DOI: 10.5061/dryad.cc2fqz6m4
title: Data from: A perilysosomal feedforward mechanism regulates starvation-induced calcium signaling

description: This dataset contains raw and processed data for a manuscript in The FEBS
 Journal. Nutrient depletion triggers a starvation-induced calcium (Ca2+)
 signal (SICS) that promotes Ca2+-dependent responses. However, the
 components and regulations of SICS are unclear. In this work, we explored
 SICS components and their regulation by the Ca2+ sensor calmodulin (CaM).
 The work provides molecular evidence of components of starvation-induced
 calcium signaling and their regulation.  All files submitted are
 named after the associated figures in the manuscript, followed by a
 specific experimental number. For example, "Fig. 1A-B_21219000 SICS
 (1)", "Fig. 1A-B_21219000 SICS (2)", etc. Thus,
 one figure panel is typically associated with multiple experimental files.
 Readme files in Word format are submitted along with the experimental
 files for convenience. Only original and processed experimental
 data are included. Drawings are not. Fluorescence images are provided in
 TIFF format. Immunoblots are provided in TIFF format. Intracellular
 signals are provided as raw calcium indicator ratios, calculation of
 fractional ratios, and areas under the curve of the signals measured, all
 in Excel format.
description: # Data from: A perilysosomal feedforward mechanism regulates
 starvation-induced calcium signaling Dataset DOI:
 [10.5061/dryad.cc2fqz6m4](https://doi.org/10.5061/dryad.cc2fqz6m4) ##
 Description of the data and file structure All sections of the files
 submitted are accompanied by README files in Word format. These README
 files are named according to the figure panels they describe.  **Figure
 1A-B README** Data for figure 1A-B were from 4 experiments: \- 21219000
 SICS (1) \- 21219002 SICS (2) \- 21219003 SICS (3) \- 21219004 SICS (4) In
 each of these files, there are three tabs: \- F340 \- F380 \- Ratio Data
 for Figure 1A are the average of all ratio data from these 4 experiments.
 Average time course was taken up to 4000s. Data for Figure 1B are the
 average of all F340 and F380 data from these 4 experiments. Average time
 course was taken up to 4000s. **Figures 1C-D README** Data for Figs. 1C-D
 are from the following experimental files: \- 21222002 SICS – Refeed (1)
 \- 21222007 SICS – Refeed (2) \- 21222008 SICS – Refeed (3) \- 21222009
 SICS – Refeed (4) Each file has two tabs: \- Fura-2 ratio F340/380 \- AUC:
 values are on top of each data trace o AUC 700 – 1200s o AUC 2000 – 2500s
 Figure 1C are the average of all traces from these four experiments, up to
 2500s. Figure 1D are the combined AUCs from all four experiments. Outliers
 are removed using Grubb’s test. **Figure 1E – README** Data for Fig. 1E
 are from the following experimental files: • 22809001 MOVAS-SICS (1) •
 22809003 MOVAS SICS (2) • 22809005 MOVAS SICS (3) Each file contains 2
 tabs: \- Raw ratio \- Relative ratio: all data points are normalized to
 the average of the first 50-s of baseline ratio. Figure 1E is the average
 of all relative data traces from these experiments, up to 1000s. **Fig.
 2E-G README** Data for Fig. 2E-G are from the following experimental
 files: \- 24129000 dCBL (1) \- 24129001 dCBL (2) \- 242129002 dCBL (3) \-
 24213000 dCBL (4) \- 24228002 dCBL (5) \- 24229004 dCBL (6) Each data file
 contains three tabs: \- Raw ratios: Per our lab’s routine, transfected
 cells were selected first, followed by the same number of non-transfected
 cells from the same microscopic field. Therefore, the first half of the
 data traces are from cells expressing mKate2-STIM1CBL, the remaining from
 the same-field, non-transfected cells (SFNT) \- Fractional ratio
 calculated from raw ratios. \- Traces & AUCs: areas under the curve of
 signals stimulated by TGN in Ca2+-free condition (ER Ca2+ release) and
 AUCs from bulk Ca2+ signal produced upon addition of extracellular Ca2+.
