Supplementary MaterialsS1 Fig: (A-A’) Confocal images from the worm intestinal cells expressing GFP-tagged recycling cargo protein GFP-FGT-1. indicates broad-spectrum intestinal autofluorescence caused by 8-Hydroxyguanine lipofuscin-positive lysosome-like organelles. Pearsons correlation coefficients for GFP and mCherry signals are calculated, error bars are 95% CIs, n = 12 8-Hydroxyguanine animals. Scale bars, 10 m. Observe S7 Table for quantitative data in this physique.(TIF) pgen.1008763.s001.tif (8.7M) GUID:?EC00616E-E634-4D29-96B1-750CB8C84E59 S2 Fig: (A-B) Confocal images of the worm intestinal cells expressing GFP-tagged organelle markers. In mutants, there was a moderate increase of GFP-RAB-11 labeled apical recycling endosome. Loss of SID-3 experienced no significant effect on the pattern of MANS-GFP-labeled Golgi or SP12-GFP-labeled ER. Black asterisks in the panels show intestinal 8-Hydroxyguanine lumen. Error bars are 95% CIs (n = 20 each, 10 animals of each genotype were sampled in whole-cell regions of two intestinal cells). Asterisks show the significant differences in the Mann-Whitney test (*** p 0.001, ns: no significance). Level bars, 10 m. Observe S8 Table for quantitative data in this physique.(TIF) pgen.1008763.s002.tif (4.7M) GUID:?864D04AA-06EC-429B-982A-35433F9F770B S3 Fig: (A) Confocal images showing GFAP that in the absence of RAB-10, SID-3-GFP and ARF-6-mCherry colocalized well at the edges of the vacuoles. In animals, SID-3-GFP no longer decorated the vacuoles edges labeled by ARF-6-mCherry. (B) Western blot showing GST pulldown with translated HA-RAB-10(Q68L). GST-SID-3 exhibited no relationship with HA-RAB-10(Q68L). (C-C’) Confocal picture displaying colocalization between EHBP-1-GFP and SID-3(K139A)-mCherry in the intestinal cells. SID-3(K139A)-mCherry located on the recycling endosome marker EHBP-1 tagged tubules. DAPI route 8-Hydroxyguanine (blue color) signifies broad-spectrum intestinal autofluorescence due to lipofuscin-positive lysosome-like organelles. Arrowheads suggest positive overlap. Pearsons relationship coefficients for GFP and mCherry indicators are calculated, mistake bar is certainly 95% CI (n = 12 pets). (D) American blot displaying GST pulldown with translated HA-EHBP-1(aa 1C223). GST-SID-3(aa 1C370) and GST-SID-3(aa 1C370 K139A) interacted with HA-EHBP-1(aa 1C223). (E-E?) Confocal pictures showing GFP-RME-1-tagged buildings in the intestinal cells. Representative pictures of wild-type, mutants, hTAC-GFP overaccumulated in enlarged intracellular buildings. There was no significant alleviation of hTAC-GFP build up upon manifestation of SID-3(K139A)-mCherry. The overexpression of SID-3(L509F)-mCherry fully rescued the hTAC-GFP build up phenotype in mutants. Black asterisks in the panels show intestinal lumen. Error bars are 95% CIs (n = 20 each, 10 animals of each genotype were sampled in whole-cell regions of two intestinal cells). Asterisks show the significant variations in the Mann-Whitney test (***p 0.001, ns: no significance). Level bars, 10 m. Observe S9 Table for quantitative data with this number.(TIF) pgen.1008763.s003.tif (7.2M) GUID:?0FA1035C-6ADD-4BA7-8CDE-CD4CB988EDE8 S4 Fig: (A-A”) Confocal images showing GFP-NCK-1 in the intestinal cells. In the middle focal aircraft, GFP-NCK-1 accumulated within the endosomal vacuoles in mutants. GFP-NCK-1 failed to label the edge of vacuoles in mutants. FOR ANY?, error bars are 95% CIs (n 8-Hydroxyguanine = 20 each, 10 animals of each genotype were sampled in whole-cell regions of two intestinal cells). FOR ANY, error bars are 95% CIs (n = 12 each, vacuoles edges were manually selected to obtain the fluorescence mean intensity). Asterisks show the significant variations in the Mann-Whitney test (***p 0.001, ns: no significance). (B) Western blot showing GST pulldown with translated HA-tagged SID-3(aa 1C544), DYN-1, and DYN-1(aa 504C838). GST-NCK-1 interacted with HA-SID-3(aa 1C544), HA-DYN-1, and HA-DYN-1(aa 504C838). (C) Western blot showing GST pulldown with translated HA-tagged NCK-1 and DYN-1. There was no connection of GST-EHBP-1 with HA-NCK-1 or HA-DYN-1. (D) Schematic diagram of the relationships between SID-3, NCK-1, and DYN-1, amino acid figures are indicated. (E-E’) Confocal image showing colocalization between mCherry-NCK-1 and SID-3-GFP or DYN-1-GFP in the intestinal cells. mCherry-NCK-1 overlapped well with both SID-3-GFP and DYN-1-GFP in punctate constructions. Arrowheads show positive overlap. DAPI channel (blue color) shows broad-spectrum intestinal autofluorescence caused by lipofuscin-positive lysosome-like organelles. Pearsons correlation coefficients for GFP and mCherry signals are calculated, error bar is definitely 95% CI (n = 12 animals). Scale pubs, 10 m. Find S10 Desk for quantitative data within this amount.(TIF) pgen.1008763.s004.tif (5.0M) GUID:?D68A29CA-D967-4744-8D5C-AF11E482EB81 S5 Fig: (A-A’) Confocal images showing SID-2-GFP in the intestinal cells. In pets, SID-2-GFP-labeled buildings overaccumulated on enlarged buildings. Light asterisks in the sections suggest intestinal lumen. (B-B’) Confocal pictures displaying PGP-1-GFP in the intestinal cells. In mutants, the Golgi-derived apical secretory cargo proteins PGP-1-GFP didn’t display a distribution irregularity. Light asterisks in the sections suggest intestinal lumen. Mistake pubs are 95% CIs (n = 20 each, 10 pets of every genotype had been sampled in whole-cell parts of two intestinal cells). Asterisks suggest the significant distinctions in the Mann-Whitney check (*** p 0.001, ns: no significance). Range.