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Rapid Recycling of Ca2+ between IP3-Sensitive Stores and Lysosomes

Abstract

Inositol 1,4,5-trisphosphate (IP3) evokes release of Ca2+ from the endoplasmic reticulum (ER), but the resulting Ca2+ signals are shaped by interactions with additional intracellular organelles. Bafilomycin A1, which prevents lysosomal Ca2+ uptake by inhibiting H+ pumping into lysosomes, increased the amplitude of the initial Ca2+ signals evoked by carbachol in human embryonic kidney (HEK) cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH) evoke Ca2+ release from distinct IP3-sensitive Ca2+ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A1 similarly exaggerated the Ca2+ signals evoked by carbachol or carbachol with PTH, indicating that Ca2+ released from distinct IP3-sensitive Ca2+ stores is sequestered by lysosomes. The Ca2+ signals resulting from store-operated Ca2+ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A1. Using Gd3+ (1 mM) to inhibit both Ca2+ entry and Ca2+ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca2+ recycling to the ER after IP3-evoked Ca2+ release. Blocking lysosomal Ca2+ uptake with bafilomycin A1 increased the amplitude of each carbachol-evoked Ca2+ signal without affecting the rate of Ca2+ recycling to the ER. This suggests that Ca2+ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca2+ released by IP3 receptors residing within distinct Ca2+ stores, but not the Ca2+ entering cells via receptor-regulated, store-operated Ca2+ entry pathways.

Introduction

Ca2+ is a ubiquitous intracellular messenger [1], [2]. The intracellular free Ca2+ concentration ([Ca2+]i) is determined by Ca2+ transport across biological membranes and by high concentrations of cytosolic Ca2+ buffers [3]. Acute regulation of the Ca2+ signals that regulate most cellular activities is achieved by regulating Ca2+ transport, most often by controlling the opening of Ca2+-permeable channels within the plasma membrane or endoplasmic reticulum (ER) [1], [4]. The receptors for inositol 1,4,5-trisphosphate (IP3Rs) are the most prominent of the intracellular Ca2+ channels [5], [6]. The large conductance of IP3Rs and their regulation by both IP3 and Ca2+ allows them to release Ca2+ rapidly from the ER in response to the many receptors that stimulate phospholipase C (PLC), and then to mediate regenerative propagation of the cytosolic Ca2+ signals [7].

The ER is unique among intracellular organelles in the extent to which it forms intimate associations with other membranes [8], [9], [10] including mitochondria [11], the nucleus [12], lysosomes [13], [14] and the plasma membrane [15], [16]. It is becoming increasingly clear that these dynamic interactions between membranes play important roles in both shaping and decoding the Ca2+ signals evoked by physiological stimuli. Furthermore, rapid gating of the Ca2+ channels that initiate most Ca2+ signals and slow diffusion of Ca2+ within the cytosol allow local Ca2+-mediated communication between closely apposed membranes. The mitochondrial uniporter (MCU) [17], [18], for example, can rapidly sequester Ca2+ released by IP3Rs when mitochondria are locally exposed to high [Ca2+]i near the mouths of open IP3Rs [11], [19], [20]. This both modulates IP3-evoked Ca2+ signals and regulates mitochondrial behaviour. Close apposition of STIM1 in ER membranes to Orai channels in the plasma membrane underlies regulation of the store-operated Ca2+ entry (SOCE) that almost invariably follows depletion of intracellular Ca2+ stores by IP3 [16]. More recently, lysosomes have also been suggested to contribute to regulation of [Ca2+]i [13], [14], [21]. A variety of Ca2+-permeable channels expressed within lysosomal membranes, including two-pore channels (TPCs) [22], TRPML1 [23] and P2X4 receptors [24] have been proposed to mediate Ca2+ release in response to such stimuli as nicotinic acid adenine dinucleotide phosphate (NAADP) [22], [25], mTOR [26], phosphatidylinositol 3,5-bisphosphate [26], [27] and luminal ATP [24]. Again there is evidence of interactions with the ER, because NAADP-evoked Ca2+ release from lysosomes can be amplified by Ca2+ release from the ER mediated by Ca2+-activation of either IP3Rs or ryanodine receptors [28], [29].

The mechanisms responsible for Ca2+ uptake into lysosomes are not known, although they require the pH gradient established across lysosomal membranes by the V-ATPase that pumps H+ into the lumen of lysosomes [14]. We [30] and others [28] recently provided evidence that lysosomes can also shape the Ca2+ signals evoked by IP3-evoked Ca2+ release from the ER. In our analysis, we demonstrated that dynamic lysosomes are associated with ER and that they selectively accumulate Ca2+ released by IP3Rs. But lysosomes do not sequester Ca2+ entering the cell via SOCE activated pharmacologically by inhibition of the SR/ER Ca2+-ATPase (SERCA) or by buffering of ER luminal Ca2+ [30]. Collectively, these observations suggest that lysosomes, like mitochondria [11], dynamically and intimately associate with ER. These associations contribute to both shaping IP3-evoked Ca2+ signals and to providing lysosomes with Ca2+ that might regulate their behaviour [30]. Here, we address three further questions relating to the interaction between lysosomes and IP3-evoked Ca2+ signals. First, we have argued that receptors, like the endogenous M3 muscarinic receptors of human embryonic kidney (HEK) cells, locally deliver IP3 to IP3Rs within signalling junctions, whereas different ‘extra-junctional’ IP3Rs release Ca2+ from distinct Ca2+ stores in response to lower concentrations of IP3 when their sensitivity is increased by cAMP [31] (Figure 1A). Do lysosomes sequester Ca2+ released from each of these IP3-sensitive Ca2+ stores? Second, does the SOCE evoked by physiological stimuli (rather than thapsigargin) direct Ca2+ to lysosomes? The answer to this question is important because it addresses whether a significant fraction of the Ca2+ entering cells via SOCE then passes through the ER and IP3Rs before re-entering the cytosol [32], [33]. Finally, and most importantly, are lysosomes ‘dead-end’ compartments for Ca2+, or is the Ca2+ they accumulate rapidly recycled to sustain refilling of ER Ca2+ stores?

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Figure 1. Lysosomes accumulate Ca2+ released from intracellular stores by IP3 alone or IP3 with cAMP.

