Apical size reduction by macropinocytosis alleviates tissue crowding
Nat Commun. 2025 Jun 23;16(1):5338. doi: 10.1038/s41467-025-60724-2.
Bresteau E, Suva EE, Revell C, Hassan OA, Grata A, Sheridan J, Mitchell J, Arvanitis C, Korobova F, Woolner S, Jensen OE, Mitchell B.
Click here to view article at Nature Communications.
Click here to view article at PubMed.
Click here to view article on Xenbase.
Abstract
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.
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.
Adapted with permission from Springer Nature s on behalf of Nature Communications: Bresteau et al. (2025). Apical size reduction by macropinocytosis alleviates tissue crowding. Nat Commun. 2025 Jun 23;16(1):5338. doi: 10.1038/s41467-025-60724-2.
This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
Last Updated: 2025-07-09