Stochastic translational activity of single SunTagx56-Ki67 polysomes. (A) Tracks of single SunTagx56-Ki67 polysomes, imaged for 45 min at a rate of 0.1 frames/s (fps). Left panel: image of the SunTag signal at the start of the video; middle panel: temporal maximal intensity projection of the video; right panel: identified tracks, and each track is color-coded. Bar, 10 µm. (B) Quantification of the intensity of individual polysomes over time. Each color is a single polysome from the tracks of A. (C and D) Measurements of translation elongation rate from ribosome run-off curves. In C, the intensity of individual polysomes was measured over time, and tracks were aligned by the last image in which the polysome was detected. In D, the mean curve (n = 10) and a fit to a model describing ribosomes running off the mRNA (gray). (E and F) Measurements of translation elongation rate by FRAP. Individual polysomes were photobleached, and fluorescence recovery was measured over time. The curves in E represent the recovery of individual polysomes, and the curve in F is the mean (n = 13), with a fit to the model in gray. a.u., arbitrary units.