(A) CCh stimulates M3 muscarinic receptors leading to activation of PLC and IP3-evoked Ca2+ release from the ER. PTH, via type 1 PTH receptors, stimulates adenylyl cyclase. Cyclic AMP sensitizes IP3Rs to IP3 and thereby potentiates the Ca2+ release evoked by CCh. We suggest that cAMP is delivered to IP3Rs at high concentrations within signalling junctions [34] and that the IP3Rs that respond to CCh alone are activated by locally delivered IP3 [31]. This local signalling allows CCh alone and CCh in combination with PTH to release Ca2+ from different stores [31]. (B) Bafilomycin A1 (Baf A1) inhibits the V-ATPase that mediates H+ accumulation by lysosomes, and thereby prevents lysosomal Ca2+ uptake. The latter may be mediated by H+-Ca2+ exchange. (C) Populations of HEK-PR1 cells were stimulated with CCh (1 mM) and then PTH (1 µM) with or without prior treatment with bafilomycin A1 (1 µM, 1 h). BAPTA (10 mM) was added as shown to chelate extracellular Ca2+. Results are means ± S.E. from 3 wells from one experiment, typical of 3 similar experiments. (D) Summary results show effects of bafilomycin A1 on the amplitudes of the peak Ca2+ signals evoked by addition of CCh, or PTH after CCh. Results (as percentages of the responses without bafilomycin A1) are means ± S.E. from 3 independent experiments. (E) Experiments similar to those in C, show the effects of bafilomycin A1 on the concentration-dependent effects of PTH on CCh-evoked Ca2+ signals. Results are means ± S.E. from 3 independent experiments. (F) The results suggest that lysosomes (LY) accumulate Ca2+ released via IP3Rs activated by IP3 alone or IP3 with cAMP.

https://doi.org/10.1371/journal.pone.0111275.g001

Results

Disruption of lysosomal Ca2+ uptake exaggerates the Ca2+ signals evoked by Ca2+ release from distinct IP3-sensitive stores

Stimulation of the endogenous muscarinic M3 receptors of HEK cells with carbachol (CCh) activates PLC. The IP3 produced then evokes Ca2+ release from intracellular stores via IP3Rs (Figure 1A) [34]. Receptors that stimulate adenylyl cyclase, including heterologously expressed type 1 PTH receptors, potentiate the Ca2+ signals evoked by CCh [34]. This potentiation is entirely mediated by cAMP, which directly sensitizes IP3Rs to IP3 [34]. Previous work established that high concentrations of cAMP are delivered directly to IP3Rs from adenylyl cyclase within cAMP signalling junctions [34]. We recently presented evidence suggesting that the Ca2+ signals evoked by CCh alone result from local delivery of IP3 to IP3Rs that are probably closely associated with PLC [31]. We propose that this spatially organized delivery of diffusible messengers allows CCh alone to evoke Ca2+ release via IP3Rs from different Ca2+ stores to those that are released by CCh in combination with PTH (Figure 1A) [31].

A previous analysis of CCh-evoked Ca2+ signals in HEK cells demonstrated that lysosomes selectively accumulate the Ca2+ released from intracellular stores by CCh [30]. In light of evidence that CCh alone and CCh with PTH evoke Ca2+ release from different stores (Figure 1A) [31], we now assess whether the latter response is also modulated by lysosomal Ca2+ uptake. For these analyses, we used bafilomycin A1 selectively to inhibit H+ uptake by lysosomes (Figure 1B) [35] and thereby to prevent them from sequestering Ca2+. Previous work established that bafilomycin A1 is the most convenient way of disrupting lysosomal Ca2+ uptake, but other means of perturbing lysosomal function using GPN to perforate lysosomal membranes or vacuolin to affect the morphology and distribution of lysosomes had similar effects on CCh-evoked Ca2+ signals [30].

Pre-incubation of HEK cells stably expressing human type 1 PTH receptor (HEK-PR1 cells) with bafilomycin A1 caused the increase in [Ca2+]i evoked by a maximally effective concentration of CCh in Ca2+-free HBS to increase by 1.5±0.2-fold (Figure 1C) [30]. PTH alone (1 µM) had no significant effect on [Ca2+]i in HEK-PR1 cells (data not shown) [34], but it potentiated the Ca2+ signals evoked by CCh (Figure 1C). The increase in [Ca2+]i evoked by addition of PTH in the continued presence of CCh was increased by 1.6±0.2-fold after pre-incubation with bafilomycin A1 (Figures 1C and 1D). The sensitivity to PTH was unaffected by bafilomycin A1: the pEC50 was 7.2±0.4 and 7.5±0.1 for control and bafilomycin A1-treated cells, respectively (where pEC50 is the -log of the half-maximally effective concentration) (Figure 1E). In these experiments, cells were first stimulated with CCh and then with PTH in the continued presence of CCh (Figure 1C). The similar effects of bafilomycin A1 on the first and second responses (Figure 1D) suggest that the capacity of lysosomes to sequester Ca2+ was unaffected by having accumulated Ca2+ during the first response to CCh. These results demonstrate that the Ca2+ signals resulting from Ca2+ release from two distinct IP3-sensitive Ca2+ stores are similarly affected by disruption of lysosomal Ca2+ uptake (Figure 1F).

Attenuation of IP3-evoked Ca2+ signals by lysosomes does not require NAADP-activated channels

In sea urchin eggs, IP3-evoked Ca2+ release triggers a rapid increase in the luminal pH of lysosomes [28]. We observed a similar response in CCh-stimulated HEK cells [30] (Figure 2A) and attributed it to an exchange of lysosomal H+ for cytosolic Ca2+ [30]. Morgan et al., however, suggest a different interpretation for their results. They argue that Ca2+ release from sea urchin lysosomes increases lysosomal pH, and that IP3-evoked Ca2+ release elicits the same response by locally stimulating formation of NAADP and perhaps also by a direct effect of cytosolic Ca2+ on NAADP-evoked Ca2+ release [28]. It is unlikely that such interactions contribute to the effects of lysosomes on IP3-evoked Ca2+ signals in HEK cells. Firstly, active lysosomes attenuate IP3-evoked Ca2+ signals in HEK cells (Figure 1), while they are proposed to amplify them in sea urchin eggs [28]. Secondly, NED-19, an antagonist of NAADP [36], had no effect on the alkalinization of lysosomal pH during stimulation of HEK cells with CCh (Figure 2B). Furthermore, NED-19 did not affect the time course of the Ca2+ signals evoked by a maximally effective concentration of CCh (Figure 2C) or the peak response to any concentration of CCh (Figure 2D).

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Figure 2. NAADP does not contribute to the effects of lysosomes on carbachol-evoked Ca2+ release in HEK cells.

