Lysosomal Ca²⁺ Homeostasis and Signaling in Health and Disease

Abstract

Calcium (Ca²⁺) signaling is an essential process in all cells that is maintained by a plethora of channels, pumps, transporters, receptors, and intracellular Ca²⁺ sequestering stores. Changes in cytosolic Ca²⁺ concentration govern processes as far reaching as fertilization, cell growth, and motility through to cell death. In recent years, lysosomes have emerged as a major intracellular Ca²⁺ storage organelle with an increasing involvement in triggering or regulating cellular functions such as endocytosis, autophagy, and Ca²⁺ release from the endoplasmic reticulum. This review will summarize recent work in the area of lysosomal Ca²⁺ signaling and homeostasis, including newly identified functions, and the involvement of lysosome-derived Ca²⁺ signals in human disease. In addition, we explore recent controversies in the techniques used for measurement of lysosomal Ca²⁺ content.

Calcium (Ca²⁺) is a ubiquitous primary signaling messenger that is essential for the function of all cells and the regulation of a diverse range of key cellular events including fertilization, cellular metabolism, growth, division, motility, and apoptosis (Berridge et al. 2000). Because of the ability of Ca²⁺ to readily precipitate with anions (e.g., carbonates, phosphates) and form insoluble salts, high Ca²⁺ concentrations are toxic to cells and Ca²⁺ homeostasis requires tight control and regulation, which has been honed during evolution (Williams 2006). The origin of the cellular use of Ca²⁺ as a signaling ion is likely to have come from its flexible chemistry and prevalence in ancient seawater (∼10 mm) (Williams 2006; Jozef et al. 2013). First, Ca²⁺ can form longer bonds than other divalent cations prevalent in seawater (e.g., Mg²⁺, 1–3 mm); for example, Ca²⁺-oxygen bonds range from 2.3 to 2.5 Å depending on whether they are monodentate or bidentate, whereas Mg²⁺-oxygen bonds are almost always 2.05 Å as Mg²⁺ will be surrounded by an octahedron of oxygen atoms (Bertini 2007). Furthermore, Ca²⁺ can form six to eight bonds, which have a more rapid rate of substitution of water molecules by 10³ compared with Mg²⁺ (Katz et al. 1996; Bertini 2007). It is therefore clear why early unicellular organisms would use Ca²⁺ over Mg²⁺ as a signaling ion. The presence of high extracellular free Ca²⁺ concentration, which is maintained in the bodily fluids of higher order organisms (e.g., ∼1–1.3 mm in human plasma; Harris et al. 2014), versus low intracellular Ca²⁺ concentration (∼100 nm in the cytosol; Berridge et al. 2000) necessitated the emergence of mechanisms to maintain this concentration gradient and avoid cellular toxicity, for example, the development of Ca²⁺ pumps such as plasma membrane (PM) Ca²⁺ ATPases (PMCAs) and Na⁺/Ca²⁺ exchangers (Williams 2006).

INTRACELLULAR AND ORGANELLAR Ca²⁺ HOMEOSTASIS

In addition to extrusion of Ca²⁺ out of cells to maintain cytosolic Ca²⁺ in the range of 100 nm, intracellular Ca²⁺ stores function to buffer and induce orders of magnitude changes in cytosolic-free Ca²⁺ concentration. Ca²⁺ levels within the nuclear and mitochondrial matrix are similar to the cytosol (Bagur and Hajnóczky 2017), at least at times when cells are not stressed (Celsi et al. 2009). The endoplasmic reticulum (ER), however, is a major intracellular Ca²⁺ store with estimations of intraorganellar Ca²⁺ ranging from 12 µm to 2000 µm (reviewed in Bygrave and Benedetti 1996). This well-defined Ca²⁺ store is maintained by a plethora of Ca²⁺ transport mechanisms, chief among these being the inositol 1,4,5-trisphosphate receptors (IP₃ receptors) and the ryanodine receptors (RyRs) (Takeshima et al. 2015), Ca²⁺ leak channels (e.g., Bax inhibitor 1, presenilin 1; Bultynck et al. 2014; Takeshima et al. 2015), and one major Ca²⁺ uptake pump, the sarcoplasmic reticulum (SR)/ER Ca²⁺ ATPase (SERCA) (Takeshima et al. 2015). Ca²⁺ release from the ER is mediated mainly by the action of second messengers including IP₃ and cyclic ADP-ribose (cADPr), which are typically generated from extracellular signals binding to G-protein-coupled receptors that activate enzymes, including phospholipase C or adenylyl cyclase (Galione and Churchill 2002). Ca²⁺ release from the ER can be amplified by processes including direct coupling of the SR Ca²⁺ channels to PM Ca²⁺ channels, or by Ca²⁺-induced Ca²⁺ release (CICR) (Galione and Churchill 2002). The estimated cellular volume of the ER is in excess of 10% (Alberts 2002), which allows for substantial cellular coverage and Ca²⁺ release within the cell. The importance of controlling and maintaining the Ca²⁺ content of organelles and the cytosol is most clearly shown via a plethora of human diseases. In certain lysosomal diseases (discussed in more detail below), the normally very tightly controlled ER Ca²⁺ release has been reported to go awry (Pelled et al. 2003, 2005). This leads to elevated concentrations of Ca²⁺ in the cytosol, which is buffered by mitochondria (Duchen 2000; Sano et al. 2009). Ultimately, the combined ER Ca²⁺ dysfunction coupled to prolonged elevation in mitochondrial Ca²⁺ triggers neuronal dysfunction and death (Duchen 2000; Tessitore et al. 2004).

In addition to the ER, a substantial amount of free Ca²⁺ (existing in an unbound state) has been measured in the endocytic system (Lloyd-Evans et al. 2010). Although endocytosis engulfs extracellular fluid, which has an estimated 1–2 mm free Ca²⁺, the initialization of the process of acidification of the endocytic system organelles via H⁺ transport by the vacuolar ATPase (vATPase) leads to the rapid extrusion of Ca²⁺. This process is thought to be mediated, at least in part, by the action of the transient receptor potential (TRP) mucolipin 3 (TRPML3) ion channel (Lelouvier and Puertollano 2011). This results in an estimated 5–40 µm free Ca²⁺ within the early endocytic system (Gerasimenko et al. 1998). In contrast to early endosomes, the intravesicular Ca²⁺ content inside lysosomes, which are the terminal point of the endocytic system, is considerably higher (see below) (Christensen et al. 2002; Lloyd-Evans et al. 2008). Indeed, over the last 10 to 15 years, lysosomes, or the so-called “acidic stores,” which encompass late endosomes, fused intermediates and lysosome-related organelles, have emerged as a new and important intracellular Ca²⁺ store (Patel and Cai 2015).

