DOI: 10.5061/dryad.95x69p8rb
title: Data from: A mechanism of lysosomal calcium entry

description: 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.
description: # A mechanism of lysosomal calcium entry Raw Data for Manuscript adk2317
 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.