(A) HEK cells loaded with dextran-conjugates of Oregon Green (OG, pH-sensitive probe) and Texas Red (TR, inert marker) were stimulated with CCh (1 mM). Results (means ± S.E. from 27 ROI on a single coverslip, representative of at least 3 independent experiments) show that CCh causes the pH of the lysosome lumen to increase. Addition of HBS did not affect OG or TR fluorescence [30]. (B) Similar experiments with and without NED-19 (10 µM, 1 h) show that it has no significant effect on the peak increase in lysosomal pH evoked by CCh. Results (means ± S.E. from 7 experiments) show the peak change in OG fluorescence (ΔRFU, relative fluorescence units). (C) [Ca2+]i was recorded from HEK cells stimulated with CCh (1 mM) alone or with NED-19 (10 µM, 1 h). Results show means ± S.E. from 3 wells in one experiment, typical of 3 experiments. (D) Summary results show the lack of effect of NED-19 on the peak Ca2+ signals evoked by CCh. Results are means ± S.E. from 3 experiments.

https://doi.org/10.1371/journal.pone.0111275.g002

In both sea urchin eggs and HEK cells, ER and lysosomes are closely apposed [28], [30], but the nature of the Ca2+-mediated ‘chatter’ between IP3Rs and lysosomes seems to be configured differently. In sea urchin eggs, IP3-evoked Ca2+ release appears to be amplified by NAADP-evoked Ca2+ release from lysosomes [28], while in HEK cells lysosomes rapidly sequester the Ca2+ released by IP3Rs (Figure 1).

Ca2+ signals resulting from carbachol-evoked Ca2+ entry are not affected by lysosomes

CCh evokes both IP3-mediated release of Ca2+ from intracellular stores (Figure 1A and 1C) and Ca2+ entry across the plasma membrane (Figure 3A). In most cells, including HEK cells (Figures 3B and 3C) [30], [37], depletion of intracellular Ca2+ stores activates SOCE [38]. But receptors that activate PLC can also stimulate additional Ca2+ entry pathways, including those that are regulated by arachidonic acid [39], [40]. Whether such Ca2+ entry pathways contribute to CCh-evoked Ca2+ entry in HEK cells is controversial [37], [41], [42]. In HEK-PR1 cells, CCh affected neither the time course of the Ca2+ signals evoked by restoration of extracellular Ca2+ to thapsigargin-treated cells, nor the amplitude of these signals when the extracellular Ca2+ concentration was varied (Figures 3B and 3C). These results suggest that the Ca2+ entry evoked by CCh in HEK-PR1 cells is mediated by SOCE.

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Figure 3. Carbachol evokes store-operated Ca2+ entry in HEK-PR1 cells.

(A) Typical responses of a population of HEK-PR1 cells stimulated with CCh (1 mM) in HBS with or without extracellular Ca2+. For the latter BAPTA (10 mM) was added with CCh. (B) HEK-PR1 cells were incubated with thapsigargin (1 µM, 15 min) in nominally Ca2+-free HBS before restoration of extracellular Ca2+ (30 mM) alone or with CCh (1 mM). Results (A and B) show means ± S.E. from 3 replicates of a single experiment, representative of at least 3 similar experiments. (C) Similar experiments show the peak amplitude of the Ca2+ signal evoked by restoration to thaspsigargin-treated cells of the indicated concentrations of extracellular Ca2+ ([Ca2+]e) alone or with CCh (1 mM). Results are means ± S.E. from 3 independent experiments.

https://doi.org/10.1371/journal.pone.0111275.g003

Our previous analysis established that lysosomes selectively accumulate Ca2+ released from the ER, but not Ca2+ entering cells via SOCE evoked by thapsigargin [30]. It is not known whether lysosomes affect SOCE evoked by CCh. The question is important because Ca2+ entering the cell via SOCE can locally regulate specific intracellular events [43], [44], but it is unclear whether it can also pass through the ER and so re-enter the cytosol via IP3Rs [32], [33]. The latter route is impossible when the SR/ER Ca2+-ATPase (SERCA) is inhibited by thapsigargin (Figure 4A). We therefore considered the possibility that CCh-evoked SOCE might be modulated by lysosomal Ca2+ uptake systems if a significant fraction of the Ca2+ entering by SOCE passed through the ER via SERCA and IP3Rs (Figure 4A). Evidence that CCh-evoked Ca2+ entry in HEK-PR1 cells is mediated by SOCE (Figure 3) [37] allows this issue to be addressed

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Figure 4. Lysosomes do not accumulate Ca2+ entering cells via store-operated Ca2+ entry evoked by carbachol.

(A) Ca2+ entering cells via SOCE evoked by CCh may pass through the ER and then re-enter the cells via IP3Rs from which some Ca2+ might then be accumulated by lysosomes (LY). That route is impossible when the SERCA is inhibited by thapsigargin. (B, C) Cells were stimulated with CCh (1 mM) in normal or Ca2+-free HBS alone (B) or with bafilomycin A1 (1 µM, 1 h) (C). The enlargements beneath the panels illustrate how the component of the Ca2+ signal attributable to Ca2+ entry (ΔΔ[Ca2+]i) was calculated. Results show means ± S.E. from 6 replicates from a single experiment, typical of 4 similar experiments. (D) Peak increases in [Ca2+]i evoked by CCh in normal or Ca2+-free HBS, with and without bafilomycin A1-treatment. Results (percentages of the responses to CCh alone in Ca2+-free HBS) are means ± S.E. from 4 experiments. *p <0.05, paired Students's t-test using the raw data. (E) Similar analysis (means ± S.E., n  =  4) shows ΔΔ[Ca2+]i recorded 2 min after CCh addition. (F) Cells were stimulated with CCh (1 mM, 15 min) in nominally Ca2+-free HBS with or without bafilomycin A1 (1 µM, 1 h) before restoration of the indicated concentrations of extracellular Ca2+. Results (means ± S.E., n  =  4) show the sustained increase in [Ca2+]i.

https://doi.org/10.1371/journal.pone.0111275.g004

The results shown in Figure 4B establish that the increase in [Ca2+]i resulting from CCh-evoked release of intracellular Ca2+ stores is complete within 2 min, whereas the small Ca2+ signal mediated by SOCE persists for much longer. We therefore analysed the increases in [Ca2+]i (Δ[Ca2+]i) detected 2 min after CCh addition in the absence and presence of extracellular Ca2+ to assess the effects of bafilomycin A1 on CCh-evoked SOCE. The difference between these values (ΔΔ[Ca2+]i  = Δ[Ca2+]i with extracellular Ca2+- Δ[Ca2+]i without extracellular Ca2+) reports the magnitude of the CCh-evoked SOCE (Figures 4B and 4C).

Bafilomycin A1 potentiated the initial peak increase in [Ca2+]i evoked by CCh in both the absence and presence of extracellular Ca2+ by 1.3±0.07 and 1.3±0.04-fold, respectively (Figures 4B–4D). This is consistent with sequestration by lysosomes of Ca2+ released by IP3Rs [30]. Because bafilomycin A1 slows the recovery of [Ca2+]i during IP3-evoked Ca2+ release [30], [Ca2+]i was still higher in bafilomycin A1-treated relative to control cells after a 2-min exposure to CCh in Ca2+-free HBS (compare the black traces in Figures 4B and 4C). More importantly, however, bafilomycin A1 had no effect on ΔΔ[Ca2+]i, which was 24±8 nM and 25±5 nM for control and bafilomycin A1-treated cells, respectively (Figure 4E). These results suggest that CCh-evoked SOCE is insensitive to bafilomycin A1.

