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

Abigail G Garrity; Wuyang Wang; Crystal MD Collier; Sara A Levey; Qiong Gao; Haoxing Xu

doi:10.7554/eLife.15887

. 2016 May 23;5:e15887. doi: 10.7554/eLife.15887

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

Abigail G Garrity ^1,^†, Wuyang Wang ^2,^†, Crystal MD Collier ², Sara A Levey ², Qiong Gao ², Haoxing Xu ^1,^2,^*

Editor: David E Clapham³

PMCID: PMC4909396 PMID: 27213518

Abstract

Impaired homeostasis of lysosomal Ca²⁺ causes lysosome dysfunction and lysosomal storage diseases (LSDs), but the mechanisms by which lysosomes acquire and refill Ca²⁺ are not known. We developed a physiological assay to monitor lysosomal Ca²⁺ store refilling using specific activators of lysosomal Ca²⁺ channels to repeatedly induce lysosomal Ca²⁺ release. In contrast to the prevailing view that lysosomal acidification drives Ca²⁺ into the lysosome, inhibiting the V-ATPase H⁺ pump did not prevent Ca²⁺ refilling. Instead, pharmacological depletion or chelation of Endoplasmic Reticulum (ER) Ca²⁺ prevented lysosomal Ca²⁺ stores from refilling. More specifically, antagonists of ER IP3 receptors (IP3Rs) rapidly and completely blocked Ca²⁺ refilling of lysosomes, but not in cells lacking IP3Rs. Furthermore, reducing ER Ca²⁺ 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 Ca²⁺for the lysosome.

DOI: http://dx.doi.org/10.7554/eLife.15887.001

Research Organism: Mouse

Introduction

A vacuolar-type H⁺-ATPase (V-ATPase) on the membrane of the lysosome maintains the acidic lumen (pH_Ly ~ 4.6), and improper acidification of lysosomes may lead to lysosomal storage diseases (LSDs) (Mindell, 2012). Like the Endoplasmic Reticulum (ER) (Clapham, 2007; Berridge, 2012), lysosomes are also intracellular Ca²⁺ stores with free [Ca²⁺]_Ly ~0.4–0.6 mM (Christensen et al., 2002; Lloyd-Evans et al., 2008), which is 3–4 orders of magnitude higher than the cytosolic [Ca²⁺] (~100 nM). A reduction in [Ca²⁺]_Ly is believed to be the primary pathogenic cause for some LSDs and common neurodegenerative diseases (Lloyd-Evans et al., 2008; Coen et al., 2012). Using the fast Ca²⁺ chelator BAPTA, Ca²⁺ release from the lysosome has been shown to be required for late endosome-lysosome fusion (Pryor et al., 2000), lysosomal exocytosis, phagocytosis, membrane repair, and signal transduction (Reddy et al., 2001; Lewis, 2007; Kinnear et al., 2004). Consistently, the principal Ca²⁺ channel in the lysosome, Mucolipin TRP channel 1 (TRPML1 or ML1), as well as lysosomal Ca²⁺ sensors such as the C2 domain–containing synaptotagmin VII, are also required for these functions (Steen et al., 2007; Lewis, 2007; Kinnear et al., 2004). Whereas human mutations of TRPML1 cause type IV Mucolipidosis, pathogenic inhibition of ML1 underlies several other LSDs (Shen et al., 2012).

How the 5000-fold Ca²⁺ concentration gradient across the lysosomal membrane is established and maintained is poorly understood. The most well understood Ca²⁺ store in the cell is the ER. Upon store depletion, the luminal sensor protein STIM1 oligomerizes to activate the highly Ca²⁺-selective ORAI/CRAC channels on the plasma membrane, refilling the ER Ca²⁺ store via the SERCA pump (Clapham, 2007; Lewis, 2007; Berridge, 2012). However, depletion of lysosomal Ca²⁺ stores does not induce extracellular Ca²⁺ entry (Haller et al., 1996b). The endocytic pathway may theoretically deliver extracellular Ca²⁺ to lysosomes. However, most Ca²⁺ taken up through endocytosis is lost quickly during the initial course of endosomal acidification prior to reaching lysosomes during endosome maturation (Gerasimenko et al., 1998). In various cell types, when the lysosomal pH gradient is dissipated, either by inhibiting the V-ATPase or by alkalizing reagents such as NH₄Cl, free luminal [Ca²⁺]_Ly was found to drop drastically (Calcraft et al., 2009; Christensen et al., 2002; Dickson et al., 2012; Lloyd-Evans et al., 2008; Shen et al., 2012), with no or very small concomitant increase in cytosolic Ca²⁺ (Christensen et al., 2002; Dickson et al., 2012). These findings have been interpreted to mean that the proton gradient in the lysosome is responsible for actively driving Ca²⁺ into the lysosome via an unidentified H⁺-dependent Ca²⁺ transporter (Morgan et al., 2011). Because these findings are consistent with studies in yeast showing that the Ca²⁺/H⁺ exchangers establish the vacuolar Ca²⁺ gradient (Morgan et al., 2011), this 'pH hypothesis' has been widely accepted (Calcraft et al., 2009; Christensen et al., 2002; Lloyd-Evans et al., 2008; Morgan et al., 2011; Shen et al., 2012). However, large, prolonged manipulations of luminal pH may interfere directly with Ca²⁺ reporters, and secondarily affect many other lysosomal processes, especially lysosome luminal Ca²⁺ buffering (Dickson et al., 2012), lysosome membrane potential, and fusion/fission of endosomes and lysosomes (Mindell, 2012). Therefore, these hypotheses about lysosomal Ca²⁺ refilling and store maintenance remain to be tested under more physiological conditions. Directly measuring lysosomal Ca²⁺ release has been made possible recently by using lysosome-targeted genetically-encoded Ca²⁺ indicators (Shen et al., 2012) (GCaMP3-ML1; see Figure 1—figure supplement 1A), which co-localized well, in healthy cells, with lysosomal associated membrane protein-1 (Lamp1), but not with markers for the ER, mitochondria, or early endosomes (Figure 1—figure supplement 1B).

Results

A physiological assay to monitor lysosomal Ca²⁺ refilling

Monitoring lysosomal Ca²⁺ store refilling requires direct activation of lysosomal Ca²⁺ channels with specific agonists to repeatedly induce Ca²⁺ release. NAADP, the only known endogenous Ca²⁺-mobilizing messenger that has been suggested to be lysosome-specific, was not useful due to its membrane impermeability and strong desensitization (Morgan et al., 2011). Using the specific, membrane–permeable synthetic agonists that we recently identified for lysosomal TRPML1 channels (ML-SA1) (Shen et al., 2012), we developed a lysosomal Ca²⁺ refilling assay as shown in Figure 1A. In HEK293 cell lines stably-expressing GCaMP3-ML1 (HEK-GCaMP3-ML1 cells), bath application of ML-SA1 (30s) in a 'zero' (low; free [Ca²⁺] <10 nM) Ca²⁺ external solution produced robust lysosomal Ca²⁺ release measured by GCaMP3 fluorescence (△F/F₀ >0.5; Figure 1A,B, Figure 1—figure supplement 1, Figure 1—figure supplement 2A). The membrane-permeable form of the fast Ca²⁺ chelator BAPTA (BAPTA-AM) completely blocked the ML-SA1 response (Figure 1—figure supplement 1D), supporting its Ca²⁺ specificity. Importantly, GCaMP3-ML1-tagged lysosomes co-localized well with LysoTracker, indicating that the pH of these lysosomes was not different from lysosomes without GCaMP3-ML1 (Figure 1—figure supplement 1E).

Figure 1. — (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—source data 1. Source data of Figure 1B, H, J: The average Ca²⁺ responses to ML-SA1 applications under control (B), Baf-A1 treatment (H), and Con-A treatment (J).
**DOI:** http://dx.doi.org/10.7554/eLife.15887.003

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DOI: 10.7554/eLife.15887.003

Figure 1—figure supplement 1. — (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—source data 1. Source data of Figure 1B, H, J: The average Ca²⁺ responses to ML-SA1 applications under control (B), Baf-A1 treatment (H), and Con-A treatment (J).
**DOI:** http://dx.doi.org/10.7554/eLife.15887.003

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DOI: 10.7554/eLife.15887.003

After release of the initial, 'naïve' Ca²⁺ store upon first application of ML-SA1, lysosomal Ca²⁺ stores are largely depleted, as immediate re-application of ML-SA1 evoked much smaller or no response (Figure 1—figure supplement 2B). The reduction in the second response was unlikely caused by channel desensitization, as surface-expressed TRPML1 mutant (TRPML1-4A [Shen et al., 2012]) showed repeated Ca²⁺ entry in Ca²⁺-containing (2 mM) external solution (Figure 1—figure supplement 2C). Notably, increasing the time interval between consecutive applications quickly and effectively restored the lysosomal ML-SA1 responses; it required 5 min for full restoration/refilling (Figure 1—figure supplement 2D–F). With 5 min of refilling time (chosen for the rest of our experiments), in healthy HEK-GCaMP3-ML1 cells, the second and third ML-SA1 responses are often slightly higher than the first, naïve response (Figure 1A,B).

