a, b, In vivo, stress granule assembly after treatment with 0.5% sodium azide was monitored by Ded1–eGFP in cells expressing untagged wild-type Dhh1 or Dhh1DQAD as the sole copy, and in a dhh1Δ background. Quantification of stress granules per cell was performed using Diatrack. Images are representative of four biological replicates, with at least 855 (WT), 755 (dhh1Δ) or 106 (Dhh1DQAD) cells per replicate. Data are mean and s.d. ***P = 0.0003 (dhh1Δ), ***P = 0.0001 (Dhh1DQAD), two-tailed unpaired t-test. Dots represent mean of individual replicates. Scale bars, 5 µm. c, d, RNA transfer between Dhh1 and Ded1 droplets. c, Forward reaction: Dhh1–mCherry droplets were assembled with Cy5-labelled RNA and added to Ded1–GFP droplets. After the addition of Not1MIF4G, Dhh1 droplets dissolve and the Cy5-RNA accumulates in the Ded1 droplets. d, Inverse reaction: Ded1–mCherry droplets were assembled with Cy5-labelled RNA and added to Dhh1–GFP droplets. After addition of recombinant eIF4GC-terminus, Ded1 droplets dissolve, but the Cy5-RNA does not accumulate in the Dhh1 droplets. In contrast to the reaction shown in Fig. 4, no stabilizing agents (such as BSA or PEG) were added in c and d to make the results in the forward and inverse reaction comparable. The fluorescence intensity scaling was adjusted for the first image (before Not1 or eIF4G addition) to account for the sample dilution after the addition of Not1 or eIF4G. However, scaling of the Cy5 channel in the first image, and in the subsequent frames (20–180 s), is identical for the forward and inverse reactions to enable a direct visual comparison. e, Quantification of the reactions presented in c and d. For each experiment, the mean Cy5-RNA intensity accumulating in six Dhh1–GFP droplets is plotted over time after the addition of Not1 or eIF4G. For background correction, six identically sized areas outside of Dhh1–GFP droplets were quantified and subtracted from the intensity measured inside the Dhh1–GFP droplets. These experiments were repeated at least three times, with comparable results. Data are mean (line) and s.d. (shaded area) of six large droplets per movie and forward and inverse reaction. At t = 180 s, 16.7 ± 2.7% of the Cy5-RNA is enriched in Ded1–GFP droplets that occupy 5–7% of the surface area (n = 3 movies). f, Line scan of the Cy5 channel (raw data), at time point t = 180 s through Ded1 droplets shown in c. In the ‘forward’ reaction, Ded1 droplets enrich Cy5-RNA 2–3-fold over background.