Fig. 2: Live-cell single mRNA imaging shows dynamic interactions between optoMCP-FUS condensates and target mRNAs.

A Schematic diagram of the mRNA dual labeling strategy for single mRNA imaging orthogonal to light-activated phase separation. 12 repeats of MBS-PBS loops were inserted at the 3′UTR of the gene of interest. Individual mRNAs can be imaged as distinct foci through the PBS-bound stdPCP-stdGFP even in the presence of the high background level of optoMCP-FUS. B Time-lapse fluorescence images of individual mRNAs, visualized with stdPCP-stdGFP, and optoMCP-FUS condensates in the MEF cell with the endogenous c-Fos gene tagged with MBS-PBS. The cell was imaged and activated every 1 min. Images are maximum-intensity projections, and post-processed through rolling-ball background subtraction, bleach-correction, and bead-based drift correction. C Time evolution of the integrated intensity of stdPCP-stdGFP within optoMCP-FUS condensates during blue light induction. Data are mean (solid line) ± SD (shaded area). D Normalized counts of the unbound mRNAs during blue light induction. Data are mean (solid line) ± SD (shaded area). n = 4 cells (C, D). E (Top) Example images of the optoMCP-FUS condensate interacting with the target mRNA. The MEF cell was imaged and activated every 50 ms after 30 s of blue light activation. Images were walking-averaged, bleach-corrected, and drift-corrected. See also Supplementary Movie S4. (Bottom) The trajectory of the mRNA and the optoMCP-FUS condensate shown in the images above. F Distances between individual mRNAs and the nearest optoMCP-FUS condensates were tracked for several target mRNAs. G (Top) Example images of the target mRNA molecule scanning the surface of the optoMCP-FUS condensate. The MEF cell was imaged and activated every 50 ms after 30 s of blue light activation. Images were walking-averaged, bleach-corrected, and drift-corrected. (Bottom) The mRNA trajectory is color-coded temporally during the scanning. Scale bars, 1 μm (B, E, and G). a.u., arbitrary units. Source data for panels C–G are provided in the Source Data file.