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Transepithelial potential difference governs epithelial homeostasis by electromechanics

Nature Physics volume 18, pages 1122–1128 (2022)Cite this article

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Abstract

Studies of electric effects in biological systems, from the work on action potential to studies on limb regeneration or wound healing, commonly focus on transitory behaviour and not on addressing the question of homeostasis. Here we use a microfluidic device to study how the homeostasis of confluent epithelial tissues is modified when a transepithelial potential difference that is different from the natural one is imposed on an epithelial layer. When the field direction matches the natural one, we can restore perfect confluence in an epithelial layer turned defective either by E-cadherin knockout or by weakening the cell–substrate adhesion; additionally, the tissue pushes on the substrate with kilopascal stress, inducing active-cell response such as death and differentiation. When the field is opposite, the tissue pulls with similar strengths, whereas homeostasis is destroyed by the perturbation of junctional actin and cell shapes, increased cell division rate and formation of mounds. Most of these observations can be quantitatively explained by an electrohydrodynamic theory involving local cytoplasmic electro-osmotic flows. We expect this work to motivate further studies on the long-time effects of electromechanical pathways with important tissue engineering applications.

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Fig. 1: AtB field induces disruption to cell shape and cell–cell junctions, whereas BtA field maintains or reinforces normal epithelial characteristics.
Fig. 2: E fields exert electromechanical stress that govern cell shape.
Fig. 3: Only AtB fields induce collective live-cell extrusions and 3D mounds in MDCK and N/TERT-1.
Fig. 4: Only BtA fields induce more cell death or differentiation and rescue intrinsically heterogeneous/non-intact tissue.
Fig. 5: Schematic showing the summary of different field conditions.

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Source data are provided with this paper.

Code availability

Custom codes for image analysis are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank M. Bornens, F. Julicher and C. Duclut for scientific discussions; C.J. Chan for critical reading of the manuscript; X. Yong for help with the experiments; D. Bhattacharjee for help with the COMSOL simulations; K. Fong-Ngern for help with the Transwell measurements; K.S. Robinson, B. Ladoux and W.J. Nelson for their cell lines; and I. Yow for help with the FUCCI lentiviral transfection. We also thank the MBI’s Wetlab, Microscopy and Microfabrication cores for support. T.B.S. acknowledges support from the Lee Kuan Yew Postdoctoral fellowship and Singapore Ministry of Education Tier 1 Academic Research Fund (grant R-397-000-320-114), and Westlake Education Foundation. X.G. acknowledges the support of the ARC Centre for Personalised Therapeutics Technologies (Australian Research Council, grant IC170100016). C.T.L. is supported by the National Research Foundation, Singapore, under the Mechanobiology Institute at the National University of Singapore, the Human Frontier Science Program (grant LIP000635/2018), and the Institute for Health Innovation and Technology at NUS.

Author information

Author notes

  1. These authors contributed equally: Thuan Beng Saw, Xumei Gao.

Authors and Affiliations

  1. Department of Biomedical Engineering, National University of Singapore (NUS), Singapore, Singapore

    Thuan Beng Saw, Muchun Li & Chwee Teck Lim

  2. Mechanobiology Institute, NUS, Singapore, Singapore

    Thuan Beng Saw, Anh Phuong Le, Supatra Marsh, Jacques Prost & Chwee Teck Lim

  3. ARC Centre for Personalised Therapeutics Technologies, University of Melbourne, Melbourne, Victoria, Australia

    Xumei Gao & Jianan He

  4. Institute of Physics, Academia Sinica, Taipei, Taiwan

    Keng-hui Lin

  5. School of Biological Sciences, Nanyang Technological University, Singapore, Singapore

    Alexander Ludwig

  6. NTU Institute of Structural Biology, Nanyang Technological University, Singapore, Singapore

    Alexander Ludwig

  7. Laboratoire Physico-chimie Curie, Institut Curie, PSL Research University–Sorbonne Universités, UPMC–CNRS, Paris, France

    Jacques Prost

  8. Institute for Health Innovation and Technology (iHealthtech), NUS, MD6, Singapore, Singapore

    Chwee Teck Lim

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Contributions

T.B.S. and J.P. conceived the project. T.B.S., X.G. and J.P. designed the research. T.B.S., X.G. and M.L. performed the experiments. T.B.S. and J.P. performed the theoretical calculations and numerical fitting. T.B.S., X.G., J.H., A.P.L., S.M., K.L. and A.L. performed the image analysis or contributed new reagents/cells and computational tools. T.B.S., X.G., A.L., J.P. and C.T.L. provided guidance and input. T.B.S., X.G. and J.P. wrote the manuscript/prepared the figures. T.B.S., J.P. and C.T.L. supervised the project. All the authors read the manuscript and commented on it.