The amplitudes of the sustained Ca2+ signals evoked by CCh in normal HBS are small relative to those resulting from IP3-evoked Ca2+ release (Figures 4B and 4C). We therefore examined the effects of bafilomycin A1 on CCh-evoked Ca2+ entry under conditions that temporally separated Ca2+ release from Ca2+ entry. We also used higher concentrations of extracellular Ca2+ to exaggerate the Ca2+ entry signals. Cells were treated with CCh in nominally Ca2+-free HBS for 15 min to deplete the ER and activate SOCE. Different extracellular Ca2+ concentrations were then restored in the continued presence of CCh. The results demonstrate that bafilomycin A1 has no effect on the sustained phase of the resulting increase in [Ca2+]i at any extracellular Ca2+ concentration (Figure 4F). These results suggest that CCh-evoked SOCE, like that evoked by thapsigargin [30], is insensitive to inhibition of lysosomal Ca2+ uptake.

Lysosomes recycle the Ca2+ accumulated after stimulation of IP3 receptors

The results so far demonstrate that in HEK cells stimulated with CCh, lysosomes selectively sequester Ca2+ released via IP3Rs, but not Ca2+ entering the cell via SOCE (Figures 1 and 4). We next assessed whether Ca2+ accumulated by lysosomes remains trapped within them or gets rapidly recycled to the ER via the cytosol (Figure S1A).

To address this issue, HEK cells were stimulated with CCh under conditions (1 mM GdCl3 in the extracellular medium) that inhibit both Ca2+ extrusion across the plasma membrane and Ca2+ entry [37] (Figure 5A inset). Comparison of the black traces in Figures 5A and 5B, where HEK cells in nominally Ca2+-free HBS were repeatedly stimulated with brief pulses of a maximally effective concentration of CCh (1 mM), demonstrates that the approach is effective, albeit without fully preventing loss of Ca2+ from stimulated cells. The incomplete inhibition of Ca2+ loss by Gd3+ contrasts with a previous analysis of HEK cells where CCh-evoked Ca2+ oscillations persisted for many minutes with undiminished amplitude in Ca2+-free medium supplemented with 1 mM Gd3+ [37]. The different results probably result from the much higher concentration of CCh used in our experiments (1 mM) relative to that used to evoke Ca2+ oscillations (1–5 µM) [37]. In Ca2+-free HBS, cells responded robustly to the first CCh challenge, but not to subsequent challenges (Figure 5A). In the same HBS supplemented with Gd3+, even the fourth challenge with CCh evoked a detectable increase in [Ca2+]i (Figures 5B and 5C). These results confirm that a substantial fraction of the Ca2+ released from intracellular stores by IP3 is normally extruded from the cell. That Ca2+ would normally be replenished by SOCE, but in the absence of extracellular Ca2+ the stores are unable to refill. A high concentration of Gd3+, by inhibiting Ca2+ exchanges across the plasma membrane (both influx and efflux), allows Ca2+ to be recycled within the cell and thereby allows the ER to respond to repeated CCh challenges (Figures 5B and 5C).

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Figure 5. Lysosomes rapidly recycle the Ca2+ sequestered after IP3-evoked Ca2+ release.

(A, B) HEK cells were repetitively stimulated with CCh (1 mM, 30 s) alone or with bafilomycin A1 (1 µM, 1 h) in nominally Ca2+-free HBS without (A) or with Gd3+ (1 mM) (B). Results show means ± S.E. for ≥ 45 cells from a single experiment, typical of at least 3 similar experiments. The inset to panel A shows how a high concentration of Gd3+ (1 mM) effectively insulates the cell from exchanging Ca2+ with the extracellular environment by blocking Ca2+ entry and extrusion [37]. Under these conditions, repetitive responses to CCh are entirely dependent on recycling of intracellular Ca2+ (dashed lines). (C) Summary results show effects of Gd3+ on the peak increase in [Ca2+]i evoked by each challenge with CCh in the absence of bafilomycin A1. (D) Predicted effects of bafilomycin A1 on the Ca2+ signals evoked by repetitive CCh challenges of Gd3+-insulated cells. The predicted results represent an idealized situation in which Gd3+ entirely insulates the cell from Ca2+ exchanges with the extracellular environment (in practise the insulation is incomplete), and then shows the results predicted for situations where lysosomes either accumulate (upper panel) or entirely recycle (lower panel) the sequestered Ca2+ (see Figure S1A). (E) Peak increases in [Ca2+]i evoked by the first CCh challenge under the conditions shown. *p <0.05, paired Students's t-test. (F) Effects of bafilomycin A1 on the peak increases in [Ca2+]i evoked by successive CCh challenges in nominally Ca2+-free HBS containing 1 mM Gd3+. Results are normalized to the first CCh challenge for each condition (the raw data and the results obtained in the absence of Gd3+ are shown in Figure S1B and S1C). Results (C, E and F) are means ± S.E. from at least 4 independent experiments.

https://doi.org/10.1371/journal.pone.0111275.g005

Some of the Ca2+ released by IP3Rs is sequestered by lysosomes (Figure 1). If that sequestered Ca2+ were only very slowly recycled to the ER (i.e. more slowly than the 5-min interval between the CCh challenges shown in Figure 5), the effect of lysosomes would be analogous to Ca2+ extrusion across the plasma membrane (Figure S1A). The lysosomes would then effectively remove Ca2+ from the recycling pool, just as Ca2+ extrusion across the plasma membrane in Ca2+-free medium effectively depletes the pool of Ca2+ available for signalling. The amplitude of the Ca2+ signals evoked by repetitive pulses of CCh under ‘Gd3+-insulating’ conditions would then be expected to decay more quickly when lysosomes are active because with each Ca2+ spike lysosomes would effectively remove some Ca2+ from the recycling pool. A cartoon representation of the predicted effects of bafilomycin A1 on the Ca2+ signals evoked by repetitive CCh challenges is shown in idealized form in Figure 5D, which assumes that Gd3+ is entirely effective in preventing Ca2+ fluxes across the plasma membrane. Bafilomycin A1 is predicted to have no effect on the run-down of CCh-evoked Ca2+ signals if Ca2+ is rapidly recycled from lysosomes, and to slow the run-down if lysosomes normally retain the sequestered Ca2+ and so remove it from the signalling pool (Figure 5D). We tested these predictions by measuring the effects of bafilomycin A1 on the responses to repeated brief (30 s) challenges with CCh in nominally Ca2+-free HBS supplemented with 1 mM Gd3+ (Figures 5A and 5B).

