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. 2022 May 23;221(7):e202111125. doi: 10.1083/jcb.202111125

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© 2022 Wang et al.

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Figure 1. — Loss of SMGL-1 impairs apical secretion of PGP-1. (A) Domain architecture of SMGL-1. SMGL-1 contains the N-terminal WD40 repeats domain, while amino acid numbers are indicated. smgl-1 open reading frame and the corresponding sequence changes of smgl-1(ycxEx1659), a heat-shock-inducible CRISPR/Cas9 conditional mutant, are indicated. Arrowheads show three single guide RNAs target locations. (B) A diagram of C. elegans intestine, showing the apical and basolateral membranes. The imaging plane where the apical membrane and intestinal lumen can be observed is defined as the middle focal plane of the confocal microscopy. (C and D) Confocal images of intestinal cells expressing GFP-tagged PGP-1. GFP::PGP-1 is explicitly localized in the apical membrane of intestinal epithelia in L4 larvae of wild-type animals and rab-10(ok1494) mutants. In contrast, GFP::PGP-1 displayed intracellular accumulation in smgl-1(ycxEx1659) and rab-8(tm2526) mutants. Overexpression of CRISPR/Cas9-editing resistant SMGL-1 rescued the localization defects of PGP-1 in smgl-1(ycxEx1659) mutants. The signals from the apical membrane were avoided by manual ROI selection. Data are shown as mean ± SD (n = 18 each, six animals of each genotype sampled in three different unit regions of each intestine defined by a 100 × 100 [pixel²] box positioned at random). Statistical significance was determined using a one-way ANOVA followed by a post-hoc test (Dunn’s Multiple Comparison Test) for multiple comparisons. ***, P < 0.001. Data distribution was assumed to be normal but this was not formally tested. (E and F) Western blot showing expressional levels of GFP::PGP-1 in wild-type animals and smgl-1(ycxEx1659) mutant. (G and H) Confocal images showing colocalization between PGP-1 and organelle markers in the intestinal cells. GFP::PGP-1 partially overlapped with PI(3)P sensor 2xFYVE in smgl-1(ycxEx1659) and rab-8(tm2526) mutants. However, GFP::PGP-1 puncta were separated from Lysotracker-labeled lysosomes in smgl-1(ycxEx1659) and rab-8(tm2526) mutants. (I and J) Quantification of colocalization of PGP-1 with 2xFYVE and Lysotracker in smgl-1(ycxEx1659) and rab-8(tm2526) mutants. Pearson’s correlation coefficients for GFP and mCherry signals were calculated (n = 12 animals). The signals from the apical membrane were avoided by manual ROI selection. Scale bars: 10 μm. Colored asterisks indicate intestinal lumen. A dotted line indicates the outline of the intestine. Source data are available for this figure: SourceData F1.