 Figure 2E: representative experiments (24129002dCBL (3)) from cells
 expressing mKate2-STIM1CBL (n = 8) and same-field non-transfected cells
 (n = 8). Figure 2F-G: AUC values from ER Ca2+ release (F) and from
 subsequence bulk Ca2+ produced upon addition of Ca2+ (G), from all cells
 from the experiments listed above. **Figure 2H-J README** Figures H-J are
 from the data files listed below. In each file, there are three tabs: \-
 Raw fura-2 ratio. SICS was induced at 200s. Max ratio was achieved in
 individual cells by addition of ionomycin & high CaCl2 concentration
 at the end of the experiments. Per our lab’s practice, signals from cells
 overexpressing WT or mutant STIM1 were selected first, constituting the
 first half of the data traces, followed by equal numbers of cells in the
 same microscopic field that did not overexpress WT or mutant STIM1. \-
 Fractional fura-2 ratio: calculated from raw fura-2 ratio \- Traces and
 AUCs: time courses of individual traces. Area under the curve values of
 the SICS signal (typically from 200 – 300s) are on top of each trace.
 Effect of WT STIM1 expression on SICS: \- 24218001 STIM1wt SICS (1) \-
 24219000 STIM1wt SICS (2) \- 24219001 STIM1wt SICS (3) \- 24219002 STIM1wt
 SICS (4) \- 24227004 STIM1wt SICS (5) Effect of STIM1CBL expression on
 SICS: \- 24218002 STIM1dCBL SICS (1) \- 24218003 STIM1dCBL SICS (2) \-
 24218004 STIM1dCBL SICS (3) \- 24219005 STIM1dCBL SICS (4) \- 24219008
 STIM1dCBL SICS (5) \- 24219009 STIM1dCBL SICS (6) \- 24227005 STIM1dCBL
 SICS (7) Figure 2H: average time course from 24227004 STIM1wt SICS (5)
 Figure 2I: average time course from 24227005 STIM1dCBL SICS (7) Figure 2J:
 AUC values from all experiments listed above. **Figure 2K-L README** Data
 show the effect of ML-9 on SICS. Data for Fig. 2K-L come from the
 following files: CONTROL: -       24o28000 CTL SICS (1) -       24n07000
 CTL SICS (2) -       24n14000 CTL SICS (3) -       24d03000 CTL SICS (4)
 ML-9: -       24o28003 ML-9 SICS (1) -       24n05000 ML-9 SICS (2)
 -       24n14001 ML-9 SICS (3) -       24d03001 ML-9 SICS (4) In each
 file, there are 3 tabs: -       Raw fura-2 ratio: SICS was induced at
 200s, at the end of the experiments, ionomycin and high concentration of
 CaCl~2~ were added to obtain Rmax in all cells. -       Fractional fura-2
 ratio: fractional ratios converted from raw ratio. Rmin and Rmax in
 individual cells are calculated as described in manuscript. -       Traces
 & AUCs: area under the curve calculated for SICS signal in each trace.
 Fig. 2K: representative average traces from CTL and ML-9 experiments Fig.
