HSP-4/BiP is required for the normal structure and barrier function of the cuticle in adults and alae formation in dauers.

(A) Confocal image of COL-19::GFP protein in the hypodermis and alae of a WT (N2) young adult hermaphrodite, fed control RNAi (empty vector, L4440) or hsp-4 RNAi. COL-19::GFP in controls is deposited in the linear circumferential pattern in the hypodermis, and in linear longitudinal pattern underlying the alae structures. hsp-4 RNAi results in abnormal deposition of COL-19::GFP protein, with large visible gaps in the circumferential pattern (arrows) and gaps and abnormal appearance of the longitudinal alae pattern (bracket). Star indicates position of the vulva (v). Lower panels: close views of boxed regions pattern. Scale bar: 20 μm. (B) Examples of disrupted COL-19::GFP underlying the alae structures in adult hsp-4(RNAi) animals. Open arrows indicate area of normal longitudinal pattern, while arrowheads point to disorganized, “spaghetti-like” pattern of COL-19::GFP. Closed arrows point to the gaps in circumferential pattern. Scale bar: 20 μm. (C) Genetic deletion of hsp-4 results in decreased deposition of the COL-19::GFP protein in the lateral hypodermis. Double-headed arrows span the lateral hypodermis, overlaying the seam; arrows point to the location of the longitudinal COL-19 structures. (D) Dauer alae are abnormal in daf-28(sa191) dauers carrying hsp-4(gk514) deletion allele. Arrows indicate individual ridges; the daf-28(sa191);hsp-4(gk514) dauer shown here has missing or flattened ridges. DIC images. (E) hsp-4 deletion increases permeability of the cuticle to small molecules. Staining of nuclei in live animals treated with DNA-binding Hoechst dye reflects the degree of leakiness of the cuticle. Staining was performed as previously described [59]. Percent animals positive for the Hoechst staining is shown as bar graph; data are mean ± SD of three independent experiments. p hsp-4, p bus-8, repeated measures ANOVA with Tukey post hoc test. BiP, immunoglobulin heavy chain-binding protein; bus-8(e2887), an allele causing leaky cuticle phenotype; COL-19, C. elegans cuticular collagen; contr, control; daf-28(sa191), an allele causing ectopic L2d entry; DIC, differential interference contrast; GFP, green fluorescent protein; HSP-4, Heat-Shock Protein 4; RNAi, RNA interference; WT, wild type.