As expected, bafilomycin A1 potentiated the increase in [Ca2+]i evoked by CCh in both the absence and presence of Gd3+ (1.8±0.4 and 1.7±0.2-fold increase, respectively) (Figure 5E). It is, however, noteworthy that the peak amplitude of the CCh-evoked Ca2+ signal was unaffected by Gd3+ (Figures 5A–5C and 5E). This suggests that Ca2+ sequestration by lysosomes is fast enough to attenuate the initial IP3-evoked Ca2+ release signal, while extrusion of Ca2+ across the plasma membrane is either too slow or too far removed from the site of Ca2+ release to detectably affect the initial rise in [Ca2+]i.

Neither bafilomycin A1 nor Gd3+ affected the number of cells responding to the initial CCh challenge (Table 1). However, responses to each successive CCh challenge were larger in the presence of bafilomycin A1 (Figures 5A and 5B). This confirms that each CCh challenge normally evokes a sequestration of Ca2+ by lysosomes. Despite the larger CCh-evoked Ca2+ signals in the presence of bafilomycin A1, the rate at which the peak amplitude of the Ca2+ signal declined with each successive CCh challenge was identical in control and bafilomycin A1-treated cells (Figure 5F, Figure S1B and S1C). These results suggest that lysosomes rapidly recycle the Ca2+ they accumulate during IP3-evoked Ca2+ release (lower panel in Figure 5D and Figure S1A).

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Table 1. Neither Gd3+ nor bafilomycin A1 affects the number of cells that respond to carbachol.

https://doi.org/10.1371/journal.pone.0111275.t001

Discussion

We have shown that lysosomes sequester Ca2+ released from the ER [30]. The present work demonstrates that different IP3-sensitive Ca2+ stores within the compartmentalized ER of HEK cells [31] are each capable of directing the Ca2+ released by IP3Rs to lysosomal Ca2+ uptake systems (Figures 1 and 6). By contrast the Ca2+ signals evoked by SOCE, whether activated pharmacologically [30] or by endogenous receptors that stimulate PLC (Figure 4), are insensitive to inhibition of lysosomes. This is not due to the small amplitude of SOCE-mediated Ca2+ signals (Figures 3A and 4) because SOCE remains insensitive to inhibition of lysosomes when SOCE-evoked increases in global [Ca2+]i are larger than those evoked by IP3Rs [30]. The insensitivity of CCh-evoked SOCE to inhibition of lysosomal Ca2+ uptake suggests two important conclusions. First, it reinforces our suggestion that lysosomes selectively sequester Ca2+ released by IP3Rs [30]. The intimacy of the relationship between ER and lysosomes is further supported by the different effects of inhibiting lysosomes (Figure 1) or Ca2+ extrusion across the plasma membrane (Figure 5C). Only the former increases the amplitude of the initial CCh-evoked increase in [Ca2+]i, suggesting that only lysosomes are both close enough to IP3Rs and accumulate Ca2+ fast enough to attenuate the initial response to IP3. Second, it suggests that during SOCE in HEK cells, there is probably no significant flux of Ca2+ from Orai channels into the ER and then back into the cytosol via IP3Rs (Figure 4A lower panel).

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Figure 6. Associations of ER with other Ca2+-sequestering organelles allows selective and reversible modulation of cytosolic Ca2+ signals.

Close association of lysosomes (LY) with ER [30], probably mediated by specific tethers (red) [46], allows them selectively to accumulate Ca2+ released by IP3Rs from distinct ER Ca2+ stores, but not Ca2+ entering the cell via SOCE. Mitochondria (right), depending on cell type, can selectively accumulate Ca2+ released from the ER, to which they are tethered, or entering the cell via SOCE [11]. For lysosomes, neither the Ca2+ uptake pathway (1) nor the efflux pathway (2) that rapidly recycles Ca2+ back to the ER via the cytosol have been identified. The equivalent pathways in mitochondria are the MCU (1) and Na+/Ca2+ or H+/Ca2+ exchangers (2) of the inner mitochondrial membrane. Rapid, reversible and selective ‘buffering’ of cytosolic Ca2+ signals by both lysosomes and mitochondria allows these organelles to both shape and decode stimulus-evoked Ca2+ signals.

https://doi.org/10.1371/journal.pone.0111275.g006

Our observations are consistent with evidence that lysosomes are both closely associated with ER [28], [30], [45], [46] and maintain their association as each organelle moves [30]. This relationship is reminiscent of that between ER and mitochondria [30], where tethering of the two organelles at mitochondria-associated membranes (MAMs) allows local exchange of Ca2+ and lipids [11], [47]. The mitochondrial uniporter (MCU) mediates Ca2+ uptake by mitochondria, whereas mitofusin 2 [48] and perhaps other proteins that may include IP3Rs [9], [11], contribute to formation of mitochondrion-ER junctions. For lysosomes, neither the Ca2+ uptake mechanism [14] nor the ER tethers are known, although both are important questions for future work. Tethering of ER to the vacuole in yeast (analogous to the acidic organelles of higher eukaryotes) is mediated by interaction of proteins anchored to ER (NVJ1) and vacuolar membranes (Vac8). These then recruit Osh1, an oxysterol-binding protein-related protein (ORP) and a lipid-synthesizing enzyme (Tcs13) [9]. Interactions between ORPs [49] or other lipid-binding proteins like STARD3 (steroidogenic acute regulatory protein domain 3) [46], and the ER protein, VAP (VAMP-related proteins), may contribute to assembly of ER-lysosome junctions in higher eukaryotes [50]. We speculate that these, or additional tethering proteins, may maintain the close association between lysosomes and ER required to allow lysosomes to accumulate Ca2+ selectively and rapidly in response to its release by IP3Rs (Figure 6).

