Spatial sequestration of misfolded proteins by a dynamic chaperone pathway enhances cellular fitness during stress

Abstract

The extensive links between proteotoxic stress, protein aggregation and pathologies ranging from ageing to neurodegeneration underscore the importance of understanding how cells manage protein misfolding. Using live-cell imaging, we determine the fate of stress-induced misfolded proteins from their initial appearance until their elimination. Upon denaturation, misfolded proteins are sequestered from the bulk cytoplasm into dynamic endoplasmic reticulum (ER)-associated puncta that move and coalesce into larger structures in an energy-dependent but cytoskeleton-independent manner. These puncta, which we name Q-bodies, concentrate different misfolded and stress-denatured proteins en route to degradation, but do not contain amyloid aggregates, which localize instead to the insoluble protein deposit compartment. Q-body formation and clearance depends on an intact cortical ER and a complex chaperone network that is affected by rapamycin and impaired during chronological ageing. Importantly, Q-body formation enhances cellular fitness during stress. We conclude that spatial sequestration of misfolded proteins in Q-bodies is an early quality control strategy occurring synchronously with degradation to clear the cytoplasm of potentially toxic species.

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Figure 1: Misfolded proteins are sequestered in Q-bodies upon heat stress.
Figure 2: Energy dependency but cytoskeleton independency of Q-body dynamics.
Figure 3: Q-body dynamics relies on an intact cortical ER.
Figure 4: The maturation and degradation of Q-bodies rely on the Hsp70–Hsp90 system.
Figure 5: Balance between addition and dissolution activities controls Q-body dynamics.
Figure 6: Different types of misfolded protein, but not amyloids, are processed together through the Q-body pathway.
Figure 7: The Q-body pathway responds to proteotoxic stress, chronological ageing and nutrient signalling.
Figure 8: Fitness advantage of spatial sequestration of misfolded proteins in Q-bodies.

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Acknowledgements

We thank C. Toret and V. Albanese for experimental advice and discussions; and C. Toret and R. Andino for critical reading of the manuscript. S.E-T. was initially supported by a fellowship from Fondation pour la Recherche Medicale (France). W.I.M.V. was supported by the Marie Curie International Outgoing Fellowship Programme. This work was supported by grants from the NIH and a Senior Scholar Award from the Ellison Foundation to J.F.

Author information

S.E-T. and J.F. conceived the project, S.E-T. performed most experiments, W.I.M.V. performed experiments in Fig. 1d,e, Fig. 3e,f and Supplementary Fig. S1b, and all authors interpreted the experiments and contributed to writing.

Correspondence to Judith Frydman.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Formation of Q-bodies.

(a) WT cells expressing Ubc9ts–GFP were grown at 28 °C in galactose medium and shifted at 33 °C or 37 °C in glucose medium. 5 min series images shows Ubc9ts–GFP signal over a 30 min movie. Scale bars equal 1 μm. (bpdr5Δ and pdr5Δatg8Δ cells expressing Ubc9ts–GFP were grown as in a. Upon temperature shift, cells were treated with 100 μM MG132 as indicated. Ubc9ts–GFP was analyzed by immunoblot using anti-GFP antibodies. (c) Images of Ubc9-GFP expressed in WT cells at 28 °C (left panel) and shifted for 15 min at 37 °C. GFP (right panel). Scale bars equal 1.5 μm.

Supplementary Figure 2 Q-bodies associate with specific sub-cellular structures.

(a) Two-color images of Z-focal plans (0.2 μm intervals; obtained after 10 min at 37 °C) of cells expressing Ubc9ts–CHFP (red) or Ubc9ts–GFP (green) and the following markers: GFP–Snc1 (early endosomes; green), GFP–Pep12 (late endosomes; green), CHFP–Atg8 (autophagic vesicles; red); the vacuole was imaged by treating cells with MDY64 (blue). Scale bars equal 1 μm. (b) Cells expressing Spc42–GFP (green) and Ubc9ts–CHFP (red) were imaged 15 min after a shift from 28 °C to 37 °C. Scale bars equal 1 μm.