 2L: combined AUC values from all experiments. **Figure 3B-D README**
 Figure 3B: Data are representative of the following experimental files: \-
 24723005-SICR-CTL (1) \- 24730000-SICR-CTL (2) \- 24730002-SICR-CTL (3)
 Figure 3C: Data are representative of the following experimental files: \-
 24723001-TGN-SICR (1) \- 24723004-TGN-SICR (2) \- 24723006-TGN-SICR (3)
 Each file contains 3 tabs: \- Raw fura-2 ratios \- Fractional fura-2
 ratios: calculated from raw ratio as described in manuscript. \- Traces
 and SICR AUCs: time courses of fura-2 signal in individual cells up to
 ~1000s, prior to the addition of ionomycin and calcium to obtain Rmax
 values in individual cells (described in Materials and Methods). Areas
 under the curves calculated for any signals following nutrient removal at
 700s. Negative AUC values indicate that the signal after nutrient removal
 falls below the value immediately before that. Figure 3D: Data are in
 excel file “Fig. 3D”. **FIGURE 3E-G README** Figure 3E is representative
 of the two experimental files: \- 23512000-MLSA1-TGN (1) \-
 23512001-MLSA1-TGN (2) \- Experiment 23509000 MLSA1 without TGN (3) did
 not have the TGN addition component but also showed the effect of 100 µM
 MLSA1 to release Ca2+ within the same time frame and was included in the
 analysis of MLSA1-induced Ca2+ release (Fig. 3G). As indicated in the
 figure and methods, in these experiments, MLSA1 (100 µM) was applied in
 Ca2+-chelated DMEM at 200s. TGN was added at 700s, followed by addition of
 high concentration of ionomycin and Ca2+ at 1100s to obtain Rmax values in
 individual cells. Each experimental file contains 3 tabs: \- Raw fura-2
 ratio \- Fractional ratio calculated from the raw ratio \- Traces &
 AUCs: Areas under the curve from 200-700s (MLSA1-induced Ca2+ release
 signal prior to addition of TGN. Figure 3F: is representative of the
 following experimental files. In these experiments, TGN was applied in
 Ca2+-chelated DMEM at 200s, followed by addition of 100 µM MLSA1 at 700s.
 High ionomycin and CaCl2 concentrations were added at 1200s to obtain Rmax
 values in individual cells. Each file contains 3 tabs: \- Raw fura-2 ratio
 \- Fractional ratio calculated from the raw ratio \- Traces & AUC:
 area under the curve values from 700 – 1200s (equal duration for the
 calculation of MLSA1-induced Ca2+ release in Fig. 3E). Many of these AUC
 values are negative, indicating absence of any Ca2+ release by addition of
 MLSA1 after TGN application and further reduction of fura-2 signal beyond
 700s. Three files: \- 23517001-TGN-MLSA1 (1) \- 23517003-TGN-MLSA1 (2) \-
 23517005-TGN-MLSA1 (3) Figure 3G: relative AUC values from these
 experiments. **Figure 3H-I README** Figure 3H: DATA from the following
 files. CTL SICS: -       24208007-CTL-SICS (1) -       24214077-CTL-SICS
 (2) -       24214079-CTL-SICS (3) -       24214081-CTL-SICS (4) ML-SI3
 SICS: -       24208005-10 µM ML-SI3-SICS (1) -       24214076-10 µM
 ML-SI3-SICS (2) -       24214078-10 µM ML-SI3-SICS (3) -       24214080-10
 µM ML-SI3-SICS (4) Cells were treated with vehicle or 10 µM ML-SI3 at 200s
 of recording. SICS was induced at 1000s. High concentrations of ionomycin
 and CaCl~2~ were added at 1500s to obtain Rmax. Each file contains 3 tabs:
 -       Raw fura-2 ratio -       Fractional fura-2 ratio: converted from
 raw fura-2 ratio as described in manuscript. -       Traces & AUC of
 SICS: areas under the curve for individual traces for SICS signal. FIGURE
 3I: combines all AUC values from CTL and ML-SI3 experiments, Grubbs’ test
 was applied to eliminate outliers. Figure 4A-B: Tiff files are included
 for \- Fig. 4A Ponceau-S image (left image) \- Fig. 4A IB TRPML1 (left
 image) \- Fig. 4B full Ponceau-S image \- Fig. 4B full IB TRPML1 image
 with annotations as in figure. Figure 4C: are from the following files:
 -       25724000 CTL SICS (1) -       25724003 CTL SICS (2) -      
 25724006 CTL SICS (3) -       25724002 DsiRNA2 SICS (1) -       25724004
 DsiRNA2 SICS (2) -       25724007 DsiRNA2 SICS (3) -       25724001
 DsiRNA3 SICS (1) -       25724005 DsiRNA3 SICS (2) Each file contains 3
 tabs: -       Raw fura-2 ratio -       Fractional ratio: calculated from
 raw fura-2 ratio & Rmax min values for individual cells -       Traces
 & AUCs of SICS in individual cells. Figure 4D: combined AUC values
 from the above experiments. Values were subjected to Grubbs’ test to
 eliminate outliers. Figure 5D-F README Files Figure 5D, Figure 5E and
 Figure 5F contains spectrofluorometric data for -       BS-TRPML1wt at
 baseline (no CaM) and 30 µM CaM in the presence of 250 µM CaCl~2~ -      
 BS-TRPML1wt at baseline (no CaM) and 30 µM CaM in the absence of CaCl~2~.