To ensure the ML-SA1-induced Ca²⁺ responses are exclusively intracellular and lysosomal, all ML-SA1 responses were measured either in the 'zero' Ca²⁺ external solution (Figure 1A) or in the presence of La³⁺ (Figure 1—figure supplement 2G,H), a membrane-impermeable TRPML channel blocker (Dong et al., 2008) that is expected to completely inhibit surface-expressed TRPML1 channels. Ca²⁺ release was completely blocked by the TRPML-specific, synthetic antagonists ML-SI1 or ML-SI3 (Figure 1—figure supplement 2I,J). In addition, pretreatment with the lysosome-disrupting reagent Glycyl-L-phenylalanine 2-naphthylamide (GPN) (Berg et al., 1994) also completely abolished the refilling either in 'zero' Ca²⁺ or in the presence of La³⁺ (Figure 1C; Figure 1—figure supplement 2G), further supporting the lysosome-specificity of the response. The effect of GPN, presumably on so-called 'lysosomal membrane permeabilization', is known to be rapid and reversible (i.e. membrane 'resealing') (Kilpatrick et al., 2013). Consistently, washout of GPN led to gradual recovery of ML-SA1 responses (Figure 1C; Figure 1—figure supplement 2K). Similar Ca²⁺ refilling of lysosomes was also observed in GCaMP3-ML1-transfected human fibroblasts (Figure 1—figure supplement 2L), Cos-7 cells (Figure 1—figure supplement 2M), primary mouse macrophages, mouse myoblasts, and DT40 chicken B cells (Figure 3D–E’). These findings support the idea that these responses are mediated by intracellular Ca²⁺ release from refilled lysosomal stores (also see Figure 1—figure supplement 1C for signals from individual lysosomes). Taken together, these results ensure that lysosomal Ca²⁺ stores can be emptied and refilled repeatedly and consistently in a time-dependent manner.

Figure 3. — (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—source data 1. Normalized ML-SA1 responses or lysosomal Ca²⁺ contents under pharmacological (C, H) or genetic manipulations (F) (Figure 3C, F, H). .
The effects of Xesto on GPN responses in GCaMP3-ML1 (G) and MEF cells (J) (Figure 3—figure supplement 1G, J). ML-SA1 responses in GCaMP3-ML1-transfected WT and IP3R-TKO DT40 cells (Figure 3—figure supplement 2B). The effects of Xesto treatment on lysosomal Ca²⁺ changes induced by ML-SA1 (Figure 3—figure supplement 3E, G).

**DOI:** http://dx.doi.org/10.7554/eLife.15887.013

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DOI: 10.7554/eLife.15887.013

Figure 3—figure supplement 1. — (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—source data 1. Normalized ML-SA1 responses or lysosomal Ca²⁺ contents under pharmacological (C, H) or genetic manipulations (F) (Figure 3C, F, H). .
The effects of Xesto on GPN responses in GCaMP3-ML1 (G) and MEF cells (J) (Figure 3—figure supplement 1G, J). ML-SA1 responses in GCaMP3-ML1-transfected WT and IP3R-TKO DT40 cells (Figure 3—figure supplement 2B). The effects of Xesto treatment on lysosomal Ca²⁺ changes induced by ML-SA1 (Figure 3—figure supplement 3E, G).

**DOI:** http://dx.doi.org/10.7554/eLife.15887.013

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DOI: 10.7554/eLife.15887.013

Studying lysosomal Ca²⁺ refilling using a lysosome-specific 'membrane-permeabilizer'

GPN is a membrane-permeable di-peptide that is broken down by the lysosome-specific enzyme Cathepsin C. GPN causes permeabilization of lysosome membranes resulting from the accumulation of its breakdown products within lysosomes (Berg et al., 1994). Because it is a lysosome-specific membrane disrupting agent, it is often used to mobilize lysosome-specific Ca²⁺ stores (Jadot et al., 1984; Morgan et al., 2011; Berg et al., 1994; Haller et al., 1996a; Haller et al., 1996b). Using Fura-2 Ca²⁺ imaging in non-transfected HEK293T cells, repeated applications of GPN resulted in a response of similar magnitude to the first, suggestive of Ca²⁺ refilling (Figure 1D). Importantly, in HEK-GCaMP3-ML1 cells, pre-treatment with GPN or BAPTA-AM abolished the initial response to ML-SA1, confirming the GCaMP3-ML1 probe’s lysosome and Ca²⁺ specificity (Figure 1C).

The GPN-mediated 'membrane permeabilizaton' causes the leakage of small solutes including Ca²⁺ and H⁺ into the cytosol (Appelqvist et al., 2012), resulting in changes in the pH (see Figure 1—figure supplement 3A) and [Ca²⁺] in both the lysosome lumen and the peri-lysosomal (juxta-lysosomal) cytosol (Berg et al., 1994; Kilpatrick et al., 2013; Appelqvist et al., 2012). We therefore tested the Ca²⁺-specificity of GPN-induced increases on the Fura-2 and GCaMP3 signals. In cells pretreated with BAPTA-AM, whereas ER-mediated Ca²⁺ responses were abolished, GPN-induced Fura-2 increases were much reduced but not abolished (Figure 1—figure supplement 3B,C). Consistently, in HEK-GCaMP3-ML1 cells pre-treated with BAPTA-AM, GPN still induced a significant increase in GCaMP3 fluorescence. However, in these BAPTA-AM-treated cells, GPN-induced increases in GCaMP3 responses were completely abolished by a pre-treatment of Bafilomycin-A (Baf-A), a specific inhibitor of the V-ATPase (Morgan et al., 2011) (Figure 1—figure supplement 3D). Given that both Ca²⁺ dyes and GFP-based Ca²⁺ indicators are known to be sensitive to other ionic factors, particularly pH (Rudolf et al., 2003), GPN-induced changes in lysosomal and peri-lysosomal pH could directly or indirectly account for the BAPTA-insensitive GCaMP3 and residual Fura-2 signals. Consistent with this prediction, in the vacuoles isolated from HEK-GCaMP3-ML1 cells, GCaMP3 fluorescence was sensitive not only to high Ca²⁺, but also to low pH (Figure 1—figure supplement 3E). Because ratiometric dyes are less susceptible to pH changes (Morgan et al., 2015), in the Fura-2 assay, GPN may induce a large Ca²⁺ signal, but also a pH-mediated contaminating non-Ca²⁺ signal (compare Figure 1—figure supplement 3B with C).

The pH gradient and V-ATPase are not required for lysosome Ca²⁺ refilling

Next, we investigated the mechanisms underlying Ca²⁺ refilling of lysosomes. Inhibition of endocytosis using dynasore and organelle mobility using cytoskeleton inhibitors such as nocodazole and trichostatin A did not block refilling (data not shown). Furthermore, disruption of Golgi function using Brefeldin-A also had no effect on refilling (Figure 2—figure supplement 1A). Hence, in agreement with previous findings, the secretory and endocytic pathways are not directly involved in Ca²⁺ refilling. PI(3,5)P₂ is a lysosome-specific phosphoinositide that regulates multiple lysosomal channels and transporters including ML1 (Xu and Ren, 2015). Pharmacologically decreasing PI(3,5)P₂ levels using two small molecule PIKfyve inhibitors: YM201636 (Jefferies et al., 2008) and Apilimod (Xu et al., 2013) did not prevent lysosomal Ca²⁺ refilling (Figure 1—figure supplement 4A,B).

Previous findings have suggested that the pH gradient in the lysosome may be important to Ca²⁺ refilling (Xu and Ren, 2015; Morgan et al., 2011), however few studies have carefully investigated this possibility. Baf-A and Concanamycin-A (Con-A), inhibitors of the V-ATPase, increase the pH of the lysosome (Morgan et al., 2011), demonstrated by abolishing LysoTracker staining within minutes after application (Figure 1E). Surprisingly, acute application of Baf-A did not affect the response to ML-SA1, and had little effect on refilling (Figure 1F), nor did pretreatment of Baf-A for 1, 3 (Figure 1G,H), or 16 hr. Similarly, pretreatment with Con-A also had no effect on Ca²⁺ refilling of lysosomes (Figure 1I, J, K). These findings suggest that contradictory to previous conclusions, the pH gradient and V-ATPase may not be required for Ca²⁺ refilling, and that an alternative mechanism is responsible for supplying Ca²⁺ to lysosomes.

