The endoplasmic reticulum, not the pH gradient, drives calcium refilling of lysosomes

Abstract

Impaired homeostasis of lysosomal Ca(2+) causes lysosome dysfunction and lysosomal storage diseases (LSDs), but the mechanisms by which lysosomes acquire and refill Ca(2+) are not known. We developed a physiological assay to monitor lysosomal Ca(2+) store refilling using specific activators of lysosomal Ca(2+) channels to repeatedly induce lysosomal Ca(2+) release. In contrast to the prevailing view that lysosomal acidification drives Ca(2+) into the lysosome, inhibiting the V-ATPase H(+) pump did not prevent Ca(2+) refilling. Instead, pharmacological depletion or chelation of Endoplasmic Reticulum (ER) Ca(2+) prevented lysosomal Ca(2+) stores from refilling. More specifically, antagonists of ER IP3 receptors (IP3Rs) rapidly and completely blocked Ca(2+) refilling of lysosomes, but not in cells lacking IP3Rs. Furthermore, reducing ER Ca(2+) or blocking IP3Rs caused a dramatic LSD-like lysosome storage phenotype. By closely apposing each other, the ER may serve as a direct and primary source of Ca(2+)for the lysosome.

Keywords: ER; calcium; cell biology; lysosome; mouse; neuroscience.

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

Figure 2.. Lysosomal Ca²⁺ refilling is dependent on the endoplasmic reticulum (ER) Ca²⁺.

(A) Ca²⁺ refilling of lysosomes requires external Ca²⁺. (B) Dissipating the ER Ca²⁺ gradient using SERCA pump inhibitor Thapsigargin (TG) blocked lysosomal Ca²⁺ refilling in HEK-GCaMP3-ML1 cells. Three representative cells from among 30–40 cells on one coverslip are shown. Note that Ca²⁺ release from the ER through passive leak revealed after blocking SERCA pumps was readily seen in HEK-GCaMP3-ML1 cells, presumably due to the close proximity between lysosomes and the ER (Kilpatrick et al., 2013). (C) The effect of acute application of TG (2 μM) on the naïve ML-SA1 response and lysosomal Ca²⁺ refilling in HEK-GCaMP3-ML1 cells. Application of TG did not affect the naïve, initial response to ML-SA1, but did abolish the refilled response (see arrow). Control naïve response 1.39 ± 0.09 (n=3); Naïve response after TG 1.08 ± 0.07 (n=3); p=0.2024). (D) LysoTracker staining was not reduced by TG (2 μM). (E) Representative Ca²⁺ imaging trace and statistical data (right panel; Figure 2—source data 1) show that TG application reduced the second responses to GPN compared to the control shown in Figure 1D. (F) Chelating ER Ca²⁺ using 2-min TPEN treatment blocked Ca²⁺ refilling of lysosomes. (G) TG (p=0.008; n=5) and TPEN (p=0.001; n=5) abolished Ca²⁺ refilling of lysosomes (Figure 2—source data 1). (H) In HEK-GCaMP3-ML1 cells that were transiently transfected with the IP3R-ligand binding domain with ER targeting sequence (IP3R-LBD-ER), which significantly reduces basal [Ca²⁺]_ER (see Figure 2—figure supplement 2E), ML-SA1 responses were reduced, compared to untransfected cells on the same coverslip. (I) The 1st (p=0.0014), 2nd (p=0.0004), and 3rd responses (p<0.0001) of GCaMP3-ML1 cells transfected with the IP3R-LBD-ER were significantly reduced compared to untransfected cells on the same coverslip (n=5; Figure 2—source data 1). (J) Lysosomes (labeled with Lamp1-mCherry) interact closely with the ER (labeled with CFP-ER). (K) Time lapse zoomed-in images of a selected region from J show the dynamics of ER-lysosome association (see an example in the boxed area). Panels A, F, H are the average responses of 30–40 cells from one representative experiment. The data in panel G represent mean ± SEM from five independent experiments. DOI: http://dx.doi.org/10.7554/eLife.15887.008

Figure 2—figure supplement 1.. The ER Ca²⁺ store regulates lysosome Ca²⁺ stores.

(A) The effect of Brefeldin-A (100 nM) pretreatment on lysosomal Ca²⁺ refilling in HEK-GCaMP3-ML1 cells. (B) The effect of La³⁺ (100 μM) pre-treatment on lysosomal Ca²⁺ refilling in HEK-GCaMP3-ML1 cells. (C) The response to the endogenous P2Y receptor agonist ATP in HEK293T cells loaded with Fura-2 was abolished after perfusing cells with 0 external Ca²⁺ for 5 min. (D, E) SERCA pump inhibitor cyclopiazonic acid (CPA) (100 μM) blocked Ca²⁺ refilling of lysosomes (Figure 2—source data 1). (F, G) CPA markedly reduced the response to GPN in the Fura-2-loading cells. Note that the remaining response could be due to GPN-induced lysosomal H⁺ release, which resulted in a change of pre-lysosomal pH that may in turn caused a Fura-2 signal (Figure 2—source data 1). (H) Tunicamycin (0.1 μg/ml) application did not affect the second responses to ML-SA1 in GCaMP3-ML1 cells. (I) Tunicamycin (0.5 μg/ml) did not induce lysosomal Ca²⁺ releases. Panels A–I and K show the average response of 30–40 cells from one representative experiment. DOI: http://dx.doi.org/10.7554/eLife.15887.010

Figure 2—figure supplement 2.. The ER Ca²⁺ store regulates lysosome Ca²⁺ stores.