Corresponding authors

Correspondence to Thuan Beng Saw, Jacques Prost or Chwee Teck Lim.

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Extended data

Extended Data Fig. 1 TEPD and TEER of MDCK.

a,b MDCK transepithelial potential difference (TEPD) and electrical resistance (TEER) as a function of days cultured on transwell systems. Cells were seeded on day 0 and became confluent on day 2. Each experiment was followed till day 5 or 7. Black lines are mean values, scatter points of different colours indicate independent experiments. Day 1 to 5: n = 4 to 6 independent experiments in 3 biological replicates; day 6 and 7: n = 2 independent experiments in 1 biological replicate.

Source data

Extended Data Fig. 2 Electric assay and microfluidic device for cell experiments.

a, 3D rendering of the whole electric assay. Picture shows four independent electric assays where the microfluidic chips are sandwiched between the cartridges and pressure-sealed with O-rings to create isolated compartments (see Methods for details). The cartridges are connected through tubes to electrodes and DC source meters for the application of external E-fields. Experiments were done on a microscope with environmental chamber maintaining 37 °C and 5% CO2 for imaging. b, The microfluidic chip is multi-layered, made from glass (cyan), 1st layer of NOA73 UV curable material with channels (red), and 2nd NOA73 layer with opening of slit geometry where the E-field emerges (blue). The chip is embedded with polyacrylamide gel (shown as chartreuse on the top of the 2nd chip layer, green within the 2nd layer, and orange within the 1st layer), coated with ECM proteins as the cell substrate. Top-down view (top, left column), layered schematic (bottom, left column), cross-section and side-view (right column). Black dotted line is cut-line for cross-section view.

Extended Data Fig. 3 Different cellular parameters under control and field conditions.

a, left, Phase-contrast, top-down view of monolayer at early and late phase. Early, cell density: mean ± s.t.d. = 18±3 cells/(100 μm)2; Late, cell density: mean ± s.t.d. = 26±4 cells/(100 μm)2. a, right, Quantification of junctional intensity relative to that of the cell body at the two phases, line is mean. Both categories: n = 60 junctions (scatter points), 6 independent field-of-views in 2 independent experiments. Two-tailed, two-sample t-test. **p = 0.01. b, left, Side view of GFP-actin cells showing typical apical surface angle at early and late phase. Early, cell density: mean ± s.t.d. = 25±3cells/(100 μm)2; Late, cell density: mean ± s.t.d. = 29±2cells/(100 μm)2. Yellow line denotes apical surface angle with respect to basal plane. Positive angle denotes convex cell shape as shown in image. b, right, Quantification of angle at the two phases, line is mean. Both categories: n = 13 cells (scatter points), 2 independent field-of-views in 2 independent experiments. Two-tailed, two-sample t-test. **p = 0.002. c, Top-down view, confocal images of GFP-actin MDCK monolayers under control (no-field), AtB and BtA field conditions. d, Quantification of corresponding relative junctional actin intensities in (c). Arrowheads in (c) point to junctional actin. (d) is average ± s.e.m. Ctrl: n = 170 junctions from 3 independent experiments in 2 biological replicates. AtB: n = 232 junctions from 3 independent experiments in 3 biological replicates. BtA: n = 199 junctions from 3 independent experiments in 3 biological replicates. Two-tailed, two-sample t-test between AtB-BtA, AtB-Ctrl. ***p < 0.001 comparing midpoints of graphs. n.s. not significant. e, Side-view, confocal images of GFP-actin cells under the three field conditions. f, Normalized junction intensity of phase contrast imaging as function of time for different field conditions. Line and shaded regions are mean ± s.e.m. Broken part of lines are without data. Each time point has between 50–93 individual junctions. Ctrl: n = 3 independent experiments in 2 biological replicates. AtB/BtA: n = 3 independent experiments in 3 biological replicates. Two-tailed, t-test against normal distribution of mean = 1 for all AtB time points. All time points p « 0.001. g,h, In-plane cell body aspect ratio and cell fluctuation speeds at different field conditions measured ~6 h after the start of experiments, line is mean. For (g) - Ctrl: n = 76 cells (scatter points) from 3 independent experiments in 2 biological replicates. AtB: n = 80 cells from 3 independent experiments in 3 biological replicates. BtA: n = 75 cells from 3 independent experiments in 3 biological replicates. Two-tailed, two-sample t-test, ***p < 0.0001 (AtB-BtA, AtB-Ctrl), *p = 0.04 (BtA-Ctrl). For (h) - Ctrl: n = 12 independent field-of-views, from 3 independent experiments in 2 biological replicates. AtB: n = 6 independent field-of-views, from 3 independent experiments in 3 biological replicates. BtA: n = 5 independent field-of-views, from 3 independent experiments in 3 biological replicates. Two-tailed, two-sample t-test, ***p = 0.0002 (AtB-BtA), ***p = 0.0006 (BtA-Ctrl), *p = 0.05 (AtB-Ctrl). All scale bars, 10 μm.