The Endoplasmic Reticulum (ER) Ca²⁺ store is required for lysosomal Ca²⁺ refilling

Lysosomal Ca²⁺ refilling was drastically reduced upon removal of extracellular Ca²⁺ during refilling time in HEK-GCaMP3-ML1 cells (Figure 2A). However, blocking Ca²⁺ entry using the generic cation channel blocker La³⁺ did not prevent refilling (Figure 2—figure supplement 1B). Because ER stores are passively, although slowly, depleted in 0 Ca²⁺ (Wu et al., 2006) (also see Figure 2—figure supplement 1C), given the demonstrated role of extracellular Ca²⁺ in ER store refilling (Lewis, 2007; Berridge, 2012), we investigated the role of the ER in lysosomal refilling. Thapsigargin (TG), a specific inhibitor of the ER SERCA pump (Thastrup et al., 1990), rapidly and completely abolished Ca²⁺ refilling to lysosomes (Figure 2B,G), but did not affect the first, naïve ML-SA1 response (Figure 2C; second response marked with arrow) or lysosomal pH (Figure 2D). In the GPN & Fura-2 assay that provides a reasonable (but not perfect; see above) measurement of lysosomal Ca²⁺ release independent of ML1, TG application also largely reduced the second GPN response (Figure 1D, 2E), which could be further reduced or abolished by Baf-A pretreatment. These results suggest that TG had no direct effect on the naïve Ca²⁺ store in lysosomes, but specifically and potently affected lysosomal Ca²⁺ refilling. A rapid and complete block of Ca²⁺ refilling was also observed for another SERCA pump inhibitor CPA (Figure 2—figure supplement 1D–G). TG may induce an unfolded protein response (UPR; Matsumoto et al., 2013). However, the UPR inducer Tunicamycin (Oslowski and Urano, 2011) did not affect refilling (Figure 2—figure supplement 1H,I).

Figure 2—figure supplement 1. — (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—source data 1. Comparisons of GPN (E) and ML-SA1 responses (G, I) under different pharmacological and genetic manipulations (Figure 2E,G,I).
Comparisons of responses to ML-SA1 (E) and GPN (G) (Figure 2—figure supplement 1E,G).

**DOI:** http://dx.doi.org/10.7554/eLife.15887.009

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DOI: 10.7554/eLife.15887.009

[Ca²⁺]_ER, but not cytosolic Ca²⁺, can be chelated by N,N,N',N'-Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a membrane-permeable metal chelator with a low affinity for Ca²⁺ (Hofer et al., 1998). Although TPEN may also enter the lysosomal lumen, the much reduced (>100 fold less) Ca²⁺-binding affinity in the acidic pH (pH_LY = 4.6) suggests that chelation of lysosomal Ca²⁺ would be minimal. Acute application of TPEN completely blocked lysosomal Ca²⁺ refilling (Figure 2F,G). A short application of TPEN also blocked ER Ca²⁺ release stimulated by the endogenous P2Y receptor agonist ATP (Figure 2—figure supplement 2B compared to Figure 2—figure supplement 2A), but not the lysosomal Fura-2 Ca²⁺ response stimulated by GPN (Figure 2—figure supplement 2C compared to Figure 2—figure supplement 2A). These findings suggest that chelation of ER Ca²⁺ stores using TPEN had no direct effect on the naïve Ca²⁺ store in lysosomes, but specifically and potently affected lysosomal Ca²⁺ refilling.

The ER Ca²⁺store can also be genetically and chronically reduced without raising intracellular Ca²⁺ levels by transfecting cells with the IP3R ligand-binding domain with an ER targeting sequence (IP3R-LBD-ER) (Várnai et al., 2005). As expected, IP3R-LBD-ER expression decreased ATP-induced IP3R-mediated Ca²⁺ release (Figure 2—figure supplement 2E). Interestingly, it also reduced the GPN induced lysosomal Ca²⁺ release in HEK293T cells (Figure 2—figure supplement 2E). Furthermore, in HEK-GCaMP3-ML1 cells transfected with IP3R-LBD-ER, lysosomal Ca²⁺ release was significantly reduced when compared to un-transfected cells on the same coverslip (Figure 2H,I). Collectively, these findings suggest that the ER, the major Ca²⁺ store in the cell, is essential for refilling and the ongoing maintenance of lysosomal Ca²⁺ stores, but not required for the naïve Ca²⁺ release from lysosomes.

A functional interaction between ER and lysosome Ca²⁺ stores was previously suggested (Haller et al., 1996a; 1996b), but these results have been largely ignored, presumably due to the lack of specific tools required for definitive interpretation. Recent findings have shown that as endosomes mature, they increase their contact with the ER (Friedman et al., 2013). Interestingly, the Ca²⁺ released from SERCA inhibition on the ER was detected on our GCaMP3-ML1 probe (Figure 2B,C, Figure 2—figure supplement 1D,F), likely due to close membrane contact between the ER and lysosomes (Eden, 2016). Similar detection of ER Ca²⁺ release by a genetically-encoded, lysosomally-targeted chameleon Ca²⁺ sensor utilizing lysosome membrane protein Lamp1 has also been reported (McCue et al., 2013). Using time-lapse confocal imaging, we found that the majority of lysosomes, marked by Lamp1-mCherry, move and traffic together with ER tubules, labeled with CFP-ER (Figure 2J,K). Thus, the ER could be the direct source of Ca²⁺ to lysosomes by forming nanojunctions with them (Eden, 2016).

IP3-receptors, not ryanodine receptors, on the ER are required for Ca²⁺ refilling of lysosomes

Ca²⁺ release from the ER is mediated primarily by two Ca²⁺ release channels, IP3Rs and ryanodine receptors (RYRs), both of which are expressed in HEK cells (Querfurth et al., 1998; Jurkovicova et al., 2008) (see also Figure 2—figure supplement 2F). Since IP3Rs are responsible for Ca²⁺ transfer to mitochondria (Hayashi et al., 2009), we examined whether IP3Rs on the ER were responsible for Ca²⁺ refilling of the lysosome. Notably, Ca²⁺ refilling of the non-naïve lysosome Ca²⁺ store was completely blocked by Xestospongin-C (Xesto; Figure 3A,C), a relatively specific IP3R blocker (Peppiatt et al., 2003) (Figure 3—figure supplement 1A–E). In addition, in the GPN & Fura-2 assay that provides a measurement of lysosomal Ca²⁺ release independent of ML1, blocking IP3 receptor by Xesto profoundly attenuated lysosomal Ca²⁺ refilling in both HEK-GCaMP3-ML1 cells and non-transfected mouse embryonic fibroblasts (MEF) cells (Figure 1D; Figure 3—figure supplement 1F–J). Acute application of Xesto after allowing lysosomal Ca²⁺ stores to refill for 5 min (hence stores are completely refilled and functionally equivalent to 'naïve' ones) also slowly (up to 10 min) reduced lysosomal Ca²⁺ release, suggesting that constitutive lysosomal Ca²⁺ release under resting conditions may gradually deplete lysosome Ca²⁺ stores if refilling is prevented (Figure 3—figure supplement 1B–E). Consistent with this hypothesis, long-term (20 min) treatment with the aforementioned ER Ca²⁺ manipulators including TG and TPEN almost completely abolished lysosomal Ca²⁺ release (Figure 2—figure supplement 2D), further supporting the interpretation that ongoing constitutive Ca²⁺ release and refilling requires ER Ca²⁺.

2-APB, a non-specific IP3R antagonist (Peppiatt et al., 2003), also blocked Ca²⁺ refilling (Figure 3C, Figure 3—figure supplement 1K). U73122 is a PLC inhibitor that blocks the constitutive production of IP3 (Cárdenas et al., 2010) and prevents ATP-induced IP3R-mediated Ca²⁺ release (Figure 3—figure supplement 1L). U73122 also completely prevented Ca²⁺ refilling of lysosomes (Figure 3C, Figure 3—figure supplement 1M), suggesting that basal production of IP3 is essential for Ca²⁺ refilling of lysosomes. In contrast, blocking the RyRs with high (>10 μM) concentrations of ryanodine (Figure 3B,C), or with the receptor antagonist 1,1′-diheptyl-4,4′-bipyridinium (DHBP) (Berridge, 2012) (Figure 3C, Figure 3—figure supplement 2A), did not affect Ca²⁺ refilling. Notably, co-application of RYR and IP3R blockers with the second ML-SA1 response did not change the amplitude of the response (Figure 3A,B). Together, these findings demonstrate that IP3Rs on the ER are specifically required for lysosomal Ca²⁺ refilling, but not for Ca²⁺ release from naïve stores or completely refilled stores.

In contrast with the pharmacological analyses described above, lysosomal store refilling occurred in both WT and IP3R triple KO (TKO) DT40 chicken B cells (Várnai et al., 2005; Cárdenas et al., 2010) that were transfected with GCaMP3-ML1 (Figure 3D–F, Figure 3—figure supplement 2B). However, unlike WT DT40 cells, in which the IP3R-specific antagonist Xesto completely blocked Ca²⁺ refilling (Figure 3D’, F), Xesto had no obvious blocking effect in IP3R-TKO cells (Figure 3E’, F). In addition, the kinetics of lysosomal refilling was markedly delayed in IP3R TKO cells compared with WT cells (Figure 3—figure supplement 2C). These results are consistent with the notion that in normal conditions, IP3Rs are the sole source of Ca²⁺ refilling of lysosomes. When IP3Rs are genetically deleted, however, IP3R-independent mechanisms contribute to lysosomal Ca²⁺ refilling, possibly as a consequence of genetic compensation. Refilling in IP3R-TKO DT40 cells was not blocked by RYR inhibitors (Figure 3—figure supplement 2D,E).