(A) In un-transfected HEK293T cells, ATP induced Ca²⁺ release through IP3-receptors on the ER, and GPN induced lysosomal Ca²⁺ release. (B) A 2-min application of TPEN, a membrane-permeable chelator of luminal ER Ca²⁺, attenuated Ca²⁺ release from IP3-receptors stimulated by ATP in HEK293T cells. (C) A 2-min TPEN application did not significantly reduce GPN-induced lysosomal Ca²⁺ release in HEK293T cells. (D) Long-term TPEN treatment (20 min) abolished ER Ca²⁺ release upon ATP stimulation and GPN-induced lysosomal Ca²⁺ release in HEK293T cells loaded with Fura-2. (E) In HEK293T cells transfected with the IP3R-ligand binding domain with ER targeting sequence (IP3R-LBD-ER), the responses to ATP and GPN were reduced compared to un-transfected cells on the same coverslip. (F) Caffeine stimulates Ca²⁺ release from ryanodine receptors and ATP stimulates Ca²⁺ release from IP3Rs in HEK-GCaMP3-ML1 cells loaded with Fura-2. Panels A–C and E show the average response of 30–40 cells from one representative experiment. DOI: http://dx.doi.org/10.7554/eLife.15887.011

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

Figure 4.. Blocking ER IP3-receptors Ca²⁺ channels refill lysosome Ca²⁺ stores to prevent lysosomal dysfunction.

(A) Upper panels: Western blotting analyses of Lamp1 in HEK293T cells treated with 2-APB (50 μM), TPEN (0.1 μM), Xesto (10 μM), and DHBP (5 μM) compared to DMSO for 24 hr (n=4 separate experiments for each condition). Lower panel: treating HEK293T cells with 2-APB (p=0.05) and Xesto (p=0.013), as well as TPEN (p=0.02), significantly increased Lamp1 expression. DHBP did not significantly change Lamp1 expression (p=0.23) (Figure 4—source data 1). (B) The effects of Xesto (10 μM, 18 hr; p=0.0001) and DHBP (50 μM, 18 hr; p=0.063) treatment compared to DMSO on the lysosomal compartments detected by LysoTracker staining in HEK293T cells (average of 20–30 cells in each of 3 experiments; Figure 4—source data 1). Scale bar = 15 μm. (C) The effect of Xesto (10 μM, 18 h) treatment on accumulation of the autofluorescent lipofuscin materials in non-transfected HEK293T cells. Autofluorescence was observed in a wide spectrum but shown at two excitation wavelengths (488 and 561 nm). ML1 KO MEFs are shown for comparison. Scale bar = 15 μm. (D) A proposed model of Ca²⁺ transfer from the ER to lysosomes. The ER is a Ca²⁺ store with [Ca²⁺]_ER ~ 0.3–0.7 mM; lysosomes are acidic (pH_Ly ~ 4.6) Ca²⁺ stores ([Ca²⁺]_Ly ~ 0.5 mM). IP3Rs on the ER release Ca²⁺ to produce a local high Ca²⁺ concentration, from which an unknown low-affinity Ca²⁺ transport mechanism refills Ca²⁺ to a lysosome. Unidentified tether proteins may link the ER membrane proteins directly with the lysosomal membrane proteins to maintain contact sites of 20–30 nm for purposes of Ca²⁺ exchange. Ca²⁺ released from lysosomes or a reduction/depletion in [Ca²⁺]_Ly may, through unidentified mechanisms, 'promote' or 'stabilize' ER-lysosome interaction (Phillips and Voeltz, 2016; Eden, 2016). At the functional ER-lysosome contact sites, Ca²⁺ can be transferred from the ER to lysosomes through a passive Ca²⁺ transporter or channel based on the large chemical gradient of Ca²⁺ that is created when lysosome stores are depleted. Baf-A and Con-A are specific V-ATPase inhibitors; Xesto and 2APB are IP3R blockers; U73122 is a PLC inhibitor that blocks the constitutive production of IP3; DHBP and Ryanodine (>10 μM) are specific RyR blockers; TG and CPA are SERCA pump inhibitors; and TPEN is a luminal Ca²⁺ chelator. DOI: http://dx.doi.org/10.7554/eLife.15887.017