Mechanical stretch triggers rapid epithelial cell division through Piezo1

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Abstract

Despite acting as a barrier for the organs they encase, epithelial cells turn over at some of the fastest rates in the body. However, epithelial cell division must be tightly linked to cell death to preserve barrier function and prevent tumour formation. How does the number of dying cells match those dividing to maintain constant numbers? When epithelial cells become too crowded, they activate the stretch-activated channel Piezo1 to trigger extrusion of cells that later die1. However, it is unclear how epithelial cell division is controlled to balance cell death at the steady state. Here we show that mammalian epithelial cell division occurs in regions of low cell density where cells are stretched. By experimentally stretching epithelia, we find that mechanical stretch itself rapidly stimulates cell division through activation of the Piezo1 channel. To stimulate cell division, stretch triggers cells that are paused in early G2 phase to activate calcium-dependent phosphorylation of ERK1/2, thereby activating the cyclin B transcription that is necessary to drive cells into mitosis. Although both epithelial cell division and cell extrusion require Piezo1 at the steady state, the type of mechanical force controls the outcome: stretch induces cell division, whereas crowding induces extrusion. How Piezo1-dependent calcium transients activate two opposing processes may depend on where and how Piezo1 is activated, as it accumulates in different subcellular sites with increasing cell density. In sparse epithelial regions in which cells divide, Piezo1 localizes to the plasma membrane and cytoplasm, whereas in dense regions in which cells extrude, it forms large cytoplasmic aggregates. Because Piezo1 senses both mechanical crowding and stretch, it may act as a homeostatic sensor to control epithelial cell numbers, triggering extrusion and apoptosis in crowded regions and cell division in sparse regions.

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Figure 1: Mechanical stretch induces epithelial monolayers to rapidly divide.
Figure 2: Stretch-induced mitosis requires the stretch-activated channel Piezo1.
Figure 3: Stretch-activation of Piezo1 triggers calcium and ERK1/2-dependent cyclin B transcription.
Figure 4: Piezo1 controls proliferation in response to stretch and extrusion and death in response to crowding.

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Acknowledgements

We thank D. Wright for design consultation and printing our new stretch device prototypes, M. Yoshigi for the previous stretch device, L. Zinn-Bjorkman for video analysis, A. Patapoutian for a GFP–Piezo1 construct, D. Morgan and B. Edgar for cell cycle experimental advice and B. Edgar for comments on the manuscript. A National Institute of Health Director’s New Innovator Award 1DP2OD002056-01, R01GM102169, and University of Utah Funding Incentive Seed Grant to J.R. and P30 CA042014 awarded to Huntsman Cancer Institute core facilities supported this work. We thank the Fluorescence Microscopy and Mutation Generation and Detection Cores in the Health Sciences Cores at the University of Utah. An NCRR Shared Equipment Grant 1S10RR024761-01 paid for microscopy equipment.

Author information

J.R. designed all of the experiments, interpreted, and analysed all data, and wrote the manuscript. J.R. and J.L. designed the new stretch device. J.L. and S.A.G. performed most stretch and wound healing experiments and FACS analysis throughout the paper. P.D.L. performed the first experiments showing stretch induces cell division. K.E. performed the immunoblots and the EdU experiments. V.K. did calcium imaging. C.F.D. produced F0 CRISPR mosaic knockout zebrafish, M.J.R. injected morpholinos into zebrafish embryos and helped with live imaging. J.R. performed the experiments using zebrafish and requirement for Piezo1 at steady state and co-quantified most of the experiments. All authors edited the manuscript.

Correspondence to J. Rosenblatt.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Profile of epithelial cell proliferation and cell density over time.

a, Daily proliferation rates of MDCK cells, as measured by H3P immunostaining, in which the asterisk indicates when confluence is reached (n = 3). Cell division slows but does not stop at day 5. b, Cell density plateaus by 5 days of growth when cells reach around 11 per 1,000 μm2 (n = 3). All values are means of 10 fields of view from 3 independent experiments; error bars denote s.e.m. c, Cell cycle profiles by FACS at days 1 and 5 after plating at high density.

Extended Data Figure 2 Epithelial cells divide in sparsest regions of epithelia.

ac, Density of cells in dividing versus non-dividing regions of epithelia, as measured by the cell length along the longest axis of the cell in four videos (except fixed colon sections) from human colon tissue (n = 8; a), developing zebrafish epidermis (n = 50; b), and MDCK cells in culture (n = 50; c). ****P < 0.0001, unpaired t-test. Error bars denote s.e.m.

Extended Data Figure 3 Stretching steady-state monolayers.

a, Left, new printable device used for stretching cells uniaxially on flexcell plates, unassembled (top) and assembled in the stretched state (bottom). Right, schematic of uniaxial stretch on an epithelium in the unassembled (top) and assembled (bottom) states (photo credit: J.R.). b, Immunostained MDCK monolayers before (top) and after (bottom) stretching. Images are representative of more than 200 captured images each. Scale bar, 10 μm.