Studying lysosomal Ca²⁺ refilling using intra-lysosomal Ca²⁺ dyes

As an additional assay to directly 'monitor' intralysosomal Ca²⁺, we employed two intraluminal Ca²⁺ indicators Fura-Dextran and Oregon 488 BAPTA-1 dextran (OG-BAPTA-dextran) (Morgan et al., 2015). After being pulse/chased into ML1-mCherry-transfected HEK293T cells or HEK-ML1 stable cells, the dyes enter the lysosome lumen (Figure 3—figure supplement 3A,B) after endocytosis (Christensen et al., 2002). Due to their pH sensitivities, these dyes can detect intra-lysosomal Ca²⁺ ([Ca²⁺]_LY) changes, but preferentially only when the intra-lysosomal pH (pH_L) remains constant below pH 5.0 (Morgan et al., 2015) (see Figure 3—figure supplement 3C). In the Fura-Dextran-loaded ML1-mCherry-transfected HEK-293T cells, ML-SA1 application induced Ca²⁺ release from the lysosome lumen (Figure 3—figure supplement 3D). As we found in our GCaMP3-ML1 assay, Xesto abolished the ML-SA1-induced decrease in [Ca²⁺]_LY(Figure 3—figure supplement 3D,E). Likewise, in HEK-ML1 stable cells loaded with OG-BAPTA-dextran, which had a much higher efficiency in loading to the lysosome (Figure 3—figure supplement 3B), TG or Xesto treatment profoundly reduced lysosomal Ca²⁺ refilling (Figure 3G—I; Figure 3—figure supplement 3F,G). Note that LysoTracker staining was not significantly reduced by ML-SA1, TG, or Xesto, suggesting that the signals were primarily mediated by changes of intralysosomal Ca²⁺, not intralysosomal pH. In contrast, treatment of cells with Baf-A1 or NH₄Cl markedly increased lysosomal pH from 4.8 to 7.0 (Figure 3—figure supplement 3J,K). Such large pH elevations may cause dramatic changes in both K_d of OG-BAPTA-dextran (see Figure 3—figure supplement 3C) and luminal Ca²⁺ buffering capability (Morgan et al., 2015; Dickson et al., 2012), preventing accurate determinations of [Ca²⁺]_LY under these pH manipulations. Taken together, these results are consistent with the conclusions that were drawn based on the aforementioned ML-SA1 & GCaMP3-ML1 assay and the GPN & Fura-2 assay.

Inhibition of ER IP3R channels and Ca²⁺ release causes lysosomal dysfunction and a LSD-like phenotype

Lysosomal Ca²⁺ is important to lysosomal function and membrane trafficking (Kiselyov et al., 2010; Shen et al., 2012; Lloyd-Evans et al., 2008). Lysosomal dysfunction is commonly associated with a compensatory increase of lysosome biogenesis, manifested as increased expression of essential lysosomal genes (Settembre et al., 2013). For example, the expression of Lamp1, a lysosomal marker, is elevated in most LSDs (Meikle et al., 1997). Lamp1 expression was significantly elevated in cells treated with low concentrations of IP3R blockers 2-APB and Xesto, as well as the ER Ca²⁺ chelator TPEN, but not in the cells treated with the RyR blocker DHBP (Figure 4A). Consistently, LysoTracker staining was significantly increased in cells treated with Xesto, but not 1,1'-diheptyl-4,4'-bipyridinium dibromide (DHBP; Figure 4B). Lysosomal dysfunction is also often associated with lysosomal enlargement and accumulation of various incompletely digested biomaterials (Shen et al., 2012; Dong et al., 2008). Notably, in cells that were treated with Xesto, but not with the vehicle control DMSO, lysosomal compartments were enlarged, and non-degradable, autofluorescent lipofuscin-like materials accumulated in puncta structures (Figure 4C), reminiscent of cells with defective lysosomal Ca²⁺ release (ML1 KO cells; Dong et al., 2008) (Figure 4C). By showing that inhibiting IP3R-mediated Ca²⁺ release from the ER results in a lysosome storage phenotype in the cell, these findings suggest that lysosome Ca²⁺ store refilling from IP3Rs ion the ER has important consequences for lysosome function and cellular health.

Figure 4. — (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

Figure 4—source data 1. Source data of Figure 4A,B: Quantifications of Lamp-1 protein levels (A) or LysoTracker staining (B) under different experimental conditions and manipulations.
**DOI:** http://dx.doi.org/10.7554/eLife.15887.018

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DOI: 10.7554/eLife.15887.018

Discussion

Using pharmacological and genetic approaches to manipulate ER Ca²⁺ levels and Ca²⁺ release and three different assays to directly measure lysosome Ca²⁺ release, we show that under normal conditions lysosome Ca²⁺ stores are refilled from the ER Ca²⁺ store through IP3 receptors independent of lysosome pH (see Figure 4D). Our findings are in contrast to several studies in the literature that suggest that inhibition of the V-ATPase is sufficient to deplete lysosome Ca²⁺ stores. Previous conclusions suggesting the importance of H⁺ gradient in regulating lysosome Ca²⁺ stores would therefore implicate the existence of an H⁺-dependent Ca²⁺ transporter in lysosomal membranes that can operate at the extremely low cytosolic free Ca²⁺ level (100 nM), representing a high affinity uptake system. Our work, however, suggest that a low affinity uptake mechanism is more likely. Hence either a low affinity Ca²⁺ transporter or rectifying Ca²⁺ channel might suffice. A putative VDAC-like channel in the lysosome, resembling mitochondrial VDAC channels (van der Kant and Neefjes, 2014), may interact directly with IP3Rs to receive Ca²⁺ from the ER. Importantly, it has been previously suggested that Ca²⁺ uptake into isolated lysosomes is mediated by a low-affinity (mM range) Ca²⁺ transporter (Lemons and Thoene, 1991).

The lysosomal pH gradient is thought to be essential for the maintenance of high free [Ca²⁺]_Ly (Calcraft et al., 2009; Christensen et al., 2002; Dickson et al., 2012; Lloyd-Evans et al., 2008; Shen et al., 2012). However, in addition to triggering lysosomal Ca²⁺ release, as proposed by Christensen et al. (Christensen et al., 2002), lysosomal pH elevation is also known to affect [Ca²⁺]_Ly or its measurement via several other mechanisms. Whereas the total [Ca²⁺]_Ly is reported to be in the low mM range (5–10 mM), free [Ca²⁺]_Ly is generally agreed to be in the high μM range (100–500 μM) (Morgan et al., 2015). Therefore, the lysosome lumen must contain substantial Ca²⁺ buffers (Morgan et al., 2015). Ca²⁺ buffers in acidic compartments and the ER are known to bind Ca²⁺ much better at neutral pH (Dickson et al., 2012). Hence increasing pH_L from 4.8 to 7.0 may effectively reduce free [Ca²⁺]_Ly without necessarily triggering lysosomal Ca²⁺ release and affecting total [Ca²⁺]_Ly. Consistent with such an interpretation, a compelling study recently demonstrated that in secretory granules and the ER, increasing luminal pH changed the Ca²⁺ buffering capacity of both Ca²⁺ containing compartments and reduced free [Ca²⁺], while causing a minimal (20 nM) increase in cytosolic Ca²⁺ (Dickson et al., 2012). Additionally, lysosomal pH may act on luminal Ca²⁺ dyes by affecting their chromophore fluorescence and Ca²⁺-binding affinity (K_d) (Morgan et al., 2015). Because K_d drops more than 1,000 foldtimes when pH_L is increased from 4.8 to 7.0, accurate calibration is currently not possible. Furthermore, prolonged lysosomal pH manipulations may also indirectly affect lysosomal Ca²⁺ homeostasis, for instance, via membrane fusion and fission between compartments containing different amounts of Ca²⁺, H⁺, and their buffers. Finally, although elevating lysosomal pH may trigger lysosomal Ca²⁺ release, the accompanying increase in cytoplasmic Ca²⁺ was rather small (20–40 nM) (Dickson et al., 2012; Christensen et al., 2002). Moreover, the instantaneous changes (following pH increase and decrease) of Ca²⁺ probe fluorescence (Dickson et al., 2012; Christensen et al., 2002) are inconsistent with the slow rates of Ca²⁺ leak and re-uptake demonstrated in the current study.

The persistence of a GPN signal even after intracellular Ca²⁺ chelation is important for understanding the limits of this lysosome-specific pharmacological tool. GPN can certainly be used in conjunction with other tools to examine lysosome specificity, but caution is necessary with its use for Ca²⁺ store measurement, as a component of the signal observed in Fura-2 loaded cells, although small, is a result of the membrane permeabilization that causes a decrease in cytosolic pH. Similarly, reagents like Baf-A and NAADP that are used to mobilize lysosomal Ca²⁺ also release H⁺ into the cytosol (Morgan and Galione, 2007; Appelqvist et al., 2012; Scott and Gruenberg, 2011; Yoshimori et al., 1991), which could have been misinterpreted as a Ca²⁺ signal in previous studies (Morgan et al., 2011). pH may affect cytosolic Ca²⁺ indicators through the chromophore fluorescence, Ca²⁺-binding affinity, or Ca²⁺-dependent conformational changes (e.g., in the case of GCaMP) (Morgan et al., 2015). Therefore, if experimental conditions are not optimized, the presumed cytosolic Ca²⁺ signals may also reflect pH changes, or unidentified pH-mediated non-Ca²⁺ signals. We propose that BAPTA-AM control experiments be routinely conducted in any lysosomal Ca²⁺ measurement. It is possible that the 'pH contaminating effect' might have resulted in numerous misinterpretations of lysosome Ca²⁺ stores in the literature, particularly those examining the interactions between ER and lysosome Ca²⁺.

