Fig. 2: Overview of the single molecule tracking experiments and diffusion analysis.

a Labeling and illumination scheme. TDP-43^Halo and G3BP1^SNAP are labeled with PA-JF646-HaloTag ligand (HTL) and TMR-SNAP-Tag ligand (STL) for 1 h at 37 °C and 5% CO₂ 24 h before imaging. TDP-43^Halo is illuminated continuously with 640 nm and the PA-JF646-dye is continuously activated with 405 nm, the TMR-dye is illuminated with 532 nm and imaged every 200 frames. b Overview of the region assignment and region-specific track assignment (cytoplasm/tracking region: white outline and green tracks; nucleus: blue outline and tracks; stress granules: red outline and tracks). Zoom-ins and exemplary tracks for all regions are shown and show both, mobile TDP-43^Halo as well as more immobile molecules. Scale bar 5 µm. c–e Shuttling of TDP-43^Halo in and out of stress granules. The c and d. Number of shuttling events significantly decreases with increasing stress duration (c: not normalized to SG size, values are displayed as median +/− interquartile range, p-value = < 0.0001, d: normalized to SG size, values are displayed as mean +/− STD, p-value = 0.0006). The ratio of TDP-43^Halo tracks going in and out of stress granules stays constant over all stress durations (e), values are displayed as median +/− interquartile range. Statistical test: Two-tailed Mann–Whitney test. The number of analyzed cells per condition (n number) is given in Supplementary Table 1, the experiments cells were examined in independent experiments. Source data are provided as a Source Data file.