LYSOSOMAL Ca²⁺ AND ION HOMEOSTASIS

Lysosomes, and vacuoles in simpler organisms, are essential acidic organelles found in all nucleated cells, serving as centers of macromolecular degradation and recycling, endocytic recycling, cellular nutrient regulation, and as a critical Ca²⁺ signaling organelle for the cell (Saftig and Klumperman 2009; Lloyd-Evans and Platt 2011; Settembre et al. 2013). The lysosome is the second-largest store of Ca²⁺ within the mammalian cell with a free Ca²⁺ concentration in the region of 500 µm–600 µm in multiple mammalian cells (Christensen et al. 2002; Lloyd-Evans et al. 2008). The Ca²⁺ concentration within the lysosome lumen has been measured using endocytosed luminal Ca²⁺-binding fluorophores conjugated to high molecular weight dextran. In mammals, lysosomes typically comprise 2%–3% of the volume of the cell, which suggests that, although lysosomes have a high intraluminal Ca²⁺ concentration, their overall contribution to global cellular Ca²⁺ signaling is relatively low in comparison to the ER (Lloyd-Evans 2016). This likely also explains the difficulty in measuring lysosomal Ca²⁺ indirectly using Ca²⁺-sensitive fluorophores (Lloyd-Evans 2016), which is expanded on in detail below. However, lysosomes have been shown to initiate global changes in cellular Ca²⁺ signaling, which may also explain discrepancies in measuring lysosomal Ca²⁺ levels (Shen et al. 2012; Vienken et al. 2017; Atakpa et al. 2019), also discussed in more detail below. In comparison, simpler organisms with single vacuoles (lysosome-like organelles), such as yeast and plants, have had their vacuolar free Ca²⁺ concentration measured using electrodes, resulting in recordings ranging between 0.2 µm and 2.3 mm (Schönknecht 2013). These measurements are in agreement with other recordings in Saccharomyces cerevisiae using the Ca²⁺-sensitive fluorophore Indo-1 (1.3 mm) (Halachmi and Eilam 1989). In contrast, total Ca²⁺ levels have been estimated in plant vacuoles, mainly using X-ray elemental microanalysis, as being considerably higher, in the range of 60–70 mm in Eudicot mesophyll cells (Conn and Gilliham 2010). The high values for vacuolar free Ca²⁺ concentration support the measurements in mammalian cells, and clearly show that these organelles can maintain high concentrations of free Ca²⁺ within this acidic environment. Unsurprisingly, the Ca²⁺ concentration is higher in vacuoles of simple organisms compared with mammalian lysosomes as the vacuole is one of the largest organelles in these organisms (e.g., yeast) comprising 10%–20% of total cellular volume (Armstrong 2010). Critical to intravesicular lysosomal Ca²⁺ quantitation is the ability to also measure pH in the same experiment (Christensen et al. 2002; Lloyd-Evans et al. 2008). pH has a critical effect on the level of free ionized Ca²⁺; in alkaline conditions, Ca²⁺ precipitates more freely with proteins, whereas acidic pH decreases protein binding leading to increased free Ca²⁺ levels (Wang et al. 2002). This supports the observations that within lysosomes there is ∼5000–6000 times the amount of free Ca²⁺ compared with the cytosol (Christensen et al. 2002; Lloyd-Evans et al. 2008). In comparison, lysosomes have ∼1000-fold more H⁺ ions, in the region of 2 times more Na⁺ (20 mm compared with 8–12 mm in the cytosol; Lodish 2000; Steinberg et al. 2010) and up to 3 times less K⁺ (50 mm compared with 140–150 mm in the cytosol; Lodish 2000; Steinberg et al. 2010) than the equivalent cytosolic concentrations of these ions. The vastly greater concentration gradient for Ca²⁺ across the lysosomal membrane is a further indication of the importance of this organelle in localized and global intracellular signaling.

Lysosomal Ca²⁺ Store Filling

For lysosomes to have a high concentration of Ca²⁺, it is likely that they have mechanisms for transporting Ca²⁺ from the cytosol, or, as recently suggested, from the ER into the lysosome lumen (Garrity et al. 2016). A variety of mechanisms mediating this process, detailed below, have been suggested, but no actual candidate transporters or pumps have yet been identified in placental mammals (Melchionda et al. 2016). In lower order organisms with vacuoles, Ca²⁺ uptake is an energy-dependent process with both Ca²⁺/H⁺ exchangers (CAX) and Ca²⁺ ATPases present on the vacuolar membrane (Pittman 2011). P-type Ca²⁺ ATPases, present across all life, are high-affinity, low-turnover transporters that sequester Ca²⁺ from the cytosol, whereas Ca²⁺ exchangers are low-affinity, high-capacity transporters (Pittman 2011). In yeast and plants, it is the acidification of vacuoles by vATPase proton pumps that generates a sufficient proton gradient to drive an exchange of Ca²⁺ ions for H⁺ (Pittman 2011). CAX genes have been identified in all species up to nonplacental mammals (Melchionda et al. 2016), indicating that further mechanisms exist for lysosomal Ca²⁺ store filling in mammals. In mammalian cells, the transport of Ca²⁺ into lysosomes requires ATP, and is inhibited by heavy metals and potentially by the long-chain base sphingosine (Lemons and Thoene 1991; Adachi et al. 1996; Colina et al. 2002; Lloyd-Evans et al. 2008). Furthermore, a recent study has suggested that acidification may not be necessary to allow Ca²⁺ entry into lysosomes and that instead the ER is the most critical cellular source of Ca²⁺ to ensure normal lysosomal Ca²⁺ store filling (Garrity et al. 2016). Despite this, the majority of studies have indicated that maintenance of resting Ca²⁺ levels within lysosomes, and indeed vacuoles, is dependent on maintaining vATPase activity (Christensen et al. 2002; Pittman 2011). Potential candidates for lysosomal transporters mediating Ca²⁺ entry include the TBHQ-inhibitable SERCA3, identified as a lysosomal Ca²⁺ transporter in platelet lysosomes (López et al. 2008), and SLC24A5, a Na⁺/K⁺/Ca²⁺ exchanger found on melanosomes, which are lysosome-related organelles responsible for pigmentation (Lamason et al. 2005). These findings could suggest that there are multiple mechanisms for lysosomal Ca²⁺ uptake that are cell-type and specialized-lysosome dependent. It should also be noted that extracellular Ca²⁺ contributes minimally to late endosomal/lysosomal Ca²⁺ content as chelation of extracellular Ca²⁺ had no significant long-term impact on intralumenal lysosomal Ca²⁺ levels (Christensen et al. 2002).