Based on our results in the current study, recent studies of ER-lysosome interactions (Phillips and Voeltz, 2016), and previous Ca²⁺ uptake studies on isolated lysosomes (Lemons and Thoene, 1991), we hypothesize that ER-refilling of lysosomal stores is a regulated, two-step process (see Figure 4D). First, lysosome store depletion may trigger establishment of ER-lysosome contacts (Phillips and Voeltz, 2016). Although lysosomes and ER are in close proximity under resting conditions, lysosome store depletion may 'stabilize' the ER-lysosome contact, and/or 'tether' and approximate both membranes (e.g., from 20–30 nm to 10 nm) (Phillips and Voeltz, 2016; Eden, 2016). Second, at the relatively stable, functional ER-lysosome contact sites, a passive Ca²⁺ transport process can occur from the ER to lysosomes, by utilizing the large Ca²⁺gradient created when lysosome stores are actively depleted. Up to 5 min may be required to complete the whole refilling process from 'initiation' through 'uptake'.

Our results not only provide an explanation for the reported sensitivity of the Ca²⁺ stores of acidic organelles to ER disrupting agents (Menteyne et al., 2006; Haller et al., 1996a), but are also consistent with the observations that lysosomes may buffer cytosolic Ca²⁺ released from the ER (López-Sanjurjo et al., 2013). The unexpected role of the ER in maintaining Ca²⁺ stores in lysosomes may help resolve the long-standing mystery of how impaired ER Ca²⁺ homeostasis is commonly seen in lysosomal storage diseases (LSDs) (Coen et al., 2012), and how manipulating ER Ca²⁺ reduces lysosome storage (Lloyd-Evans et al., 2008; Mu et al., 2008). In addition, our work reveals that, depending on the treatment conditions (acute versus prolonged treatment), many assumed-to-be ER-specific reagents may indirectly affect lysosome Ca²⁺ stores. This may impact the interpretations of a large body of literature on Ca²⁺ signaling. Although we demonstrated a central role of IP3Rs in lysosomal Ca²⁺ refilling, other ER Ca²⁺ channels may also participate under certain conditions, as seen in the IP3R TKO cells.

Accumulating evidence suggests that the ER forms membrane contact sites with other organelles, including plasma membrane, mitochondria (Cárdenas et al., 2010), endosomes (Alpy et al., 2013), and lysosomes (van der Kant and Neefjes, 2014). ER-endosome membrane contact, although currently difficult to study, was proposed to facilitate cholesterol transport from endosomes to the ER (Rocha et al., 2009; van der Kant and Neefjes, 2014; Drin et al., 2016). Given the established role of lysosomal Ca²⁺ release in cholesterol transport (Shen et al., 2012), lysosomal Ca²⁺ release may have a direct role in regulating ER-lysosome interaction (see Figure 4D). In ER-mitochondria contact sites, the tethering protein GRP-75 links IP3Rs with VDAC channels on mitochondria to regulate Ca²⁺ homeostasis and ATP production (Cárdenas et al., 2010). Similar unidentified tethers may also link IP3Rs with the putative lysosomal Ca²⁺ transporter for store refilling (see Figure 4D). The importance of lysosomal Ca²⁺ in regulating a variety of intracellular signaling pathways is becoming increasingly recognized (Medina et al., 2015). ER-lysosome interaction may serve as a hub for Ca²⁺ signaling to regulate cellular homeostasis through coordinating the primary anabolic and catabolic pathways in the cell. Studying the two-step lysosomal Ca²⁺ refilling process may prove important for future identification of the low-affinity Ca²⁺ uptake transporter/channel in the lysosome, and for studying the molecular mechanisms that regulate the functional ER-lysosome interaction.

Materials and methods

Molecular biology

Genetically-encoded Ca²⁺ indicator GCaMP3 was fused directly to the N-terminus of ML1 (GCaMP3-ML1) as described previously (Shen et al., 2012). The IP3R-LBD-ER construct (Várnai et al., 2005) was a kind gift from Dr. Thomas Balla (National Institute of Child Health and Human Development, NIH). The pECFP-ER plasmid was obtained from CLONTECH. Lamp1-mCherry was made by fusing mCherry with the C terminus of Lamp1.

Western blotting

Standard Western blotting protocols were used. HEK293T cells were treated every 4 hr for 24 hr with IP3R antagonists 2-APB and Xestospongin-C, ER Ca²⁺ chelator TPEN, and RyR antagonist DHBP. Lamp1 antibody was from Developmental Studies Hybridoma Bank (Iowa).

Mammalian cell culture

Immortalized cell lines (HEK293 and Cos-7) were purchased from ATCC and cultured following standard culture protocols. DT40-WT and IP3R-TKO cells were a generous gift from Dr. Darren Boehning (The University of Texas Health Sciences Center at Houston). Human fibroblasts were obtained from the Cornell Institute for Medical Research (NJ, USA). HEK 293 cells stably expressing GCaMP3-ML1 (HEK-GCaMP3-ML1 cells) were generated using the Flip-In T-Rex 293 cell line (Invitrogen). All these cells were neither authenticated nor tested for mycoplasma contamination. HEK293 cells are on the list of frequently misidentified or cross-contaminated cell lines. All cells were cultured in a 37°C incubator with 5% CO₂. HEK293T cells, Tet-On HEK293 cells stably expressing GCaMP3-ML1 (HEK-GCaMP3-ML1 cells), Cos-7 cells, and human fibroblasts were cultured in DMEM F12 (Invitrogen) supplemented with 10% (vol/vol) FBS or Tet-free FBS. DT40 cells were kept in suspension in RPMI 1640 (Invitrogen) supplemented with 450 µL β-mercaptoethanol, 2 mM L-glutamine, 10% FBS, and 1% chicken serum (Várnai et al., 2005; Cárdenas et al., 2010). We noted that lysosomal Ca²⁺ store refilling was often compromised in high-passage or poorly-maintained cell cultures.

Human fibroblasts and DT40 cells were transiently transfected using the Invitrogen Neon electroporation kit (1200 V, 1 pulse, 30 s). HEK293T cells, HEK-GCaMP3-ML1 cells, and Cos-7 cells were transfected using Lipofectamine 2000 (Invitrogen). All cells were used for experiments 24–48 hr after transfection.

Confocal imaging

Live imaging of cells was performed on a heated and humidified stage using a Spinning Disc Confocal Imaging System. The system includes an Olympus IX81 inverted microscope, a 100X Oil objective NA 1.49 (Olympus, UAPON100XOTIRF), a CSU-X1 scanner (Yokogawa), an iXon EM-CCD camera (Andor). MetaMorph Advanced Imaging acquisition software v.7.7.8.0 (Molecular Devices) was used to acquire and analyze all images. LysoTracker (50 nM; Invitrogen) was dissolved in culture medium and loaded into cells for 30 min before imaging. MitoTracker was dissolved in culture medium and loaded into cells for 15 min before imaging (25 nM). Coverslips were washed 3 times with Tyrode’s and imaged in Tyrode’s.

GCaMP3-ML1 Ca²⁺ imaging

GCaMP3-ML1 expression was induced in Tet-On HEK-GCaMP3-ML1 cells 20-24h prior to experiments using 0.0 1µg/mL doxycycline. GCaMP3-ML1 fluorescence was monitored at an excitation wavelength of 470 nm (F₄₇₀) using a EasyRatio Pro system (PTI). Cells were bathed in Tyrode’s solution containing 145 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM Glucose, and 20 mM Hepes (pH 7.4). Lysosomal Ca²⁺ release was measured in a zero Ca²⁺ solution containing 145 mM NaCl, 5 mM KCl, 3 mM MgCl₂, 10 mM glucose, 1 mM EGTA, and 20 mM HEPES (pH 7.4). Ca²⁺ concentration in the nominally free Ca²⁺ solution is estimated to be 1–10 μM. With 1 mM EGTA, the free Ca²⁺ concentration is estimated to be <10 nM based on the Maxchelator software (http://maxchelator.stanford.edu/). Experiments were carried out 0.5 to 6 hr after plating. 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.

Fura-2 Ca²⁺ imaging

Cells were loaded with Fura-2 (3 µM) and Plurionic-F127 (3 µM) in the culture medium at 37°C for 60 min. Florescence was recorded using the EasyRatio Pro system (PTI) at two different wavelengths (340 and 380 nm) and the ratio (F₃₄₀/F₃₈₀) was used to calculate changes in intracellular [Ca²⁺]. All experiments were carried out 1.5 to 6 hr after plating.