Mitochondrial Ca2+ uptake plays an important role in buffering cytosolic Ca2+ signals [11]. The capacity of mitochondria to modulate [Ca2+]i is abrogated when mitochondrial Ca2+ efflux is inhibited [51], [52]. Furthermore, temporal changes of [Ca2+] within mitochondria faithfully track even quite rapid oscillations in [Ca2+]i [53]. These observations suggest that mitochondria can rapidly recycle at least some of the Ca2+ they accumulate from the cytosol, and that rapid shuttling of Ca2+ between the ER and mitochondria contributes to both cytosolic Ca2+ oscillations [54] and mitochondrial activity [53]. We suggest a similar situation for lysosomes (Figure 6), although neither the Ca2+ uptake nor efflux pathways are resolved for lysosomes. It is clear from experiments where cells were first stimulated with CCh and then with CCh and PTH (Figure 1) that the ability of lysosomes to sequester Ca2+ is unaffected by prior Ca2+ sequestration. This suggests that lysosomes have a considerable capacity to accumulate Ca2+, or that having sequestered Ca2+ they can rapidly recycle it, via the cytosol, to other organelles. We used Gd3+ to ‘insulate’ cells from Ca2+ exchanges with the extracellular environment and so force them into relying on recycling of intracellular Ca2+ pools to generate increases in [Ca2+]i [37]. Under these conditions, we demonstrated that successive responses to CCh were each exaggerated by inhibition of lysosomes, but the rate at which Ca2+ was lost from the recycling pool of Ca2+ was unaffected (Figure 5 and Figure S1). These results suggest that lysosomes rapidly recycle the Ca2+ that they accumulate (Figure 6). This conclusion is consistent with evidence that inhibition of lysosomes increases the amplitude, but decreases the frequency, of the Ca2+ spikes evoked by low concentrations of CCh [30]. The latter reflecting the slower, but still effective, recycling of Ca2+ from the cytosol to ER when lysosomes are active.

We conclude that lysosomes rapidly, reversibly and selectively accumulate Ca2+ released by IP3Rs, even when the IP3Rs reside in distinct Ca2+ stores, but they are unable to accumulate Ca2+ entering cells via SOCE (Figures 14). The behaviour of lysosomes provides a striking analogy with mitochondria [30]. Both organelles rapidly accumulate Ca2+ from microdomains surrounding specific Ca2+ channels and thereby shape cytosolic Ca2+ signals [11], [30] (Figures 1, 4 and 5), and both are capable of rapidly recycling the accumulated Ca2+ [51], [52], [53], [54] (Figure 5). Finally, for both organelles the increase in luminal [Ca2+] regulates their activity: enzyme activity, apoptosis and motility for mitochondria [11], and endo-lysosomal trafficking [55] and perhaps ion channel activity [56] for lysosomes (Figure 6).

Materials and Methods

Materials

Dulbecco's modified Eagle's/Ham's F-12 (DMEM/F-12), fluo-4-AM, fura-2-AM, dextran-conjugates of Oregon Green (Mr = 10,000) and Texas Red (Mr = 10,000), and Ca2+ standard solutions were from Invitrogen (Paisley, U.K.). NED-19 was from Enzo Life Sciences (Exeter, U.K.). G418 was from Formedium (Norfolk, U.K.). Cell culture plastics and 96-well plates were from Greiner (Stonehouse, Gloucestershire, U.K.). Imaging dishes (35-mm diameter with a 7-mm No. 0 glass insert) were from MatTek Corporation (Ashland, MA, U.S.A.) or PAA Laboratories (Yeovil, U.K.). Carbamyl choline chloride (carbachol, CCh), DMSO, foetal bovine serum (FBS), poly-l-lysine, Pluronic F127 and Triton-X-100 were from Sigma-Aldrich (Poole, Dorset, U.K.). BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) was from Molekula (Dorset, U.K.). Bafilomycin A1 was from AG scientific (San Diego, CA, U.S.A.). Parathyroid hormone (PTH, residues 1–34) was from Bachem (St. Helens, U.K.). Ionomycin was from Merck Eurolab (Nottingham, U.K.). Thapsigargin was from Alomone Labs (Jerusalem, Israel).

Cell culture

HEK cells and HEK cells stably expressing human type 1 PTH receptors (HEK-PR1 cells) were cultured at 37°C in DMEM/F-12 medium with GlutaMAX-1, FBS (10%) and G418 (800 µg/ml for HEK-PR1 cells) in humidified air with 5% CO2. For experiments, cells were seeded into 96-well plates or onto 22-mm round glass coverslips coated with 0.01% (w/v) poly-l-lysine.

Measurements of [Ca2+]i

[Ca2+]i in populations of confluent cells loaded with fluo-4 was measured at intervals of 1.44 s using a fluorescence plate-reader as described previously [34]. Cells were incubated at 20°C in HEPES-buffered saline (HBS: NaCl 135 mM, KCl 5.9 mM, MgCl2 1.2 mM, CaCl2 1.5 mM, HEPES 11.6 mM and glucose 11.5 mM, pH 7.3). Ca2+ was omitted from nominally Ca2+-free HBS, and replaced by BAPTA (10 mM) in Ca2+-free HBS. Fluorescence (F) was calibrated to [Ca2+]i from [Ca2+]i = KD(F-Fmin)/(Fmax-F), where KD is the dissociation constant of fluo-4 for Ca2+ (345 nM), Fmin and Fmax are the fluorescence signals recorded after treatment of parallel wells with Triton X-100 (0.1% v/v) in HBS supplemented with 10 mM BAPTA or 10 mM CaCl2, respectively. Concentration-effect relationships were fitted to Hill equations using non-linear curve-fitting (GraphPad Prism, version 5).

For single-cell imaging, confluent cultures of HEK cells on 22-mm round, poly-l-lysine-coated glass coverslips were loaded with fura-2-AM (2 µM, 1 h) supplemented with Pluronic F127 (0.02% v/v), washed and incubated for a further 1 h in HBS. Fluorescence, detected at>510 nm after alternating excitation at 340 and 380 nm, was detected using an Olympus IX71 inverted fluorescence microscope with a Luca EMCCD camera (Andor Technology, Belfast, U.K.). After correction for background fluorescence by addition of MnCl2 (10 mM) and ionomycin (1 µM) at the end of the experiment, fluorescence ratios (F340/F380) were calibrated to [Ca2+]i using Ca2+ standard solutions [34]. Only cells that responded to the first stimulation with CCh (typically>80% of cells) were included in the analysis of Ca2+ signals evoked by successive CCh challenges (see Figure 5).

Measurement of lysosomal pH

Almost confluent cultures of HEK cells grown on poly-l-lysine-coated, glass-bottomed dishes were incubated in culture medium with dextran-conjugates of Texas Red (TR, 0.1 mg/ml, an inert marker) and Oregon Green (OG, 0.1 mg/ml, a pH indicator) for 12 h at 37°C to allow uptake of the indicators by endocytosis. After a further incubation (4 h) without indicators, the cells were washed with HBS and fluorescence was recorded in HBS at 20°C using an Olympus IX81 microscope with a 60x/1.45 NA objective. Cells were illuminated with a mercury xenon lamp using alternating filter sets: U-MNIBA (Olympus, λex 470–495 nm, λem 510–550 nm for OG) and LF561A (Semrock, λex 550–570 nm, λem 580–630 nm for TR). Images were captured at 2-s intervals using an EMCCD camera (Andor iXon 897) and analyzed using Cell∧R software (Olympus, Milton Keynes, U.K.). Records were corrected for background fluorescence determined under identical conditions from cells without indicators. Fluorescence changes from defined regions of interest (ROI) are expressed as F/F0, where F0 and F denote the average fluorescence within the ROI at the start of the experiment (F0) and at each time point (F).