Supplementary Figure 3 Hsp70–Hsp90 chaperones promote the maturation and degradation of Q-bodies.

(a) Schematic of the Hsp70 chaperone system and its connection to the Hsp90 system. (b) Ubc9ts–GFP was expressed in WT and ssa1-4 ssa2Δssa3Δssa4Δ (herein ssa1ts ssa2Δssa3Δssa4Δ) background at 28 °C in galactose medium and shifted at 37 °C in glucose medium. 5 min series of images shows Ubc9ts–GFP in these strains over 30 min. Scale bars equal 1 μm. (c) Representation of the average number of puncta per cell in the WT (purple squares) and the ydj1Δ (orange circles) strains over time. Puncta assessed from a total population of n = 38 cells over three independent experiments (1 field counted per experiment). (*) p<0.05 (**) p<0.005 compared to WT for the same indicated time. (d) Ubc9ts–GFP expressed in WT, hlj1Δ and hlj1Δydj1-151 cells was imaged as in b. Scale bar equals 1 μm. (e) Average number of puncta per cell in the WT (purple squares) and the sse1Δ (green triangles) strains over timePuncta assessed from a total population of n = 16 cells over three independent experiments (1 field counted per experiment). (**) p<0.005 compared to the WT for the same indicated time.

Supplementary Figure 4 Hsp104 does not colocalize with perinuclear Q-bodies.

Images of a fixed cell expressing Hsp104–GFP (green) and Ubc9ts–CHFP (red) and stained with DAPI (blue) after a 15 min shift from 28 °C to 37 °C. Merged represents the overlay of the three channels. Scale bar equals 1 μm.

Supplementary Figure 5 Different types of misfolded proteins, but not amyloids, co-localize to and are processed together via the same Q-bodies.

(a) WT cells expressing Ubc9ts–CHFP and Luc-GFP were grown at 28 °C in galactose medium and imaged at 37 °C in glucose medium. 5 min series of images show Ubc9ts–CHFP in red and Luc-GFP in green. Cells expressing Ubc9-GFP were similarly prepared and imaged 15 min after the shift. Scale bar equals 1 μm. (b) WT cells expressing CHFP-VHL were grown as in a. 5 min series of images shows CHFP-VHL over 30 min. Scale bar equals 1 μm. (c) WT cells expressing GFP-VHL (green) and Rnq1-CHFP (red) were grown and imaged as in a. Scale bar equals 1 μm. (d) Cells expressing Hsp42–GFP and Htt-Q97-CHFP were grown at 28 °C in galactose medium and shifted for 10 min at 37 °C in glucose medium. Two-color images (deconvolved in the lower panel) show Hsp42–GFP signal in green and Htt-Q97-CHFP signal in red. Scale bar equals 1 μm. (e) Cells expressing Hsp104–GFP (green) and Htt-Q97-CHFP (red) were grown and imaged as in d. Scale bars equal 1 μm.

Supplementary Figure 6 Aging impairs the Q-body pathway.

Cells expressing Ubc9ts–GFP were grown at 28 °C for 5 hours (young) or for 7 days (aged) and imaged at 37 °C. 5 min series of a 30 min movie show the GFP signal. Scale bars represent 1 μm.

Supplementary Figure 7 Deletion of Hsp42, Hsp26 or Hsp104 does not cause any appreciable growth defects at 37 °C.

A suspension of WT, hsp104Δ, hsp42Δ, and hsp26Δ cells were serially diluted and dropped on YPD at 28 °C and 37 °C.

Supplementary Figure 8 Full scans of original immunoblot data presented in this study.

Red outlines represent the immunoblot sections presented in the corresponding main figures.

Supplementary Table 1 Chaperones involved in the Q-body pathway organized by family and corresponding yeast names. Previously described activities and phenotypes of each chaperone family in the Q-body pathway are included.

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Supplementary Table 1

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Misfolded Ubc9ts forms Q-bodies.