Local Requirement for Lysosomal Ca²⁺ Signaling

The process of endocytosis is critical to all cells and is regulated by a host of machinery and signals, one of which is Ca²⁺. Indeed, in yeast, it has been shown that before membrane tethering by SNARE proteins there are three key requirements to allow membrane fusion: (1) release of Ca²⁺ from the vacuole, (2) calmodulin, and (3) protein phosphatase 1 (Peters and Mayer 1998; Peters et al. 1999). Yeast mutants deficient in the ability of calmodulin to bind Ca²⁺, but are otherwise functional, can release Ca²⁺ from vacuolar stores but cannot trigger vesicular fusion, illustrating the critical requirement for Ca²⁺-bound calmodulin (Ca²⁺/calmodulin) as a sensor in mediating fusion events (Peters and Mayer 1998). In mammalian cells and cell-free systems, it has been shown that an approximate elevation of 0.5 µm cytosolic Ca²⁺ in the vicinity of endosomes, lysosomes, and autophagosomes is an absolute essential component mediating trafficking and fusion between these compartments (Gordon et al. 1993; Pryor et al. 2000; Lloyd-Evans et al. 2008). This is in contrast to the effect of high Ca²⁺ concentrations (0.1–1 mm), which inhibits endocytosis (Pryor et al. 2000). Furthermore, fusion between purified late endosomes is inhibited by the action of the fast Ca²⁺ chelator BAPTA, but not the slow Ca²⁺ chelator EGTA (Pryor et al. 2000). Taken together, these data indicate that only local changes in Ca²⁺ in the vicinity of lysosomes governs lysosomal transport and fusion events. As with yeast, in mammalian cells, one of the key effectors of vesicular fusion that uses this local elevation in Ca²⁺ to complete vesicular docking and promote fusion is calmodulin (Colombo et al. 1997; Peters and Mayer 1998; Pryor et al. 2000). Calmodulin inhibitors (e.g., by W7, calmidazolium, and chlorpromazine; Masson et al. 1992) have been shown to retard endosome fusion and alter cellular lipid metabolism in a manner that resembles the lysosomal storage disease (LSD), Niemann-Pick C1 (NPC1) (Masson et al. 1992), in which lysosomal Ca²⁺ levels and release are reduced (discussed below) (Lloyd-Evans et al. 2008). It has been suggested that the mechanisms by which Ca²⁺/calmodulin mediates vesicular fusion likely involves SNARE proteins that promote vesicle association through tethering (Burgoyne and Clague 2003; Luzio et al. 2005). Of these, syntaxin 13 is a likely candidate as a direct interaction with Ca²⁺/calmodulin has been shown and it has previously been implicated in early endosome fusion (Mills et al. 2001).

Further evidence that lysosomal transport and fusion events require the release of Ca²⁺ from the lysosome lumen have come from studies into lysosomal diseases (reviewed below) and lysosomal ion channel function, particularly the two-pore channels (TPCs). Overexpression of TPC1, TPC2, and TPC3 in mammalian cells leads to potentiated (TPC1 and TPC2) and inhibited (TPC3) endolysosomal Ca²⁺ release in response to a membrane permeant version of the lysosomal Ca²⁺ releasing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP-AM) (Ruas et al. 2010). In all three cases, the endocytosis of lipids and their transport between the late endosomal system and the Golgi was impaired with endocytosed ganglioside GM1 bound to the B subunit of cholera toxin (a specific GM1 ganglioside marker) accumulating in the lysosomes of the TPC-overexpressing cells compared with Golgi localization in the controls (Ruas et al. 2010). This was accompanied by lysosomal expansion and intra-lysosomal accumulation of multiple lipids in the TPC1- and TPC2-overexpressing cells (Ruas et al. 2010), mimicking the phenotypes observed in Niemann-Pick C (NPC) disease cells, presumably as a result of lowered intralysosomal Ca²⁺ content (Lloyd-Evans et al. 2008). Further evidence of the important role of maintaining normal lysosomal Ca²⁺ signaling and lysosomal ion channel function comes via the TRPML1 and CACNA1A lysosomal Ca²⁺ channels. Alterations in the functions of these channels has been shown to impair endolysosomal–autolysosomal transport and fusion events, and are discussed in more detail below. It is therefore clear that maintaining physiological Ca²⁺ homeostasis and release from lysosomal/vacuolar Ca²⁺ stores is critical for the normal function of the endolysosomal system.

Global Requirement for Lysosomal Ca²⁺ Signaling

Membrane contact sites between organelles, for example, the ER and mitochondria (Patergnani et al. 2011) and the ER and the PM (Elbaz and Schuldiner 2011), have been well studied in terms of Ca²⁺ signaling and the potential for amplification of Ca²⁺ signals (Penny et al. 2015). Contact sites between lysosomes and the ER exist but are not yet particularly well characterized. However, there appears to be a key role for ER–lysosome contact sites in potentiating local lysosomal Ca²⁺ release events into global Ca²⁺ release events (Fig. 1; Kilpatrick et al. 2013; Morgan et al. 2013) and in providing a source of Ca²⁺ to refill the depleted lysosomal Ca²⁺ store (Garrity et al. 2016; Atakpa et al. 2018). (For a detailed review of ER–lysosome contact points, see Penny et al. 2015.)

Figure 1.

Interplay between lysosomal and endoplasmic reticulum (ER) Ca²⁺ release. Lysosomes and the ER are now well known to have contact sites that bring them together in physical space. This ensures that Ca²⁺ uptake and release from lysosomes is further modulated by the ER. Indicated are different examples of endogenous (1,2) and pharmacological (3,4) release of Ca²⁺ from lysosomal stores and the subsequent impact that this may have on Ca²⁺ release from the ER. Movement of Ca²⁺ ions (blue for lysosomal Ca²⁺, red for ER Ca²⁺) is indicated by the arrows either through channels (rectangles and triangles) or via lysosomal membrane perforation induced by lysosomal cathepsin-mediated hydrolysis of Gly-Phe-β-naphthylamide (GPN).