Oregon green 488 BAPTA-1 dextran imaging

Cells were loaded with Oregon Green 488 BAPTA-1 dextran (100 μg/ml) at 37°C in the culture medium for 4–12 hr, and then pulsed/chased for additional 4–16 hr. Fluorescence imaging was performed at 37°C. In vitro calcium-binding (K_d) affinities of OG-BAPTA-dextran were determined using KCl-based solutions (140 mM KCl, X mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, 10 mM MES, 0 or 1 mM BAPTA) adjusted to different pH (pH 4.5, 5.0, 6.0, and 7.0). By varying the amount of added Ca²⁺ (X= 0- 10 mM), solutions with different pH and free [Ca²⁺] were made based on the Maxchelator software (http://maxchelator.stanford.edu/). OG-BAPTA-dextran (5 μg/ml) fluorescence for each solution was obtained to plot the calibration curve (Morgan et al., 2015; Dickson et al., 2012; Christensen et al., 2002). In cells that were pre-treated with ionomycin, nigericin, and valinomycin (Morgan et al., 2015; Dickson et al., 2012; Christensen et al., 2002), in vivo minimal and maximal Fluorescence (F_min and F_max) were determined by perfusing the cells with 0 or 10 mM Ca²⁺ external solutions, respectively. Lysosomal [Ca²⁺] at different pH were determined using the following calibration equation: [Ca²⁺] = K_d × (F-F_min)/ (F_max-F).

Lysosomal pH measurement

Cells were pulsed with OG-488-dextran for 6 hr, and chased for additional 12 hr (Johnson et al., 2016). Cells were then bathed in the external solutions (145 mM KCl, 5 mM glucose, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM HEPES, 10 mM MES, adjusted to various pH values ranging from 4.0 to 8.0) that contained 10 μM nigericin and 10 μM monensin (Johnson et al., 2016). Images were captured using an EasyRatio Pro system. A pH standard curve was plotted based on the fluorescence ratios: F₄₈₀/ F₄₃₀.

Cytosolic pH sensitivity of GCaMP3-ML1

GCaMP3-ML1-positive vacuoles were isolated from vacuolin-1-treated HEK-GCaMP3-ML1 cells, as described previously (Zhang et al., 2012). Briefly, cells were treated with 1 μM vacuolin-1 for up to 12 hr to increase the size of late endosomes and lysosomes (Cerny et al., 2004). Vacuoles were released into the dish by mechanical disruption of the cell membrane with a small glass electrode. After vacuoles were released into the dish, patch pipettes containing either a 'high-Ca²⁺' (10 mM) internal solution or a 'low-pH' solution (140 mM KCl, 1 mM EGTA, 20 mM MES, 10 mM Glucose, pH adjusted to 2.0) were placed close to 'puff' the vacuoles. Images were captured using a CCD camera connected to the fluorescence microscope.

Reagents

All reagents were dissolved and stored in DMSO or water and then diluted in Tyrode’s and 0 Ca²⁺ solutions for experiments. 2-APB, ATP, Con-A, CPA, Doxycycline, DHBP, TG, TPEN were from Sigma; GPN and U73122 were from Santa Cruz; Ryanodine was from Abcam; LysoTracker, Fura-2, Mitotracker, Plurionic F-127, and Fura-Dextran were from Invitrogen; Baf-A was from LC Laboratories; ML-SA1 was from Chembridge; and Xestospongin-C was from Cayman Chemical, AG Scientific, and Enzo; Oregon Green 488 BAPTA-1 dextran was from life technologies. ML-SI compounds were identified from a Ca²⁺-imaging-based highthroughput screening conducted at NIH/NCATS Chemical Genomics Center (NCGC;https://pubchem.ncbi.nlm.nih.gov/bioassay/624414#section=Top). ML-SI compounds are available upon request.

Data analysis

Data are presented as mean ± SEM. All statistical analyses were conducted using GraphPad Prism. Paired t-tests were used to compare the average of three or more experiments between treated and untreated conditions. A value of p<0.05 was considered statistically significant. In the cases only individual traces were shown, the traces are representative from at least 30–40 cells, or from at least independent repeats.

Acknowledgement

We thank Dr. Darren Boehning for DT40-WT and IP3R-TKO cells, and Thomas Balla for the IP3R-LBD-ER construct. We also thank Richard Hume and Edward Stuenkel for comments on the manuscript, and appreciate the encouragement and helpful comments of other lab members in the Xu lab.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Funding Information

This paper was supported by the following grants:

NIH Office of the Director NS062792 to Abigail G Garrity, Wuyang Wang, Crystal MD Collier, Sara A Levey, Qiong Gao, Haoxing Xu.
NIH Office of the Director AR060837 to Abigail G Garrity, Wuyang Wang, Crystal MD Collier, Sara A Levey, Qiong Gao, Haoxing Xu.

Additional information

Competing interests

The authors declare that no competing interests exist.

Author contributions

AGG, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents.

WW, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents.

CMDC, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents.

SAL, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents.

QG, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents.

HX, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents.

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eLife. 2016 May 23;5:e15887. doi: 10.7554/eLife.15887.020

Decision letter

Editor: David E Clapham¹

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "The Endoplasmic Reticulum, Not the pH Gradient in the Lysosome, Are the Source of Calcium to Lysosomes" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by David Clapham as the Reviewing Editor and Eve Marder as the Senior Editor. One of the three reviewers, Murali Prakriya, has agreed to share his identity.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

Garrity et al. challenge the previously held view that lysosomal V-ATPase driven-acidification provides the driving force for lysosomal Ca²⁺ refilling. Using various manipulations designed to inhibit the proton pump (Baf-A or Con-A) and several Ca²⁺ imaging tools that directly and indirectly monitor lysosomal Ca²⁺, the authors show that the proton pump by is likely not responsible for refilling of the lysosomal Ca²⁺ store. Rather the authors report that manipulations that deplete ER Ca²⁺ stores also affect refilling of the lysosomal compartment, suggesting a functional and possibly physical coupling of the ER and lysosomal Ca²⁺ stores. The authors include new results using OG-BAPTA-dextran showing that depletion of ER Ca²⁺ stores by thapsigargin, or inhibition of IP3Rs by the membrane permeable Xestospongin inhibited the lysosomal Ca²⁺ content. An interesting point is that the previously postulated H⁺-dependent Ca²⁺ transporter must operate with cytosolic free Ca²⁺ level of 100 nM and therefore be extremely high affinity. The present mechanism, although not identified, would be a low affinity uptake mechanism.

Overall the data represent quite a challenging and intriguing new concept. Although most of the data appear convincing, some major open questions remain to be addressed. We feel these can be remedied within a month by additional experiments.

Essential revisions:

1) You need at least one convincing experiment showing that inhibiting the V-ATPase increases pH (needs to be measured) but doesn't affect store [Ca²⁺]. In the Christensen studies, inhibiting V-ATPase with Baf (similar to the treatment used in this paper) increased luminal pH to ~ 7.0 (where the Ca²⁺ imaging dye should work well) and drastically decreased [Ca²⁺]lys from ~ mM to μM. Although you added new OG-BAPTA imaging, it needs calibration. You should show the data measuring the pH and [Ca²⁺]. The bar graphs (Figure 3H and Figure 3—figure supplement 3F) do not provide information about the kinetics of the Ca²⁺ change and how they may (or may not) correlate with the Ca²⁺ changes reported by the GCaMP ML1 indicator shown in the majority of the data. Example traces of the lysosomal Ca²⁺ indicator in response to lysosomal and ER Ca²⁺ store depletion should be shown.

2) The authors contend that previous [Ca²⁺]lumen measurements were unreliable because of the pH-sensitivity of the dyes. In the Christensen et al. studies, however, the pH of each lysosome was monitored and used to calibrate the K_d of the dye. The Christensen studies (using lysosomes that do not overexpress any Ca-releasing channel) suggest that [Ca²⁺]lumen faithfully follows [pH]lumen. Other studies using 45Ca uptake measurement also support the importance of pH gradient (e.g. Lemons & Thoene 1991, JBC 266: 14378). However, the pH sensitivity of the indicators used in the present paper were not tested in situ. We suggest you test the pH sensitivity of your Ca²⁺ indicator in situ by placing a small mouth pipette electrode containing low pH solution+ x [Ca²⁺] next to tagged channel indicator and measure its sensitivity to Ca²⁺ changes. Please provide more detail in the Discussion why you think the previously published measurements were erroneous.

