Data from: A mechanism of lysosomal calcium entry
Data files
Feb 02, 2024 version files 11.28 GB
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Raw_Data_for_Repository.zip
11.24 GB
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README.md
3.15 KB
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Whole_Cell_Electrophysiology_Raw_Data.zip
38.21 MB
Abstract
Lysosomal calcium (Ca2+) release is critical to cell signaling and is mediated by well-known lysosomal Ca2+ channels. Yet, how lysosomes refill their Ca2+ remains hitherto undescribed. Here, from an RNAi screen in C. elegans we identify an evolutionarily conserved gene, lci-1, that facilitates lysosomal Ca2+ entry in C. elegans and mammalian cells. We found that its human homolog TMEM165, previously designated as a Ca2+/H+ exchanger (CAX), imports Ca2+ pH-dependently into lysosomes. Using two-ion mapping and electrophysiology we show that TMEM165, hereafter referred to as human LCI, acts as a proton-activated, lysosomal Ca2+ importer. Defects in lysosomal Ca2+ channels cause several neurodegenerative diseases, and knowledge of lysosomal Ca2+ importers may provide new avenues to explore the physiology of Ca2+ channels.
Raw Data for Manuscript adk2317
https://doi.org/10.5061/dryad.95x69p8rb
Dataset contains fluorescent images, gels, plates, and quantitation from those data.
Description of the data and file structure
Data is organized by type of experiment. Within each experimental folder, there is an excel spreadsheet that contains the data plotted in the relevant figure (named on the excel sheet tab). There are also subfolders for individual data sets, which contain raw images, gels, and plates.
The "Electrophysiology" folder and "Whole Cell Electrophysiology Raw Data" folders contain spreadsheets of I-V plot data points for lysosome and whole-cell electrophysiology experiments, respectively. The "Gels" folder contains images of full gels from indicated RT-PCR experiments.
The "Mammalian Cell Cytosolic Ca" folder contains raw images of HeLa or COS7 cells labeled with the calcium-sensitive fluorophore Fura Red, under the indicated treatments. It also contains spreadsheets showing the calculation of relative Ca2+ levels from whole-cell fluorescence intensity in the 440 and 488 channels.
The "Mammalian Cell Lysosome pH and Ca" contains raw images of HeLa cells (WT and TMEM165 KO) labeled with the pH-correctable lysosomal Ca2+ reporter, CalipHluor. It also includes spreadsheets showing the calculation of lysosomal pH and Ca2+ from fluorescence intensity of each lysosome in the relevant channels.
The "Subcellular Localization" folder contains images showing the localization or expression of TMEM165 in mammalian cells or yeast. This includes its endogenous or overexpressed localization with lysosomal or Golgi markers, cell surface expression, and localization on swollen lysosomes. The folder also contains images showing the endolysosomal localization of CalipHluor, as well as spreadsheets showing calculations of Pearson Correlation Coefficients and relative expression levels.
The "Worm Lysosome pH and Ca" folder contains images of coelomocytes in N2 and lci-1 knockout worms labeled with the pH-correctable lysosomal Ca2+ reporter, CalipHluor, expressing the indicated variant of human TMEM165. It also contains spreadsheets showing the calculation of lysosomal pH and Ca2+ from fluorescence intensity of each lysosome in the relevant channels.
The "Worm Lysosomal Size Assay" folder contains images of coelomocytes in N2 and lci-1 knockout worms labeled with Alexa647-dsDNA or ssGFP, expressing the indicated variant of human TMEM165. It also contains spreadsheets showing the calculation of the area of large lysosomes.
The "Worm Survival Assay" folder contains images of worm plates showing the brood size following the indicated treatment. This includes N2 worms, cup-5 knockout worms after knockdown of potential lysosomal Ca2+ importers, and lci-1 knockout worms after expression of human TMEM165 variants. The folder also includes a spreadsheet showing the number of worms on the plates for each condition.
The "Yeast Phenotypes" folder contains images of yeast strain K665 cultured on plates containing varying levels of Ca2+, after expression of human TMEM165 variants.