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Proteotoxic stress is a driver of the loser status and cell competition

Nature Cell Biology volume 23, pages 136–146 (2021)Cite this article

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Abstract

Cell competition allows winner cells to eliminate less fit loser cells in tissues. In Minute cell competition, cells with a heterozygous mutation in ribosome genes, such as RpS3+/− cells, are eliminated by wild-type cells. How cells are primed as losers is partially understood and it has been proposed that reduced translation underpins the loser status of ribosome mutant, or Minute, cells. Here, using Drosophila, we show that reduced translation does not cause cell competition. Instead, we identify proteotoxic stress as the underlying cause of the loser status for Minute competition and competition induced by mahjong, an unrelated loser gene. RpS3+/− cells exhibit reduced autophagic and proteasomal flux, accumulate protein aggregates and can be rescued from competition by improving their proteostasis. Conversely, inducing proteotoxic stress is sufficient to turn otherwise wild-type cells into losers. Thus, we propose that tissues may preserve their health through a proteostasis-based mechanism of cell competition and cell selection.

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Fig. 1: Reduced protein synthesis does not confer the loser status.
Fig. 2: Prospective losers display defective autophagic flux.
Fig. 3: Autophagy impairment does not confer the loser status.
Fig. 4: Prospective losers display proteotoxic stress.
Fig. 5: Alleviating proteotoxic stress rescues the loser status.
Fig. 6: Proteotoxic stress is sufficient to confer the loser status.

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Data availability

The following publicly available databases were used in this study: FlyBase (https://flybase.org) and the UniProt D. melanogaster proteome (https://www.uniprot.org/proteomes/UP000000803). Source data are provided with this paper. All other data supporting the findings of this study are available upon reasonable request.

Code availability

The Fiji-based custom-made script can be made available to individuals upon reasonable request while we seek to publish it independent of this study.

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Acknowledgements

We thank the Piddini group for input on the project and manuscript and R. Carazo Salas for feedback and discussions on the data. We thank the Wolfson Bioimaging Facility for access to microscopes and for assistance in performing the electron microscopy. We thank the University of Bristol Proteomics Facility for performing the TMT proteomics experiments and for proteomics bioinformatics support. We are grateful to T. E. Rusten for the generous gift of the p62 antibody. This work was supported by Wellcome Trust PhD studentships to M.P.D. and I.K., a Cancer Research UK Programme grant to E.P. (A12460), a Cancer Research UK Programme Foundation Award to E.P. (grant C38607/A26831) and a Royal Society University Research Fellowship to E.P. (UF0905080). E.P. is a Wellcome Trust Senior Research Fellow (205010/Z/16/Z).

Author information

Author notes

  1. Michael P. Dinan

    Present address: University of Cambridge, Cambridge, UK

  2. Iwo Kucinski

    Present address: Wellcome and MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK

  3. Iwo Kucinski

    Present address: Department of Haematology, University of Cambridge, Cambridge, UK

  4. These authors contributed equally: Michael E. Baumgartner, Michael P. Dinan.

Authors and Affiliations

  1. School of Cellular and Molecular Medicine, University of Bristol, Bristol, UK

    Michael E. Baumgartner, Michael P. Dinan, Paul F. Langton & Eugenia Piddini

  2. The Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK

    Michael P. Dinan & Iwo Kucinski

  3. Zoology Department, University of Cambridge, Cambridge, UK

    Michael P. Dinan & Iwo Kucinski

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  1. Michael E. Baumgartner
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Contributions

E.P. led the project. All authors conceived of the experiments. M.P.D., M.E.B., I.K. and P.F.L. performed and analysed the experiments. M.P.D., M.E.B., P.F.L. and E.P. wrote the manuscript.

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Correspondence to Eugenia Piddini.

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Extended data

Extended Data Fig. 1 Protein synthesis and its regulation in Rps3+/− cells.