Supporting Information

Figure S1.

Responses to repetitive challenges with carbachol reveal that Ca2+ rapidly recycles from lysosomes. (A) A fraction of the Ca2+ released from the ER via IP3Rs is normally lost to the extracellular space as Ca2+ pumps in the plasma membrane (PM) extrude it from the cytosol. When Ca2+ is present in the extracellular medium, this loss is replenished by store-operated Ca2+ entry (SOCE). Removal of extracellular Ca2+ or blockade of SOCE by Gd3+ prevents this recycling of Ca2+. Lysosomes also sequester Ca2+ released by IP3Rs [30], but it is important to resolve whether that Ca2+ is also rapidly recycled via the cytosol to the ER. The experiments shown in Figure 5 address this issue. (B) The Ca2+ signals evoked by repetitive challenges with CCh (1 mM, 30 s) were recorded from HEK cells in Ca2+-free HBS with 1 mM Gd3+ (as shown in Figure 5B). The peak amplitudes of the Ca2+ signals are shown for control cells and cells treated with bafilomycin A1 (means ±S.E., n = 6). These raw data were used to produce Figure 5F. (C) Summary data (means ±S.E., n = 6) from experiments similar to those shown in (B), but in Ca2+-free HBS, show that in the absence of high concentrations of Gd3+, cells respond robustly to the first CCh challenge, but not to subsequent challenges.

https://doi.org/10.1371/journal.pone.0111275.s001

(TIF)

Author Contributions

Conceived and designed the experiments: CILS SCT CWT. Performed the experiments: CILS. Analyzed the data: CILS SCT CWT. Wrote the paper: CILS SCT CWT.

