FIGURE 1: Knockdown of
snap-29 causes accumulation of the transmembrane and secreted cargo proteins in cytoplasmic small vesicles. (A-D) In the wild-type intestine, GFP-UNC-64 and GFP-SYN-1 localize to the apical and basolateral PM, respectively. In the
snap-29(RNAi) animals, GFP-UNC-64 and GFP-SYN-1 targeted to the PM are reduced and the GFP signals are observed in fine-vesicular cytoplasmic structures. Autofluorescence of lysosome-like organelles can be seen in both the green and red channels, whereas GFP appears only in the green channel. Merged images of green and red channels are shown in A-D, in which autofluorescence is seen as yellow. An enlarged (2) image of the boxed area is shown in the inset (B and D). (A', B') Nomarski images corresponding to A and B, respectively. The wild-type intestine is filled with granules (A', arrowheads). In the intestine of
snap-29(RNAi) animals (B'), there are fewer granules (arrowheads) and the cytoplasm appears grainy (arrows). The lumen of the intestine accumulates undigested E. coli (asterisk). (E-F'') Accumulation of GFP-tagged yolk protein YP170-GFP in the intestine of
snap-29(RNAi) animals. In the wild-type animal, YP170-GFP is secreted from the intestine and taken up by growing oocytes, resulting a weak GFP signal in the intestine (E, arrowhead).
snap-29(RNAi) causes a high accumulation of YP170-GFP in the intestine (F, arrowheads). High-magnification images of the intestine are also shown in E'' (mock) and F'' [
snap-29(RNAi)]. (E', F') Nomarski images corresponding to E and F, respectively. (G, H) Subcellular localization of the yolk receptor RME-2 in oocytes. In wild-type worms, RME-2-GFP localizes to cortical endosomes and the PM (G), whereas RME-2-GFP accumulates in deep cytoplasmic vesicles in the oocytes of
snap-29(RNAi) animals (H). An enlarged (1.5) image of the boxed area is shown in the inset (G and H). All images were observed in living animals. Bars, 10 um (B, D, F'', and H) or 50 um (F'). (I) Detection of SNAP-29 protein. Total lysates were prepared from N2 wild-type worms treated with RNAi of either control vector or
snap-29. They were examined by Western blotting using anti-SNAP-29 antibody. The same membrane was also probed with anti-actin antibody.