LYSOSOMAL Ca²⁺ CHANNELS

In recent years, a plethora of ion channels have been identified as functioning to release Ca²⁺ from lysosomes, chief among these, and the most well characterized, are the TPCs and TRPML1. TPCs have been extensively reviewed elsewhere, but it is worth reiterating that a recent controversy surrounding the ion selectivity of TPC2 has been addressed with this ion channel clearly releasing Ca²⁺ in response to the second messenger NAADP (Ruas et al. 2015). Other Ca²⁺ permeable channels found in lysosomes include the neuronal voltage-gated Ca²⁺ channel CACNA1A (Tian et al. 2015), and the ATP-gated multi-ion channel P2X₄ (Huang et al. 2014; Murrell-Lagnado 2018). The mechanisms by which these channels function in the lysosome is not well understood. CACNA1A, a voltage-gated Ca²⁺ channel, has been identified to reside on lysosomes where it is thought to mediate neuron-specific lysosomal fusion with endosomes and autophagic vacuoles (Tian et al. 2015). P2X₄ on the other hand is inhibited at low pH and is therefore not active in lysosomes, but is thought to become active on induction of lysosomal exocytosis, for example, to facilitate secretion of surfactant by alveolar type II cells (Murrell-Lagnado 2018), or phagocytosis, in which it cooperates with PM P2X₇ to facilitate killing of mycobacteria (Fairbairn et al. 2001; Qureshi et al. 2007). In this review, we are going to predominantly focus on another lysosomal ion channel, namely TRPML1.

TRPML1

TRPML1, or mucolipin-1, is a six-transmembrane domain channel belonging to the TRP superfamily and located on late endosomes and lysosomes (Waller-Evans and Lloyd- Evans 2015). It is evolutionarily conserved in Drosophila and Caenorhabditis elegans (Treusch et al. 2004; Wong et al. 2012).

TRPML1 Ion Selectivity

Despite being referred to as the major Ca²⁺ efflux channel of the lysosome, TRPML1 has actually been reported to be permeable to several additional cations including H⁺, K⁺, Na⁺, Fe²⁺, Mn²⁺, and Zn²⁺ (Waller-Evans and Lloyd- Evans 2015). TRPML1 was first identified as an inwardly rectifying (i.e., ions move from the lysosome to the cytoplasm) cation channel permeable to Ca²⁺, K⁺, and Na⁺ by LaPlante et al. (2002) using Xenopus oocytes overexpressing human TRPML1. K⁺ permeability has also been observed by Raychowdhury et al. (2004), using TRPML1 in planar lipid bilayers, and Kiselyov et al. (2005), using whole-cell recordings of HEK293T cells overexpressing TRPML1. It has also been suggested that TRPML1 acts as a lysosomal H⁺ leak channel (Soyombo et al. 2006).

Following generation of a constitutively active TRPML1, based on the activating varitint-waddler TRPML3 mutation, by Xu et al. (2007) and Grimm et al. (2007), Ca²⁺ permeability has been confirmed several times (Dong et al. 2010; Yamaguchi et al. 2011; Shen et al. 2012; Chen et al. 2017; Schmiege et al. 2017). This variant has also been used to detect Fe²⁺ (but not Fe³⁺), Zn²⁺, and Mn²⁺ permeability via patch-clamping of lysosomes swollen using vacuolin (Dong et al. 2008). However, as the effects of vacuolin on endosomal membranes and ion content have yet to be determined, these experiments may not reflect TRPML1 ion selectivity under physiological conditions. There is some supporting evidence in that anemia is common among patients suffering from mucolipidosis type IV (caused by loss of TRPML1 function), although this could be caused by reduced absorption through the gut (Chen et al. 2014). Additionally, Zn²⁺ homeostasis is altered in the absence of TRPML1 (Eichelsdoerfer et al. 2010; Kukic et al. 2013; Chen et al. 2014). However, these experiments were performed with nonphysiologically high concentrations of extracellular Zn²⁺ so, again, may not reflect normal physiology. In summary, there is substantial evidence that TRPML1 is permeable to Ca²⁺ and K⁺, but it may also be permeable to other cations.

TRPML1 Regulation

Phosphatidylinositols, a group of signaling lipids, have been identified as the native agonists and antagonists of TRPML1. Phosphatidyl-(3,5)-bisphosphate (PI(3,5)P₂), an endolysosome-specific phosphatidylinositol generated from phosphatidyl-(3)-phosphate (PI3P) by PIK-fyve, activates TRPML1, while PI(4,5)P₂, which is abundant at the PM, inhibits TRPML1, as do PI(3,4)P₂ and PI(3,4,5)P₃ (Dong et al. 2010). A motif rich in basic amino acids in the amino terminus of TRPML1 is necessary for phosphatidylinositol-mediated regulation (Zhang et al. 2012). TRPML, the Drosophila TRPML1 ortholog, is also activated by PI(3,5)P₂ and inhibited by PI(4,5)P₂ (but not by other phosphatidylinositols) when expressed in HEK293T cells, suggesting an evolutionarily ancient regulatory mechanism (Feng et al. 2014a). It is very easy to see how this activation and inhibition by different phosphatidylinositols found in different cellular compartments could prevent aberrant triggering of TRPML1 at the PM, where it is occasionally found following lysosomal exocytosis (Medina and Ballabio 2015).

Several synthetic agonists and antagonists have been developed for TRPML1, with MLSA1 (mucolipin synthetic agonist 1) the best known. This was identified by Shen et al. (2012), who screened analogs of SF-51, a TRPML3 agonist identified in an earlier high-throughput screen (Grimm et al. 2010), using changes to whole-cell and whole-endosome currents in HEK293T cells overexpressing TRPML1. MLSA1 interacts directly with the pore region of TRPML1 and forces it into an open conformation (Fine et al. 2018). This is in contrast to the endogenous ligand, PI(3,5)P₂, which acts as an allosteric activator. There is significant synergy between MLSA1 and PI(3,5)P₂, with TRPML1 more sensitive to MLSA1 activation in the presence of PI(3,5)P₂. There are also differences in the way MLSA1 affects mammalian and Drosophila TRPML1. Although MLSA1 can directly activate mammalian TRPML1, even in the absence of PI(3,5)P₂, it can only act to increase the sensitivity of Drosophila TRPML to PI(3,5)P₂ (Feng et al. 2014b). Since the discovery of MLSA1, several other TRPML1 agonists have been discovered (Wang et al. 2015; Zhang et al. 2016), as have antagonists (Samie et al. 2013; Wang et al. 2015), although the mechanism of action of these compounds is unknown.

