Basolateral Endocytic Recycling Requires RAB-10 and AMPH-1 Mediated Recruitment of RAB-5 GAP TBC-2 to Endosomes
Fig 4
Rescue of the cargo-recycling defect of tbc-2 mutants requires intact RAB-10 and AMPH-1 interaction sequences.
All images are from deconvolved 3D confocal image stacks acquired in intact living animals expressing GFP- and RFP-tagged proteins. (A-E) Representative confocal images of the worm intestine expressing a GFP-tagged recycling cargo protein, the human transferrin receptor (hTFR-GFP). Loss of TBC-2 caused accumulation of hTFR-GFP on abnormally enlarged endosomal structures. The tbc-2 mutant phenotype in cargo recycling is rescued by expression of RFP-tagged full-length TBC-2 in the worm intestine. However, expression of mutant forms of RFP-tagged TBC-2 defective either in AMPH-1-binding (TBC-2[P150A]) or in RAB-10-binding (TBC-2[288–292 AAAAA]) in the worm intestine failed to rescue the tbc-2 mutant phenotype. (C') Confocal image of the worm intestine expressing RFP-tagged wild-type TBC-2. (D') Confocal image of the worm intestine expressing RFP-tagged mutant form of TBC-2 with alanine substitution at proline P150. (E') Confocal image of the worm intestine expressing RFP-tagged mutant form of TBC-2 with five alanines replacing amino acids 288–292. (Scale bar: 10 μm) (F) Quantification of the average total intensity of hTFR-GFP. In each image autofluorescent lysosome-like organelles appears in blue in all three channels, whereas GFP appears only in the green channel and RFP shows up only in the red channel. Signals observed in the green or red channels that do not overlap with signals in the blue channel are considered bona fide GFP or RFP signals respectively. (n = 18 each, 6 animals of each genotype sampled in three different regions of each intestine.) Error bars represent SEM. **P<0.01, ***P<0.001(student's t test).
