Figure 1. Cytosolic GFP accumulates at higher levels and is produced more rapidly than transmembrane tethered GFP in mid L3 larval uterine cells (UCs):(A) A schematic diagram of constructs for cytosolic GFP and the transmembrane tethered GFP generated using the PAT-3 (b integrin subunit) signal sequence (PAT-3SS) and PAT-3 transmembrane domain (PAT-3TM). The PAT-3 transmembrane domain retains the protein in the secretory apparatus, allowing assessment of protein production as the GFP signal is easily quantified within the secretory apparatus of individual cells. Both constructs were driven by the ubiquitous promoter
eef-1A.1. Table shows amino acid sequence length and average codon frequency of the codons used to encode the protein or protein region indicated (see Methods). (B) Graph shows cytosolic (Cyto) and transmembrane (TM) tethered GFP fluorescence levels in the anchor cell (AC) and neighboring UCs (ventral UCs, see Methods). n > 10, **** p < 0.0001, ns (not significant) p > 0.05, one-way ANOVA, Tukey's multiple comparison test. (C) (Left) Fluorescence micrographs show the AC (arrow) and UCs (green brackets) over the vulval precursor cells (yellow brackets) prior to photobleaching the AC (Pre-Bleach), right after photobleaching (blue dashed line, Bleach), and then after 3 hours (h) of recovery (3 h Recovery). Note that prior to photobleaching, the levels of cytosolic GFP are lower in the AC compared to the uniform high expression in other UCs and that transmembrane tethered GFP levels are uniform in all UCs (n > 50/50 observed for each). Bar = 5µm. (Right) Masking of the AC after 3 h of recovery highlights differences in GFP recovery after photobleaching. Bar = 2.5µm. Graph displays GFP recovery rates. n > 10, **** p < 0.0001, ** p < 0.01, ns p > 0.05, one-way ANOVA, Tukey's multiple comparisons test. (D) AC/neighboring UC cytosolic GFP ratio after control RNAi (empty vector) or RNAi treatment against autophagy genes. n > 13, ns p > 0.05, one-way ANOVA, Dunnett's multiple comparisons test. (E) The autophagosome marker GFP::LGG-1 driven by its own promoter. GFP::LGG-1 puncta mark autophagosomes in the AC (blue dashed line) and neighboring UC (green dashed line). Bar = 5µm. Graph displays number of GFP::LGG-1 puncta. n = 5 each, ns p > 0.05, Student's t test.