 -       BS-TRPML1qm at baseline (no CaM) and 30 µM CaM in the presence of
 250 µM CaCl~2~. **Figures 6A-C**: included are -        16-bit image of
 TRPML1wt-mKate2 -        RGB image of TRPML1wt-mKate2 -        16-bit
 image of fura-2 loaded in the same cells -        RGB image of fura-2
 loaded in the same cells. -        Merged RGB images of fura-2 and
 TRPML1wt-mKate2 images Figures 6G-H: Data are separated into two
 subfolders: WT ML1 (9 individual experiments) and QM ML1 (7 individual
 experiments) Each file contain multiple tabs: \- Raw ratios: Per our
 laboratory routine, regions of interest (ROIs) for cells that
 heterologously expressed TRPML1 WT or TRPML1qm were selected first,
 followed by selection of equal numbers of same-field non-transfected cells
 (SFNT). Therefore, the first half of raw ratio columns are from
 transfected cells, and the second half, from same-field non-transfected
 cells. \- Fractional ratios: calculated from raw ratios, using formulas
 previously established. \- Traces: fractional data from 0 – 700s (without
 the Rmax portion) are used. “WT ML1” folder includes the following
 experiments: \- 24605002-ML1wt-SICS (1) \- 24607002-ML1wt-SICS (2) \-
 24705000-ML1wt-SICS (3) \- 24708002-ML1wt-SICS (4) \- 24711073-ML1wt-SICS
 (5) \- 24719001-ML1wt-SICS (6) \- 24719002-ML1wt-SICS (7) \-
 24719004-ML1wt-SICS (8) \- 24802002ML1wt-SICS (9) File Fig. 6G contains
 average and SEM for SFNT time course and WT ML1 time course in figure. “QM
 ML1” folder includes the following experiments: \- 24605001-qmML1-SICS (1)
 \- 24605003-qmML1-SICS (2) \- 24607003-qmML1-SICS (3) \-
 24705001-qmML1-SICS (4) \- 24705004-qmML1-SICS (5) \- 24708001-qmML1-SICS
 (6) \- 24717001-qmML1-SICS (7) File Fig. 6H contains average and SEM for
 SFNT time course and QM ML1 time course in figure. README – Figure 7
 **Fig. 7A.** Responses of ML1*~WT~*-GG and ML1*~QM~*-GG biosensors to
 nutrient removal. ML1*~WT~*-GG data are from the following experimental
 files: -        25902000 ML1wt-GG (1) -        25902002 ML1wt-GG (2)
 -        25902006 ML1wt-GG (3) -        25904006 ML1wt-GG (4) -       
 25904007 ML1wt-GG (5) ML1*~QM~*-GG data are from the following
 experimental files: -        25902001 ML1qm-GG (1) -        25902003
 ML1qm-GG (2) -        25902005 ML1qm-GG (3) -        25904001 ML1qm-GG (4)
 -        25904002 ML1qm-GG (5) -        25904003 ML1qm-GG (6) -       
 25904004 ML1qm-GG (7) -        25904005 ML1qm-GG (8) Each file contains 3
 tabs: -        Raw: EGFP fluorescence intensity -        F/Fo: data is
 normalized to baseline fluorescence intensity -        Traces & AUC:
 areas under the curve in individual cells calculated from the induction of
 nutrient removal ## Code/software Any program that will open a
 spreadsheet, such as Excel is recommended. TIFF- can be viewed using Paint
 or Paint 3D or any Tif file viewer ## Access information Other publicly
 accessible locations of the data: * None Data was derived from the
 following sources: * None