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. 2022 Apr 28;13:2317. doi: 10.1038/s41467-022-29988-w

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a Live cell imaging MDCK-II cells stably expressing SNAP-tagged Na⁺/K⁺ ATPase (NKA) a-subunit and stained with TMR-STAR dye in the MFKP. Time-lapsed confocal images of cells’ lateral side in three consecutive conditions: zero pressure (ΔP = 0), pressure gradient (ΔP = 200 Pa) and pressure released (ΔP = 0). The arrowhead indicates disruption of lateral NKA expression in regions of interest (ROIs) at various time stamps. Scale bar = 10 mm. b A zoomed confocal slice of an ROI in MDCK-II cells expressing SNAP-tagged NKA and stained with TMR-STAR dye. c NKA intensity is quantified in a red band centered between two cells in b. The intensity profile in c is the vertical average intensity along the red box in b, the peak (indicated by black arrow) is the maximum intensity of NKA. d Mean of normalized peak lateral NKA intensity for the same cell over the course of three pressure conditions in a. Shading represents the SEM. (n = 25, N = 3, cells, biological replicates). e Live cell imaging of transiently transfected MDCK-II cells expressing GFP-tagged F-actin (Ftractin). Time-lapsed confocal images of the baso-lateral side of the cells were taken under three consecutive conditions: ΔP = 0, ΔP = 200 Pa, and pressure released (ΔP = 0). Scale bar = 10 mm. Pressure application induces rapid high-frequency invaginations in the baso-lateral domain. White arrowhead indicates F-actin rings, which are cross-section of invaginations. f Number of invaginations per unit cell over the course of three pressure conditions in e (n = 8, N = 3, cells, biological replicates). g Mean area of invaginations as a function of time under pressure. Shading represents the SEM. (n = 10, N = 3, cells, biological replicates). The invaginations have an average lifetime of ~15 s and disappear when pressure is released as shown in f. h Schematic showing co-localization of F-actin (green lines) and NKA (red dots) in cells in MFKP at ΔP = 0. i Hydrostatic pressure gradient results in high-frequency invaginations on the baso-lateral F-actin cortex and a reduction in NKA localization in the lateral domain.