Source data

Extended Data Fig. 4 Stress fibers and deformation of single cells and small cell patches do not correlate with field conditions.

left, Typical image of stress fibers at the basal side of confluent cells. right, Quantification of “Coherency” and “Strength” parameters for stress fiber conditions of each cell, line is mean, scatter points are different cells. Two-tailed, two-sample t-test. For “Coherency”, **p = 0.01 (BtA-Ctrl), **p = 0.005 (BtA-AtB), p = 0.57 (AtB-Ctrl). n.s. non-significant. For “Strength”, ***p = 0.00002 (BtA-Ctrl, BtA-AtB), p = 0.2 (AtB-Ctrl). Scale bar, 10 μm.

Source data

Extended Data Fig. 5 Burst of division rate precedes significant mound formation.

Quantification of RUDM value and division rate as function of time under AtB fields, mean ± s.t.d. Each time point has n between 6–8 independent field-of-views, 3 independent experiments in 3 biological replicates. Two-tailed, two-sample t-test. *p = 0.02 shows the earliest respective time points to be significantly different from the first time points.

Source data

Extended Data Fig. 6 Effect of different field conditions on intact but heterogenous layers on fibronectin-coated gel.

a, Top-down view of nucleus/cell distributions of MDCK on fibronectin-coated gel under different field conditions for ~2 days. Scale bars, 50 μm. b, RUDM values of such experiments. Line is mean ± s.t.d., scatter points are independent experiments. Ctrl: n = 6 independent experiments in 3 biological replicates. AtB: n = 3 independent experiments in 2 biological replicates. BtA: n = 5 independent experiments in 3 biological replicates. One-tailed, two-sample t-test. *p = 0.008 (Ctrl-BtA). *p = 0.01 (AtB-BtA). n.s. non-significant. c, Local division rates of dense and sparse regions under BtA fields. Line is mean ± s.t.d., scatter points are independent field-of-views. Dense/Sparse: n = 6 independent field-of-views, 3 independent experiments in 3 biological replicates. Two-tailed, paired t-test. p = 0.09.

Source data

Extended Data Fig. 7 Effect of different field conditions on E-cad KO cells and layers.

a, Measure of tissue integrity of different experiments with Ecad KO cells grown without fields for ~200 h, then applied with different field conditions for another ~100 h. Line is mean ± s.t.d., scatter points are independent experiments. Ctrl/AtB: n = 3 independent experiments in 2 biological replicates. BtA: n = 6 independent experiments in 3 biological replicates. One-tailed, t-test. *p = 0.03. b, Schematic showing no local gel deformation for sparse cell conditions under different E-field conditions. c, Montages showing imaging focus of cells before and (at least 1 hour) after field application, and control (field strength = 0) under sparse cell conditions. n = 5 independent cells/doublets in at least 2 biological replicates for each condition (corresponding to different columns). d, Montage of rescue of E-cad KO layer under BtA fields. Time = 0 h is when the field is first turned on. e, Montage of reversal of a rescue process after the field is turned off (time = 0 h). Bright parts of the cells show clumps. Boxes show out-of-focus areas, asterisks show gapped areas. Scale bars, c: 10 μm d, e: 100 μm.

