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Replication stress promotes cell elimination by extrusion

Abstract

Cell extrusion is a mechanism of cell elimination that is used by organisms as diverse as sponges, nematodes, insects and mammals1,2,3. During extrusion, a cell detaches from a layer of surrounding cells while maintaining the continuity of that layer4. Vertebrate epithelial tissues primarily eliminate cells by extrusion, and the dysregulation of cell extrusion has been linked to epithelial diseases, including cancer1,5. The mechanisms that drive cell extrusion remain incompletely understood. Here, to analyse cell extrusion by Caenorhabditis elegans embryos3, we conducted a genome-wide RNA interference screen, identified multiple cell-cycle genes with S-phase-specific function, and performed live-imaging experiments to establish how those genes control extrusion. Extruding cells experience replication stress during S phase and activate a replication-stress response via homologues of ATR and CHK1. Preventing S-phase entry, inhibiting the replication-stress response, or allowing completion of the cell cycle blocked cell extrusion. Hydroxyurea-induced replication stress6,7 triggered ATR–CHK1- and p53-dependent cell extrusion from a mammalian epithelial monolayer. We conclude that cell extrusion induced by replication stress is conserved among animals and propose that this extrusion process is a primordial mechanism of cell elimination with a tumour-suppressive function in mammals.

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Fig. 1: Cell-cycle genes control cell extrusion cell autonomously.
Fig. 2: Cells undergoing extrusion arrest in S phase.
Fig. 3: Replication stress is coincident with and promotes cell extrusion.
Fig. 4: Replication stress promotes cell extrusion from a simple mammalian epithelial layer.

Data availability

Data supporting all figures are available within the paper and in the associated Source Data files. Raw microscopy data are available upon request from the corresponding author. Source data are provided with this paper.

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Acknowledgements

We thank S. van den Heuvel and the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440), for providing strains; L. Hufnagel and X. Trepat for providing MDCK-Fucci cells; G. van Meer and the ECACC for providing MDCK-II cells (ECACC 62107); N. An for strain management; S. Luo, S. R. Sando, E. L. Q. Lee, A. Doi, A. Corrionero and other members of the Horvitz laboratory for helpful discussions; and D. Ghosh, C. L. Pender, M. G. Vander Heiden, P. W. Reddien, and R. O. Hynes for suggestions regarding the manuscript. This work was supported by the Howard Hughes Medical Institute and by NIH grant R01GM024663. V.K.D. was a Howard Hughes Medical Institute International Student Research fellow. C.P.-P. was the recipient of Human Frontiers Science Program postdoctoral fellowship LT000654/2019-L. J.N.K. was supported by NIH grant R01GM024663. N.T. was supported by NIH Pre-Doctoral Training Grant T32GM007287. D.P.D. was supported by postdoctoral fellowships from the Damon Runyon Cancer Research Foundation and from the Charles A. King Trust. J.R. and C.P.-P. were funded by King’s College London startup funds. H.R.H. is the David H. Koch Professor of Biology at MIT and an Investigator at the Howard Hughes Medical Institute.

Author information

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Authors

Contributions

H.R.H. supervised the project. V.K.D. and H.R.H. conceptualized the project. V.K.D. and H.R.H. designed the experiments that used C. elegans. V.K.D., R.D., J.N.K. and N.T. performed the experiments that used C. elegans. V.K.D., R.D. and D.P.D. generated reagents. C.P.-P. and J.R. designed the experiments that used mammalian cells. C.P.-P. performed the experiments that used mammalian cells. V.K.D., D.P.D. and H.R.H. wrote the original and revised manuscript drafts. All authors contributed to data analysis, interpretation, and reviewing and editing of the manuscript.

Corresponding author

Correspondence to H. Robert Horvitz.

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Peer review information Nature thanks Joan Brugge and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 A genome-wide RNAi screen for the Tex phenotype revealed control of cell extrusion by cye-1 and cdk-2.

a, Schematic representation of the genome-wide RNAi screen for the Tex phenotype. RNAi using pL4440 empty vector was used as negative control and pig-1(RNAi) was used as positive control3. b, Time-lapse confocal fluorescence micrographs of ced-3(lf); stIs10026[his-72::GFP]; nIs632[Pegl-1::mCherry::PH] embryos after indicated RNAi treatment at the indicated times. tve, time point of ventral enclosure. Arrowheads, ABplpappap. Scale bars, 10 μm.

Extended Data Fig. 2 Genetically mosaic cye-1(lf); ced-3(lf) animals with the Tex phenotype lack a cye-1-rescuing transgene in ABplpappap.

ai, Confocal micrographs showing the presence of the cye-1(+)-rescuing transgene in the excretory cell but not in ABplpappap (ah) or in neither the excretory cell nor ABplpappap (i) of cye-1(eh10); ced-3(n3692); nIs434[Ppgp-12::4xNLS-GFP]; nEx3043[cye-1(+); Psur-5::RFP] animals with the Tex phenotype. Scale bars, 10 μm.

