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.https://doi.org/10.7554/eLife.15887.001
A vacuolar-type H+-ATPase (V-ATPase) on the membrane of the lysosome maintains the acidic lumen (pHLy ~ 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).
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).
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.
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).
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.
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).
[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 (pHLY = 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).
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).
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 (pHL) 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 Kd 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.
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.
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 pHL 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 (Kd) (Morgan et al., 2015). Because Kd drops more than 1,000 foldtimes when pHL 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.
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.
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).
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.
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.18.104.22.168 (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 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.
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.
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 (Kd) 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 (Fmin and Fmax) 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+] = Kd × (F-Fmin)/ (Fmax-F).
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.
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.
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 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.
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David E ClaphamReviewing Editor; Howard Hughes Medical Institute, Boston Children's Hospital, United States
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.
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.
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 Kd 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.https://doi.org/10.7554/eLife.15887.020
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+].
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 (pHL), 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 . At the resting pHL 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 pHL 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. . However, in addition to triggering lysosomal Ca2+ release, as proposed by Christensen et al. , 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) . Therefore, the lysosome lumen must contain a substantial amount of Ca2+ buffers . 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) . Hence increasing pHL 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) . Because Kd is dropped more than 1,000 times when pHL 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 pHLis ~ pH 3.8 . When pHLwas increased from 3.8 to 5.0, the free [Ca2+]Ly was already dropped to ~ 10 μM (see Figure 3 of ref. ). However, in most studies including the current study, lysosomal pH is between 4.5 to 5.0 . Hence, the mechanism proposed by Christensen et al., might not apply to most cell types whose basal pHLis 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 pHLis 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.  and Figure 3 in ref. ) 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 Kd 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 [Ca2+]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 pHLfrom 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 pHLis 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).
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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.
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- Abigail G Garrity
- Wuyang Wang
- Crystal MD Collier
- Sara A Levey
- Qiong Gao
- Haoxing Xu
- Abigail G Garrity
- Wuyang Wang
- Crystal MD Collier
- Sara A Levey
- Qiong Gao
- Haoxing Xu
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
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.
- David E Clapham, Howard Hughes Medical Institute, Boston Children's Hospital, United States
© 2016, Garrity et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.