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. 2022 Oct 14;25(11):105357. doi: 10.1016/j.isci.2022.105357

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© 2022 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

GMAP-1 is secreted from hypodermis and contributes to the cuticular ultrastructure

(A) The adult cuticle is organized in several layers, including coat, epicuticle, cortical, medial, and basal layers. The furrows are circumferential indentations within the cortical layer labeled by DPY-7-GFP. The struts are collagen pillars within the medial layer labeled by BLI-1-GFP (Chisholm and Xu, 2012; Lints and Hall, 2009; Thein et al., 2003; Tong et al., 2009).

(B) Representative electron micrographs of 4 N2 and 7 gmap-1 individuals are presented for adult worms cut at midbody, including the alae. The blue star indicates the fluid accumulation in the medial layer below the alae in N2 and its reduction in gmap-1. The blue arrowheads indicate the fluid accumulation in the medial layer of N2 and its absence in gmap-1. Scale bars = 3 μm.

(C) The mean thickness of the whole cuticle and of each of its layer was quantified at the midbody of 4 N2 and 7 gmap-1 individuals. Unpaired t-test.

(D) Left, the mean thickness of each of the cuticle layers was quantified at the midbody of 4 N2 and 7 gmap-1 individuals observed at high magnification. It shows a reduced medial layer in gmap-1 mutants compared to N2. two-Way ANOVA with Sidak’s correction for multiple comparisons test. Right, representative electron microgram of the same cuticle area in gmap-1 mutants compared to N2. The blue arrowhead indicates the fluid accumulation in the medial layer, red arrowhead indicates the strut in N2. Scale bars = 500 nm.

(E) Marked by DPY-7-GFP, the annular furrows are observed as ventral and dorsal annular stripes along the body of the animal and were similarly organized in ∼20 gmap-1 and N2 observed at high magnification. Bellow, zoom ups show higher magnification of the cuticle. Scale bar = 20 μm.

(F) The potentially additive effects of dpy-10 and gmap-1 were assessed in dpy-10;gmap-1 double mutants. Survival in deionized water was quantified for N2, gmap-1, dpy-10 and gmap-1;dpy-10 double mutants over 1 h. At 5 min, we observe additive effects of gmap-1 and dpy-10 on survival to deionized water. The p values are indicated for single mutants compared to the double mutant (RM two-way ANOVA with Geisser’s correction, and Tukey’s for multiple comparisons).

(G) Marked by BLI-1-GFP, the struts are observed as regularly spaced puncta, forming two rows on each side of each furrow and were similarly organized in ∼20 gmap-1 and N2 observed at high magnification. Bellow, zoom ups show higher magnification of the cuticle. The vulva location is indicated by v. Scale bar = 20 μm.

(H) Confocal imaging shows the subcellular localization of the GMAP-1-EGFP (green) and CTNS-1-wrmScarlet (Magenta) proteins in the hypodermis. In the lateral hyp7 syncytium, GMAP-1-EGFP accumulates in mixed-size cytoplasmic vesicles (green arrow). In the same confocal plane, CTNS-1-wrmScarlet marker for lysosomes was observed in separate cytoplasmic vesicles (magenta arrows) not marked with EGFP. Scale bar = 20 μm.

(I) GMAP-1-EGFP also marks the surface of the animal, likely the cuticle, including the longitudinal alae (arrow). As controls N2 animals not expressing GMAP-1-EGFP are used in the same imaging conditions. Scale bar = 20 μm.

(J) In the bli-1 strain, GMAP-1-mCherry appears outside the hypodermis, within the blister (magenta asterisk). The right insets show single medial confocal images, stressing GMAP-1-mCherry sublocalization within the cuticle. In N2 it marks the cuticle, in bli-1 it marks the surface of the blisters (magenta arrowheads) as well as the fluid inside the blisters (magenta asterisk). GMAP-1-mCherry also weakly marked the coelomocytes (surrounded in dotted red): scavenger cells known to endocytose proteins secreted in pseudocoelom, suggesting a small fraction is secreted toward the pseudocoelomic cavity. Scale bar = 20 μm.

(K) The size of day 1 adults blisters was evaluated in bli-1 and in gmap-1;bli-1 double mutants. Individuals are categorized according to the percentage of their body covered by blister(s) or absence of blisters. Wilcoxon rank-sum test with continuity correction. All data are represented as Mean +/− SEM.