(a-c) AHA (grey) protein synthesis assay in wing discs harboring either Rps3+/− clones (GFP-positive) (a) or clones overexpressing 4EBPTA (GFP-positive) (b), and corresponding quantification (n = 7 and 7, respectively, two-sided paired t-test without p-adjustment for multiple comparisons) (c). (d-e) OPP (green) protein synthesis assay in a wing disc expressing mahj-RNAi in the P compartment (positively labelled with RFP) (d) and corresponding quantification (n = 10, two-sided Wilcoxon signed-rank test) (e). (f-g) An RpS3+/− wing disc expressing GADD34 in the P compartment and labelled with phospho-eIF2α (red) (f), and corresponding quantification (n = 10, two-sided paired t-test) (g). (h-i) GST-GFP reporter (green) activation in an RpS3+/− wing disc expressing GADD34 in the P compartment (h), and corresponding quantification (n = 10, two-sided paired t-test) (i). For all micrographs, scale bars correspond to 50 µm. For all quantifications provided, the horizontal line represents the mean and whiskers indicate 95% confidence intervals. All n numbers refer to the number of individual wing discs.

Source data

Extended Data Fig. 2 The role of autophagy in prospective losers.

(a) GstD1-GFP signal (green) in a RpS3+/− wing disc expressing Puc in P cells (labelled by the absence of Ci, magenta). (b-c) Apoptotic cell death, as detected by anti-cleaved Caspase-3 reactivity (red), in wing discs of an atg8+/− heterozygote (b, left), RpS3+/− heterozygote (b, middle), or atg8+/−, RpS3+/− transheterozygote (b, right) and corresponding quantification (n = 9, 8, and 9 respectively, two-sided two sample Kolmgorov-Smirnov test without p-adjustment for multiple comparisons) (c). (d) Apoptotic cell death, as detected by anti-cleaved Caspase-3 reactivity (red), in wing discs of an atg13+/− heterozygote (d, left), RpS3+/− heterozygote (d, middle), or atg13+/−, RpS3+/− transheterozygote (d, right). (e-f) Apoptotic cell death, as detected by anti-cleaved dcp1 antibody staining (red), in wing discs of a p62+/− heterozygote (f, left), RpL27A+/− heterozygote (f, middle), or RpL27A+/−, p62+/− transheterozygote (f, right) and corresponding quantification (n = 10, 10, and 12 respectively, two-sided Mann-Whitney U test without p-adjustment for multiple comparisons) (e). (g-i) Wing discs harboring RpS3+/− clones (GFP-positive) (h, left), RpS3+/− clones expressing atg1-RNAi (GFP-positive) (h, middle), or RpS3+/− clones expressing atg9-RNAi (GFP-positive) (h, right) stained with cleaved-dcp1 (red) and corresponding quantification of border cell death (n = 16, 12, and 9 respectively, two-sided Mann-Whitney U test without p-adjustment for multiple comparisons) (g) and clone coverage (n = 16, 12, and 9 respectively, two-sided student’s t-test without p-adjustment for multiple comparisons) (i). For all micrographs, scale bars correspond to 50 µm. For all quantifications provided, the horizontal line represents the mean and whiskers indicate 95% confidence intervals. All n numbers refer to the number of individual wing discs.

Source data

Extended Data Fig. 3 Autophagy flux in ribosome mutants and upon translation inhibition.

(a-c) GFP-p62 ReFlux signal (green) in wing discs expressing RNAi against the autophagy gene atg1 specifically in P cells (labelled by the absence of Ci, magenta), immediately after heat shock (a) or three hours later (b), and corresponding signal quantifications (n = 7 and 6 respectively, two-sided two sample Kolmgorov-Smirnov test) (c). (d-f) GFP-p62 ReFlux signal (green) in a wing disc harboring RpS3+/− clones (dsRed-positive) three hours after heat-shock (d) and corresponding quantification of GFP-p62 signal intensity (e) and number of GFP-p62 foci per area (f) (for both measurements, n = 5, two-sided paired t-test). (g) GFP-p62 ReFlux signal (green) in wing discs harboring RpS3+/− A cells and wild-type P cells, three hours after heat-shock, with or without addition of chloroquine, as indicated. (h) GFP-p62 ReFlux signal (green) in wing discs harboring RpS3+/− A cells (dsRed-positive) and wild-type P cells (dsRed-negative) twenty-four hours after heat-shock. (i-k) GFP-p62 ReFlux signal (green) in wing discs harboring wild-type A cells and 4E-BPTA-expressing P cells (labelled by the absence of Ci, magenta), immediately after heat shock (i) or three hours later (j), and corresponding signal quantifications relative to wing discs containing an RpS3+/− A compartment and wildtype P compartment (images not shown) (n = 9 and 8 for 0 and 3 hour 4E-BPTA, and n = 7 and 8 for 0 and 3 hour RpS3+/−, respectively; two-sided two-sample Kolmgorov-Smirnov test without p-adjustment for multiple comparisons) (k). For all micrographs, scale bars correspond to 50 µm. For all quantifications provided, the horizontal line represents the mean and whiskers indicate 95% confidence intervals. All n numbers refer to the number of individual wing discs.

