EMB-9 and LET-2 glycine substitution mutants mimicking Gould syndrome accumulate at higher levels in body wall muscle BM. (A) Schematic indicating the location of glycine-to-aspartic acid mutations in EMB-9 and LET-2 proteins. (B) Schematic of the C. elegans body wall muscles, which are the primary site of type IV collagen production. Secreted type IV collagen is incorporated into body wall muscle BM or trafficked in extracellular fluid and incorporated into the BM of distant tissues (such as the pharyngeal BM). (C) Schematic showing BM locations analyzed in L1 larvae. The green box corresponds to images in Fig. 8 D, and the pink box corresponds to images in Fig. 8 E. (D) Left: Single z-slice confocal fluorescence images in the body wall muscles of LET-2::mNG (C) and EMB-9::mNG wild-type animals, LET-2::mNG G1287D and EMB-9::mNG (C) G1173D mutant animals, and animals with a wild-type fluorescently tagged protein paired with an untagged mutant protein: EMB-9 G1173D; LET-2::mNG (C) and EMB-9::mNG (C); LET-2 G1173D in the body wall muscles. Yellow arrows indicate body wall muscle BM, and yellow arrowheads indicate aggregates in body wall muscle cells (see Fig. S5 G). Right: Boxplots showing mean fluorescence intensity measurements of the body wall muscle BM (n > 10 animals per condition, **P < 0.01, ***P < 0.001, Kruskal–Wallis test followed by Dunn’s multiple comparisons test) (E) Left: Single z-slice confocal fluorescence images in pharynx of LET-2::mNG (C) and EMB-9::mNG (C) wild-type animals, LET-2::mNG (C) G1287D and EMB-9::mNG (C) G1173D mutant animals, and animals with a wild-type fluorescently tagged protein paired with an untagged mutant protein: EMB-9 G1173D; LET-2::mNG (C) and EMB-9::mNG (C); LET-2 G1173D. Yellow arrows indicate the body wall muscle BM (note lack of intracellular aggregates in the pharynx, which does not express type IV collagen). Right: Boxplots showing mean fluorescence intensity measurements of pharyngeal BM (n > 10 animals per condition, *P < 0.1, ***P < 0.001, ****P < 0.0001, Kruskal–Wallis test followed by Dunn’s multiple comparisons test). The scale bars are 1 μm, and all animals were grown at 25°C (restrictive temperature for emb-9 and let-2 mutants).