Beclin 1 ortholog BEC-1 mediates endocytic retrograde transport in addition to autophagy in C. elegans - PubMed (original) (raw)

Figure 3

bec-1 mutants display a defect in retrograde transport. Confocal images in a wild-type background are shown for GFP::RAB-5 (A), GFP::RAB-7 (B), LMP-1::GFP (C), GFP::hTfR (D), MIG-14::GFP (E), GFP::RME-8 (F), GFP::SNX-1 (G), GFP::VPS-35 (H). Confocal images of bec-1(ok691) mutants are shown for GFP::RAB-5 (A'), GFP::RAB-7 (B'), LMP-1::GFP (C'), GFP::hTfR (D'), MIG-14::GFP (E'), RME-8::GFP (F'), GFP::SNX-1 (G'), GFP::VPS-35 (H'). (B) GFP::RAB-7 positive puncta (marked by yellow arrow) labels maturing endosomes and late endosomes (marked by yellow arrowhead). bec-1(ok691) mutants accumulate maturing endosomes marked with GFP::RAB-7, and display a decrease in late endosomes marked with GFP::RAB-7 (compare B and B'; See Fig. 2I). White arrowheads indicate enlarged intestinal endosomes (abnormal vacuoles) labeled by GFP::RAB-5 and GFP::RAB-7. In bec-1(ok691) animals, LMP-1::GFP also accumulates in the membrane of the enlarged endosomes (C'; marked by white arrowheads). In wild-type animals (D), the basolateral plasma membrane and basolateral endocytic compartments are labeled by hTfR::GFP (human transferrin receptor). The accumulation of hTfR::GFP is not affected in bec-1(ok691) mutants (D'), and the recycling endosome cargo hTfR::GFP does not accumulate in the enlarged endosomes (abnormal vacuoles) of bec-1(ok691) mutants. Confocal images of the worm intestine expressing the GFP-tagged endocytic transmembrane cargo marker MIG-14::GFP in wild-type (E), and bec-1(ok691) (E') animals. The retromer-dependent cargo protein Wntless MIG-14::GFP normally localizes basolaterally in the intestine and colocalizes with the early endosomal marker RAB-5. In bec-1 mutants, MIG-14::GFP transmembrane cargo protein accumulates in the enlarged endosomes (abnormal vacuoles; E'). We observed a decrease in the number of MIG-14::GFP positive puncta (I). The retromer subunit RME-8::GFP (F'), GFP::SNX-1 (G') and GFP::VPS-35 (H'), do not accumulate in the enlarged intestinal endosomes (abnormal vacuoles). A decrease in the number of GFP::RME-8 positive puncta is observed in bec-1(ok691) mutants (F'), and its intensity is markedly decreased. For all images, scale bars represent 10 µm. Quantification of endosome number as visualized by the positive labeling with endocytic markers is shown in (I). In the quantification of endosomal compartments, we saw no significant difference in RAB-5 early endosomes, we observed an increase in RAB-7 maturing endosomes, and a dramatic decrease in RAB-7-positive late endosomes. In addition, we observed a decrease in MIG-14 as well as RME-8 labeled compartments, and a slight increase in SNX-1 as well as in VPS-35 labeled compartments. Error bars represent standard deviation from the mean (n = 30 each, 10 animals of each genotype were sampled in three different regions of the intestine). In (J and K), we analyzed whether lysosomal degradation of GFP::MIG-14 occurs in bec-1 mutant animals. RNAi mediated depletion of lysosome biogenesis protein CUP-5/mucolipin1 increased the number and the intensity of MIG-14::GFP positive puncta (J and Fig. S5). After cup-5 RNAi, we saw a 4X increase in the number of MIG-14 positive puncta in bec-1 mutants, when compared to control RNAi, indicating that there is lysosomal processing of the MIG-14 cargo in bec-1 mutants (J, left panel). Similarly, we observed a significant increase in the intensity of MIG-14::GFP in bec-1 mutants after cup-5 RNAi (J, right panel). Asterisks indicate a significant difference in the one-tailed Student's t-test (*p < 0.05, **p < 0.005, ***p < 0.0005, n.s.: not significant).