RAB-8– and RAB-11–positive endosomes serve as intermediate carries in the unconventional apical protein transport. (A) Confocal images and quantification showing colocalization pattern between intracellular GFP::PGP-1 with MC::RAB-8, RFP::RAB-11, Golgi marker AMAN-2::RFP, and MC::GOLG-4 in wild-type animals kept at 10°C for 12 h. Intracellular GFP::PGP-1 puncta overlapped with subsets of RAB-8 and RAB-11 and were separated from AMAN-2–labeled cis- and medial-Golgi and GOLG-4–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. White asterisks indicate intestinal lumen. Scale bars: 10 μm. (B) Transgenic animals of GFP::PGP-1 and single-copy mScarlet::RAB-11 or mScarlet::RAB-8 or MC::GOLG-4 were imaged live by spinning disk confocal microscope at 20°C after a 12 h incubation at 10°C. Images were captured every 0.5 s for 2 min. Series of images that depict mScarlet::RAB-11– and mScarlet::RAB-8–labeled vesicles transporting GFP-labeled PGP-1 towards the apical surface are shown (see Videos 1 and 2). RAB-11 puncta can be observed to either interact with the RAB-11–positive endosomal subpopulation located along the apical surface (indicated by arrowheads) or disappear below the PGP-1–labeled apical membrane (indicated by long arrows). Image series show the movement of GFP::PGP-1 vesicles (indicated by arrows), with a neighboring MC::GOLG-4–labeled Golgi (indicated by arrowheads) remaining stationary at the time interval (see Video 3). Scale bars: 4 μm.