Guide to source data for: Journal of Cell Biology article DOI: 10.1083/jcb.202112101 Title: Drosophila SPG12 ortholog, Reticulon-like 1, governs presynaptic ER organization and Ca2+ dynamics Authors: Juan José Pérez-Moreno, Rebecca C. Smith, Megan K. Oliva, Filomena Gallo, Shainy Ojha, Karin H. Müller, and Cahir J. O’Kane Article Description: Neuronal endoplasmic reticulum (ER) appears continuous throughout the cell. Its shape and continuity are influenced by ER-shaping proteins, mutations in which can cause distal axon degeneration in Hereditary Spastic Paraplegia (HSP). We therefore asked how loss of Rtnl1, a Drosophila ortholog of the human HSP gene RTN2 (SPG12), which encodes an ER-shaping protein, affects ER organization and the function of presynaptic terminals. Loss of Rtnl1 depleted ER membrane markers at Drosophila presynaptic motor terminals and appeared to deplete narrow tubular ER while leaving cisternae largely unaffected, thus suggesting little change in resting Ca2+ storage capacity. Nevertheless, these changes were accompanied by major reductions in activity-evoked Ca2+ fluxes in the cytosol, ER lumen, and mitochondria, as well as reduced evoked and spontaneous neurotransmission. We found that reduced STIM-mediated ER-plasma membrane contacts underlie presynaptic Ca2+ defects in Rtnl1 mutants. Our results show the importance of ER architecture in presynaptic physiology and function which are, therefore, potential factors in the pathology of HSP. Dataset Description: Files are organized within folders and subfolders, ordered by Figure and Figure Panel, containing progressive steps in data processing, from raw data to final version used in Figure Panels. Original Figures shown in the article, in .pdf formats, were made using Adobe Illustrator or Affinity Designer from the images described below. Confocal microscopy images: * .nd2 files are the raw data collected with Nikon EZ-C1 acquisition software; .tif files, generated with Fiji from .nd2 files, are progressive steps in data processing. * Each different date indicates an independent experiment. The data processing performed is described in the corresponding file name. In some cases, we provide microscopy images from additional examples and/or controls to those shown in the article. Widefield microscopy calcium videos: * Raw data collected with Micro-Manager acquisition software saved as multilayer .tif files; further steps in data processing, generated with Fiji, are also saved as multi-layer .tif files. These multi-layer .tif files were downsized from 16-bit 512x512 pixels to 8-bit 256x256 pixels in Fiji for repository upload. * Each larvae indicates an independent experiment; each larvae has its own folder, containing sub folders, one for each stimulation frequency tested. Each sub folder contains original and processed videos in .tif files; ROIs used in .roi files; time traces graphs showing raw graphs, along with graphs showing bleach corrected, delta F/F, and rolling average transformations in .pdf files exported from R; raw time traces data exported from R in .csv files; bleach corrected and rolling average transformed data in .txt files; extrapolated intensity and time data exported from these time traces from R in .csv files; and R files used to quantify this data in .R files. Quantifications and statistical analysis: * The quantifications performed are annotated in .xlsx or .pzfx, generated with Microsoft Excel or Prism; .txt files, generated from the .xlsx files, were used for statistical analysis using RStudio. All the R libraries and the specific functions used are indicated in the scripts, .R files. Graphs were exported as .pdf files from RStudio or Prism. Analysis of nucleotide and amino acid sequences: * .dna files were generated by SnapGene software from reference genome sequences. The subsequent annotations, selections or actions performed are described in the corresponding file name. Alignments were save as .dnaln files. Genomic maps were exported as .png files. Electron microscopy images: * TIFF files acquired with FEI MAPS software were imported to Fiji and aligned and reconstructed with TrakEM2 plugin. The resulting files were imported to Meshlab to generate .ply files. All the information about each Figure can be found in the article “DOI: 10.1083/jcb.202112101” (open access). Software * Windows OS Software: Nikon EZ-C1 acquisition software. Micro-Manager acquisition software. FEI MAPS acquisition software. * Mac OSX Software: Adobe Illustrator CC 2017 Affinity Designer 1.10.0 Fiji 2.1.0/1.53c Microsoft Excel 2019 Prism 9.1.2 2021 RStudio Desktop 1.3.1093 SnapGene 4.2.11 Meshlab 2022.02 File Formats: .dna - SnapGene file containing annotated nucleotide sequence and its products. .dnaln - SnapGene file containing an alignment of nucleotide sequences. .fa - Fasta format .nd2 - Nikon confocal files, open with Fiji - LOCI BioFormats. .pdf - Layered Adobe pdf format. Keep edition capabilities from Adobe Illustrator. .png - png image file. .R - R script file. .tif - tif image file. Note that some tif files are multilayered. .txt - text document. .xlsx - Spreadsheet file created by Microsoft Excel. .csv - Spreadsheet file created by R (also compatible with Microsoft Excel). .pzfx - Spreadsheet and graph file created by Prism9. .zip - Files are zipped because their filenames contain characters disallowed on some file systems, or due to file size. You will need to decompress before accessing them. .roi - Region of interest file created by Fiji. .ply - 3D rendering of EM sections generated with Meshlab.