(a) Ubc9ts-GFP fate upon heat stress. (b) Ubc9ts-GFP expressed in WT cells at 28°C was shifted for 15 min at 37°C. GFP (upper panel) and DIC (lower panel) are shown. Scale bars equal 1.5 μm. (c) pdr5Δ cells expressing Ubc9ts-GFP were grown at 28°C in galactose medium and shifted to 37°C in glucose medium with (+) or without (-) 100 μM MG132. Images show Ubc9ts-GFP at the shift (t=0) and after 60 min (t=60). Dotted lines highlight cell outlines. Scale bar equals 1μm. (d) As in c, Ubc9ts-GFP was immunoblotted with anti-GFP antibodies and quantified as the relative ratio to the initial amount for each condition. Results represent mean and Standard Deviation (SD) of three independent experiments. (e) pdr5Δ and pdr5Δatg8Δ cells expressing Ubc9ts-GFP were grown as in c. Ubc9ts-GFP was immunoblotted with anti-GFP antibodies () and quantified relative to the initial amount for each condition. Results represent mean and SD of three independent experiments. (f) WT cells expressing Ubc9ts-GFP at 28°C in galactose medium were shifted at 37°C in glucose medium. Time series images show GFP signal in a cell 1 min (1′) to 30 min (30′) after the shift (). Scale bar equals1 μm. (g) Inset in f, highlights the coalescence of Ubc9ts-GFP Q-bodies (arrows), from 10 to 13 min after the shift. (h) Number of puncta in the medial focal plane over time in WT (main panel; puncta assessed from a total population of n=63 cells over three independent experiments, 1 field counted per experiment) or in pdr5Δ cells (puncta assessed from a total population of n=36 cells over three independent experiments, 1 field counted per experiment) with or without MG132 (secondary panel). Results represent mean of puncta for n cells and SD. (**) p<0.005, compared to untreated cells for the same indicated time. (i) Trajectories of three Ubc9-GFP Q-bodies in WT cells. Positions of particles are represented from each frame of a movie (1 frame/15 sec) at the medial focal plane. Consecutive positions are connected by lines: black, before coalescence; grey, after coalescence. Green and red dots indicate the initial and final positions, respectively. Initial, final and coalescing times are indicated, and average speed (+/- SD) over the entire trajectory.

(a) WT cells expressing Ubc9ts-GFP or Abp1-GFP (lower panel) were grown at 28°C in galactose medium and treated with (right panel) or without (left panel) 200 μM Latrunculin A (LatA) for 10 min prior to a shift to 37°C in glucose medium. 5 min series of images show Ubc9ts-GFP or Abp1-GFP signal (). Scale bars equal 1 μm. Trajectories of three Ubc9-GFP puncta in WT cells with or without LatA are represented (lower panel) as described in . (b) WT cells expressing Ubc9ts-GFP and Abp1-GFP were grown as in a but treated with (right panel) or without (left panel) LatA for 2h prior to the shift. Scale bars equal 1 μm. (c) WT cells expressing Ubc9ts-GFP or Tub1-GFP were grown as in a but treated with (lower panel) or without (upper panel) 15 μg/ml Nocodazole (Noc) for 10 min prior to the shift. 5 min series of images show Ubc9ts-GFP or Tub1-GFP signal. Scale bars equal 1 μm. (d) WT cells expressing Ubc9ts-GFP were grown as in a but treated for 30 min with (right panel) or without (left panel) 10 mM azide and deoxyglucose prior to the shift. 5 min series of images show Ubc9ts-GFP signal (). Scale bars equal 1 μm. Trajectories of three Ubc9-GFP puncta in cells with and without azide and deoxyglucose treatment are represented (lower panel) as described in .

(a) Cells expressing Npl3-RFP and Ubc9ts-GFP were imaged after 10 min at 37°C. Two-color deconvolved images show Npl3-RFP signal (nucleus) in red and Ubc9ts-GFP signal in green. Scale bar equals 1 μm. (b) Cells expressing Hmg1-GFP (perinuclear ER, green) and Ubc9ts-CHFP (red) were imaged as in a. Arrow indicates Q-body in proximity to the ER. Scale bar equals 1 μm. (c) Cells expressing Rtn1-GFP and Ubc9ts-CHFP were imaged after 10 min at 37°C. Two-color images of three Z-focal plans (0.2 μm intervals) show Rtn1-GFP signal (cortical ER) in green and Ubc9ts-CHFP signal in red (). Arrows indicate Q-bodies in proximity to the cortical ER. Scale bar equals1 μm. (d) Inset from c representing a series of 0.2 μm Z-section. Scale bar equals 1 μm. (e) Ubc9ts-GFP was expressed in WT and rnt1Δrtn2Δyop1Δ cells at 28°C in galactose medium and shifted at 37°C in glucose medium. 5 min series of images show Ubc9ts-GFP signal over 30 min. Scale bars equal 1 μm. (f) As in e, cells were harvested at indicated time from the shift and Ubc9ts-GFP was immunoblotted with anti-GFP antibodies. Ubc9ts-GFP was quantified as the relative ratio to the initial amount from one experiment representative of four independent experiments. (g) Schematic of ER-associated Q-body processing of misfolded Ubc9ts (red star). Green represents the ER network throughout the cell.