Extended Data Figure 4 Piezo1 morphants have reduced cell division in zebrafish epidermis at the steady state.

a, Cell division in zebrafish reaches a low steady state at days 4–5 after fertilization. n = 50 fish each day; error bars denote s.e.m. b, Photo-activation of zebrafish injected with a Piezo1 translation-blocking morpholino (MO) results in knockdown of Piezo1 protein, as shown by an immunoblot. See Supplementary Fig. 1 for full blots. c, d, Zebrafish Piezo1 morphants have notably reduced epidermal mitoses at 5 days post-fertilization when cells homeostatic growth rate. Values in c are means from 3 separate experiments, error bars denote s.e.m. ****P < 0.005, unpaired t-test. Each micrograph in d is representative of approximately 75 samples. Scale bars, 100 μm. Source data

Extended Data Figure 5 A single calcium spark occurs around 1 h before cells divide.

a, Blocking stretch-induced proliferation with gadolinium (Gd3+) at 2 h after stretch does not affect the percentages of cells in the S phase, as measured by EdU incorporation. Data are mean and s.e.m. from 6 independent experiments. P > 0.05, t-test (compared to non-stretched control). b, Knockdown (KD) of Piezo1 with Gd3+ or inhibition of transcription with α-amanitin blocks stretch-induced cytoplasmic cyclin B accumulation and mitosis (H3P). Micrographs are representative of more than 100 samples, except for α-amanitin (40 samples). Scale bar, 10 μm. c, Quantification of time from calcium spark to cell rounding, measured from 10 mitotic events in 8 videos. Error bars denote s.e.m. d, Sample graph measuring the time (min) from calcium spark (arrow) to cell rounding (arrowhead) in MDCK cells expressing the calcium indicator CMV-R-GECO1 using Nikon Elements Software.

Extended Data Figure 6 Models for how Piezo1 controls cell division in response to stretch, and cell extrusion in response to crowding.

a, Theoretical graph of density-dependent Piezo1 function for cell division and cell death. Epithelia trend to a steady-state density, X. If density is reduced, stretching causes Piezo1 to activate cell division; if density increases, crowding causes Piezo1 to activate cells to extrude and die. b, Schematic showing how Piezo1 (green) localizes to the plasma membrane in sub-regions of the epithelia in which cells are sparser, and divides and accumulates into cytoplasmic aggregates in sub-regions in which cells are older, crowded, and prone to extrude.

Extended Data Figure 7 Stretch causes cells at the steady state to enter mitosis.

a, Characterization of the Piezo1 antibody using a Piezo1 shRNA construct tagged with mCherry indicates that cells expressing the mCherry shRNA construct (red) lack Piezo1 (green). Micrographs are representative of approximately 50 samples. Scale bar, 10 μm. b, High-density MDCK monolayers (more than 500 cells per 40× field) in which the intrinsic proliferation rate is low (less than 50 H3P-positive cells per 10,000 cells) proliferate in response to mechanical stretch (left) or wounding (right). Error bars denote s.e.m. ***P = 0.0007, unpaired t-test. c, Low-density MDCK monolayers just reaching confluence with a higher intrinsic rate of proliferation (more than 50 H3P-positive cells per 10,000 cells) do not significantly proliferate in response to stretch or wounding. P > 0.05, t-test. n = 12 experiments for each case.

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Supplementary information

Supplementary Information

This file contains the uncropped scans with size marker indications. (PDF 838 kb)

Cells of MDCK monolayer dividing just after reaching confluence

Phase time-lapse video of MDCK monolayer growing for three days that have just reached confluence but are still sparse and rapidly dividing. Frames are taken every 10 minutes for 18 hours. (MOV 6277 kb)

Cells of MDCK monolayer divide more slowly once they become denser

Phase time-lapse video of MDCK monolayer grown for four days (two days after reaching confluence) divide less frequently when more densely populated. Note cells are dividing in sparsest regions and extruding in most crowded regions. Frames are taken every 10 minutes for 18 hours. (MOV 7290 kb)

Cells dividing within zebrafish larval epidermis

Time-lapse video from a spinning disc confocal microscope of a KRT4:GFP zebrafish larva that expresses GFP in the epidermis. Cell division occurs in sparsest regions. One sample used for quantification in Extended Data Figure 2. (MOV 4040 kb)

Wound closure and cell divisions in a wild type MDCK monolayer

Phase time-lapse video of an MDCK monolayer grown for four days and scratched with a pipet tip. Notice that most of the cell divisions take place after the wound has completely closed. One of ten videos quantified in Figure 1 A. Frames are taken every 10 minutes for 18 hours. (MOV 5312 kb)

Wound closure and cell divisions in a Piezo1 knockdown MDCK monolayer

Phase time-lapse video of an MDCK monolayer grown for four days and scratched with a pipet tip. Notice that few cell divisions occur and cells continue to migrate after wound closure. One of 10 videos quantified in Figure 2. Frames are taken every 10 minutes for 24 hours. (MOV 2391 kb)

Wound closure and cell divisions in a wild type MDCK monolayer

Phase time-lapse video of an MDCK monolayer grown for four days and scratched with a pipet tip. Note that most of the cells divide after the wound has completely closed and start to extrude when they become crowded later. Frames are taken every 10 minutes for 24 hours. (MOV 3425 kb)

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Gudipaty, S., Lindblom, J., Loftus, P. et al. Mechanical stretch triggers rapid epithelial cell division through Piezo1. Nature 543, 118–121 (2017) doi:10.1038/nature21407

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