MINFLUX/DΝΑ-PΑΙΝΤ raw data of PIEZO1-ALFA-mGL in N2a cells and Matlab analysis scripts

PIEZO1 MINFLUX analysis Matlab scripts and MINFLUX raw datasets for "Cluster nanoarchitecture and structural diversity of PIEZO1 at rest and during activation in intact cells" by Clement Verkest, Lucas Roettger, Nadja Zeitzschel & Stefan Lechner Contact: [e-mail] / [e-mail] This set of Matlab scripts are used to analyse and visualize PIEZO1 DNA-PAINT/MINFLUX published in Verkest et al., 2025 (10.1101/2024.11.26.625366). The scripts and data are provided for academic and visualization purposes only. Commercial usage of the provided Minflux data (reproduction outside of the publication, etc. ) is forbidden. Please contact the authors for further inquiries. System requirements The scripts were generated and tested with Matlab R2023b and R2024a and require additional Matlab toolboxes such as the Signal_Processing_Toolbox and Image_Processing_Toolbox. Matlab scripts provided in this repository: For the data analysis and reproduction of the MINFLUX-related panels in Fig. 1–3 in Verkest et al., the following scripts are required: MFX_PIEZO1_ALFA_01_main_script.m MFX_PIEZO1_ALFA_02_TrimerInPlaneProjection.m For the data analysis and reproduction of the MINFLUX-related panels in Fig. 5 and 7 in Verkest et al., the following scripts are required: PIEZO1_GFP_ClusterAnalysis_1 PIEZO1_GFP_ClusterAnalysis_2 PIEZO1_GFP_ClusterAnalysis_3 In addition the following subroutines are required for all analysis. DBSCAN dbscan2 FitInterbladeDistribution PlotClusterAnalysisResult PlotRawData SetGFPanalysisParameters_V2 CalcTrimerAngle1 CalculateTraceMean PIEZO1Superparticle PlotTrimerAnalysisResult SetALFAanalysisParameters MINFLUX raw data provided in this repository: confocal images as tif files, with region selected for Minflux scan highlighted Matlab structure files with raw, unfiltered minflux localizations data Raw data for ALFA-tag experiments: Soma_Yoda1.mat, Soma_Stoml3.mat, Soma_cytoD.mat, Soma_cytoD_Yoda1.mat, Soma_CTL.mat, Soma_Blebbistatin.mat, Neurite_Yoda1.mat, Neurite_CTL.mat. Files contain a matlab structure called MFXdata with raw data from all imaged cells in the repsective experimental condition (col1: X coord /col2: Y coord / col3: Z coord / col4: TID (trace identification number) / col5: efo (Hz) / col6: cfr / col7: background (Hz) / col8: time (s). ) Raw data for mGL-tag experiments: PIEZO1mGL_CTL_RawData, PIEZO1mGL_OSMO_RawData.Matlab structure files with selected PIEZO clusters -GFP_CTL_all_selected_clusters.mat and GFP_OSMO_all_selected_clusters.mat contain data array (X rows and 4 cols) with the cluster coordinates (X,Y,Z) and trace-ID (TID) How to use: I - Analysis of data and reproduction of MINFLUX-related panels shown in Fig. 1–3. 1.) Download folder ‘Figure_1_to_3’ with subfolders and add the path to these folders to Matlab. 2.) Open ‘MFX_PIEZO1_ALFA_01_main_script.m’ 3.) Chose which dataset to analyse (set Variable ‘DataScource’ to value between 1 and 8) 4.) Set additional analysis options (default options reproduced data from paper) 5.) Run the script. 6.) ! Optional !: Open and run the script ‘MFX_PIEZO1_ALFA_02_TrimerInPlaneProjection.m’. This script will generate 3D, 2D as well as probabilty density plots of all identified trimers II - Analysis of data and reproduction of MINFLUX-related panels shown in Fig. 5 and 7. 1.) Download folder ‘Figure_5_and_7’ with subfolders and add the path to these folders to Matlab. 2.) Run PIEZO1_GFP_ClusterAnalysis_1.m -This script is used to visualize Minflux unfiltered and filtered data from DNA-PAINT GFP -Select options in the 'CHOOSE OPTIONS' section (set 'true' or 'false') -Select the data source (Control or hypo-osmotic) in 'Load data' section, (set 'DataSource = 0', '1', '2', '3' or '4') 3.) Run PIEZO1_GFP_ClusterAnalysis_2.m -This script is used to plot panels in Fig 1f-g, as well as 3D plot of selected PIEZO cluster -Select options in the 'set options' section ! Warning ! Plotting the full cluster dataset might slow down things. -Select the data source ('DataSource = 1', '2', '3' or '4') 4.) Run PIEZO1_GFP_ClusterAnalysis_3.m -This script is used to plot panels in Fig 7c-f