(a) WT cells co-expressing GFP-Ssa1 and Ubc9ts-CHFP, or GFP-Ssa1 and Ubc9-CHFP, were grown at 28°C in galactose medium and shifted to 37°C in glucose medium. Two-color images show GFP-Ssa1 signal in green and Ubc9ts-CHFP signal in red at 0, 30 and 60 min after the shift. Scale bars equal 1.5 μm. (b) Cells expressing Ubc9ts-GFP at 28°C in galactose medium were shifted at 37°C (t=0) in glucose medium. Cells were harvested at indicated times and Ubc9ts-GFP immunoblotted using anti-GFP antibodies. (c) Ubc9ts-GFP was expressed in WT and ssa1Δssa2Δ cells at 28°C in galactose medium and shifted at 37°C in glucose medium. 5 min series of images show Ubc9ts-GFP signal over 30 min (i). Cells expressing Ubc9-GFP were similarly prepared and imaged 15 min after the shift (ii). Scale bars represent 1 μm. (d) Average number of puncta per cell in WT (black squares) and ssa1Δssa2Δ (blue diamonds) cells over time. Puncta assessed from a total population of n=35 cells over three independent experiments (1 field counted per experiment).(**) p<0.005 compared to WT for the same indicated time. (e) WT and hsp82ts cells expressing Ubc9ts-GFP or Ubc9-GFP were prepared and imaged as in c. Scale bars represent 1 μm. (f) Average number of puncta per cell in WT (black triangles) and hsp82ts (red diamonds) cells over time. Puncta assessed from a total population of n=25 cells over two independent experiments (1 field counted per experiment).(*) p<0.05 (**) p<0.005 compared to WT for the same indicated time. (g) ydj1Δ strains expressing Ubc9ts-GFP alone, and in combination with YDJ1, ydj1(C406S) or empty vector was prepared and imaged as in c. Shown an inset of a ydj1(C406S)-expressing cell. Scale bars represent 1 μm. (h) WTorsse1Δ cells expressing Ubc9ts-GFP or Ubc9-GFP were prepared and imaged as in c. Scale bars represent 1 μm. (i) Role of the Hsp70-Hsp90-Hsp110 system in Q-bodies pathway for misfolded Ubc9ts (red star). Green represents the ER network throughout the cell.

(a) Cells expressing Hsp104-GFP and Ubc9ts-CHFP or Ubc9-GFP were grown at 28°C in galactose medium and shifted at 37°C in glucose medium. 5 min series of a two-color movie show Hsp104-GFP signal in green and Ubc9ts-CHFP signal in red over 60 min (i). Cells expressing Hsp104-GFP and Ubc9-CHFP were imaged 15 min after the shift (ii). Scale bar equals 1 μm. (b) As in a, but cells were imaged 10 min after the shift. Two-color deconvolved images show Hsp104-GFP signal in green and Ubc9ts-CHFP or Ubc9-CHFP signal in red. Scale bars represent 1 μm. (c) Cells expressing Hsp42-GFP (green) and Ubc9ts-CHFP or Ubc9-CHFP (red) were imaged as in b. Scale bars equal 1 μm. (d) WT, hsp104Δ, hsp42Δ, and hsp26Δ cells expressing Ubc9ts-GFP (i) or Ubc9-GFP (ii) were prepared as in a. 5 min series of images show Ubc9ts-GFP signal over 30 min. Ubc9-GFP signal is shown 15 min after the shift. Scale bars represent 1 μm. (e) Average number of puncta per cell in the WT (black squares), hsp104Δ (orange diamonds), hsp26Δ (gray circles) strains over time. Puncta assessed from a total population of n=35 cells over three independent experiment (1 field counted per experiment). (*) p<0.05 (**) p<0.005 compared to WT for the same indicated time. (f) Indicated strains expressing Ubc9ts-GFP were grown as in a and harvested at indicated times from the shift (t=0). Ubc9ts-GFP was immunoblotted using anti-GFP antibodies. (g) WT and hsp42Δhsp104Δ cells expressing Ubc9ts-GFP and Ubc9-GFP were prepared and imaged as in d. Scale bars represent 1 μm. (h) WT and the hsp42Δsse1Δ cells expressing Ubc9ts-GFP and Ubc9-GFP were prepared and imaged as in d. Scale bars represent 1 μm. (i) Dissolution-addition balance between Hsp104 and Hsp42: Q-body coalescence results from an Hsp104-mediated process of dissolution followed by Hsp42 stimulated re-addition of the released misfolded protein into existing inclusions.