Extended Data Fig. 3 ABplpappap, which is generated by an unequal cell division, arrests in S phase and is extruded.

a, b, Confocal fluorescence micrographs of tDHB–GFP fluorescence in ABplpappap (arrowheads) before (a) and after (b) ventral enclosure in heSi192[Peft-3::tDHB-GFP]; ced-3(lf); nIs861[Pegl-1::mCherry::PH] embryos after the indicated RNAi treatment. Dotted outline, ABplpappap nucleus, as identified by Nomarski optics. c, d, Confocal fluorescence micrographs of GFP–PCN-1 fluorescence in ABplpappap (arrowheads) before (c) and after (d) ventral enclosure in ced-3(lf); isIs17[Ppie-1::GFP::pcn-1]; nIs861 embryos after the indicated RNAi treatment. e, Time-lapse confocal fluorescence micrographs of GFP–PCN-1 fluorescence in ABplpappap (arrowheads) in a ced-3(lf); isIs17; nIs861; pig-1(RNAi) embryo at the indicated times. tve, time point of ventral enclosure. fh, Micrographs of virtual lateral section of ced-3(lf); nIs861; stIs10026 embryos showing either ABplpappap (arrowhead) or its daughter cells (arrowheads) after indicated RNAi treatment. im, Confocal fluorescence micrographs of ced-3(lf); ltIs44[Ppie-1::mCherry::PH]; stIs10026 embryos showing the relative sizes of ABplpappap and its sister cell, ABplpappaa, in embryos after the indicated RNAi treatment. Insets, ABplpappap (ad); ABplpappap or its daughters (e–h); magnified view of the region indicated, which includes ABplpappap (†) and ABplpappaa (*) (im). Scale bars, 10 μm.

Extended Data Fig. 4 All extruded cells display features of cell cycle entry, S-phase arrest, and replication stress.

ad, tDHB–GFP fluorescence in unidentified extruded cells from the anterior sensory depression (b), the ventral pocket (c), and the posterior tip (d) of a comma stage embryo of the genotype heSi192; ced-3(lf); nIs861 after RNAi against empty vector control. Nuclei of extruded cells, as identified by Nomarski optics, are marked by dotted outlines. e, f, Micrographs of GFP–PCN-1 fluorescence in unidentified extruded cells (arrowhead) at the ventral pocket (e) or the anterior sensory depression (f) from ced-3(lf); isIs17; nIs861 embryos after RNAi against empty vector control (e) or no RNAi (f). Insets, extruded cells marked by arrowheads in micrographs. gj, RPA-1–YFP fluorescence in unidentified extruded cells from the anterior sensory depression (h, i) and ventral pocket (j) in a ced-3(lf); ltIs44; opIs263[Prpa-1::rpa-1::YFP] embryo after RNAi against empty vector control. Scale bars, 10 μm.

Extended Data Fig. 5 The replication-stress response, probably caused by lrr-1 and nucleotide insufficiency, promotes cell extrusion.

ad, Confocal fluorescence micrographs showing the localization of RPA-1–YFP in ABplpappap (arrowheads) in ced-3(lf); ltIs44; opIs263 embryos after the indicated RNAi treatment. Insets, magnified views of ABplpappap. e, Genes identified as suppressors of the sterility of lrr-1(lf) mutants19 were tested for suppression of cell extrusion. f, Nomarski micrograph showing a cell extruded (arrow) from a wild-type embryo after gmpr-1(RNAi) treatment. Scale bars, 10 μm.

Extended Data Fig. 6 Inhibitors of HU-induced replication-stress response and pan-caspase inhibitors do not alter stochastic cell extrusion.

a, c, d, Representative micrographs of anti-γH2AX (a), anti-pATR (c) and anti-p53 immunofluorescence signal (d) in vehicle- or HU-treated MDCK-II cells (c), and in vehicle-, HU- or Nutlin3-treated MDCK-II cells (a, d). DNA is stained with Hoechst. Scale bars, 20 μm. b, Quantification of extrusions per hour after the indicated treatments. n = 13, 6, 5, 5 and 5 (biological replicates) each for control, PFT, zVAD-FMK, SB 218078 and PF477736 treatments, respectively. Each data point represents a separate experiment. These data were collected and analysed for statistical significance with the data in Fig. 4g. P values are indicated; n.s., not significant. e, Quantification of anti-p53 immunofluorescence signal in MDCK-II cells treated with vehicle, HU, or Nutlin-3. n = 9, 7 and 5 (biological replicates) for vehicle, HU and Nutlin3, respectively. Each data point represents mean fluorescence intensity signal from one image of hundreds of cells. Kruskal–Wallis one-way ANOVA followed by Dunn’s correction was performed. P values are indicated. Data in b, e are represented as mean ± s.d.

