Figure 1. The proton gradient of the lysosome is not required for lysosomal Ca²⁺ store refilling.

(A) In HEK293 cells stably expressing GCaMP3-ML1 (HEK-GCaMP3-ML1 cells), bath application of the ML1 channel agonist ML-SA1 (20 µM) in a low or 'zero' Ca²⁺ (free [Ca²⁺]<10 nM) external solution induced an increase in GCaMP3 fluorescence (F₄₇₀). After washout for 5 min, repeated applications of ML-SA1 induced responses that were similar to or larger than the first one. Note that because baseline may drift during the entire course of the experiment (up to 20 min), we typically set F₀ based on the value that is closest to the baseline. (B) The average Ca²⁺ responses of three ML-SA1 applications at intervals of 5 min (n=26 coverslips; Figure 1—source data 1). (C) Pre-treatment with lysosome-disrupting agent GPN for 30 min abolished the response to ML-SA1 in HEK-GCaMP3-ML1 cells. Washout of GPN resulted in a gradual re-appearance of ML-SA1 responses. See quantitation in Figure 1—figure supplement 2K. (D) Repeated applications of GPN resulted in Ca²⁺ release that was measured with the Ca²⁺-sensitive dye Fura-2 (F₃₄₀/F₃₈₀) in non-transfected HEK293T cells. (E) Application of Bafilomycin-A (Baf-A, 5 µM) and Concanamycin-A (Con-A, 1 µM) quickly (<5 min) abolished LysoTracker staining, an indicator of acidic compartments. (F) Acute application of Baf-A (5 µM) for 5 min did not block Ca²⁺ refilling of lysosomes in HEK-GCaMP3-ML1 cells. (G) Prolonged pre-treatment (3 hr) with Baf-A did not block Ca²⁺ refilling of lysosomes. (H) Quantification of 1^st (p value = 0.11), 2^nd (p=0.01), and 3^rd (p=0.004) ML-SA1 responses upon Baf-A treatment (n=8) compared to control traces (n=6; Figure 1—source data 1). (I) Prolonged treatment (1 hr) with Con-A did not prevent lysosomes from refilling their Ca²⁺ stores. (J) Quantification of 1st (p=0.90), 2nd (p=0.33), and 3rd (p=0.66) ML-SA1 responses with Con-A pre-treatment (n=3; Figure 1—source data 1). (K) Con-A did not reveal differences in Ca²⁺ refilling responses to repeated applications of GPN in untransfected HEK293T cells. Panels A, C, D, F, G, I, and K are the average of 30–40 cells from one representative coverslip/experiment. The data in panels B, H, and J represent mean ± SEM from at least three independent experiments.

DOI: http://dx.doi.org/10.7554/eLife.15887.002

Figure 1—figure supplement 1. A lysosome-targeted genetically-encoded Ca²⁺ indicator to measure lysosomal Ca²⁺ release, store depletion, and refilling.

(A) Detection of lysosomal Ca²⁺ release by a genetically-encoded Ca²⁺ indicator (GCaMP3) fused directly to the N-terminus of ML1 (GCaMP3-ML1). (B) Co-localization analyses between GCaMP3-ML1 and various organellar markers, including Lamp1-mCherry, CFP-ER, Mito-tracker, and EEA1-mCherry. Scale bars = 5 μm. (C) In an HEK-GCaMP3-ML1 cell, both ML-SA1 and subsequent ionomycin induced GCaMP3 fluorescence increases in lysosomes shown by both fluorescence imaging and Ca²⁺ imaging. (D) BAPTA-AM pre-treatment abolished ML-SA1-induced responses in HEK-GCaMP3-ML1 cells. Panel C shows the average response of 30–40 cells from one representative experiment. (E) Cos7 cells transfected with GCaMP3-ML1 show strong co-localization with LysoTracker, which is highly selective for acidic organelles.

DOI: http://dx.doi.org/10.7554/eLife.15887.004

Figure 1—figure supplement 2. An assay to monitor lysosomal Ca²⁺ store depletion and refilling.

(A) Raw traces of ML-SA1-induced GCaMP3 Ca²⁺ responses of individual HEK-GCaMP3-ML1 cells on one coverslip. (B) Immediate re-application of ML-SA1 showed a nearly-abolished lysosomal Ca²⁺ release. (C) In HEK293T cells transfected with surface-expressed TRPML1-4A channels, repeated applications of ML-SA1 induced comparable responses. (D) After 1 min refilling time, application of ML-SA1 in HEK-GCaMP3-ML1 cells induced responses that were smaller than the first one. (E) The amount of Ca²⁺ released after 8 min of washout and refilling was similar to the amount released after 5 min. (F) Time-dependence of lysosomal Ca²⁺ store refilling. (G) In the presence of La³⁺ (100 μM), a membrane-impermeable TRPML blocker, GPN pretreatment abolished ML-SA1-induced responses in HEK-GCaMP3-ML1 cells. (H) Lysosomal Ca²⁺ refilling in the presence of La³⁺. (I) ML-SI3 (5 μM) reversibly inhibited ML-SA1-induced Ca²⁺ responses in HEK-GCaMP3-ML1 cells. (J) Normalized ML-SA1 responses with and without co-application of MI-SI3. (K) Quantification of ML-SA1 responses shown in Figure 1C. (L) Lysosomal Ca²⁺ refilling in human fibroblasts transfected with GCaMP3-ML1. (M) Lysosomal Ca²⁺ refilling in Cos-7 cells transfected with GCaMP3-ML1. Panel B, D, E, G, H, I, L and M show the average response of 30–40 cells from one representative experiment out of at least independent repeats.

DOI: http://dx.doi.org/10.7554/eLife.15887.005

Figure 1—figure supplement 3. GPN and ML-SA1 have different effects on lysosome pH and GCaMP3 fluorescence.

(A) LysoTracker staining was not affected by ML-SA1 (20 μM), but was abolished by GPN (400 μM). Scale bar = 15 µm. (B) Pretreatment of a membrane-permeable form of Ca²⁺ chelator BAPTA (BAPTA-AM) for 2h abolished Fura-2 response to ATP in HEK293T cells. GPN still induced small increases in the Fura-2 signal in the same cells (also see panel C). (C) ATP and GPN Fura-2 responses in HEK293T cells. (D) GPN (400 μM) induced increases of GCaMP3 fluorescence in HEK-GCaMP3-ML1 cells that were pre-treated and kept in continuous presence of BAPTA-AM, and the increases were abolished by Baf-A1 co-treatment. Note that ionomycin was still able to induce GCaMP3 increases. Panels D show the average response of 30–40 cells from one representative experiment. (E) Representative traces showing GCaMP3 sensitivities to patch-electrode-based 'puffing' of low-pH and high-Ca²⁺ solutions to GCaMP3-ML1-expressing vacuoles isolated from HEK-GCaMP3-ML1 cells.

DOI: http://dx.doi.org/10.7554/eLife.15887.006

Figure 1—figure supplement 4. Inhibition of PI(3,5)P₂ production does not prevent lysosomal Ca²⁺ refilling.

Representative Ca²⁺ imaging traces showing lysosomal Ca²⁺ refilling in GCaMP3-ML1 cells pretreated with Apilimod (A) or YM201636 (B). Panels A and B show the average response of 30–40 cells from one representative experiment.

DOI: http://dx.doi.org/10.7554/eLife.15887.007