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Nat Commun
2025 Jun 23;161:5338. doi: 10.1038/s41467-025-60724-2.
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Apical size reduction by macropinocytosis alleviates tissue crowding.
Bresteau E
,
Suva EE
,
Revell C
,
Hassan OA
,
Grata A
,
Sheridan J
,
Mitchell J
,
Arvanitis C
,
Korobova F
,
Woolner S
,
Jensen OE
,
Mitchell B
.
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Tissue crowding represents a critical challenge to epithelial tissues, which often respond via the irreversible process of live cell extrusion. We report that apical size reduction via macropinocytosis serves as a malleable and less destructive form of tissue remodeling that can alleviate the need for cell loss. We find that macropinocytosis is triggered by tissue crowding via mechanosensory signaling, leading to substantial internalization of apical membrane. This drives a reduction in apical surface which alleviates crowding. We report that this mechanism regulates the long-term organization of the developing epithelium and controls the timing of proliferation-induced cell extrusion. Additionally, we observe a wave of macropinocytosis in response to acute external compression. In both scenarios, inhibiting macropinocytosis induces a dramatic increase in cell extrusion suggesting cooperation between cell extrusion and macropinocytosis in response to both developmental and external compression. Our findings implicate macropinocytosis as an important regulator of dynamic epithelial remodeling.
R01GM089970 U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS), 225408/Z/22/Z Wellcome Trust (Wellcome), RPG-2021-394 Leverhulme Centre for Integrative Research on Agriculture and Health (LCIRAH), BB/T001984/1 RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)
Fig. 1: Constitutive macropinocytosis occurs in the apical surface of the Xenopus embryonic epidermis. a Actin ruffles (arrowheads) in the epidermis of Xenopus embryo stained with phalloidin. Scale bar 20 μm. b Timelapse of a single ruffle event in the epidermis of Xenopus embryo expressing Lifeact-fluorescent protein (FP). Scale bar 10 μm. c SEM images of actin ruffles as circular (top) or dome-shaped (bottom). Scale bar 5 μm. d–f Quantification of actin ruffle duration (d), peak area (e), and percentage of apical area (f) (n = 3 exp. and 90 ruffles; center line = mean; error bars = SD). g Quantification of actin ruffle localization within the cell (n = 9 exp. and 361 ruffles; bar = mean; error bars = SD). h Time lapse image of the Lifeact-FP (cyan) showing macropinocytotic internalization of fluorescent dextran (red) from the media, h’ is a z projection of the dotted line. Scale bar 10 μm. i Time lapse image of the Lifeact-FP (cyan) showing internalization of apical membrane (red) during macropinocytosis, i’ is a z projection of the dotted area. Scale bar 10 μm. j Circular actin ruffle (cyan, Lifeact-FP) surrounded by an outer ring of activated myosin II (red; SF9 intrabody). The dotted arrow shows an example of linescan averaged in (k). Scale bar 10 μm. k Localization of Lifeact and activated myosin II at the peak of an actin ruffles (n = 2 exp. and 15 ruffles; point = mean; error bars = SD). l Quantification of macropinocytosis (MP) events in embryos treated with DMSO or Blebbistatin (n = 3 exp. and 6 embryos; two-sided paired t-test, p-value: 0.0116). Source data are provided as a Source Data file.
Fig. 2: Macropinocytosis is associated with tissue crowding and low levels of tension. a Image of Xenopus stained with phalloidin (white) at ST34 (left) and ST42 (right, asterisk highlight actin ruffles). Scale bar 10 μm. b Quantification of cell density (blue; right axis) and macropinocytotic events (MP, red; left axis) at ST34 and ST42 (n = 3 exp. and 15 embryos per stage for cell density, p-value = 3.43e-10; 4 exp. and 6 embryos for macropinocytotic events at ST34 and 5 exp. and 16 embryos at ST42, p-value = 0.0282). c Quantification of cell density (blue; right axis) and macropinocytotic events (red; left axis) in embryo treated or not with Roscovitine for 24 h (n = 4 exp. and 18 control embryos and 21 Roscovitine-treated embryos for MP events, p-value = 0.0019; 3 exp. and 29 control embryos and 28 Roscovitine-treated embryos for goblet cell density, p-value = 0.0045). d Representation of the generation of 3D organoids or 2D explant plated onto fibronectin coated coverslip. Adapted from61,62. e Representative images of the surface of 2D explant (left) or 3D organoids (right) expressing Lifeact-FP. Scale bar 30 μm. f Quantification of cell density (blue; right axis) and macropinocytosis (red; left axis) in 2D explants or 3D organoids (n = 6 experiments with 22 explants and 23 organoids; MP p-value = 2.00e-06 and density p-value = 5.97e-07). g Image of actin ruffles (asterisk) in the epithelium of an embryo expressing Lifeact-FP before (up) and after (down) treatment with GsMTx4. Scale bar 30 μm. h Quantification of the fold difference in macropinocytosis level between 1 h before and 1 h after treatment with DMSO (mock treatment, n = 9 embryo), GsMTx4 (n = 5 embryo; p-value = 2.03e-06), NaDeoxy (n = 5 embryo; p-value = 0.0012), Mβ Cyclodextrin (n = 11 embryo; p-value = 0.0260), or Yoda1 (n = 5 embryo; p-value 3.00e-05). All tests are two-sided unpaired t-tests. Source data are provided as a Source Data file.