In addition to activation and inhibition by phosphatidylinositols, TRPML1 is subject to several other layers of regulation. It is regulated by pH, although there is contradictory evidence as to whether TRPML1 is more active in an acidic environment, or is inhibited by it. Earlier single-channel studies by LaPlante et al. (2002) and Raychowdhury et al. (2004) found that TRPML1 conductance was inhibited at low pH. However, later studies using the constitutively active varitint TRPML1 mutant have reported increased activation of TRPML1 at a more acidic pH (Dong et al. 2008; Feng et al. 2014a), with optimal TRPML1 activity at pH4.5 followed by inhibition with increasing acidity. Interestingly, the same effect has been seen with Drosophila TRPML, but with a slightly higher pH optimum of 5.2 (Feng et al. 2014a). These pH optima correspond with the lysosomal pH of mammals and Drosophila, respectively, suggesting that TRPML1 is most active in lysosomes (Venkatachalam et al. 2015). Recent structural studies identified aspartate residues in the luminal pore that are important for pH regulation. These are thought to be protonated at low pH, which would prevent the inhibition of TRPML1 by high luminal Ca²⁺ concentrations that occurs at neutral pH (Li et al. 2017). These structural studies therefore support the idea that TRPML1 is more likely to be active in an acidic environment such as the lysosome.

As well as direct action of protons on the channel, TRPML1 is indirectly regulated by pH as it is inactivated through cathepsin B-dependent cleavage (Kiselyov et al. 2005). Addition of purified cathepsin B to TRPML1-overexpressing HEK293T cells drastically reduced TRPML1-mediated current and the amount of full-length TRPML1 present in cells. As cathepsin B is a lysosomal enzyme with a pH optimum of 4.5–5.5 (Linebaugh et al. 1999; Lloyd-Evans and Haslett 2016), it is therefore unlikely to be involved in inactivation of TRPML1 at the PM, but rather to regulate TRPML1 activity within lysosomes, possibly by restricting the time during which TRPML1 is active (Kiselyov et al. 2005). Finally, TRPML1 has been suggested to be inhibited by high lysosomal concentrations of sphingomyelin (Shen et al. 2012) and adenosine (Zhong et al. 2017), although the relevance of this inhibition in normal physiological function is not clear. What is clear from the multiple layers of regulation is that it is very important for the cell to tightly control TRPML1 activity.

Cellular Function of TRPML1

One of the major cellular roles for TRPML1 is as a regulator of autophagy (Di Paola et al. 2018). Autophagy is the process by which defective organelles and macromolecules are engulfed by specialist membrane-bound compartments called autophagosomes. These fuse with late endosomes to form amphisomes, which then fuse with lysosomes where their contents can be degraded. Autophagy is a constitutive process needed to maintain organelle quality control, but is a vital component in the cellular response to stress, allowing the cell to use itself as a nutrient source (Onyenwoke et al. 2015). Defects in autophagy have been observed in C. elegans, Drosophila, and mammalian models following loss of TRPML1 function, as well as in mucolipidosis type IV patients (Venkatachalam et al. 2008; Vergarajauregui et al. 2008; Curcio-Morelli et al. 2010; Sun et al. 2011). Amphisome-like structures are observed accumulating within cells, suggesting that TRPML1 is needed for amphisome–lysosome fusion.

TRPML1 interacts with the major regulators of autophagy, mammalian target of rapamycin complex 1 (mTORC1) and transcription factor EB (TFEB) (Di Paola et al. 2018). The lysosomal membrane amino acid sensor mTORC1 suppresses autophagy under basal conditions. In the absence of TRPML1, mTORC1 activity is diminished, causing increased formation of autophagosomes and build-up of amphisomes. Reactivation of mTORC1 can alleviate this amphisome accumulation, even in the absence of TRPML1 (Wong et al. 2012). As well as regulating mTORC1 activity, TRPML1 is regulated by mTORC1, and can be directly phosphorylated by mTOR in vivo, which is thought to result in inhibition of TRPML1 activity under basal conditions (Onyenwoke et al. 2015). This is supported by evidence that TRPML1 is up-regulated following starvation, or initiation of autophagy by Torin treatment, which inhibits mTORC1 (Wang et al. 2015). TRPML1 activity is also needed for the translocation of TFEB to the nucleus that occurs on induction of autophagy (Medina et al. 2015). This process is mediated by calcineurin. Ca²⁺ released via TRPML1 activates calcineurin, which dephosphorylates TFEB. This dephosphorylation promotes nuclear translocation of TFEB. TFEB is a master regulator of lysosomal and autophagy genes, and movement of TFEB to the nucleus up-regulates lysosome biogenesis and autophagy genes, including TRPML1 (Palmieri et al. 2011). It is suggested that nuclear translocation of TFEB during autophagy, dependent on TRPML1 activity, is responsible for the up-regulation of TRPML1 during autophagy, in another example of reciprocal regulation (Wang et al. 2015). It is clear that TRPML1 is intimately involved in autophagy via a number of complex regulatory mechanisms. (For a more detailed review on TRPML1 in autophagy, see Di Paola et al. 2018.)

Another major cellular role for TRPML1 is in lysosomal exocytosis, which is the fusion of lysosomes with the PM, important for processes including PM repair and phagocytosis (Samie and Xu 2014). Lysosomal exocytosis is controlled by TFEB. Overexpression of TFEB causes lysosomes to move close to the PM along microtubules, and also increases lysosomal Ca²⁺ release, needed for lysosome–PM fusion, via up-regulation of TRPML1 (Medina et al. 2011; Samie and Xu 2014). TFEB cannot induce lysosomal exocytosis in the absence of TRPML1. This has knock on consequences for phagocytosis, which requires the extra membrane derived from lysosomal exocytosis to engulf material (Samie et al. 2013). Potentially related to its role in lysosomal exocytosis, TRPML1 has also been shown to be required for gastric acid secretion by parietal cells in the stomach (Chandra et al. 2011; Sahoo et al. 2017). TRPML1 is located on tubulovesicles, specialized organelles found in these cells that are rich in the K⁺/H⁺ ATPase that pumps acid into the stomach following stimulation. On stimulation of G-protein-coupled receptors on the PM of parietal cells, the tubulovesicles fuse with the extensive secretory network, canaliculi, to secrete acid. This process, in a similar manner to lysosomal exocytosis, is dependent on TRPML1-mediated Ca²⁺ release, and can be induced by directly activating TRPML1 (Sahoo et al. 2017). It is likely that this failure to fuse tubulovesicles with canaliculi underlies the achlorydia observed in mucolipidosis type IV patients (Schiffmann et al. 1998).

