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. 2021 Feb 5;10:e60577. doi: 10.7554/eLife.60577

Figure 4. Rad52 and Rfa1 behavior strongly differs inside foci (A) Simplified view of a DSB repaired by HR where RPA is shown.

(B) Typical S/G2-phase haploid cells harboring Rfa1-Halo/JF646 after 2 hr if galactose induction. From left to right: transmission image; Time-projection of the whole movie; Rfa1 density map: each spot represents a single detection of Rfa1-Halo/JF646, the color map indicates the number of Rfa1 neighbors inside a 50 nm radius disk; Rfa1 displacement map: each line represents the trajectory of a detection, the color map indicates the distance in mm covered in 20 ms. The bar scale represents 1 μm. (C) Displacement histograms of individual Rfa1 molecules (yellow) and individual Rad52 molecules (red) inside foci. The time interval is 20 ms. 29 S/G2 cells were analyzed, representing 621 traces (mean length of 12.4 frames) and 7095 displacement of 20 ms time intervals. (D) Displacement histograms of individual Rfa1 molecules inside foci (yellow) and whole Rad52 foci (grey). The time interval is 20 ms. (E) MSD of individual Rfa1 molecules inside foci (yellow), individual Rad52 molecules inside foci (red, same data as Figure 3E) and the whole Rad52 focus (green, same data as Figure 3E). MSDs of Rfa1 molecules and the whole Rad52 focus are fitted with an anomalous model while the MSD of individual Rad52 is fitted with a confined model. (F) Typical haploid cells expressing Rad52-RFP and an I-SceI inducible DSB at LYS2, observed by wide field microscopy. First panel: a DSB is induced for 2 hr; second panel: a DSB is induced for 2 hr including 30 min with 10% hexanediol and 10 μg/ml of digitonin; third panel: a DSB is induced for 2 hr and cells are fixed with fixation with 4% paraformaldehyde or 10 min; fourth panel: quantification of the images presenting the percentage of S/G2 phase cells with a Rad52 focus. 200 cells are analyzed in each condition. The bar scale represents 1 μm. (G) Typical haploid cells expressing Rfa1-RFP and an I-SceI inducible DSB at LYS2, observed by wide field microscopy. Cells are images in the same conditions as in figure (F). Since Rfa1 form several foci per nucleus, the quantification shows the number of foci per nucleus (0bar 3 foci). The bar scale represents 1 μm.

Figure 4.

Figure 4—figure supplement 1. Dissolution of P-bodies with 10% hexanediol teatment.

Figure 4—figure supplement 1.

The efficiency of 1.6- hexanediol treatment was tested using a strain expressing Edc3-GFP as a control. GFP-labelled Edc3 cells were grown in four conditions: (i) glucose containing media (no stress), (ii) after 1 hr in glucose-depleted media (stress), (iii) after 1 hr in glucose-depleted media and the addition of 10% hexanediol and 10 μg/ml of digitonin for 30 min (stress + Hexa), and (iv) after 1 hr in glucose-depleted media and 10 min fixation with 4% paraformaldehyde (Stress + PAF). The bar scale represents 1 μm. The right panel shows the quantification of the images: the number of Edc3 foci per cell is shown for each condition. 200 cells were imaged in each condition on an inverted wide field microscope (left panel) and the number of foci per cells was quantified (right panel). P-values (Wilcoxon-Mann-Whitney test) are given below: p No stress / Stress = 4 10−15 ; p Stress / Stress + Hexa = 6 10−19 ; p No stress + Hexa / Stress + Hexa = 0.5 p No stress / Stress + PAF = 0.01; p Stress / Stress + PAF = 2 10−8.