Source data

Supplementary information

Supplementary Information

Supplementary Discussions 1–5, Figs. 1–4 and video captions 1–10.

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Supplementary Data 1

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Supplementary Video 1

Cell–cell junction brightness increases shortly after AtB field is applied. Phase-contrast imaging of an MDCK monolayer shows the cell–cell junction contrast before and after the AtB field is applied. The experiment is started at no-field (0 V) conditions and the AtB field is switched on at t = 20 min. Scale bar, 10 μm.

Supplementary Video 2

Cell–cell junction brightness remains low under BtA fields. Phase-contrast imaging of an MDCK monolayer shows the cell–cell junction contrast during 6 h of BtA-field application. Scale bar, 15 μm.

Supplementary Video 3

Junctional E-cadherin intensity increases after switching from no field to BtA fields within few hours. Top-down and side views of confocal imaging of GFP–E-cadherin MDCK layers. There was no field in the first frame, but the BtA field was subsequently turned on, and the E-cadherin intensity steadily increased during this time. Scale bar, 10 μm.

Supplementary Video 4

Junctional actin intensity increases after switching from AtB to BtA fields within few hours. Top-down and side views of confocal imaging of GFP–actin MDCK layers. The cells have been under AtB fields for ~10 h before the imaging started at 0 min, and the field direction is switched to the BtA direction at t = 100 min. Scale bar, 10 μm.

Supplementary Video 5

Collective live-cell extrusions are incurred under long-term AtB-field application. Epifluorescence imaging of an H1–GFP MDCK monolayer shows the evolution of nucleus/cellular distributions over days during AtB-field application. Bright patches are collective live-cell extrusions. Scale bar, 50 μm.

Supplementary Video 6

Three-dimensional mounds are formed in the N/TERT-1 human skin layers under long-term AtB-field application. Phase-contrast and epifluorescence imaging of a FUCCI N/TERT-1 cell line under AtB fields over 61 h. Basal progenitor cells are largely grey (with minimal red FUCCI signal indicative of the G1 phase). Non-terminally differentiated, suprabasal cells have a clear red FUCCI signal in their nucleus. Large patches of connected red nuclei show the 3D mounds (blue box). Scale bar, 50 μm.

Supplementary Video 7

MDCK cells stay in the monolayer and undergo high cell death rates under long-term BtA-field application. Epifluorescence imaging of H1–GFP MDCK monolayer shows the evolution of cell fate over days during BtA-field application. The yellow arrowheads point to fragmented/condensed nuclei indicative of cell death events. Scale bar, 50 μm.

Supplementary Video 8

N/TERT-1 skin cells undergo high terminal differentiation rates under long-term BtA-field application. Phase-contrast and epifluorescence imaging of an FUCCI N/TERT-1 cell line under BtA fields over 49 h. Basal stem cells are largely grey (with minimal red FUCCI signal indicative of the G1 phase). Non-terminally differentiated, suprabasal cells have a clear red FUCCI signal in their nucleus. Events of disappearance of large, red nuclei that are replaced by grey condensed nuclei (with bright halo) are differentiation events (verified with involucrin staining; Fig. 4b). Scale bar, 20 μm.

Supplementary Video 9

BtA fields rescue heterogeneous distribution of MDCK cells on fibronectin-coated soft PA gel. Imaging of FUCCI MDCK cells shows the evolution of cell distribution over days on fibronectin-coated gel. The AtB field is applied for the first ~100 h before being switching to the BtA direction. Bright patches are dense cell regions. Scale bar, 50 μm.

Supplementary Video 10

BtA fields rescue the integrity of E-cadherin KO layers on fibronectin-coated soft PA gel, whereas switching to AtB fields disrupt them within few hours. Phase-contrast imaging of E-cadherin KO MDCK cells under BtA fields first, before the field direction is switched to AtB at t ≈ 166 h. Smooth, dark regions are gaps without cells. Scale bar, 30 μm.

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Saw, T.B., Gao, X., Li, M. et al. Transepithelial potential difference governs epithelial homeostasis by electromechanics. Nat. Phys. 18, 1122–1128 (2022). https://doi.org/10.1038/s41567-022-01657-1

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  • DOI: https://doi.org/10.1038/s41567-022-01657-1

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