Live-cell movie of Ubc9ts–GFP in a WT cell at 37 °C. Interval between frames is 15 s. Total time of acquisition was 30 min. Refers to Fig. 1f. (AVI 2678 kb)

Actin cytoskeleton does not affect the Q-body pathway.

Live-cell movie of cells expressing Ubc9ts–GFP (left panel) or Abp1–GFP (right panel) at 37 °C with (lower panel) or without (upper panel) LatA treatment. Interval between frames is 15 s. Total time of acquisition was 30 min. Refers to Fig. 2a. (AVI 13424 kb)

The Q-body pathway is energy dependent.

Live-cell movie of Ubc9ts–GFP in WT cell at 37 °C with (right panel) or without (left panel) sodium azide and deoxyglucose (Az/Deox) treatment. Interval between frames is 15 s. Total time of acquisition was 30 min. Refers to Fig. 2d. (AVI 6903 kb)

Q-bodies localize in proximity to the cortical ER.

3D projection of a live-cell expressing Ubc9ts–CHFP Q-bodies (red) and Rtn1-GFP (green, cortical ER) at 37 °C. 0.2 μm intervals. Refers to Fig. 3c. (AVI 2388 kb)

The Q-body pathway depends on an intact cortical ER.

Live-cell movie of Ubc9ts–GFP in WT and rtn1Δrtn2Δyop1Δ cells at 37 °C. Interval between frames is 15 s. Total time of acquisition was 30 min. Refers to Fig. 3e. (AVI 5395 kb)

Hsp70 is required for Q-body formation, dynamics and clearance.

Live-cell movie of Ubc9ts–GFP in WT, ssa1Δssa2Δ and sse1Δ cells at 37 °C. Interval between frames is 15 s. Total time of acquisition was 30 min. Refers to Fig. 4c and h. (AVI 9091 kb)

Hsp82 is required for Q-body maturation and clearance.

Live-cell movie of Ubc9ts–GFP in HSP82 and hsp82ts cells at 37 °C. Interval between frames is 15 s. Total time of acquisition was 30 min. Refers to Fig. 4e. (AVI 6113 kb)

Ydj1 participates in the formation and localization of Q-bodies.

Live-cell movie of Ubc9ts–GFP expressed in ydj1Δ cells with an empty vector, YDJ1, or ydj1(C406S) at 37 °C. Interval between frames is 15 s. Total time of acquisition was 30 min. Refers to Fig. 4g. (AVI 14555 kb)

Hsp104 co-localizes with peripheral Ubc9ts-forming Q-bodies.

Live-cell movie of a cell co-expressing Ubc9ts–CHFP (red) and Hsp104–GFP (green) at 37 °C. Interval between frames is 15 s. Total time of acquisition was 60 min. Refers to Fig. 5a. (AVI 19157 kb)

Q-body pathway relies on the balance between Hsp104 and Hsp42 activities.

Live-cell movie of Ubc9ts–GFP in WT, hsp104Δ, hsp42Δ, and hsp42Δhsp104Δ cells at 37 °C. Interval between frames is 15 s. Total time of acquisition was 30 min. Refers to Fig. 5d and g. (AVI 13544 kb)

VHL, but not Htt-Q97, is concentrated to Q-bodies.

Live-cell movie of Ubc9ts–GFP (green) co-expressed with CHFP-VHL (red) or Htt-Q97-CHFP (red) in WT cell at 37 °C. Interval between frames is 15 s. Total time of acquisition was 30 min. Refers to Fig. 6a and b. (AVI 16100 kb)

TOR signaling regulates the Q-body pathway.

Live-cell movie of Ubc9ts–GFP in WT cell with or without 0.2 μg ml−1 rapamycin (Rap) treatment at 37 °C. Total time of acquisition was 30 min. Refers to Fig. 7c. (AVI 5395 kb)

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Escusa-Toret, S., Vonk, W. & Frydman, J. Spatial sequestration of misfolded proteins by a dynamic chaperone pathway enhances cellular fitness during stress. Nat Cell Biol 15, 1231–1243 (2013) doi:10.1038/ncb2838

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