3) A triple IP3R knockout cell line was used to assess the proposed role of IP3Rs in refilling the lysosomal store. However, in contrast to the authors' theory, this does not have a lysosomal Ca²⁺ defect. The authors attribute this to an unknown compensatory mechanism. Please examine whether the Xestospongin abolishes ML1-SA1 responses in the triple IP3R KO cells and show the kinetics of the response. If the responses in the IP3R KO are not affected, and the kinetics now differ, the results would provide an additional layer of confidence for the proposed role of IP3Rs in lysosomal refilling.

eLife. 2016 May 23;5:e15887. doi: 10.7554/eLife.15887.021

Author response

We performed the experiments as suggested by the reviewers. First, we monitored lysosomal pH under different conditions using Oregon green 488-dextran, a commonly- used luminal pH indicator [1, 2] (see Methods and new Figure 3—figure supplement 3I). Using the calibration that we made for OG-488-dextran (see Figure 3—figure supplement 3I), we showed that lysosomal pH (pH_L), which is ~4.8 under the resting condition, increased dramatically within minutes to ~ pH 7.0 upon treatment of Baf-A1 (5 μM) or NH4Cl (10 mM) in HEK293 cells stably expressing ML1 (see Figure 3—figure supplement 3J, K). Second, based on the calibration for the luminal Ca²⁺ dye OG-BAPTA-dextran (see Figure 3—figure supplement 3C), we found that the intra-lysosomal [Ca²⁺] ([Ca²⁺]Ly), which was ~ 360 μM under the resting condition, dropped quickly to ~ 100 μM upon ML-SA1 application (see Figure 3H, I). Note that at pH 4.5, OG-BAPTA- dextran’s Kd is ~300 μM (Figure 3—figure supplement 3C), allowing measurement of Ca²⁺ ranging from 30 (0.1 Kd) to 3,000 (10 Kd) μM [3]. At the resting pH_L of 4.8 in the HEK cells, OG-BAPTA- dextran’s working range is 130-1,300 μM (Kd ~ 130 μM). Overall, all these values are in rough agreement with previous measurements [1, 3, 4].

Upon Baf-A1 and NH4Cl treatment to increase lysosomal pH, the fluorescence intensity of OG-BAPTA-dextran increased mildly, following the pH_L changes (Figure 3—figure supplement 3J, K). If the effect of lysosomal pH is exclusively on the Kd of OG-BAPTA-dextran, these results would suggest that [Ca²⁺]Ly dropped significantly, as concluded by Christensen et al. [5]. However, in addition to triggering lysosomal Ca²⁺ release, as proposed by Christensen et al. [5], lysosomal pH elevation is known to affect [Ca²⁺]Ly or its measurement via several additional mechanisms. Therefore, the interpretation offered by Christensen et al. might be too simplistic.

First, a change in lysosomal pH may cause a significant change in luminal Ca²⁺ buffering. In most studies dealing with cytosolic Ca²⁺, the total versus free [Ca²⁺] in the intracellular organelles (stores) are often not separately-considered since these two parameters are interrelated. Whereas the total [Ca²⁺]Ly is reported to be in the low mM range (5-10 mM), free [Ca²⁺]Ly is generally agreed to be in the high μM range (100-500 μM; 360 μM in the current study) [3]. Therefore, the lysosome lumen must contain a substantial amount of Ca²⁺ buffers [3]. Ca²⁺ buffers in the acidic compartments and ER are known to bind Ca²⁺ much better at neutral pH (H⁺ and Ca²⁺ compete with each other for Ca²⁺ buffers) [4]. Hence increasing pH_L from 4.8 to 7.0 may effectively reduce free [Ca²⁺]Ly without necessarily triggering lysosomal Ca²⁺ release and affecting total [Ca²⁺]Ly.

Second, lysosomal pH may act on luminal Ca²⁺ dyes by affecting their chromophore fluorescence and Ca²⁺-binding affinity (Kd) [3]. Because Kd is dropped more than 1,000 times when pH_L is increased from 4.8 to 7.0 (Figure 3—figure supplement 3C), perfect calibration is currently impossible. Note that in the Christensen studies, which were conducted in the activated macrophages, the basal pH_Lis ~ pH 3.8 [5]. When pH_Lwas increased from 3.8 to 5.0, the free [Ca²⁺]Ly was already dropped to ~ 10 μM (see Figure 3 of ref. [5]). However, in most studies including the current study, lysosomal pH is between 4.5 to 5.0 [1]. Hence, the mechanism proposed by Christensen et al., might not apply to most cell types whose basal pH_Lis close to pH 5.0. In addition, because of high levels of luminal Ca²⁺ buffers, the amount of releasable Ca²⁺ is high. Hence, a depletion of free [Ca²⁺]Ly to low μM is expected to produce a large cytosolic Ca²⁺ increase, which did not occur (see below). In the current study, upon ML-SA1 application to deplete the stores, free [Ca²⁺]Ly dropped from 360 to 100 μM (Figure 3I). Hence, within physiological ranges of lysosomal pH (4.8 -7.0) and Ca²⁺, the fluorescence signals of OG-BAPTA- dextran dyes might be nearly saturated, and this would prevent accurate determinations of [Ca²⁺]Ly changes when pH_Lis increased from 4.8 to 7.0 upon Baf-A1 or NH4Cl application. Note that at pH 7.0, OG-BAPTA-dextran’s Kd is ~0.1 μM (Figure—figure supplement 3C), which is not suitable for measuring Ca²⁺ over 10 μM.

Third, prolonged lysosomal pH manipulations may also indirectly affect lysosomal Ca²⁺ homeostasis, for instance, via membrane fusion and fission between compartments containing different amounts of Ca²⁺, H⁺, and their buffers.

Fourth,although elevating lysosomal pH might trigger lysosomal Ca²⁺ release, the accompanied increase in cytoplasmic Ca²⁺ was rather small (20-40 nM) (see refs. [4, 5]). This minor increase is more consistent with the interpretation that other pH-dependent processes contribute to the changes of OG-BAPTA fluorescence upon Baf-A1 and NH4Cl application. In addition, the instantaneous changes (following pH increase and decrease) of Ca²⁺ probe fluorescence (see Figure 5 in ref. [5] and Figure 3 in ref. [4]) are inconsistent with the slow rates of Ca²⁺ leak (τ = 6 min; see Figure 3—figure supplement 1E) and re-uptake (τ = 2 min; see Figure 1—figure supplement 2F & Figure 3—figure supplement 2C). Furthermore, given the pH-sensitivity of the Fura-2 dyes, even the observed minor increase (20-40 nM) in cytosolic Ca²⁺, based on instantaneous increases of Fura-2 ratios, could be mostly due to the effects of lysosomal proton release on the Fura-2 dyes (see Figure 1—figure supplement 3).

Because of the reasons listed above, in the current study, we used the luminal Ca²⁺ dyes for free [Ca²⁺]Ly measurement only in the experiments during which the pH remains unchanged.

The bar graphs (Figure 3H and Figure 3—figure supplement 3F) do not provide information about the kinetics of the Ca2+ change and how they may (or may not) correlate with the Ca2+ changes reported by the GCamP ML1 indicator shown in the majority of the data. Example traces of the lysosomal Ca2+ indicator in response to lysosomal and ER Ca2+ store depletion should be shown.

The example traces were now added into new Figure 3I and Figure 3—figure supplement 3H. Note that due to the photo-bleaching and small signal, we had to minimize the problem by reducing data acquisition. With the calibration curve, we measured lysosomal Ca²⁺ changes under different experimental manipulations. Whereas ML-SA1 application significantly depleted lysosomal Ca²⁺ contents (see new Figure 3I), TG treatment only slightly reduced lysosomal Ca²⁺ after 5- 10 min (Figure 3—figure supplement 3H). Although a quick drop in [Ca²⁺]Ly may cause a transient increase in cytosolic [Ca²⁺], it may also liberate certain amount of Ca²⁺ from the Ca²⁺-bound buffers, resulting in a delayed reduction in free [Ca²⁺]Ly. Therefore, as suspected by the reviewer, the kinetics of the GCaMP3-ML1 and OG-BAPTA-dextran were not perfectly correlated.

This is an excellent suggestion – thank you! See response #1 regarding [Ca²⁺]Ly measurement.

In GCaMP3-ML1-positive vacuoles that were isolated from HEK-GCaMP3-ML1 cells and released into the dish, we moved patch pipettes containing either a “high Ca²⁺” (10 mM) internal solution or a “low pH” solution (140 mM KCl, 1 mM EGTA, 20 mM MES, 10 mM Glucose, pH adjusted to pH 2.0) close to the vacuoles to produce a “puffing” effect. We showed that GCaMP3- ML1 responded to both Ca²⁺ and pH (see Figure 1—figure supplement 3E), but not to the “puffing” of the standard internal solution. These data suggest that our GCaMP3-ML1 probe, in addition to being a good indicator for measuring Ca²⁺ released from lysosomes, may also respond to lysosomal proton release, for instance, upon GPN and Baf-A1 application. Our results suggest that caution is necessary in designing the experiments to study the interaction between lysosomal Ca²⁺ and lysosomal pH. We propose that BAPTA-AM control experiments should be routinely performed in any lysosomal Ca²⁺ studies.

Please provide more detail in the Discussion why you think the previously published measurements were erroneous.

We added a new paragraph in the Discussion (second paragraph), and included a discussion in the Results.