References

  1. 1. Berridge MJ, Bootman MD, Roderick HL (2003) Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4: 517–529.
  2. 2. Rizzuto R, Pozzan T (2006) Microdomains of intracellular Ca2+: molecular determinants and functional consequences. Physiol Rev 86: 369–408.
  3. 3. Schwaller B (2012) Cytosolic Ca2+ buffers. Cold Spring Harb Persp Biol 2: a004051.
  4. 4. Taylor CW, Dale P (2012) Intracellular Ca2+ channels - a growing community. Mol Cell Endocrinol 353: 21–28.
  5. 5. Foskett JK, White C, Cheung KH, Mak DO (2007) Inositol trisphosphate receptor Ca2+ release channels. Physiol Rev 87: 593–658.
  6. 6. Taylor CW, Tovey SC (2010) IP3 receptors: toward understanding their activation. Cold Spring Harb Persp Biol 2: a004010.
  7. 7. Smith IF, Parker I (2009) Imaging the quantal substructure of single IP3R channel activity during Ca2+ puffs in intact mammalian cells. Proc Natl Acad Sci USA 106: 6404–6409.
  8. 8. English AR, Voeltz GK (2013) Endoplasmic reticulum structure and interconnections with other organelles. Cold Spring Harb Persp Biol 5: a013227.
  9. 9. Elbaz Y, Schuldiner M (2011) Staying in touch: the molecular era of organelle contact sites. Trends Biochem Sci 36: 616–623.
  10. 10. Toulmay A, Prinz WA (2011) Lipid transfer and signaling at organelle contact sites: the tip of the iceberg. Curr Opin Cell Biol 23: 458–463.
  11. 11. Rizzuto R, De Stefani D, Raffaello A, Mammucari C (2012) Mitochondria as sensors and regulators of calcium signalling. Nat Rev Mol Cell Biol 13: 566–578.
  12. 12. Gomes DA, Leite MF, Bennett AM, Nathanson MH (2006) Calcium signaling in the nucleus. Can J Physiol Pharm 84: 325–332.
  13. 13. Patel S, Ramakrishnan L, Rahman T, Hamdoun A, Marchant JS, et al. (2011) The endo-lysosomal system as an NAADP-sensitive acidic Ca2+ store: Role for the two-pore channels. Cell Calcium 50: 157–167.
  14. 14. Morgan AJ, Platt FM, Lloyd-Evans E, Galione A (2011) Molecular mechanisms of endolysosomal Ca2+ signalling in health and disease. Biochem J 439: 349–374.
  15. 15. Orci L, Ravazzola M, Le Coadic M, Shen WW, Demaurex N, et al. (2009) STIM1-induced precortical and cortical subdomains of the endoplasmic reticulum. Proc Natl Acad Sci USA 106: 19358–19362.
  16. 16. Lewis RS (2012) Store-operated calcium channels: new perspectives on mechanism and function. Cold Spring Harb Persp Biol: a003970.
  17. 17. De Stefani D, Raffaello A, Teardo E, Szabo I, Rizzuto R (2011) A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature 476: 336–340.
  18. 18. Baughman JM, Perocchi F, Girgis HS, Plovanich M, Belcher-Timme CA, et al. (2011) Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter. Nature 476: 341–345.
  19. 19. Hajnóczky G, Hager R, Thomas AP (1999) Mitochondria suppress local feedback activation of inositol 1,4,5-trisphosphate receptors by Ca2+. J Biol Chem 274: 14157–14162.
  20. 20. Olson ML, Chalmers S, McCarron JG (2010) Mitochondrial Ca2+ uptake increases Ca2+ release from inositol 1,4,5-trisphosphate receptor clusters in smooth muscle cells. J Biol Chem 285: 2040–2050.
  21. 21. Kiselyov K, Yamaguchi S, Lyons CW, Muallem S (2010) Aberrant Ca2+ handling in lysosomal storage disorders. Cell Calcium 47: 103–111.
  22. 22. Calcraft PJ, Ruas M, Pan Z, Cheng X, Arredouani A, et al. (2009) NAADP mobilizes calcium from acidic organelles through two-pore channels. Nature 459: 596–600.
  23. 23. Yamaguchi S, Jha A, Li Q, Soyombo AA, Dickinson GD, et al. (2011) TRPML1 and two-pore channels are functionally independent organellar ion channels. J Biol Chem 286: 22934–22942.
  24. 24. Huang P, Zou Y, Zhong XZ, Cao Q, Zhao K, et al. (2014) P2X4 forms functional ATP-activated cation channels on lysosomal membranes regulated by luminal pH. J Biol Chem 289: 17658–17667.
  25. 25. Pitt SJ, Lam AK, Rietdorf K, Galione A, Sitsapesan R (2014) Reconstituted human TPC1 is a proton-permeable ion channel and is activated by NAADP or Ca2+. Sci Signal 7: ra46.
  26. 26. Cang C, Zhou Y, Navarro B, Seo YJ, Aranda K, et al. (2013) mTOR regulates lysosomal ATP-sensitive two-pore Na+ channels to adapt to metabolic state. Cell 152: 778–790.
  27. 27. Dong XP, Shen D, Wang X, Dawson T, Li X, et al. (2010) PI(3,5)P2 controls membrane traffic by direct activation of mucolipin Ca2+ release channels in the endolysosome. Nat Commun 1: 38.
  28. 28. Morgan AJ, Davis LC, Wagner SK, Lewis AM, Parrington J, et al. (2013) Bidirectional Ca2+ signaling occurs between the endoplasmic reticulum and acidic organelles. J Cell Biol 18: 789–805.
  29. 29. Brailoiu E, Rahman T, Churamani D, Prole DL, Brailoiu GC, et al. (2010) An NAADP-gated two-pore channel targeted to the plasma membrane uncouples triggering from amplifying Ca2+ signals. J Biol Chem 285: 38511–38516.
  30. 30. Lopez Sanjurjo CI, Tovey SC, Prole DL, Taylor CW (2013) Lysosomes shape Ins(1,4,5)P3-evoked Ca2+ signals by selectively sequestering Ca2+ released from the endoplasmic reticulum. J Cell Sci 126: 289–300.
  31. 31. Tovey SC, Taylor CW (2013) Cyclic AMP directs inositol (1,4,5)-trisphosphate-evoked Ca2+ signalling to different intracellular Ca2+ stores. J Cell Sci 126: 2305–2313.
  32. 32. Courjaret R, Machaca K (2014) Mid-range Ca2+ signalling mediated by functional coupling between store-operated Ca2+ entry and IP3-dependent Ca2+ release. Nature Commun 5: 3916.
  33. 33. Suzuki J, Kanemaru K, Ishii K, Ohkura M, Okubo Y, et al. (2014) Imaging intraorganellar Ca2+ at subcellular resolution using CEPIA. Nat Commun 5: 4153.
  34. 34. Tovey SC, Dedos SG, Taylor EJA, Church JE, Taylor CW (2008) Selective coupling of type 6 adenylyl cyclase with type 2 IP3 receptors mediates a direct sensitization of IP3 receptors by cAMP. J Cell Biol 183: 297–311.
  35. 35. Yoshimori T, Yamamoto A, Moriyama Y, Futai M, Tashiro Y (1991) Bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J Biol Chem 266: 17707–17712.
  36. 36. Naylor E, Arredouani A, Vasudevan SR, Lewis AM, Parkesh R, et al. (2009) Identification of a chemical probe for NAADP by virtual screening. Nat Chem Biol 5: 220–226.
  37. 37. Bird GSJ, Putney JW (2004) Capacitative calcium entry supports calcium oscillations in human embryonic kidney cells. J Physiol 562: 697–706.
  38. 38. Putney JW (2009) Capacitative calcium entry: from concept to molecules. Immunol Rev 231: 10–22.
  39. 39. Broad LM, Cannon TR, Taylor CW (1999) A non-capacitative pathway activated by arachidonic acid is the major Ca2+ entry mechanism in rat A7r5 smooth muscle cells stimulated with low concentrations of vasopressin. J Physiol 517: 121–134.
  40. 40. Shuttleworth TJ (2004) Receptor-activated calcium entry channels - who does what, and when? Science STKE pe40.
  41. 41. Shuttleworth TJ, Thompson JL (1999) Discriminating between capacitative and arachidonate-activated Ca2+ entry pathways in HEK293 cells. J Biol Chem 274: 31174–31178.
  42. 42. Mignen O, Thompson JL, Shuttleworth TJ (2001) Reciprocal regulation of capacitative and arachidonate-regulated noncapacitative Ca2+ entry pathways. J Biol Chem 276: 35676–35683.
  43. 43. Kar P, Nelson C, Parekh AB (2012) CRAC channels drive digital activation and provide analog control and synergy to Ca2+-dependent gene regulation. Curr Biol 22: 242–247.
  44. 44. Willoughby D, Cooper DMF (2007) Organization and Ca2+ regulation of adenylyl cyclases in cAMP microdomains. Physiol Rev 87: 965–1010.
  45. 45. Kilpatrick BS, Eden ER, Schapira AH, Futter CE, Patel S (2013) Direct mobilisation of lysosomal Ca2+ triggers complex Ca2+ signals. J Cell Sci 126: 60–66.
  46. 46. Alpy F, Rousseau A, Schwab Y, Legueux F, Stoll I, et al. (2013) STARD3/STARD3NL and VAP make a novel molecular tether between late endosomes and the ER. J Cell Sci 126: 5500–5512.
  47. 47. de Brito OM, Scorrano L (2010) An intimate liaison: spatial organization of the endoplasmic reticulum-mitochondria relationship. EMBO J 29: 2715–2723.
  48. 48. de Brito OM, Scorrano L (2008) Mitofusin 2 tethers endoplasmic reticulum to mitochondria. Nature 456: 605–610.
  49. 49. Rocha N, Kuijl C, van der Kant R, Janssen L, Houben D, et al. (2009) Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150 Glued and late endosome positioning. J Cell Biol 185: 1209–1225.
  50. 50. Helle SC, Kanfer G, Kolar K, Lang A, Michel AH, et al. (2013) Organization and function of membrane contact sites. Biochim Biophys Acta 1833: 2526–2541.
  51. 51. Naghdi S, Waldeck-Weiermair M, Fertschai I, Poteser M, Graier WF, et al. (2010) Mitochondrial Ca2+ uptake and not mitochondrial motility is required for STIM1-Orai1-dependent store-operated Ca2+ entry. J Cell Sci 123: 2553–2564.
  52. 52. Malli R, Frieden M, Trenker M, Graier WF (2005) The role of mitochondria for Ca2+ refilling of the endoplasmic reticulum. J Biol Chem 280: 12114–12122.
  53. 53. Hajnóczky G, Robb-Gaspers LD, Seitz MB, Thomas AP (1995) Decoding cytosolic calcium oscillations in the mitochondria. Cell 82: 415–424.
  54. 54. Ishii K, Hirose K, Iino M (2006) Ca2+ shuttling between endoplasmic reticulum and mitochondria underlying Ca2+ oscillations. EMBO Rep 7: 390–396.
  55. 55. Luzio JP, Pryor PR, Bright NA (2007) Lysosomes: fusion and function. Nat Rev Mol Cell Biol 8: 622–632.
  56. 56. Pitt SJ, Funnell T, Sitsapesan M, Venturi E, Rietdorf K, et al. (2010) TPC2 is a novel NAADP-sensitive Ca2+-release channel, operating as a dual sensor of luminal pH and Ca2+. J Biol Chem 285: 35039–35046.