LYSOSOMAL Ca²⁺ SIGNALING IN DISEASE

Lysosomal Storage Diseases

Lysosomal storage disorders are a group of individually rare inherited metabolic disorders that together are the most common cause of childhood neurodegeneration with a combined frequency of 1:5,000 live births (Cox and Cachón-González 2012). These diseases are predominantly caused by mutations in genes encoding lysosomal enzymes, or their accessory proteins, leading to primary intralysosomal accumulation of the enzyme substrate, and a plethora of downstream alterations in cellular function that ultimately result in cell death and neuronal loss (Cox and Cachón-González 2012). In addition, a second group of lysosomal diseases is caused by mutations in lysosomal transmembrane proteins that include transporters, ion channels, and proteins of unknown function (Cox and Cachón-González 2012). Although the precise function of many of these proteins remains to be elucidated, almost all associated diseases are characterized by similar changes in cell biology. These include intralysosomal accumulation of various substrates including lipids and proteins, and defects in endocytosis, autophagy, and mitochondrial function (Platt and Walkley 2004; Cox and Cachón-González 2012). In addition, changes in Ca²⁺ signaling has emerged as a contributing factor in the pathophysiology of these diseases (Vitner et al. 2010; Lloyd-Evans 2016). Earlier work indicated a role for abnormal ER Ca²⁺ signaling in the pathophysiology of Gaucher disease, Sandhoff disease, and GM1 gangliosidosis (Pelled et al. 2003, 2005; Tessitore et al. 2004). In these diseases, the accumulating lysosomal lipid (e.g., glucosylceramide in Gaucher disease) is presumed to exit the lysosome and is delivered to the ER, where they interfere with the function of ER Ca²⁺ channels (Vitner et al. 2010). Although some evidence exists for this mechanism, another potential explanation may be the potential for lysosomal storage lipids to move between lysosomes and other organelles including peroxisomes and the ER via the contact sites discussed above. For example, syntaxin 7 has recently been shown to assist cholesterol transport between lysosomes and peroxisomes (Chu et al. 2015). Perhaps these transport pathways could be hijacked by other lipids as well.

Niemann–Pick Disease Type C

It is only in the last decade that lysosomal Ca²⁺ signaling has emerged as a contributor in the pathogenic mechanisms underlying human disease, with the lysosomal disease NPC1 providing the first key evidence for a role of altered lysosomal Ca²⁺ levels and signaling in contributing to pathogenesis (Lloyd-Evans et al. 2008). NPC disease is predominantly a childhood disease in which patients present with a plethora of symptoms including ataxia, supranuclear gaze palsy, hallmarks of Alzheimer's pathology, and speech and swallowing impairment (Lloyd-Evans and Platt 2010). At the cellular level, almost all cells and tissues present with characteristic intralysosomal accumulation of lipids including free unesterified cholesterol, sphingolipids, and lyso-(bis)phosphatidic acid (LBPA), as well as defects in endocytic trafficking (Lloyd-Evans and Platt 2010). In 2008, Lloyd-Evans and colleagues reported reduced lysosomal Ca²⁺ levels and altered lysosomal Ca²⁺ signaling mediated by NAADP in mouse, human, and hamster cells either null for or carrying mutations in the NPC1 protein, as well as cells in which NPC1 function had been inhibited using the chemical U18666a. These defects in lysosomal Ca²⁺ in NPC disease were measured by both a direct in situ approach using Ca²⁺-sensitive fluorophores conjugated to dextran that had been calibrated for the approximate lysosomal pH as well as an indirect method of perforating the lysosomes with the small molecule cathepsin C substrate Gly-Phe-β-naphthylamide (GPN) and measuring the subsequent cytosolic Ca²⁺ elevation. Both methods supported one another and were further strengthened by the presence, in the NPC disease cells, of (1) reduced NAADP-mediated lysosomal Ca²⁺ release, (2) an observed reduction in lysosomal Ca²⁺ uptake following emptying of the ER store with thapsigargin, and (3) a complete phenocopy of NPC disease-like lysosomal storage in healthy cells following chelation of intraluminal Ca²⁺. The reduced lysosomal Ca²⁺ content and release in NPC disease cells was identified as the cause of the documented retardation in endocytic trafficking, and contributed to the resulting lipid storage characteristic of this disease. The lysosomal Ca²⁺-signaling defect could be compensated for following treatment with the weak SERCA antagonist curcumin, which elevated cytosolic Ca²⁺, reduced lipid storage, and improved function in Npc1^−/− mice and astroglial cells. These data indicate that the lysosomal Ca²⁺ defect contributes directly to NPC disease pathogenesis. Subsequent reports have shown that the NPC disease lysosomal Ca²⁺ transport and signaling defects contribute to abnormalities in immune system function, neuronal function, can be used as a drug screening phenotype and can be ameliorated using an adenosine A2A receptor agonist that elevates cytosolic Ca²⁺ (Lee et al. 2010; Xu et al. 2012; Visentin et al. 2013; Speak et al. 2014).