Results: “In contrast, treatment of cells with Baf-A1 or NH₄Cl markedly increased lysosomal pH from 4.8 to 7.0 (Figure 3—figure supplement 3J, K). Such large pH elevations may cause dramatic changes in both K_d of OG-BAPTA-dextran (see Figure 3—figure supplement 3C) and luminal Ca²⁺ buffering capability (Dickson et al., 2012; Morgan et al., 2015), preventing accurate determinations of [Ca^2+]LY under these pH manipulations”.

Discussion:“Lysosomal pH gradient is thought to be essential for the maintenance of high free [Ca²⁺]Ly (Calcraft et al., 2009; Christensen et al., 2002; Dickson et al., 2012; Lloyd-Evans et al., 2008; Shen et al., 2012). However, in addition to triggering lysosomal Ca²⁺ release, as proposed by Christensen et al. (Christensen et al., 2002), lysosomal pH elevation is also known to affect [Ca²⁺]Ly or its measurement via several other mechanisms. Whereas the total [Ca²⁺]Ly is reported to be in the low mM range (5-10 mM), free [Ca²⁺]Ly is generally agreed to be in the high μM range (100-500 μM) (Morgan et al., 2015). Therefore, lysosome lumen must contain substantial amount of Ca²⁺ buffers (Morgan et al., 2015). Ca²⁺ buffers in the acidic compartments and ER are known to bind Ca²⁺ much better at neutral pH (Dickson et al., 2012). Hence increasing pH_Lfrom 4.8 to 7.0 may effectively reduce free [Ca²⁺]Ly without necessarily triggering lysosomal Ca²⁺ release and affecting total [Ca²⁺]Ly. Consistent with such interpretation, a compelling study recently demonstrated that in secretory granules and the ER, increasing luminal pH changed the Ca²⁺ buffering capacity of both Ca²⁺ containing compartments intraluminally to reduce free [Ca²⁺], while causing a minimal (20 nM) increase in cytosolic Ca²⁺ (Dickson et al., 2012). Additionally, lysosomal pH may act on luminal Ca²⁺ dyes by affecting their chromophore fluorescence and Ca²⁺-binding affinity (Kd) (Morgan et al., 2015). Because Kd is dropped more than 1,000 times when pH_Lis increased from 4.8 to 7.0, perfect calibration is near impossible. Furthermore, prolonged lysosomal pH manipulations may also indirectly affect lysosomal Ca²⁺ homeostasis, for instance, via membrane fusion and fission between compartments containing different amounts of Ca²⁺, H⁺, and their buffers. Finally, although elevating lysosomal pH may trigger lysosomal Ca²⁺ release, the accompanied increase in cytoplasmic Ca²⁺ was rather small (20- 40 nM) (Christensen et al., 2002; Dickson et al., 2012). Moreover, the instantaneous changes (following pH increase and decrease) of Ca²⁺ probe fluorescence (Christensen et al., 2002; Dickson et al., 2012) are inconsistent with the slow rates of Ca²⁺ leak and re-uptake demonstrated in the current study”.

3) A triple IP3R knockout cell line was used to assess the proposed role of IP3Rs in refilling the lysosomal store. However, in contrast to the authors' theory, this does not have a lysosomal Ca2+ defect. The authors attribute this to an unknown compensatory mechanism. Please examine whether the Xestospongin abolishes ML1-SA1 responses in the triple IP3R KO cells and show the kinetics of the response. If the responses in the IP3R KO are not affected, and the kinetics now differ, the results would provide an additional layer of confidence for the proposed role of IP3Rs in lysosomal refilling.

This is another excellent suggestion. We performed these experiments as suggested, and showed that although lysosomal Ca²⁺ refilling still occurred in IP3R TKO cells, the refilling kinetics were much slower than WT cells (see Figure 3—figure supplement 2C), and more importantly, refilling were completely insensitive to IP3R inhibitions (see Figure 3D-F).

References:

1) Johnson, D.E., et al., The position of lysosomes within the cell determines their luminal pH. J Cell Biol, 2016. 212(6): p. 677-92.

2) DiCiccio, J.E. and B.E. Steinberg, Lysosomal pH and analysis of the counter ion pathways that support acidification. J Gen Physiol, 2011. 137(4): p. 385-90.

3) Morgan, A.J., L.C. Davis, and A. Galione, Imaging approaches to measuring lysosomal calcium. Methods Cell Biol, 2015. 126: p. 159-95.

4) Dickson, E.J., et al., Orai-STIM-mediated Ca2+ release from secretory granules revealed by a targeted Ca2+ and pH probe. Proceedings of the National Academy of Sciences of the United States of America, 2012. 109(51): p. E3539-48.

5) Christensen, K.A., J.T. Myers, and J.A. Swanson, pH-dependent regulation of lysosomal calcium in macrophages. Journal of cell science, 2002. 115(Pt 3): p. 599-607.

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure 1—source data 1. Source data of Figure 1B, H, J: The average Ca²⁺ responses to ML-SA1 applications under control (B), Baf-A1 treatment (H), and Con-A treatment (J).

DOI: http://dx.doi.org/10.7554/eLife.15887.003

elife-15887-fig1-data1.xlsx^{(17.4KB, xlsx)}

DOI: 10.7554/eLife.15887.003

Figure 2—source data 1. Comparisons of GPN (E) and ML-SA1 responses (G, I) under different pharmacological and genetic manipulations (Figure 2E,G,I).

Comparisons of responses to ML-SA1 (E) and GPN (G) (Figure 2—figure supplement 1E,G).

DOI: http://dx.doi.org/10.7554/eLife.15887.009

elife-15887-fig2-data1.xlsx^{(13.3KB, xlsx)}

DOI: 10.7554/eLife.15887.009

Figure 3—source data 1. Normalized ML-SA1 responses or lysosomal Ca²⁺ contents under pharmacological (C, H) or genetic manipulations (F) (Figure 3C, F, H). .

The effects of Xesto on GPN responses in GCaMP3-ML1 (G) and MEF cells (J) (Figure 3—figure supplement 1G, J). ML-SA1 responses in GCaMP3-ML1-transfected WT and IP3R-TKO DT40 cells (Figure 3—figure supplement 2B). The effects of Xesto treatment on lysosomal Ca²⁺ changes induced by ML-SA1 (Figure 3—figure supplement 3E, G).

DOI: http://dx.doi.org/10.7554/eLife.15887.013

elife-15887-fig3-data1.xlsx^{(13.8KB, xlsx)}

DOI: 10.7554/eLife.15887.013

Figure 4—source data 1. Source data of Figure 4A,B: Quantifications of Lamp-1 protein levels (A) or LysoTracker staining (B) under different experimental conditions and manipulations.

DOI: http://dx.doi.org/10.7554/eLife.15887.018

elife-15887-fig4-data1.xlsx^{(15.1KB, xlsx)}

DOI: 10.7554/eLife.15887.018

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PERMALINK

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

Abigail G Garrity

Wuyang Wang

Crystal MD Collier

Sara A Levey

Qiong Gao

Haoxing Xu

Roles

Abstract

Introduction

Results

A physiological assay to monitor lysosomal Ca2+ refilling

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

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

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

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

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

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

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

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

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

Studying lysosomal Ca2+ refilling using a lysosome-specific 'membrane-permeabilizer'

The pH gradient and V-ATPase are not required for lysosome Ca2+ refilling

The Endoplasmic Reticulum (ER) Ca2+ store is required for lysosomal Ca2+ refilling

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

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

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

IP3-receptors, not ryanodine receptors, on the ER are required for Ca2+ refilling of lysosomes

Studying lysosomal Ca2+ refilling using intra-lysosomal Ca2+ dyes

Inhibition of ER IP3R channels and Ca2+ release causes lysosomal dysfunction and a LSD-like phenotype

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

Discussion

Materials and methods

Molecular biology

Western blotting

Mammalian cell culture

Confocal imaging

GCaMP3-ML1 Ca2+ imaging

Fura-2 Ca2+ imaging

Oregon green 488 BAPTA-1 dextran imaging

Lysosomal pH measurement

Cytosolic pH sensitivity of GCaMP3-ML1

Reagents

Data analysis

Acknowledgement

Funding Statement

Funding Information

Additional information

Competing interests

Author contributions

References

Decision letter

Roles

Author response

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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Cited by other articles

Links to NCBI Databases

A physiological assay to monitor lysosomal Ca²⁺ refilling

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

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

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

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

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

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

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

Studying lysosomal Ca²⁺ refilling using a lysosome-specific 'membrane-permeabilizer'

The pH gradient and V-ATPase are not required for lysosome Ca²⁺ refilling

The Endoplasmic Reticulum (ER) Ca²⁺ store is required for lysosomal Ca²⁺ refilling

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

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

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

IP3-receptors, not ryanodine receptors, on the ER are required for Ca²⁺ refilling of lysosomes

Studying lysosomal Ca²⁺ refilling using intra-lysosomal Ca²⁺ dyes

Inhibition of ER IP3R channels and Ca²⁺ release causes lysosomal dysfunction and a LSD-like phenotype

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

GCaMP3-ML1 Ca²⁺ imaging

Fura-2 Ca²⁺ imaging