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

Nature Cell Biology volume 15, pages 1231–1243 (2013)Cite this article

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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.

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  1. Department of Biology, Stanford University, Stanford, California 94305, USA

    Stéphanie Escusa-Toret, Willianne I. M. Vonk & Judith Frydman

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  1. Stéphanie Escusa-Toret
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Contributions

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.

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Correspondence to Judith Frydman.

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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. (b) pdr5Δ 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

Supplementary Information (XLSX 15 kb)

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). https://doi.org/10.1038/ncb2838

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