Juvenile Neuronal Ceroid Lipofuscinosis/CLN3 Disease

Since the discovery of altered lysosomal Ca²⁺ transport and storage in NPC disease other lysosomal diseases have also been associated with aberrant lysosomal Ca²⁺ homeostasis (Lloyd-Evans et al. 2010; Lloyd-Evans 2016). However, only one lysosomal disease has been associated with elevated lysosomal Ca²⁺, namely, juvenile neuronal ceroid lipofuscinosis (CLN3 disease) (Chandrachud et al. 2015). CLN3 disease is caused by mutations in the CLN3 gene encoding the CLN3 protein, a six-transmembrane protein of the lysosome-limiting membrane of currently unknown function (Mole et al. 2011). Patients present with blindness, seizures, walking abnormalities, and neurodegeneration with death usually occurring in the second to third decades of life (Mole et al. 2011). At the cellular level, CLN3 patient cells present with intralysosomal accumulation of autofluorescent lipofuscin-like aggregating material that is characterized by the presence of subunit C of the mitochondrial ATP synthase (Mole et al. 2011). Defects in endocytic transport, autophagy, mitochondrial, and ER function have all been described, although the precise mechanisms leading from cellular dysfunction to neuronal loss remain poorly understood. Recent work from our research group has shown the presence of a number of Ca²⁺ signaling defects in Cln3 mutant mouse cerebellar cells carrying the common human disease-causing 1 kb deletion in exon 7–8 (Chandrachud et al. 2015). Most interesting is that these cells have elevated resting lysosomal Ca²⁺ measured using GPN, which we have confirmed by in situ Ca²⁺-sensitive dextran-conjugated probes (M Walker and E Lloyd-Evans, unpubl.), a phenotype that we have also observed in human CLN3 disease patient fibroblasts (H Waller-Evans, AS Alshehri, and E Lloyd-Evans, unpubl.). Although the cause of this elevated lysosomal Ca²⁺ remains to be elucidated, it may contribute to the abnormally enhanced ER Ca²⁺ release we observed in the Cln3 disease cerebellar cells in which either (1) low concentrations of thapsigargin elicited greater Ca²⁺ leak from the ER, or (2) caffeine induced considerably greater Ca²⁺ release from the ER RyR. These abnormalities in Ca²⁺ signaling in CLN3 disease had a clear effect on stimulating autophagy, leading to considerable accumulation of autophagosomes within the cells. How elevated lysosomal Ca²⁺ in CLN3 disease affects lysosomal Ca²⁺ release and global Ca²⁺ remains to be elucidated, but recent reports indicate alterations in both glial and neuronal Ca²⁺ signaling in CLN3 disease (Parviainen et al. 2017) and other NCLs, including CLN1 (Lange et al. 2018). Furthermore, a number of studies have suggested that Ca²⁺-channel modulators could be effective treatments for CLN3 disease (An Haack et al. 2011; Warnock et al. 2013). It is clear that further work is required to fully elucidate the role of lysosomal, ER, and global Ca²⁺ signaling on CLN3 disease pathogenesis, but it would appear that Ca²⁺ plays an important role in the pathogenesis of this disease in some way.

Lysosomal Ca²⁺ Homeostasis Defects in Diseases of Aging

Lysosomal Ca²⁺-signaling abnormalities have now been reported in a number of diseases of aging, including Parkinson's disease (PD), Alzheimer's disease (AD), and frontotemporal dementia (FTD) (Bae et al. 2014; Schondorf et al. 2014; Lee et al. 2015; Zou et al. 2015), and are described in detail elsewhere (Lloyd-Evans and Haslett 2016). Briefly, alterations in lysosomal Ca²⁺ homeostasis have been described in various forms of PD, including reduced lysosomal Ca²⁺ levels caused by mutations in the GBA1 gene (Sidransky et al. 2009; Kilpatrick et al. 2016a) (whose complete loss-of-function leads to Gaucher disease, the most common LSD; Cox and Cachón-González 2012) and also an involvement of TPC2 channels in PD caused by mutations in the LRRK2 gene (Hockey et al. 2015). Regarding AD, reduced lysosomal Ca²⁺ levels (Coen et al. 2012; Lee et al. 2015) and altered Ca²⁺ signaling (Lee et al. 2015) have been observed in familial AD in which mutations in PSEN1 lead to lysosomal deacidification (Lee et al. 2015) and in dementia caused by HIV in which TRPML1 activation is beneficial (Bae et al. 2014). Finally, mutations in FIG4 associated with FTD have been shown to disrupt lysosomal TRPML1 signaling, which can be corrected by activating Ca²⁺ release from TRPML1 with the synthetic agonist MLSA1 (Zou et al. 2015).

METHODS FOR ACCURATELY MEASURING LYSOSOMAL Ca²⁺

Accurate measurement of lysosomal Ca²⁺ content is complicated by the acidic pH and signaling cross talk between lysosomes and other intracellular Ca²⁺ stores, particularly the ER. The most accurate quantitation is undertaken by direct intravesicular measurements using probes endocytosed into lysosomes over a long timecourse of 24–48 h, confirmed to predominantly load lysosomes using antibody markers. These probes have a relatively low affinity for Ca²⁺, such as Rhod dextran, fura-dextran, or Oregon Green BAPTA 5N (Christensen et al. 2002; Lloyd-Evans et al. 2008; Lelouvier and Puertollano 2011). However, the Ca²⁺-binding affinity of all these probes are affected by pH. Therefore, to accurately estimate lysosomal Ca²⁺ content, it is also necessary to measure lysosomal pH, and to calibrate the Ca²⁺-binding probe appropriately. This should be performed with a pH-sensitive and -insensitive dextran combination, such as lysotracker yellow-blue dextran, and not a non-dextran pH-sensitive probe whose emissions do not always overlap with lysosomes. Using these methods, lysosomal free Ca²⁺ concentration has been measured as ∼500–600 µm in macrophages and human skin fibroblasts (Christensen et al. 2002; Lloyd-Evans et al. 2008).

It is also possible to undertake indirect measurements of lysosomal Ca²⁺. Perilysosomal Ca²⁺ can be measured using genetically encoded Ca²⁺ reporters such as cameleons or GCaMPs, conjugated to a lysosomal membrane protein. Cameleons and GCaMPs both use a conformational change brought about by Ca²⁺ binding to elicit changes in fluorescence (Kotlikoff 2007). When tethered to a lysosomal membrane protein, these probes can detect changes in cytosolic Ca²⁺ in the area immediately surrounding the lysosome, which should reduce interference by Ca²⁺ released from other intracellular stores. So far, the lysosomal membrane proteins LAMP-1 and TRPML1 have been used to create lysosome-specific Ca²⁺ reporters (Shen et al. 2012; McCue et al. 2013; Cheng et al. 2014). These can detect changes in perilysosomal Ca²⁺ concentration following addition of agents known to release lysosomal Ca²⁺, such as the cathepsin C substrate GPN, whose product causes osmotic lysis of lysosomes (Srinivas et al. 2002). Note that a recent study using cathepsin C inhibitors and null cells has raised questions about the ability of GPN to induce lysosomal Ca²⁺ release (Atakpa et al. 2019). However, other lysosomal proteases such as DPP7 can also hydrolyze Gly-Phe peptides (Rouf et al. 2013), suggesting that GPN is not a specific cathepsin C substrate and therefore remains a viable mechanism to induce lysosomal rupture. The most important consideration of the genetically encoded lysosomal near membrane Ca²⁺ sensors is that their use can potentially lead to overexpression of the protein. There is the potential that in expressing exaggerated levels of a protein, for example, TRPML1, above the normal range could in fact interfere with resting lysosomal Ca²⁺ levels. This could well be the case with the curious results obtained in NPC cells of no lysosomal Ca²⁺ defect when using the TRPML1-GCaMP, particularly when TRPML1 activation is supposed to improve NPC cellular function (Shen et al. 2012).

