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. 2024 Nov 16;14:28254. doi: 10.1038/s41598-024-79352-9

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© The Author(s) 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — Elevated Piezo levels cause a severe nephrocyte phenotype in adult pericardial nephrocytes, which is mediated by the mechanosensitive domain. (A) Visualization of the nephrocyte diaphragm with Duf and Pyd antibodies shows a significant reduction of nephrocyte diaphragm length in adult pericardial cells expressing Piezo wildtype, while cells expressing the mutant without the mechanosensitive channel activity do not exhibit a phenotype. Experiments have been done at 25 °C resulting in medium expression levels. Scale bar = 5 μm. 1way-ANOVA with Tukey’s multiple comparison test: *: p < 0.05. Control: w; sns-Gal4/ + ;UAS-dicer2/ + . Piezo WT: w; sns-Gal4/ + ;UAS-dicer2/UAS-piezo-flag. Piezo w/o MS: w; sns-Gal4/ + ;UAS-dicer2/UAS-piezo-2306MYC.flag. (B) Immunofluorescence staining to visualize the nephrocyte diaphragm at 28 °C (high expression levels) also reveal a severe and significant phenotype upon expression of Piezo wildtype, while pericardial cells expressing the mutant exhibit a normal morphology. Scale bar = 5 μm. 1way-ANOVA with Tukey’s multiple comparison test: *: p < 0.05. (C, D) FITC-Albumin uptake assays reveal a severe phenotype in adult pericardial cells overexpressing wildtype Piezo at 25 °C (medium levels) and 28 °C (high levels). Expression of the mutant variant of Piezo does not reveal a functional defect, neither at 25 °C nor at 28 °C. Scale bar = 25 μm. 1way-ANOVA with Tukey’s multiple comparison test: **: p < 0.01; ***: p < 0.001. (E) Visualization of actin fibres by phalloidin immunofluorescence reveals a significant increase of cortical actin fibres in Piezo wildtype overexpressing adult pericardial cells, while surface levels are not altered. Scale bar = 25 μm. Student’s t-test: *: p < 0.05.