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Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia

Nature volume 484, pages 546–549 (2012)Cite this article

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Abstract

For an epithelium to provide a protective barrier, it must maintain homeostatic cell numbers by matching the number of dividing cells with the number of dying cells. Although compensatory cell division can be triggered by dying cells1,2,3, it is unknown how cell death might relieve overcrowding due to proliferation. When we trigger apoptosis in epithelia, dying cells are extruded to preserve a functional barrier4. Extrusion occurs by cells destined to die signalling to surrounding epithelial cells to contract an actomyosin ring that squeezes the dying cell out4,5,6. However, it is not clear what drives cell death during normal homeostasis. Here we show in human, canine and zebrafish cells that overcrowding due to proliferation and migration induces extrusion of live cells to control epithelial cell numbers. Extrusion of live cells occurs at sites where the highest crowding occurs in vivo and can be induced by experimentally overcrowding monolayers in vitro. Like apoptotic cell extrusion, live cell extrusion resulting from overcrowding also requires sphingosine 1-phosphate signalling and Rho-kinase-dependent myosin contraction, but is distinguished by signalling through stretch-activated channels. Moreover, disruption of a stretch-activated channel, Piezo1, in zebrafish prevents extrusion and leads to the formation of epithelial cell masses. Our findings reveal that during homeostatic turnover, growth and division of epithelial cells on a confined substratum cause overcrowding that leads to their extrusion and consequent death owing to the loss of survival factors. These results suggest that live cell extrusion could be a tumour-suppressive mechanism that prevents the accumulation of excess epithelial cells.

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Figure 1: Both apoptotic and non-apoptotic epithelial cells extrude at locations of high crowding during homeostasis and development.
Figure 2: Characterization of experimental overcrowding.
Figure 3: Overcrowding MDCK monolayers promotes extrusion of non-apoptotic cells.
Figure 4: Disrupting the stretch-activated channel Piezo1 in vivo blocks live cell extrusion and causes epithelial mass formation.

References

  1. Fan, Y. & Bergmann, A. Apoptosis-induced compensatory proliferation. The Cell is dead. Long live the Cell! Trends Cell Biol 18, 467–473 (2008)

    Article  CAS  Google Scholar 

  2. Fan, Y. & Bergmann, A. Distinct mechanisms of apoptosis-induced compensatory proliferation in proliferating and differentiating tissues in the Drosophila eye. Dev. Cell 14, 399–410 (2008)

    Article  CAS  Google Scholar 

  3. Ryoo, H. D., Gorenc, T. & Steller, H. Apoptotic cells can induce compensatory cell proliferation through the JNK and the Wingless signaling pathways. Dev. Cell 7, 491–501 (2004)

    Article  CAS  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  Google Scholar 

  5. Gu, Y., Forostyan, T., Sabbadini, R. & Rosenblatt, J. Epithelial cell extrusion requires the sphingosine-1-phosphate receptor 2 pathway. J. Cell Biol. 193, 667–676 (2011)

    Article  CAS  Google Scholar 

  6. Slattum, G., McGee, K. M. & Rosenblatt, J. P115 RhoGEF and microtubules decide the direction apoptotic cells extrude from an epithelium. J. Cell Biol. 186, 693–702 (2009)

    Article  CAS  Google Scholar 

  7. Monks, J., Smith-Steinhart, C., Kruk, E. R., Fadok, V. A. & Henson, P. M. Epithelial cells remove apoptotic epithelial cells during post-lactation involution of the mouse mammary gland. Biol. Reprod. 78, 586–594 (2008)

    Article  CAS  Google Scholar 

  8. Riedl, J. et al. Lifeact: a versatile marker to visualize F-actin. Nature Methods 5, 605–607 (2008)

    Article  CAS  Google Scholar 

  9. Frisch, S. M. & Francis, H. Disruption of epithelial cell-matrix interactions induces apoptosis. J. Cell Biol. 124, 619–626 (1994)

    Article  CAS  Google Scholar 

  10. Gilmore, A. P. Anoikis. Cell Death Differ. 12 (suppl. 2), 1473–1477 (2005)

    Article  CAS  Google Scholar 

  11. Reddig, P. J. & Juliano, R. L. Clinging to life: cell to matrix adhesion and cell survival. Cancer Metastasis Rev. 24, 425–439 (2005)

    Article  Google Scholar 

  12. Andrade, D. & Rosenblatt, J. Apoptotic regulation of epithelial cellular extrusion. Apoptosis 16, 491–501 (2011)

    Article  CAS  Google Scholar 

  13. Tournier, C. et al. Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway. Science 288, 870–874 (2000)

    Article  ADS  CAS  Google Scholar 

  14. Coste, B. et al. Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science 330, 55–60 (2010)

    Article  ADS  CAS  Google Scholar 

  15. Olsen, S. M., Stover, J. D. & Nagatomi, J. Examining the role of mechanosensitive ion channels in pressure mechanotransduction in rat bladder urothelial cells. Ann. Biomed. Eng. 39, 688–697 (2011)

    Article  Google Scholar 

  16. Bhattacharya, M. R. et al. Radial stretch reveals distinct populations of mechanosensitive mammalian somatosensory neurons. Proc. Natl Acad. Sci. USA 105, 20015–20020 (2008)

    Article  ADS  CAS  Google Scholar 

  17. Yang, X. C. & Sachs, F. Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions. Science 243, 1068–1071 (1989)

    Article  ADS  CAS  Google Scholar 

  18. Haas, P. & Gilmour, D. Chemokine signaling mediates self-organizing tissue migration in the zebrafish lateral line. Dev. Cell 10, 673–680 (2006)

    Article  CAS  Google Scholar 

  19. Tallafuss, A. et al. Turning gene function ON and OFF using sense- and antisense-photo-morpholinos in zebrafish. Development (in the press)

  20. Holgate, S. T. The airway epithelium is central to the pathogenesis of asthma. Allergol. Int. 57, 1–10 (2008)

    Article  CAS  Google Scholar 

  21. Knight, D. A. & Holgate, S. T. The airway epithelium: structural and functional properties in health and disease. Respirology 8, 432–446 (2003)

    Article  Google Scholar 

  22. Swindle, E. J., Collins, J. E. & Davies, D. E. Breakdown in epithelial barrier function in patients with asthma: identification of novel therapeutic approaches. J. Allergy Clin. Immunol. 124, 23–34,–quiz 35–36 (2009)

    Article  Google Scholar 

  23. Shraiman, B. I. Mechanical feedback as a possible regulator of tissue growth. Proc. Natl Acad. Sci. USA 102, 3318–3323 (2005)

    Article  ADS  CAS  Google Scholar 

  24. Yoshigi, M., Clark, E. B. & Yost, H. J. Quantification of stretch-induced cytoskeletal remodeling in vascular endothelial cells by image processing. Cytometry A 55A, 109–118 (2003)

    Article  Google Scholar 

  25. Yoshigi, M., Hoffman, L. M., Jensen, C. C., Yost, H. J. & Beckerle, M. C. Mechanical force mobilizes zyxin from focal adhesions to actin filaments and regulates cytoskeletal reinforcement. J. Cell Biol. 171, 209–215 (2005)

    Article  CAS  Google Scholar 

  26. Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. & Schilling, T. F. Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253–310 (1995)

    Article  CAS  Google Scholar 

  27. Davison, J. M. et al. Transactivation from Gal4-VP16 transgenic insertions for tissue-specific cell labelling and ablation in zebrafish. Dev. Biol. 304, 811–824 (2007)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank B. Welm, M. Redd and K. Ullman for valuable input on our manuscript and T. Marshall and D. Andrade for Lifeact- and BCL2-expressing MDCK lines. The custom-designed cell culture device was made with the support of National Institute of Biomedical Imaging and Bioengineering Grant EB-4443 to M.Y. (patent pending). This work was supported by a NIH-NIGMS NIH Director’s New Innovator Award 1 DP2 OD002056-01 and a Laura and Arthur Colwin Endowed Marine Biology Laboratories Summer Research Fellowship Fund to J.R., and a P30 CA042014 awarded to The Huntsman Cancer Institute for core facilities. An NIH Multidisciplinary Cancer Training Program Grant 5T32 CA03247-8 and American Cancer Society Salt Lake City Postdoctoral Fellowship (120464-PF-11-095-01 CSM) supported G.T.E., the University of Utah Undergraduate Research Opportunities Program Parent Fund Assistantship supported P.D.L., and an NIH R01 Grant MH092256 supported H.O. and C.B.C.

Author information

Author notes

  1. George T. Eisenhoffer and Patrick D. Loftus: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, 2000 Circle of Hope Drive, Salt Lake City, Utah 84112, USA,

    George T. Eisenhoffer, Patrick D. Loftus & Jody Rosenblatt

  2. Department of Pediatrics, University of Utah, 295 Chipeta Way RM 2S010, Salt Lake City, Utah 84108, USA,

    Masaaki Yoshigi

  3. Department of Neurobiology and Anatomy, University of Utah, 401 MREB, 20N 1900E, Salt Lake City, Utah 84132, USA,

    Hideo Otsuna & Chi-Bin Chien

  4. Gene Tools, LLC, 1001 Summerton Way, Philmath, Oregon 97370, USA ,

    Paul A. Morcos

Authors
  1. George T. Eisenhoffer
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  7. Jody Rosenblatt
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Contributions

G.T.E. and P.D.L. both contributed to the study design and performed all the experiments. M.Y. designed the stretching apparatus and consulted in the study design. J.R. found preliminary results of live cell extrusion in vivo and contributed to the design of the study. G.T.E., J.R. and P.D.L. analysed the data and wrote the paper. P.A.M. designed the photo-cleavable morpholinos, and H.O. and C.-B.C. provided zebrafish with epidermal Kaede expression. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Jody Rosenblatt.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-11. (PDF 1169 kb)

Supplementary Movie 1

This movie shows MDCK cells within a monolayer expressing Lifeact-GFP at 1 hour after overcrowding and illustrates extrusions occurring within the monolayer due to contracting actin rings. Images were captured every 2 minutes over the course of 30 minutes. (MOV 1349 kb)

Supplementary Movie 2

This movie shows a lateral view of a developing Tg(cldnb:lynGFP) zebrafish embryo at 51-54 hours post-fertilization, every 2 minutes over the course of 3 hours. Epithelial cells migrate towards the edge of the tail, where they eventually extrude from the tissue. (MOV 3204 kb)

Supplementary Movie 3

This movie shows a rotating confocal projection of a 10 mM Gd3+-treated zebrafish larvae immunostained with actin (red) and DNA (turquoise) (shown in Figure 4g) and highlights the cell masses protruding from the epidermis on either side of the fin. (MOV 1860 kb)

Supplementary Movie 4

This movie shows a lateral view, anterior to the left, of 60 hpf Tg(cldnb:lynGFP) zebrafish treated with 10 mM Gd3+ shows the epidermal masses arise from failed extrusion at the edge of the tail fin, where two areas of tissue converge. Cell migration occurs every 2 minutes over the course of 3 hours. (MOV 1160 kb)

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Eisenhoffer, G., Loftus, P., Yoshigi, M. et al. Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia. Nature 484, 546–549 (2012). https://doi.org/10.1038/nature10999

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