Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia (original) (raw)

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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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.

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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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  2. Patrick D. Loftus
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  3. Masaaki Yoshigi
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  4. Hideo Otsuna
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  5. Chi-Bin Chien
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  6. Paul A. Morcos
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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 toJody Rosenblatt.

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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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Editorial Summary

Crowd control in epithelia

For an epithelial-cell layer to retain its structure and provide a protective barrier, it needs to maintain a balance between the number of cells dividing and the number dying. Buzz Baum and colleagues study this process in Drosophila tissues and demonstrate a direct link between physical forces in a tissue and the rates of cell loss. In regions of tissue that are overcrowded, some of the cells undergo a loss of cell-adhesive junctions and are squeezed out by neighbouring cells. This process of live-cell delamination buffers epithelial cells against variations in growth and contributes to normal tissue homeostasis. As a link between epithelial hyperplasia and cell invasion, it may have relevance to the early stages of cancer development. In a second paper, Jody Rosenblatt and colleagues study epithelial-cell monolayers and find that epithelia extrude live but not dying cells at sites of high strain. The extruded cells undergo cell death owing to loss of survival factors. Hence, extrusion could provide a tumour-suppressive mechanism that could be used to eliminate excess cells. In carcinomas with high levels of survival signalling pathways, extrusion may promote tumour-cell invasion.