Figure 7.
VTI-1 interacts with RAB-8 and is involved in unconventional apical protein transport. (A) Confocal images and quantification showing colocalization pattern between intracellular GFP::PGP-1 and several endosomal markers in rab-11(RNAi) knockdown animals. Intracellular GFP::PGP-1 puncta overlapped with subsets of MC::RAB-8. Intracellular GFP::PGP-1 puncta were separated from RAB-7–labeled late endosomes, and SDPN-1– and SNX-3–labeled early/sorting endosomes. (B) Confocal images and quantification showing colocalization pattern between intracellular GFP::PGP-1 and several endosomal markers in rfip-1; rfip-2 (RNAi) knockdown animals. Intracellular GFP::PGP-1 puncta overlapped with subsets of RFP::RAB-11 and MC::RAB-8. Intracellular GFP::PGP-1 puncta were separated from RAB-7–labeled late endosomes, SNX-3– and SDPN-1–labeled early/sorting endosomes, GOLG-4 and SYX-16–labeled trans-Golgi. In each set of images, broad-spectrum intestinal autofluorescent lysosome-like organelles can be seen in blue. Pearson’s correlation coefficients for GFP and MC/RFP signals were calculated (n = 12 animals). The signals from the apical membrane were avoided by manual ROI selection. (C) Confocal images and quantification showing the abnormal intracellular GFP::PGP-1 in vti-1(RNAi) and snap-29(RNAi) knockdown animals, in comparison with wild-type animals. For quantification, 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. (D) Confocal images and quantification showing the abnormal localization and decreased fluorescent intensity of GFP::VTI-1 in rab-8(RNAi) knockdown animals, in comparison with wild-type animals. Statistical significance was determined using a two-tailed, unpaired Student’s t test. ***P < 0.001. Data distribution was assumed to be normal but this was not formally tested. (E) Confocal images and quantification showing the intact localization of GFP::SNAP-29 in rab-8(RNAi) knockdown animals, in comparison with wild-type animals. Statistical significance was determined using a two-tailed, unpaired Student’s t test. ns, no significance. Data distribution was assumed to be normal but this was not formally tested. (F) GFP::VTI-1 precipitated MC::RAB-8 in a representative Co-IP assay. (G) Confocal images and quantification showing noticeable colocalization between GFP::VTI-1 and MC::RAB-8. Manders’ correlation coefficients for GFP and MC signals were calculated (n = 12 animals). Scale bars: 10 μm. White and yellow asterisks indicate intestinal lumen. Dashed lines indicate the outline of the intestine. Source data are available for this figure: SourceData F7.

VTI-1 interacts with RAB-8 and is involved in unconventional apical protein transport. (A) Confocal images and quantification showing colocalization pattern between intracellular GFP::PGP-1 and several endosomal markers in rab-11(RNAi) knockdown animals. Intracellular GFP::PGP-1 puncta overlapped with subsets of MC::RAB-8. Intracellular GFP::PGP-1 puncta were separated from RAB-7–labeled late endosomes, and SDPN-1– and SNX-3–labeled early/sorting endosomes. (B) Confocal images and quantification showing colocalization pattern between intracellular GFP::PGP-1 and several endosomal markers in rfip-1; rfip-2 (RNAi) knockdown animals. Intracellular GFP::PGP-1 puncta overlapped with subsets of RFP::RAB-11 and MC::RAB-8. Intracellular GFP::PGP-1 puncta were separated from RAB-7–labeled late endosomes, SNX-3– and SDPN-1–labeled early/sorting endosomes, GOLG-4 and SYX-16–labeled trans-Golgi. In each set of images, broad-spectrum intestinal autofluorescent lysosome-like organelles can be seen in blue. Pearson’s correlation coefficients for GFP and MC/RFP signals were calculated (n = 12 animals). The signals from the apical membrane were avoided by manual ROI selection. (C) Confocal images and quantification showing the abnormal intracellular GFP::PGP-1 in vti-1(RNAi) and snap-29(RNAi) knockdown animals, in comparison with wild-type animals. For quantification, 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. (D) Confocal images and quantification showing the abnormal localization and decreased fluorescent intensity of GFP::VTI-1 in rab-8(RNAi) knockdown animals, in comparison with wild-type animals. Statistical significance was determined using a two-tailed, unpaired Student’s t test. ***P < 0.001. Data distribution was assumed to be normal but this was not formally tested. (E) Confocal images and quantification showing the intact localization of GFP::SNAP-29 in rab-8(RNAi) knockdown animals, in comparison with wild-type animals. Statistical significance was determined using a two-tailed, unpaired Student’s t test. ns, no significance. Data distribution was assumed to be normal but this was not formally tested. (F) GFP::VTI-1 precipitated MC::RAB-8 in a representative Co-IP assay. (G) Confocal images and quantification showing noticeable colocalization between GFP::VTI-1 and MC::RAB-8. Manders’ correlation coefficients for GFP and MC signals were calculated (n = 12 animals). Scale bars: 10 μm. White and yellow asterisks indicate intestinal lumen. Dashed lines indicate the outline of the intestine. Source data are available for this figure: SourceData F7.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal