Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Replication stress promotes cell elimination by extrusion

Nature volume 593, pages 591–596 (2021)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

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.

References

  1. Ohsawa, S., Vaughen, J. & Igaki, T. Cell extrusion: a stress-responsive force for good or evil in epithelial homeostasis. Dev. Cell 44, 284–296 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. De Goeij, J. M. et al. Cell kinetics of the marine sponge Halisarca caerulea reveal rapid cell turnover and shedding. J. Exp. Biol. 212, 3892–3900 (2009).

    Article  PubMed  Google Scholar 

  3. Denning, D. P., Hatch, V. & Horvitz, H. R. Programmed elimination of cells by caspase-independent cell extrusion in C. elegans. Nature 488, 226–230 (2012).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  4. Rosenblatt, J., Raff, M. C. & Cramer, L. P. An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism. Curr. Biol. 11, 1847–1857 (2001).

    Article  CAS  PubMed  Google Scholar 

  5. Gudipaty, S. A. & Rosenblatt, J. Epithelial cell extrusion: pathways and pathologies. Semin. Cell Dev. Biol. 67, 132–140 (2017).

    Article  CAS  PubMed  Google Scholar 

  6. Timson, J. Hydroxyurea. Mutat. Res. 32, 115–132 (1975).

    Article  CAS  PubMed  Google Scholar 

  7. Koç, A., Wheeler, L. J., Mathews, C. K. & Merrill, G. F. Hydroxyurea arrests DNA replication by a mechanism that preserves basal dNTP pools. J. Biol. Chem. 279, 223–230 (2004).

    Article  PubMed  Google Scholar 

  8. Takeda, D. Y. & Dutta, A. DNA replication and progression through S phase. Oncogene 24, 2827–2843 (2005).

    Article  CAS  PubMed  Google Scholar 

  9. Fay, D. S. & Han, M. Mutations in cye-1, a Caenorhabditis elegans cyclin E homolog, reveal coordination between cell-cycle control and vulval development. Development 127, 4049–4060 (2000).

    Article  CAS  PubMed  Google Scholar 

  10. van Rijnberk, L. M., van der Horst, S. E. M., van den Heuvel, S. & Ruijtenberg, S. A dual transcriptional reporter and CDK-activity sensor marks cell cycle entry and progression in C. elegans. PLoS One 12, e0171600 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Brauchle, M., Baumer, K. & Gönczy, P. Differential activation of the DNA replication checkpoint contributes to asynchrony of cell division in C. elegans embryos. Curr. Biol. 13, 819–827 (2003).

    Article  CAS  PubMed  Google Scholar 

  12. Zerjatke, T. et al. Quantitative cell cycle analysis based on an endogenous all-in-one reporter for cell tracking and classification. Cell Rep. 19, 1953–1966 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Teuliere, J. & Garriga, G. Size matters: how C. elegans asymmetric divisions regulate apoptosis. Results Probl. Cell Differ. 61, 141–163 (2017).

    Article  CAS  PubMed  Google Scholar 

  14. Stergiou, L., Eberhard, R., Doukoumetzidis, K. & Hengartner, M. O. NER and HR pathways act sequentially to promote UV-C-induced germ cell apoptosis in Caenorhabditis elegans. Cell Death Differ. 18, 897–906 (2011).

    Article  CAS  PubMed  Google Scholar 

  15. Dinant, C. et al. Activation of multiple DNA repair pathways by sub-nuclear damage induction methods. J. Cell Sci. 120, 2731–2740 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Wu, Y. C., Stanfield, G. M. & Horvitz, H. R. NUC-1, a Caenorhabditis elegans DNase II homolog, functions in an intermediate step of DNA degradation during apoptosis. Genes Dev. 14, 536–548 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Toledo, L. I. et al. ATR prohibits replication catastrophe by preventing global exhaustion of RPA. Cell 155, 1088–1103 (2013).

    Article  CAS  PubMed  Google Scholar 

  18. Stevens, H., Williams, A. B. & Michael, W. M. Cell-type specific responses to DNA replication stress in early C. elegans embryos. PLoS One 11, e0164601 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Ossareh-Nazari, B., Katsiarimpa, A., Merlet, J. & Pintard, L. RNAi-based suppressor screens reveal genetic interactions between the CRL2LRR-1 E3-ligase and the DNA replication machinery in Caenorhabditis elegans. G3 (Bethesda) 6, 3431–3442 (2016).

    Article  CAS  Google Scholar 

  20. Sonneville, R. et al. CUL-2LRR-1 and UBXN-3 drive replisome disassembly during DNA replication termination and mitosis. Nat. Cell Biol. 19, 468–479 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Dewar, J. M., Low, E., Mann, M., Räschle, M. & Walter, J. C. CRL2Lrr1 promotes unloading of the vertebrate replisome from chromatin during replication termination. Genes Dev. 31, 275–290 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Merlet, J. et al. The CRL2LRR-1 ubiquitin ligase regulates cell cycle progression during C. elegans development. Development 137, 3857–3866 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Meek, D. W. Tumour suppression by p53: a role for the DNA damage response? Nat. Rev. Cancer 9, 714–723 (2009).

    Article  CAS  PubMed  Google Scholar 

  24. Jones, M. C., Askari, J. A., Humphries, J. D. & Humphries, M. J. Cell adhesion is regulated by CDK1 during the cell cycle. J. Cell Biol. 217, 3203–3218 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Grieve, A. G. & Rabouille, C. Extracellular cleavage of E-cadherin promotes epithelial cell extrusion. J. Cell Sci. 127, 3331–3346 (2014).

    CAS  PubMed  Google Scholar 

  26. Wernike, D., Chen, Y., Mastronardi, K., Makil, N. & Piekny, A. Mechanical forces drive neuroblast morphogenesis and are required for epidermal closure. Dev. Biol. 412, 261–277 (2016).

    Article  CAS  PubMed  Google Scholar 

  27. Eisenhoffer, G. T. et al. Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia. Nature 484, 546–549 (2012).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  28. Gaillard, H., García-Muse, T. & Aguilera, A. Replication stress and cancer. Nat. Rev. Cancer 15, 276–289 (2015).

    Article  CAS  PubMed  Google Scholar 

  29. Olive, K. P. et al. Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome. Cell 119, 847–860 (2004).

    Article  CAS  PubMed  Google Scholar 

  30. Lang, G. A. et al. Gain of function of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndrome. Cell 119, 861–872 (2004).

    Article  CAS  PubMed  Google Scholar 

  31. Singh, S. et al. Mutant p53 establishes targetable tumor dependency by promoting unscheduled replication. J. Clin. Invest. 127, 1839–1855 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Yeo, C. Q. X. et al. p53 maintains genomic stability by preventing interference between transcription and replication. Cell Rep. 15, 132–146 (2016).

    Article  CAS  PubMed  Google Scholar 

  33. Karagiannis, G. S. et al. Neoadjuvant chemotherapy induces breast cancer metastasis through a TMEM-mediated mechanism. Sci. Transl. Med. 9, eaan0026 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Brodigan, T. M., Liu, Ji., Park, M., Kipreos, E. T. & Krause, M. Cyclin E expression during development in Caenorhabditis elegans. Dev. Biol. 254, 102–115 (2003).

    Article  CAS  PubMed  Google Scholar 

  36. Wu, Y. C. & Horvitz, H. R. C. elegans phagocytosis and cell-migration protein CED-5 is similar to human DOCK180. Nature 392, 501–504 (1998).

    Article  ADS  CAS  PubMed  Google Scholar 

  37. Hsieh, J. et al. The RING finger/B-box factor TAM-1 and a retinoblastoma-like protein LIN-35 modulate context-dependent gene silencing in Caenorhabditis elegans. Genes Dev. 13, 2958–2970 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Grishok, A., Sinskey, J. L. & Sharp, P. A. Transcriptional silencing of a transgene by RNAi in the soma of C. elegans. Genes Dev. 19, 683–696 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Fischer, S. E. J. et al. Multiple small RNA pathways regulate the silencing of repeated and foreign genes in C. elegans. Genes Dev. 27, 2678–2695 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Boeck, M. E. et al. Specific roles for the GATA transcription factors end-1 and end-3 during C. elegans E-lineage development. Dev. Biol. 358, 345–355 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Mello, C. C., Kramer, J. M., Stinchcomb, D. & Ambros, V. Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, 3959–3970 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Rual, J.-F. et al. Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library. Genome Res. 14 (10B), 2162–2168 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Fraser, A. G. et al. Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408, 325–330 (2000).

    Article  ADS  CAS  PubMed  Google Scholar 

  44. Kamath, R. S. et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231–237 (2003).

    Article  ADS  CAS  PubMed  Google Scholar 

  45. Sulston, J. E., Schierenberg, E., White, J. G. & Thomson, J. N. The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev. Biol. 100, 64–119 (1983).

    Article  CAS  PubMed  Google Scholar 

  46. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  Google Scholar 

  47. Hansson, G. C., Simons, K. & van Meer, G. Two strains of the Madin-Darby canine kidney (MDCK) cell line have distinct glycosphingolipid compositions. EMBO J. 5, 483–489 (1986).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Streichan, S. J., Hoerner, C. R., Schneidt, T., Holzer, D. & Hufnagel, L. Spatial constraints control cell proliferation in tissues. Proc. Natl Acad. Sci. USA 111, 5586–5591 (2014).

    Article  ADS  CAS  PubMed  Google Scholar 

  49. Sakaue-Sawano, A. et al. Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 132, 487–498 (2008).

    Article  CAS  PubMed  Google Scholar 

  50. McQuin, C. et al. CellProfiler 3.0: next-generation image processing for biology. PLoS Biol. 16, e2005970 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  51. Kipreos, E. T., Gohel, S. P. & Hedgecock, E. M. The C. elegans F-box/WD-repeat protein LIN-23 functions to limit cell division during development. Development 127, 5071–5082 (2000).

    Article  CAS  PubMed  Google Scholar 

Download references

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

Authors and Affiliations

  1. Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

    Vivek K. Dwivedi, Rita Droste, Ji Na Kong, Nolan Tucker, Daniel P. Denning & H. Robert Horvitz

  2. Randall Centre for Cell & Molecular Biophysics, King’s College London, London, UK

    Carlos Pardo-Pastor & Jody Rosenblatt

  3. Novartis Institutes for BioMedical Research, Cambridge, MA, USA

    Daniel P. Denning

Authors
  1. Vivek K. Dwivedi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Carlos Pardo-Pastor
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rita Droste
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ji Na Kong
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Nolan Tucker
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Daniel P. Denning
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jody Rosenblatt
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. H. Robert Horvitz
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

a–i, Confocal micrographs showing the presence of the cye-1(+)-rescuing transgene in the excretory cell but not in ABplpappap (a–h) 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. f–h, 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. i–m, 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 (a–d); ABplpappap or its daughters (e–h); magnified view of the region indicated, which includes ABplpappap (†) and ABplpappaa (*) (i–m). Scale bars, 10 μm.

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

a–d, 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. g–j, 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.

a–d, 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
Full size table
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
Full size table

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.

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-021-03526-y

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer