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. 2017 Aug 2;6:e26376. doi: 10.7554/eLife.26376

Figure 1. Ribosome Imaging Based On split GFP (RIBOS).

(A) Schematics of endogenous rps-18 locus on chromosome IV and single-copy transgenes on chromosome II. Black box: exon, Grey box: untranslated region, Red box: deletion. (B) Schematic of RIBOS for mechanosensory neurons. The rps-18 fused to the smaller part of split GFP (GFP11) is replaced to the endogenous rps-18 (juSi94[GFP11::rps-18]; rps-18(0), left). The larger part of split GFP (GFP1-10) is overexpressed using a high-copy transgene in a targeted tissue (middle). After crossing these strains, GFP1-10 binds to the GFP11::RPS-18 in the targeted tissue and visualize RPS-18 (right). (C) Epidermis-specific Pcol-19-RIBOS (juSi94[GFP11::rps-18]; rps-18(0); juEx5375[Pcol-19-GFP1-10]). The RIBOS signals were excluded from the nuclei (Left panel, arrowheads) and reticular structures (magnified image). Although GFP11::RPS-18 is expressed in the intestine, it had no signals. The negative control only expressed GFP1-10 (juEx5375) in the epidermis. Scale bars: 20 µm. (D) Diffuse IFE-2 expression visualized by juEx5809[Pcol-19-IFE-2::GFP]. (E) Representative images of the FRAP experiment using Pcol-19-RIBOS. The fluorophore was bleached in the area (arrowheads) at 0 s. Scale bars: 2 µm. (F) Fluorescent recovery after photobleaching was plotted for juEx5809[Pcol-19-IFE-2::GFP], juEx5811[Pcol-19-IFE-4::GFP], Pcol-19-RIBOS, and juSi123[RPL-29::GFP]; rpl-29(0). The line represents the one-phase fit to an exponential function for each plot. The inset shows the magnified graph for IFE-2::GFP and IFE-4::GFP. Error bars indicate S.E.M. (G and H) t1/2 and mobile fraction calculated from (F). n= 5 or 6. Error bar indicates S.E.M., Statistics: One-way ANOVA, ns: p>0.05, p**<0.01, p****<0.0001.

Figure 1.

Figure 1—figure supplement 1. GFP::RPS-18 visualizes ribosomes in the whole body.

Figure 1—figure supplement 1.

(A) RPS-18 overexpression in mechanosensory neurons with juEx5815[Pmec-4-RPS-18] caused abnormal axon (left panels) and soma (middle panels) morphologies. ALM neurons were visualized by muIs32[Pmec-7-GFP]. Scale bars: 20 and 10 µm in left and right panels, respectively. (B) Representative images of the indicated regions of juSi83[GFP::rps-18]-expressing worms in rps-18(0)/nT1 background at the young adult stage. All images were taken from the live animals except for the gonad, which was dissected out from the body. The magnified image in the middle shows the cell bodies of motor neurons. Scale bars: 20 µm, 10 µm in the magnified image. (C) Brood size of worms for genotypes indicated. Single-copy transgenes, juSi83[GFP::rps-18] or juSi94[GFP11::rps-18], were used to rescue the sterility of rps-18(0). N: numbers of parental worms. Error bars: S.E.M. Statistics: 1-way ANOVA, compared to the wild type. ns: not significant (p>0.05), p***<0.001. (D) Differential interference contrast (DIC) and fluorescent images of juSi83-expressing worms at the L3 stage in the wild-type, heterozygous (rps-18(0)/nT1), or homozygous (rps-18(0)) background. Arrowhead: pharyngeal GFP in the nT1 balancer. (E) Quantification of fluorescence intensity of juSi83-carrying worms in the indicated backgrounds. Numbers of worms are shown in the bars. Error bars indicate S.E.M. Statistics: One way ANOVA, ***p<0.001.
Figure 1—figure supplement 2. Ribosome visualization with RPL-29::GFP.

Figure 1—figure supplement 2.

(A) Pan-neuronal RPL-29 overexpression with juEx5243[Prgef-1-RPL-29::GFP] caused abnormal aggregates of RPL-29 in the nucleus. Head ganglia and mechanosensory ALM neurons are shown. Scale bars: 20 µm (left); 10 µm (right). (B) Schematics of the rpl-29 locus and a transgene. Black box: exon, Grey box: untranslated region, Red box: deletion. (C and D) The juSi123[rpl-29::GFP]; rpl-29(tm3555) strain showed the expression of RPL-29 in the cytosol (D, scale bar = 20 µm) throughout development (C, scale bar = 200 µm). (E) Quantification of the fluorescence intensity in the indicated genetic background. Error bars indicate S.E.M. Statistics: Student’s t-test, ***p<0.001.
Figure 1—figure supplement 3. RIBOS visualizes ribosomes in muscle arms.

Figure 1—figure supplement 3.

(A) Ribosome distribution in muscles visualized by Pmyo-3-RIBOS (juSi94[GFP11::rps-18]; rps-18(0); juEx5377[Pmyo-3-GFP1-10]). Ventral nerve cord runs horizontally in the middle of the image. Ribosomes in muscle arms are shown by arrows. Arrowheads indicate autofluorescence in the intestine. Scale bar: 20 µm. (B) Electron micrographs of the ventral nerve cord and muscle arm. Right panel is the enlarged muscle arm (dotted square in the middle panel), filled with ribosomes detected as 20 nm electron-dense structures (white arrowheads). Black arrowheads indicate microtubules in neurons, which have a similar diameter to ribosomes. Scale bar: 400 nm; 200 nm in the magnified image.