hep, Mkk4, and Tak1 protein levels were reduced, consistent with previous reports showing that Cdc37 is important for the stability of various client kinases. Protein levels of the integrin β subunit and its wound-induced protein expression were also reduced, reflecting the disruption of JNK activation, which is crucial for expression of integrin β during wound closure. These results are consistent with a role of Cdc37 in maintaining the stability of the JNK pathway kinases, thus mediating cell growth and migration during Drosophila wound healing.

FIGURE 1:

cdc37-knockdown larvae display defects in wound closure. (A–E) The epidermis was examined in late third instar larvae of the indicated genotypes, either before wounding (A) or 24 h after injury to the dorsal epidermis (B–E). Cell boundaries were stained red with anti-FasIII antibody, and the nuclei were stained blue with DAPI. The dotted line indicates the wound hole. (A, B) A58-GAL4-only control. (C) A58-GAL4, UAS-cdc37-RNAi¹²⁰¹⁹, UAS-GFP.nls (A58>cdc37-i¹²⁰¹⁹, GFP.nls, hereafter). The strong blue staining patterns show hemocytes attached to the wound site. (D) A58>cdc37-i¹²⁰¹⁹, cdc37^D.moj. (E) A58>bsk^DN. (F) Quantification of the wound closure phenotype. For each genotype, 13 or more larvae were examined. Scale bar: 100 μm (A–E). (G, H) RNAi knockdown of cdc37 was confirmed in the larval epidermis by immunohistochemistry using anti-Cdc37 antibody, as shown in the heat map. cdc37-RNAi was driven using e16E-GAL4 in the posterior half of each segment, leaving the anterior half as an internal control. The dotted line indicates the anterior–posterior compartment boundary. Anterior is up. (G) e16E-only control. (H) e16E>cdc37-i¹²⁰¹⁹. Scale bar: 25 μm (G, H).

FIGURE 2:

cdc37 is required for cell polarization during wound healing. Localization of the GFP-Zip fusion protein on the rear side of cells was analyzed 10 h after injury. GFP-Zip is shown in green. Cell boundaries were stained red with anti-FasIII antibody, and the cell nuclei were visualized by DAPI staining in blue. The dotted line indicates the wound margin. (A) A58-GFP-zip, control. (B) A58>GFP-zip, cdc37-i¹²⁰¹⁹. The arrows indicate the directionality of the cells, based on the localization of GFP-Zip and the position of the nucleus. Scale bar: 25 μm.

FIGURE 3:

Activation of the JNK pathway is disrupted in cdc37 knockdown larvae. Activation of the JNK pathway in larvae was monitored using the msn-lacZ reporter at 4 h after injury (A–D) or without wounding (E, F). β-Galactosidase expression was visualized with X-gal staining in blue. The asterisk indicates the wound hole. (A) A58-only control. (B) A58>cdc37-i¹²⁰¹⁹. (C) A58>cdc37-i¹²⁰¹⁹, cdc37^D.moj. (D) A58>bsk^DN. (E) A58>cdc37-i¹²⁰¹⁹, cdc37^D.moj. (F) A58>cdc37^D.mel. Scale bar: 100 μm.

FIGURE 4:

Overexpression of Jra does not rescue the wound healing defects displayed by cdc37-knockdown larvae. (A–E) Examination of JNK pathway activation using the msn-lacZ reporter in the larvae of the indicated genotypes, 4 h after injury. (A) A58>luc-i (control). (B) A58>Jra. (C) A58>hep. (D) A58>cdc37-i, luc-i (control). (E) A58>cdc37-i, Jra. β-Galactosidase expression was visualized with X-gal staining in blue. The white-dotted line indicates the wound margin. (F–G′) Wound closure analysis in the larvae of the indicated genotypes, 30 h after injury. (F, F′) A58>cdc37-i, luc-i (control). (G, G′) A58>cdc37-i, Jra. (F, G) Cell boundaries were stained red with anti-FasIII antibody, and the nuclei were stained blue with DAPI. The white-dotted line indicates the epidermal wound margin, 30 h after injury, and the yellow-dashed line indicates the original wound margin, left as impressions on the cuticle. (F′, G′) Bright-field micrographs of the cuticle layer. Scale bar: 100 μm. (H) Quantification of the size of the wound hole measured in the bright-field micrograph (original) or in the fluorescent micrograph (recovered). (I) Ratios of the area of the epidermal wound hole (white-dotted) to the area of the cuticle impression (yellow-dotted), which may indicate wound recovery. The difference is not significant. The error bars represent SEM.

FIGURE 5:

cdc37 knockdown disrupts the wound-induced growth of cells and the nuclei. Nuclear width was assessed based on DAPI fluorescence. Nuclei were ranked by size, and those in the top 15% were chosen for additional analysis. (A) The average width of a nucleus that was within the top 15% in unwounded epidermis. (B) The average width of a nucleus that was within the top 15% selected from nonwounded (nw) and wounded (w) segments of the epidermis 7 h after injury. (C) The average width of a cell that was chosen for the analysis indicated in B. In B and C, luc-i was used as a control, and all the RNAi constructs were driven using A58-GAL4. At least eight larval epidermal samples were analyzed for each genotype. *, p < 0.01; Wilcoxon rank-sum test.

FIGURE 6:

The JNK pathway kinase and βPS integrin protein levels are reduced in cdc37-knockdown larvae. (A) Western blotting was performed on epidermal samples from control (luc knockdown) and cdc37-knockdown late third instar larvae at the indicated times after wounding. β-Tub was used as a loading control. (B–H) Quantification of three independent Western blotting results. Error bars represent the SEM. (I, J) Protein levels of βPS integrin were analyzed by immunostaining using an anti-βPS antibody 7 h after wounding. Cell nuclei were stained blue using DAPI. The dotted line indicates the wound margin. Scale bar: 100 μm.