Figure 2.
Live-cell imaging of ribosomal RNAs via LiveArt. (A) Representative images to show the co-localization of MS2-tagged rRNAs with rDNA and GC marker NPM1 in clonal HeLa cells. Fluorescently labeled 3′-ETS (from clone 1, named 3′-ETS-1), ITS1, and 5.8S rRNA were shown in the second, third, and fourth rows, respectively. Insert magnification: 5.2×. (B) Line scan of the relative fluorescence of the signal indicated by the dotted lines in A. (C) Quantification of MS2-tagged rRNA accumulation in the three rRNA tagging clones (3′-ETS-1, ITS1, and 5.8S) revealed by the total intensity of stdMCP-tdTomato spots in the absence or presence of ActD. Each dot represents a single cell (n = 100). Black line indicates mean ± SEM. (D) Live-cell imaging snapshots of cells showing accumulation of ITS1 rRNA indicated by stdMCP-tdTomato in the absence or presence of ActD. BFP-NPM1 was imaged to reveal the nucleoli. See Video 1 for dynamics. (E) Quantitative analysis of three representative cells (including one from D) showing the dynamic change of ITS1 rRNA defined by the total intensity of stdMCP-tdTomato spots in the absence or presence of ActD, respectively. (F and G) Representative images and line scan of fluorescent intensity showing the colocalization of stdMCP-tdTomato labeled rRNA signal (red) and RNA-FISH (green). RNA-FISH was performed using probes that do not recognize (F, negative control) or specifically recognize (G) MS2-tagged rRNAs. Colocalization ratios are indicated on the corresponding images. n ≥ 50 cells. All images in Fig. 2 are maximum intensity projections from z stacks. Scale bars, 10 µm (large-field image) and 5 µm (single-cell image).