Source data

Extended Data Table 1 Penetrances of the Tex phenotype produced by RNAi against cell cycle genes (and non-cell-cycle cyclins and CDKs) in ced-3(lf) animals
Extended Data Table 2 Penetrances of the Tex phenotype produced in wild-type animals by RNAi against cell-cycle genes with potential roles in cell extrusion

Supplementary information

Supplementary Information

This file contains the Supplementary Methods, including the sequences of the RNAi constructs - the targets of which affect cell extrusion.

Reporting Summary

Video 1

A control embryo extrudes ABplpappap. Time-lapse video of a ced-3(lf); stIs10026[his-72::GFP]; nIs632[Pegl-1::mCherry::PH] embryo after RNAi against empty vector control over a 50-minute period ending in ventral enclosure shows ABplpappap (circled at the beginning and end of video) was extruded from this embryo. All nuclei are labeled with GFP (green) and membranes of egl-1–expressing cells are labeled with mCherry (magenta). Time-lapse images used to generate this video were obtained using confocal microcopy. Video playback is at 600x real speed.

Video 2

A cye-1(RNAi) embryo does not extrude ABplpappap. Time-lapse video of a ced-3(lf); stIs10026[his-72::GFP]; nIs632[Pegl-1::mCherry::PH]; cye-1(RNAi) embryo over a 50-minute period ending in ventral enclosure shows ABplpappap (circled at the beginning and end of video) was not extruded from this embryo. All nuclei are labeled with GFP (green) and membranes of egl-1–expressing cells are labeled with mCherry (magenta). Time-lapse images used to generate this video were obtained using confocal microcopy. Video playback is at 600x real speed.

Video 3

A cdk-2(RNAi) embryos does not extrude ABplpappap. Time-lapse video of a ced-3(lf); stIs10026[his-72::GFP]; nIs632[Pegl-1::mCherry::PH]; cdk-2(RNAi) embryo over a 50-minute period ending in ventral enclosure shows ABplpappap (circled at the beginning and end of video) was not extruded from this embryo. All nuclei are labeled with GFP and membranes of egl-1–expressing cells are labeled with mCherry (magenta). Time-lapse images used to generate this video were obtained using confocal microcopy. Video playback is at 600x real speed.

Video 4

ABplpappap arrests in S phase and is extruded in a control embryo. Time-lapse video of a ced-3(lf); isIs17[Ppie-1::GFP::pcn-1]; nIs861[Pegl-1::mCherry::PH] embryo after RNAi against empty vector control over a 35-minute period ending in ventral enclosure shows ABplpappap (circled at the beginning) arrests in S phase and is extruded (circled at the end of video). All cells express GFP–PCN-1 (green) and membranes of egl-1–expressing cells are labeled with mCherry (magenta). Time-lapse images used to generate this video were obtained using confocal microcopy. Video playback is at 600x real speed.

Video 5

ABplpappap completes the cell cycle and is not extruded in a pig-1(RNAi) embryo. Time-lapse video of a ced-3(lf); isIs17[Ppie-1::GFP::pcn-1]; nIs861[Pegl-1::mCherry::PH]; pig-1(RNAi) embryo over an 80-minute period ending in ventral enclosure shows ABplpappap (circled at the beginning) completed the cell cycle and divided to generate daughters (circled at the end of video) that were not extruded. All cells express GFP–PCN-1 (green) and membranes of egl-1–expressing cells are labeled with mCherry (magenta). Time-lapse images used to generate this video were obtained using confocal microcopy. Video playback is at 600x real speed.

Video 6

A vehicle-treated MDCK monolayer extrudes a few cells. A time-lapse video of mammalian MDCK monolayer treated with vehicle control for 21.25 h obtained using phase contrast imaging shows that a few cells are extruded during this period. Extruded cells can be identified as bright, white, rounded spots rising from the epithelial plane. Video playback is at 7200x real speed. Scale bar, 100 μm.

Video 7

A HU-treated MDCK monolayer extrudes a large number of cells. A time-lapse video of mammalian MDCK monolayer exposed to HU for 21.25 h obtained using phase contrast imaging shows that many more cells are extruded during this period as a result of HU treatment. Extruded cells can be identified as bright, white, rounded spots rising from the epithelial plane. Video playback is at 7200x real speed. Scale bar, 100 μm.

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Dwivedi, V.K., Pardo-Pastor, C., Droste, R. et al. Replication stress promotes cell elimination by extrusion. Nature 593, 591–596 (2021). https://doi.org/10.1038/s41586-021-03526-y

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