(a) WT cells expressing Ubc9ts-GFP and CHFP-VHL was grown at 28°C in galactose medium and imaged at 37°C in glucose medium. 5 min series of images show Ubc9ts-GFP signal in green and CHFP-VHL signal in red (i). Cells expressing Ubc9-GFP and CHFP-VHL were similarly prepared and imaged 15 min after the shift (ii). Scale bar equals 1 μm. (b) 15 sec interval series of an inset from cell represented in a (top panel) and from a cell co-expressing Luc-GFP and Ubc9-CHFP (bottom panel, and ). Scale bars equal 1 μm (c). WT cells expressing Htt-Q97-CHFP (red) and Ubc9ts-GFP (green) were prepared and imaged as in a. Scale bar equals 1 μm. (d) 15 sec interval series of an inset from cell represented in c (bottom panel) and from a cell co-expressing GFP-VHL and Rnq1-CHFP (top panel, and ). Scale bars equal 1 μm. (e) Two-color Z-stack projection of cells expressing both Htt-Q97-GFP (in green) and Rnq1-CHFP (in red). Scale bar equals 1 μm. (f) GFP signal of WT, ssa1Δssa2Δ, hsp42Δ, hsp104Δ cells expressing Ubc9ts-GFP or Luc-GFP at 37°C; or Htt-Q97-GFP or Rnq1-GFP at 28°C. Scale bars equal 1 μm.(g-h) Percentages of cells with puncta from a population analyzed in f. Respective total population sizes for WT, ssa1ssa2, hsp42, and hsp104 cells are [ (g): orange n=(2418, 375, 3013, 591) cells; yellow n=(1035, 472, 442, 681) cells; (h): purple, n=(971, 519, 1047, 617) cells; green n=(1165, 410, 1235, 853) cells] over three independent experiments.(6 fields counted per experiment) (*) p<0.05 (**) p<0.005 compared to WT for the same indicated time.

(a) Ubc9ts-GFP-expressing cells were grown at 28°C in galactose medium and Hsp104-GFP-expressing at 28°C in glucose medium. Cells were imaged at 28°C in glucose medium after a 10 min treatment with 10% ethanol (EtOH), 30 min treatment with 5 mg/mL AZC, or at 37°C with no treatment. GFP signal is shown in 5 min series movies over 30 min. Scale bars equal 1 μm. (b) Cells expressing Ubc9ts-GFP (i) or Hsp104-GFP (ii) were grown at 28°C for 5 hrs (young) or for 3 days (aged) and imaged at 37°C. GFP signal is shown in 5 min series movies. Scale bars equal 1 μm. (c) Ubc9ts-GFP-expressing cells were grown at 28°C in galactose medium and imaged at 37°C in glucose medium in the absence (-) or presence (+) of 0.2 μg/mL rapamycin 10 min prior the shift. GFP signal is shown in 5 min series movies over 30 min. Scale bars equal 1 μm. (d) Quantification of the average number of puncta per cell in untreated (-, circles) or rapamycin-treated (+, triangles) cells over time as presented in c. Puncta assessed from a total population of n=40 cells over three independent experiment (3 fields counted per experiment).(*) p<0.05 (**) p<0.005 compared to untreated cells for the same indicated time.

(a) Schematic of competition assay; [KanS], kanamycin-sensitive phenotype; [KanR], kanamycin-resistant phenotype. (b) Percentage of KanR hsp26Δ colony-forming units (CFU) over KanS WT CFU was quantified at 0 and 5 days. (c-d) Percentage of KanR hsp42Δ CFU (c) or hsp104Δ CFU (d) over KanS WT CFU was quantified over time at 28°C (left panels) or 37°C (right panels). Data represent three independent experiments (n = 350 CFU for each day of the experiment).