Source data

Extended Data Fig. 4 Proteasome defects are linked to the prospective loser status but not to translation inhibition.

(a-b) Apoptosis as detected by anti-cleaved caspase-3 reactivity (green), in Prosβ2+/− (a, left), RpS3+/− (a, middle), or Prosβ2+/−, RpS3+/− transheterozygote (a, right) wing discs and corresponding quantification (n = 10, 10, and 10 respectively, two-sided two sample Kolmgorov-Smirnov test without p-adjustment for multiple comparisons) (b). (c-d) Apoptotic cell death as detected by cleaved-dcp1 (red) in Prosβ2+/− (c, left), a RpL27A+/− (c, middle), or a RpL27A+/−, prosβ2+/− transheterozygote (c, right) wing discs, and corresponding quantification (n = 8, 13, and 10 respectively, two-sided Mann-Whitney U test without p-adjustment for multiple comparisons) (d). (e-g) ProteoFLUX CL1-GFP signal (green) in wing discs expressing mahj-RNAi in the P compartment (RFP-positive), immediately after heat shock (e) or two hours later (f) and corresponding signal quantifications (n = 9 and 7 respectively, two-sided two sample Kolmgorov-Smirnov test) (g). (h-j) ProteoFLUX CL1-GFP signal (green) in wing discs harboring wild-type A cells and 4E-BPTA-expressing P cells (labelled by the absence of Ci, magenta), immediately after heat shock (h) or two hours later (i), and corresponding signal quantifications relative to wing discs containing an RpS3+/− A compartment and wildtype P compartment (images not shown) (n = 9 and 10 for 0 and 2 hour 4E-BPTA, and n = 7 and 7 for 0 and 2 hour RpS3+/−, respectively; two-sided two-sample Kolmgorov-Smirnov test without p-adjustment for multiple comparisons) (j). (k) Transmission Electron microscopy images of a wing disc with wildtype P (left panel) and RpS3+/− A compartments (right panel). Red arrows indicate phago-lysosomal structures containing ribosomes. The scale bar is 500 nm. (l-m) Phospho-eIF2α staining (red) in wing discs harboring RpS3+/− A cells (GFP-positive) and wild-type P cells (GFP-negative) (l) and corresponding signal quantifications (n = 6, two-sided Wilcoxon ranked-sum test) (m). (n-o) A wing disc harboring RpS3+/− clones (GFP-positive) and stained for phospho-eIF2α (red) (n) and corresponding signal quantification (n = 9, two-sided paired t-test) (o). For all micrographs other than those in (k), scale bars correspond to 50 µm. For all quantifications, the horizontal line represents the mean and whiskers indicate 95% confidence intervals. All n numbers refer to the number of individual wing discs.

Source data

Extended Data Fig. 5 Proteostasis and the oxidative stress response.

(a-c) GstD1-GFP signal (green) in wild type (a) or RpS3+/− wing discs (b) fed DMSO control or 10μM bortezomib, as indicated, and corresponding quantification (n = 7, 8, 12, and 12, two-sided Mann-Whitney U test without p-adjustment for multiple comparisons) (c). (d-f) Wing discs harboring GFP-positive clones expressing MJDQ27 (d) or MJDQ78 (e) and stained with cleaved-dcp1 (red) and corresponding quantification of cell death (n = 17 and 15 respectively, two-sided Wilcoxon signed-rank test without p-adjustment for multiple comparisons) (f). For all micrographs, scale bars correspond to 50 µm. For all quantifications provided, the horizontal line represents the mean and whiskers indicate 95% confidence intervals. All n numbers refer to the number of individual wing discs.

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Baumgartner, M.E., Dinan, M.P., Langton, P.F. et al. Proteotoxic stress is a driver of the loser status and cell competition. Nat Cell Biol 23, 136–146 (2021). https://doi.org/10.1038/s41556-020-00627-0

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