Finally, lysosomal Ca²⁺ can be measured indirectly using cytosolic Ca²⁺ probes such as Fura2-AM or Fluo dyes. Changes in cytosolic Ca²⁺ can be recorded following release of lysosomal Ca²⁺. This can also be achieved by osmotic lysis of lysosomes using the cathepsin C substrate GPN, by inducing lysosomal deacidification using bafilomycin A1, nigericin, or monensin, which exposes lysosomal Ca²⁺ leak, or by using agonists of lysosomal Ca²⁺ channels such as MLSA1 (TRPML1 agonist) or NAADP-AM (TPC2 agonist) (Shen et al. 2012; Morgan et al. 2015). However, as lysosomal Ca²⁺ release can trigger Ca²⁺ release from the ER, it is important to prevent ER Ca²⁺ release (Figs. 1 and 2). This can be achieved via pretreatment with ionomycin, a Ca²⁺ ionophore that releases Ca²⁺ from all intracellular stores apart from lysosomes, or inhibition of IP₃ receptor-mediated Ca²⁺ release using xestospongin C (Davis et al. 2012; Kilpatrick et al. 2016b). It is easy to misinterpret lysosomal Ca²⁺ measurements if Ca²⁺ release from other intracellular stores is not clamped. This is best shown by measurements of lysosomal Ca²⁺ content in NPC disease cells, which was directly measured as 60% reduced compared with control cells (Lloyd-Evans et al. 2008). This reduced lysosomal Ca²⁺ content finding has been replicated by several other groups (Lee et al. 2010; Xu et al. 2012; Visentin et al. 2013; Speak et al. 2014; Höglinger et al. 2015), but in a few studies has not been replicated (Shen et al. 2012; Vienken et al. 2017). The two aspects that these studies have in common is (1) a lack of clamping of ER Ca²⁺ stores, which does not prevent a much larger ER Ca²⁺ release (Kilpatrick et al. 2013) that masks the NPC Ca²⁺ defect (Fig. 2), and (2) no direct intravesicular measurement, which should always be the gold standard when attempting to replicate others’ findings, as was the case with Coen et al. (2012). The cause of the NPC lysosomal Ca²⁺ defect has been attributed to the accumulation of sphingosine (Lloyd-Evans et al. 2008), a simple amino alcohol sphingolipid that is produced in the lysosome from the degradation of ceramide by the enzyme acid ceramidase. A recent study using sphingosine 1-phosphate lyase null cells to elevate intracellular sphingosine levels via S1P phosphatase has suggested that elevating intracellular/lysosomal sphingosine levels, as observed in NPC disease, does not cause defects in lysosomal Ca²⁺ (Vienken et al. 2017). However, it is important to note that in this study no direct measurements of lysosomal Ca²⁺ were performed, and with the indirect measurements no clamping of the ER Ca²⁺ store was performed; as such, the measurement of lysosomal Ca²⁺ content using GPN is most likely “drowned out” by a much greater associated ER Ca²⁺ release (Fig. 2). It is clear, therefore, that in a manner similar to the autophagy arena (Klionsky et al. 2016), the lysosomal Ca²⁺ field also requires a consensus on the best methodologies used to measure lysosomal Ca²⁺, otherwise this area of research will struggle to move forward.

Figure 2.

Accurate lysosomal Ca²⁺ measurements are impaired by failure to clamp intracellular stores and by whichever Ca²⁺-sensitive probe is used. (A–E) Comparison of quantification of release of lysosomal Ca²⁺ into the cytoplasm using 5 µm Fura-2, AM on a Zeiss Colibri LED widefield fluorescence system with AxioCam MRm charge-coupled device (CCD) camera (A–C) or 5 µm Calcium Green-1, AM in combination with Fura Red, AM on a Zeiss LSM510 confocal (D,E) all in Ca²⁺ free buffer. A comparison is made between the use of Gly-Phe-β-naphthylamide (GPN) to perforate lysosomes in the absence of intracellular store clamping (A,D) or following an initial addition of ionomycin (B,E) to clamp intracellular stores other than lysosomes (H Waller-Evans and AS Alshehri, unpubl.). As can be seen, in the absence of clamping (A,D) GPN releases considerably more Ca²⁺ than even the subsequent addition of ionomycin, which is not in keeping with the estimation of lysosomal volume (3%) in comparison with the endoplasmic reticulum (ER) (>10%). However, when the other intracellular stores are clamped (B,E), the amount of Ca²⁺ that is then released using GPN is considerably smaller. In the Fura 2 recordings (A–C), the Ca²⁺ released using GPN in clamped cells (B, inset) amounts to only ∼7% of the total Ca²⁺ released (C), which is in keeping with the estimation of the small cellular volume of lysosomes. In contrast, in the absence of clamping (A), GPN releases ∼82% of the total Ca²⁺ (C), which is clearly an overestimation and presumably reflects the induction of Ca²⁺ release from both lysosomes and other stores. Furthermore, a failure to clamp intracellular stores results in an underestimation of the lysosomal Ca²⁺ defect (D,E) reported in Niemann–Pick C (NPC) disease (red traces). In human fibroblast cells that are not clamped (D), the reduction in Ca²⁺ released from NPC cells on addition of GPN is only 21% compared with control. However, when intracellular stores are initially clamped with ionomycin the reduction in lysosomal Ca²⁺ content in the NPC human fibroblasts is now ∼58%, which is in keeping with the ∼55% reduction in intravesicular Ca²⁺ concentration in NPC cells measured directly with Ca²⁺-sensitive dextrans.

CONCLUDING REMARKS

The lysosomal Ca²⁺ field has expanded dramatically in the last decade, particularly in the area of human disease. However, some major questions remain, most importantly perhaps are issues concerning the best methods for measuring lysosomal Ca²⁺ content accurately, and what proteins mediate lysosomal Ca²⁺ uptake. Future directions in this field may focus on those areas, but also it is likely that advancement in our understanding of the proteins and mechanisms involved in maintaining ER–lysosome contact sites are going to have a big impact on our understanding of lysosomal Ca²⁺ homeostasis and how lysosomal Ca²⁺ regulates local and global Ca²⁺ signaling.