Figure 3. IP3-receptors on the ER are required for lysosomal Ca²⁺ store refilling.

(A) The IP3-receptor (IP3R) antagonist Xestospongin-C (Xesto, 10 μM) prevented Ca²⁺ refilling of lysosomes in HEK-GCaMP3-ML1 cells (p=0.007). Note that Xesto was co-applied with ML-SA1. (B) Ryanodine (100 μM), which blocks Ryanodine receptors at high concentrations, did not block Ca²⁺ refilling to lysosomes. Note that Ryanodine was co-applied with ML-SA1. (C) Quantification of the responses to ML-SA1 in HEK-GCaMP3-ML1 cells after treatment with Xesto, 2-APB (Figure 3—figure supplement 1K), U73122 (Figure 3—figure supplement 1L,M), Ryanodine (Ry), and DHBP (Figure 3—figure supplement 2A) (Figure 3—source data 1).(D) DT40 WT cells transiently transfected with GCaMP3-ML1 show Ca²⁺ refilling. (D’) IP3R antagonist Xesto completely blocked Ca²⁺ refilling of lysosomes in DT40 WT cells. (E) DT40 IP3R triple KO (TKO) cells transiently transfected with GCaMP3-ML1 also show Ca²⁺ refilling. (E’) Xesto did not block Ca²⁺ refilling of lysosomes in IP3R-TKO cells. (F) Quantification of ML-SA1 responses with or without Xesto in WT and IP3R-TKO DT40 cells (Figure 3—source data 1). (G) Representative images showing the effects of Xesto on the recovery of ML-SA1-induced responses in HEK-ML1 stable cells loaded with OG-BAPTA-dextran. La³⁺ was used to block external Ca²⁺ influx that could be mediated by surface-expressed ML1 in the overexpression system (see Figure 1—figure supplement 2G). (H) The effects of TG and Xesto on intralysosomal Ca²⁺ contents measured by OG-BAPTA-dextran (Figure 3—source data 1). (I) The effects of ML-SA1 on [Ca²⁺]_Ly measured by OG-BAPTA-dextran. Panels A, B, D, D’, E, E’, F, F’ and H are the average of 30–40 cells from one representative experiment. The data in panels C, F and H represent mean ± SEM from at five independent experiments. The scale bar in panel G = 10 μm.

DOI: http://dx.doi.org/10.7554/eLife.15887.012

Figure 3—figure supplement 1. ER IP3-Receptors regulate Ca²⁺ refilling of lysosomes.

(A) Xesto application (5 min) blocked ER Ca²⁺ release in HEK293T cells loaded with Fura-2. (B) After 5 min of refilling, which is expected to fully refill the lysosomal Ca²⁺ stores, acute treatment of Xesto (10 μM) for 2 min did not significantly reduce lysosomal Ca²⁺ release. Lysosomal Ca²⁺ release was induced by ML-SA1 in HEK-GCaMP3-ML1 cells. (C) After 5 min of refilling of lysosomal Ca²⁺ stores, subsequent acute treatment of Xesto (10 μM) for 5 min slightly reduced lysosomal Ca²⁺ release. (D) After 5 min of refilling of lysosomal Ca²⁺ stores, acute treatment of Xesto (10 μM) for 10 min abolished lysosomal Ca²⁺ release. (E) Time-dependent depletion of lysosomal Ca²⁺ stores by pharmacological inhibition of IP3-receptors. (F, G) In contrast to control (Figure 1C), application of Xesto dramatically reduced Fura-2 responses to GPN in GCaMP3-ML1 cells loaded with Fura-2 (Figure 3— source data 1). (H) Fura-2 Ca²⁺ imaging of GPN responses in MEF cells. (I) Xesto application dramatically reduced the second response to GPN in MEF cells. (J) Average effects of Xesto on lysosomal Ca²⁺ refilling in MEF cells (Figure 3—source data 1). (K) IP3R antagonist 2-APB (200 μM) blocked lysosomal Ca²⁺ refilling (p=0.013; also see Figure 3C). (L) PLC inhibitor U73122 (10 μM) blocked Ca²⁺ release from IP3Rs stimulated by ATP. (M) U73122 treatment abolished Ca²⁺ refilling of lysosomes (p=0.0070). Panels A–D, F, H, I, K, L and M show the average response of 30–40 cells from one representative experiment.

DOI: http://dx.doi.org/10.7554/eLife.15887.014

Figure 3—figure supplement 2. Lysosomal Ca2+ refilling is compromised in IP3R TKO DT40 cells.

(A) Ryanodine receptor blocker DHBP (50 μM) did not block Ca²⁺ refilling of lysosomes. (B) Quantification of the 1st, 2nd and 3rd ML-SA1 responses in GCaMP3-ML1-transfected WT and IP3R-TKO DT40 cells (Figure 3—source data 1). (C) Time-dependence of lysosomal Ca²⁺ store refilling in WT and IP3R TKO DT40 cells. (D) GCaMP3-ML1-transfected IP3R-TKO DT40 cells still showed refilling after 5 min of DHBP application to block RYRs. (E) RYR inhibitors Diltiazem (50 µM) and Dantrolene (50 µM) did not block lysosomal Ca²⁺ refilling in GCaMP3-ML1-transfected IP3R-TKO DT40 cells. Panels A, D and E show the average response of 30–40 cells from one representative experiment.

DOI: http://dx.doi.org/10.7554/eLife.15887.015

Figure 3—figure supplement 3. Measuring lysosomal Ca²⁺ release with lysosome-targeted luminal Ca²⁺ indicators.

(A) Fura-Dextran was pulse/chased into HEK293T cells transfected with Lamp1-mCherry. Fura-Dextran dyes were co-localized well with Lamp1-mCherry after 12 hr pulse and 4 hr chase, although not all lysosomes were loaded with the dye, evidenced by many Lamp1-mCherry vesicles without Fura-Dextran co-localization. Scale bar = 5 μm. (B) OG-BAPTA-dextran displayed better loading to lyssosomes and a high level of co-localization with LysoTracker. Scale bar =10 μm. (C) pH-dependence of the measured K_d values for OG-BAPTA-dextran. (D) Compared with the control, Xesto (25 μM) treatment for 5 min prevented Ca²⁺ refilling to lysosomes measured with Fura-Dextran, a lysosome-targeted luminal Ca²⁺ indicator. Right panels show the zoom-in images of ML-SA1-induced responses before and after Xesto treatment. (E) Quantification of ML-SA1 responses with or without Xesto in cells loaded with Fura-Dextran. Xesto significantly (p=0.026) blocked refilling as shown by no response to ML-SA1 after Xesto application during refilling (n=3; Figure 3— source data 1). (F) Representative images showing the effect of TG treatment on the recovery of ML-SA1-induced responses in OG-BAPTA-dextran loaded HEK-ML1 stable cells. (G) Average effects of TG on lysosomal Ca²⁺ refilling in OG-BAPTA-dextran loaded HEK-ML1 cells. Lysosomal Ca²⁺ release was induced by ML-SA1 in zero Ca²⁺ external solution (Figure 3—source data 1). (H) The effects of TG treatment on intra-lysosomal Ca²⁺ levels, measured by OG-BAPTA-dextran imaging. (I) A calibration curve for the pH-sensitive dye OG-488-dextran. (J, K) Baf-A1 (5 μM; J) and NH₄Cl (10 mM; K) induced changes in both lysosomal pH and OG-BAPTA-dextran fluorescence.

DOI: http://dx.doi.org/10.7554/eLife.15887.016