Fig. 3: Macropinocytosis leads to cell size reduction. a Time lapse of a macropinocytotic event in tissue labeled with Lifeact-FP. The dotted line represents cell original size, highlighting the cell size reduction. Scale bar 10 μm. b Quantification of cell size change in cells with (blue) or without (red) a macropinocytotic event (MP) using cells synchronized to the onset of macropinocytosis and normalized to original cell size (n = 3 exp. and 43 cells total; mean and SD). c Correlation between the apical size reduction and the size of actin ruffle (n = 3 exp. and 43 cells). d Quantification of the change in junction length in cells with and without macropinocytosis (n = 4 exp. and 40 cells; two-sided unpaired t-test, p-value = 1.23e-07). e Graphical depiction of a cell with different junction types labeled (N1-N4) in different colors based on their distance from the macropinocytotic event. f CellFit analysis of time lapse movies quantifying the change in relative junctional tension before and after macropinocytosis (n = 25 events; see Fig. S1f; box = interquartile, center line = median, error bars = min/max; two-sided paired t-test of before and after the event p-value N1 = 0.0001, N2 = 0.0281, N3 = 0.4890, N4 = 0.3492). g Model comparing the effect of cell extrusion (center) or macropinocytosis (right) occurring in the cell (asterisk) of a crowded epithelium (left). Blue color highlights the putative effect on cells/neighbors. Source data are provided as a Source Data file.
Fig. 4: Macropinocytosis regulates tissue crowding during epithelial development. a Representative images of multiciliated cells (MCCs, marked with acetylated tubulin ab) in ST47 embryo after treatment with DMSO or Roscovitine. Scale bar 300 μm. b Quantification of MCC number in embryo treated for 24 h with either DMSO or Roscovitine (n = 4 exp. and 50 DMSO and 51 Roscovitine embryos; two-sided unpaired t-test, p-value = 1.42e-014). c Representative image of macropinocytic events (arrowheads) occurring next to MCCs in the epithelium of a TubA1A-membrane RFP transgenic embryo labeling multiciliated cells (red) and injected with Lifeact-FP (cyan). Dashed lines represent rosette-like structure around MCCs. Scale bar 30 μm. d Representative SEM image showing a macropinocytotic event (arrowhead) adjacent to an MCC. Scale bar 5 μm. e Quantification of the number of events (MP) per cell per hour for cells adjacent or not adjacent to MCCs (n = 4 exp. 191 macropinocytotic events; two-sided paired t-test, p-value = 0.0034). f, h, j Images of a simulated epithelium with multiciliated cells (asterisk). Cells are color-coded for effective pressure (f), tension (h) or shear stress (j). Dashed lines represent rosette-like structure around MCCs. g, i, k Percentage of non-MCC neighbors and MCC neighbors that are in the highest decile of effective pressure (g) and shear stress (k) or in the lowest decile of tension (i) (n = 10 simulations; two-sided paired t-test; effective pressure p-value = 0.0004, tension p-value = 0.0021, shear stress p-value = 5.4e-05). l Quantification of the fold difference in macropinocytotic events comparing 1 h before and 1 h after treatment with DMSO (n = 9 exp.), 5 μM LY294002 (n = 4 exp.; p-value = 8.0e-06), or 1 mM Amiloride (n = 7 exp.; p-value = 0.0002); two-sided unpaired t-tests. m Representative images of MCCs (marked with acetylated tubulin ab; red/white) in ST46 embryos stained with phalloidin (cyan) after treatment with DMSO, LY294002 or Amiloride. Scale bar 300 μm. n Quantification of the number of MCCs after treatment with DMSO (n = 115 embryos), 5 μM LY294002 (n = 81 embryos; p-value = 0.0457) or 1 mM Amiloride (n = 49 embryos; p-value < 0.0001); two-sided unpaired t-tests. Source data are provided as a Source Data file.
Fig. 5: A wave of macropinocytosis remodels the epithelium in response to external tissue compression. a Representative image of an embryo injected with Cadherin-FP (cyan) and Lifeact-FP (red) before (left), during (middle), and after (right) a wave of macropinocytosis induced by tissue compression. Scale bar 30 μm. b Quantification of the wave of macropinocytic events (MP) upon compression (n = 80 scan areas from 32 embryos from 12 exp.; bars = mean, error bars = SD). c, f Representative images of embryos injected with Cadherin-FP (cyan/white) and Lifeact-FP (red) 5 min after compression in control (c) or Amiloride treated (f) embryos. Scale bar 10 μm. d, g Representation of tissue remodeling over 10 min using a temporal color code of control (d) or Amiloride (g) treated embryos. Scale bar 10 μm. e, h Quantification of tissue remodeling (i.e., overall change in cell apical area) (blue; left axis) and macropinocytotic events (red; right axis) in DMSO (e) and Amiloride treated (h) embryos upon compression (n = 3 exp.; bar = mean, error bars = SD). i Quantification of macropinocytotic events (red; left axis) and cell extrusion events (blue; right axis) in non-compressed (n = 9 embryos; MP p-value 5.00e-05, extrusion p-value = 0.0168) and compressed embryos in either DMSO (n = 32 embryos) or treated with amiloride (n = 13 embryos; two-sided unpaired t-tests; MP p-value = 1.31e-07, extrusion p-value = 0.0028) or LY294002 (n = 19 embryos; MP p-value = 4.19e-06, extrusion p-value = 0.0272). j Representative image of an embryo injected with Cadherin-FP (cyan) and Lifeact-FP (red) having both macropinocytotic events and cell extrusion (arrowhead) upon compression. Scale bar 30 μm. Source data are provided as a Source Data file.
