Analysis of live-cell videos

Videos were projected along Z using pixels of maximal intensity and corrected for photobleaching using the histogram matching method in ImageJ (National Institutes of Health). Individual spots were identified and tracked with the TrackMate plugin in ImageJ, using the DoG detector, subpixel localization, a blob diameter of 0.8 µm, and a threshold manually adapted for each video. For fast imaging conditions, tracks were reconstructed using the simple LAP tracker option, using a maximal linking distance of 1.5 µm, a gap-closing distance of 1 µm, and a maximal frame gap of 2.

Tracks were imported and analyzed in R. Instant 1D displacements between frames were calculated along the x and y axis, and the resulting histograms were fitted to a Gaussian function, for which variance is directly proportional to the diffusion coefficient (D). The values obtained for the x and y displacements were treated independently and within 1% for the SunTagx56-Ki67 and SunTag-POLR2A polysomes and within 7% for the SunTagx32-DYNC1H1. We also calculated a mean MSD as a function of time, by aligning all tracks at their start and averaging the resulting 2D displacements. The segment of the resulting curves comprised between 0 and 18 s was fit to a linear model, and the slope was used to calculate D. These values were approximately twofold lower than the ones calculated from instant displacements, which may be a result of errors in pointing accuracy for the instant displacements (mean instant displacement was between 100 and 300 nm) or to a subdiffusive behavior for the particles. We also analyzed single individual tracks, for tracks longer than 36 s. For each track, we calculated all instant 1D displacements in x and y, pooled them, and estimated D for each polysome from the variance of the resulting data (>150 data points). We then calculated the SD of individual diffusion coefficients. If all particles were diffusing identically, they should have the same D, and the measured SD between individual particles should be D/(nbp)^0.5, with nbp being the number of data points used to calculate D for each track (in this study, 150). For the three polysomes analyzed in this study (Ki67, POLR2A, and DYNC1H1), the measured SD was 5–10-fold higher than expected, indicating that particles are heterogeneous in terms of diffusional properties.

To quantify the fluctuation of polysomes intensity over time, the tracking data were used to locate the particles in the videos. The videos were bleach corrected, projected along Z using the pixels of maximal intensities, and cropped around the spot location at each time point. The resulting images were then fit to 2D Gaussian, and particle intensity was measured from the integrated intensity above background.