Dataset DOI: 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 CaCl₂ 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 CaCl₂ 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 CaCl₂

-       BS-TRPML1wt at baseline (no CaM) and 30 µM CaM in the absence of CaCl₂.

-       BS-TRPML1qm at baseline (no CaM) and 30 µM CaM in the presence of 250 µM CaCl₂.

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