Figure 5. Imaging translation of single molecules of endogenous POLR2A mRNAs. (A) Schematic of the recombination cassette used to edit the POLR2A gene. (B) Colocalization of the bright protein foci labeled by the scFv-sfGFP with tagged POLR2A mRNAs. Panels represent microscopy images of cells stably expressing the scFv-sfGFP and containing the SunTagx56-POLR2A allele and labeled with smFISH probes recognizing the SunTag-Puro sequences. (right) Color overlay of the smFISH image (red, left) and the SunTag signal (green, middle). (inset) Zoom of the boxed area (4.5 × 4.5 µm). Bar, 4.5 µm. (C) Single-cell, single-molecule quantification of POLR2A gene expression. Legend as in Fig. 2 (n = 94 cells for the left and middle panels; n = 326 mRNAs for the right panel). (D) FRAP recovery curves of SunTagx56-POLR2A polysomes. The curve is the mean of the bleaching of 16 single polysomes, with a fit to the model in gray. (E and F) Diffusion of POLR2A polysomes. Legend as in Fig. 4. Data are from 5,888 particles. a.u., arbitrary units; fps, frames per second.
Figure 5.

Imaging translation of single molecules of endogenous POLR2A mRNAs. (A) Schematic of the recombination cassette used to edit the POLR2A gene. (B) Colocalization of the bright protein foci labeled by the scFv-sfGFP with tagged POLR2A mRNAs. Panels represent microscopy images of cells stably expressing the scFv-sfGFP and containing the SunTagx56-POLR2A allele and labeled with smFISH probes recognizing the SunTag-Puro sequences. (right) Color overlay of the smFISH image (red, left) and the SunTag signal (green, middle). (inset) Zoom of the boxed area (4.5 × 4.5 µm). Bar, 4.5 µm. (C) Single-cell, single-molecule quantification of POLR2A gene expression. Legend as in Fig. 2 (n = 94 cells for the left and middle panels; n = 326 mRNAs for the right panel). (D) FRAP recovery curves of SunTagx56-POLR2A polysomes. The curve is the mean of the bleaching of 16 single polysomes, with a fit to the model in gray. (E and F) Diffusion of POLR2A polysomes. Legend as in Fig. 4. Data are from 5,888 particles. a.u., arbitrary units; fps, frames per second.

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