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

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.

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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.

Author information

Authors and Affiliations

Authors

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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