DOI: 10.5061/dryad.dbrv15f7h
title: Data from: ESCRT-III-dependent adhesive and mechanical changes are triggered by a mechanism detecting alteration of Septate Junction integrity in Drosophila epithelial cells

description: Barrier functions of proliferative epithelia are constantly challenged by
 mechanical and chemical constraints. How epithelia respond to and cope
 with disturbances of barrier functions to allow tissue integrity
 maintenance is poorly characterized. Cellular junctions play an important
 role in this process and intracellular traffic contribute to their
 homeostasis. Here, we reveal that, in Drosophila pupal notum, alteration
 of the bi- or tricellular septate junctions (SJs) triggers a mechanism
 with two prominent outcomes. On one hand, there is an increase in the
 levels of E-cadherin, F-Actin and non-muscle Myosin II in the plane of
 adherens junctions. On the other hand, β-integrin/Vinculin-positive cell
 contacts are reinforced along the lateral and basal membranes. We found
 that the weakening of SJ integrity, caused by the depletion of bi- or
 tricellular SJ components, alters ESCRT-III/Vps32/Shrub distribution,
 reduces degradation, and instead favours recycling of SJ components, an
 effect that extends to other recycled transmembrane protein cargoes
 including Crumbs, its effector β-Heavy Spectrin Karst, and β-integrin. We
 propose a mechanism by which epithelial cells, upon sensing alterations of
 the septate junction, reroute the function of Shrub to adjust the balance
 of degradation/recycling of junctional cargoes and thereby compensate for
 barrier junction defects to maintain epithelial integrity.
description: LIve imaging acquisition: LSM Zeiss 880 AiryScan equipped with a 63X N.A.1.4. objective and
 controlled by ZEN software. Fixed specimens: LSM Zeiss 880 AiryScan equipped
 with a 63X N.A.1.4. objective and controlled by ZEN software or LEICA SPE
 equipped with a 63X N.A. 1.4 Laser ablations: were performed on live pupae aged for 16h to
 19h APF using a Leica SP5 confocal microscope equipped with a 63X N.A. 1.4
 objective or a LSM Zeiss
 880 AiryScan equipped with
 a 63X N.A. 1.4 objective.
 Ablation was carried out on epithelial cell membranes at AJ level with a
 two-photon laser-type Mai-Tai HP from Spectra Physics set to 800 nm and a
 laser power of 2.9W. Images were then analyzed and quantitated using
 Fiji Statistical analyses: All information concerning
 the statistical details is provided in the main text and in figure
 legends, including the number of samples analyzed for each experiment.
 Prism 8 software and R 4.2.1 were used
 to perform the analyses. 
description: # ESCRT-III-dependent adhesive and mechanical changes are triggered by a
 mechanism detecting alteration of Septate Junction integrity in Drosophila
 epithelial cells
 [https://doi.org/10.5061/dryad.dbrv15f7h](https://doi.org/10.5061/dryad.dbrv15f7h) #### Contributors \- Thomas Esmangart de Bournonville, current address: Global Health Institute, School of Life Science, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland \- Mariusz Jaglarz, Department of Developmental Biology and Invertebrate Morphology, Institute of Zoology and Biomedical Research, Jagiellonian University in Krakow, Gronostajowa 9, 30-387 Krakow, Poland \- Emeline Durel, Univ Rennes, CNRS UMR 6290-IGDR, 35000 Rennes, France \- Roland Le Borgne, Univ Rennes, CNRS UMR 6290-IGDR, 35000 Rennes, France #### Overview This supplementary dataset, integral to our research article, contains data crucial for understanding the key findings of our study. It comprises stacks of confocal microscopy images used to assemble the main and supplementary figures, and those used for quantitative analysis of phenotypes and different fluorescent markers. The raw data from confocal microscope acquisitions are in their original format, promoting transparency and allowing for varied data processing and analysis methods, thus facilitating diverse scientific interpretations and collaborative research. ## Description of the data and file structure **Data Format** Metadata and binary Files:.czi (Carl Zeiss group)or  .tif : raw confocal image data Excel Files .xlsx/.xls: utilized for oganizing and presenting data summaries and summaries of quantitative analyses .csv Files utilized for oganizing and presenting data summaries and summaries of quantitative analyses (generated by R and which can be opened by Excel or Numbers) R Files: .R: macro files containing scripts for statistical processing of data. The output files are in the format .csv Matlab Files: .m: matlab macro files containing scripts for anlayzing the length of bicellular junctions (files named Matlab lengh analysis), and to monitor the evolution of vertex position following laser abaltion (files named ablation point picker). Output files are in the .fig and .txt format. **Recommended software for data analysis:** Confocal data analysis: FiJi/ImageJ for visualizing and processing .tif, .lif and .czi files Zen (Carl Zeiss group) for visualizing and processing .czi files Statistical analysis:  R Numerical Calculation: Matlab scripting language for numerical calculation ## **Usage, Compatibility, and Accessibility** This dataset includes a wide range of file formats, meeting various research needs in the fields of confocal imaging and the analysis of microscopy data. By providing data in their original formats, we provide full access and maintain the integrity of the information. We recognise that the dataset includes some proprietary file types, such as .czi and .lif files, which are specific to the microscopes and software used in our study. Although these files are not essential to the analysis of the primary data, they are included to