Fig. 3: Spatial distribution of the polysome and orientation of translating mRNA.
From: Direct observation of translational activation by a ribonucleoprotein granule
a, Left: schematic of the live imaging setup. Right: a live image of germplasm. Blue, Vasa; green, scFv–GFP. b, Spatiotemporal tracking of germ granules and attached translation foci. Two pairs of germ granules (arrowheads) and translation foci (arrows) were tracked for 400 s, and showed co-movement throughout the movie. c–f, Orientation of translating mRNA. Translating suntag-nanos mRNAs in embryos from Vasa-mApple/+; suntag-nanos, scFv-GFP/Df(nanos) flies are detected with smFISH against suntag (c) and nanos 3′ UTR (e). Example germplasm images are shown in c and e. The orthogonal views of the outlined regions are shown on the right. Scale bar for the orthogonal views: 0.3 µm. Blue, Vasa; magenta, mRNA smFISH; green, scFv–GFP. The distributions of scFv–GFP and smFISH foci were mapped and plotted in relative frequency histograms overlaid with KDEs in d and f. The x axis refers to the distance of foci centroids to the border of the closest granule; zero marks the granule border; a negative value denotes being inside a granule and a positive value denotes outside. In total, 12,684 smFISH foci and 12,733 scFv–GFP foci from images of 7 embryos were mapped in d. A total of 5,663 smFISH foci and 5,649 scFv–GFP foci from images of 3 embryos are mapped in f. g, Detecting the 5′ and 3′ sequence of native nanos mRNA in wild-type (WT) embryos. The schematic shows the probes used for smFISH. 5′ probe signal (green) coats around the 3′ probe signal (magenta) and germ granule marker Oskar–GFP (blue). The orthogonal views of the outlined region are shown on the right. Scale bar, 0.3 µm. h, Averaging germ granule images showed the distribution pattern of 5′ and 3′ of nanos mRNA relative to germ granules. Using the Oskar channel as a reference, 40 images of germ granules from 3 embryos were randomly picked, made into a z-stack and averaged. i, PCC measurement showing a stronger co-localization of Oskar with nanos 3′ signal than 5′. n = 4 embryos for both conditions. The data are the mean ± s.d. Statistics: two-tailed Welch’s t-test.