provide full transparency of all data generated during the course of the research. This approach ensures that researchers have access to the most complete dataset possible from the image acquisition properties, providing an in-depth view of the study's methodologies and results. Researchers are encouraged to use the original data formats for their analyses to ensure the accuracy and reliability of their results. For assistance with data conversion and analysis, researchers are invited to contact the authors. We are committed to providing support and advice to facilitate the effective use of this comprehensive dataset for further research and analysis. ### Content: This file contain the data presented in the main article and supplemenary figures (Dataset A_images_figures), and provides comprehensive support for our research findings, and it includes measurements and statistical analysis (Dataset B_data_analysis). These datasets serve to support the conclusions drawn in our study and are useful for further scientific exploration and verification. The date on which the experiments were performed is indicated in the raw data files as year/month/day (for example 190118 means the experiment was performed in 2018/01/18). ### Supplementary File 1: DatasetA\_Images\_Figures The folder which can be find on Zenodo, contains the raw confocal data files for each panel of Figures 1 to 6 and 1S1 to 6S1. The data/confocal stacks (.tif or .czi format) are organized in the form of a sub-folder for each figure or supplementary figures (12 subfolders in total). Each sub-folder contains a .txt file format providing a brief description of the Figure panels and a stack of confocal images from the corresponding panels. The markers used are named and appear in order of acquisition wavelength (488 (GFP), 531 (RFP, Cherry) and 635 nm). ### Supplementary File 2: Dataset B\_Original Data\_Analysis The dataset comprises raw data_original stacks of confocal images used for the quantifications, the quantifications and statistical tests carried out in this article. The data is organized in four folders as follows: #### dataset\_Quantification Fluorescence Signal Intensity’ This folder contains 19 subfolders named according to the corresponding panels of each figure in the article. Each sub-folder contains the raw data images in .tif or .czi format named data set 1 to n, and the quantification of the fluorescent signal in an .xlsx file. In the .xlsx file, each data set (1 to n) is quantified in a separate sheet. In each sheet, the date of the experiment, the genotype of the cells (WT = control, Mut = cell homozygote mutant for the given gene), the fluorescent markers and the ROI used for quantification are indicated. Raw grey value column corresponds to the grey value measured using Fiji with the ROI described. Raw grey value - background gray value column corresponds to the grey value corrected for the background. Normalized grey value corresponds to the raw data corrected for the background, normalized and used for quantification and plots. Mean row corresponds to the mean of the data in the dedicated column. #### Dataset\_apical cell area measurement This folder contains 3 subfolders named according to the corresponding panels of each figure in the article. Each sub-folder contains the raw data images in .tif or .czi format named data set 1 to n, and the result of the signal quantification and the statistical analysis are entered in the corresponding .csv and .R files, respectively. The cell area for the different conditions (WT, akaL200, akaL200,Crb11A22 or akaL200, RNAi mys clones) is in µm² and the perimeter is in µm.The date on which the experiments were performed is indicated in the raw data files as year/month/day (for example 190118 means the experiment was performed in 2018/01/18). #### dataset\_Measurement of Cell Junction Length\_Fig. 2 G This folder contains 3 subfolders. The first sub-folder contains the quantified data compiled in an .xlsx file, and the second contains the MatLab script (file in .m format) utilized for calculating the change in junction length over time. The third sub-folder entitled raw data for MatLab ‘length analysis’ is itself subdivided in 11 sub-folders, each containing the raw data in .tif format, the distance measured in .txt format, and the evolution of the distance over time in .fig and .tiff format. in each sub-folder, the file names indicate the genotype of the dividing cell for which the length of the new junction is measured (at the middle), and the genotype of its two neighbours (for example WT-Mut-WT means that the dividing cell is homozygous mutant and surrounded by two control (WT) cells. #### dataset\_Recoil Velocity Measurement This folder contains 3 subfolders. the first sub-folder entitled 'ablation_pointpicker_v6b', in .m format, contains the Matlab script used to measure vertex spacing after laser ablation.  the other two sub-folders contain the raw data and the quantifications performed in the mutant situations for nrv2 and aka, corresponding to panel D of figure 1 S1. Each of these subfolders is then subdivided into four subfolders, one containing the quantified data in .xlsx format, the other the raw data (confocal stacks) in .czi format, and the other two the measurements carried out on the control and mutant cells. The latter two are themselves subdivided into subfolders (one number for each cell that underwent ablation) showing the measurements obtained by point picker (.txt format) with the representation of vertex deviation over time (.tiff and .fig format files). ## Sharing/Access information This is a section for linking to other ways to access the data, and for linking to sources the data is derived from, if any. ##