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Research Article
A computational model of lysosome–ER Ca2+ microdomains
Christopher J. Penny, Bethan S. Kilpatrick, Jung Min Han, James Sneyd, Sandip Patel
Journal of Cell Science 2014 127: 2934-2943; doi: 10.1242/jcs.149047

ABSTRACT

Acidic organelles form an important intracellular Ca2+ pool that can drive global Ca2+ signals through coupling with endoplasmic reticulum (ER) Ca2+ stores. Recently identified lysosome–ER membrane contact sites might allow formation of Ca2+ microdomains, although their size renders observation of Ca2+ dynamics impractical. Here, we generated a computational model of lysosome–ER coupling that incorporated a previous model of the inositol trisphosphate (IP3) receptor as the ER Ca2+ ‘amplifier’ and lysosomal leaks as the Ca2+ ‘trigger’. The model qualitatively described global Ca2+ responses to the lysosomotropic agent GPN, which caused a controlled but substantial depletion of small solutes from the lysosome. Adapting this model to physiological lysosomal leaks induced by the Ca2+ mobilising messenger NAADP demonstrated that lysosome–ER microdomains are capable of driving global Ca2+ oscillations. Interestingly, our simulations suggest that the microdomain [Ca2+] need not be higher than that in the cytosol for responses to occur, thus matching the relatively high affinity of IP3 receptors for Ca2+. The relative distribution and overall density of the lysosomal leaks dictated whether microdomains triggered or modulated global signals. Our data provide a computational framework for probing lysosome–ER Ca2+ dynamics.

INTRODUCTION

Changes in cytosolic Ca2+ ion concentration ([Ca2+]) form the basis of a ubiquitous signalling pathway responsible for regulating a multitude of disparate cellular events ranging from fertilisation to cell death (Berridge et al., 2000). Specificity is realised through temporal and spatial complexities in the Ca2+ signals, which can be achieved by controlled mobilisation of not only endoplasmic reticulum (ER) Ca2+ stores, but also those in acidic organelles such as lysosomes (Patel and Docampo, 2010). The ubiquitous inositol 1,4,5 trisphosphate receptor (IP3R), together with the ryanodine receptor (RyR), are the principal intracellular Ca2+ release channels, which localise predominantly to the sarcoplasmic reticulum (SR) and ER (Clapham, 2007). These channels can be activated by second messengers (IP3 and cyclic ADP ribose) and are regulated by cytosolic [Ca2+] in a biphasic manner. Both support Ca2+-induced Ca2+ release (CICR) whereby modest rises in [Ca2+] generate a Ca2+ spike (Roderick et al., 2003). Subsequent negative feedback terminates the spike, allowing clearance by ‘off’ mechanisms such as the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) (Berridge et al., 2000).

Membrane contact sites are regions of close membrane apposition (<30 nm) between organelles, allowing congregation of Ca2+ signalling proteins in restricted spaces, forming the anatomical basis for the encoding and decoding of functional ‘Ca2+ microdomains’ (Berridge, 2006; Helle et al., 2013). Ca2+ signalling between the ER and the plasma membrane or organelles, such as mitochondria, is a well-characterised constituent of the Ca2+ signalling network (Helle et al., 2013). For example, mitochondria form tight (∼10 nm) physical junctions with the ER in many cell types allowing mitochondrial uptake of ER-released Ca2+, thereby matching mitochondrial ATP production to cellular demand (Csordás et al., 2006; Szabadkai et al., 2006; Tarasov et al., 2012). Furthermore, in cardiac muscle, a strongly coupled Ca2+ microdomain in the dyadic cleft junction (∼15-nm wide) between L-type Ca2+ channels on the plasma membrane and clusters of ryanodine receptors on the SR is crucial for excitation–contraction coupling (Bers, 2002; Franzini-Armstrong et al., 1999; Schendel et al., 2012). ER–mitochondria and SR/ER–plasma membrane associations provide a precedent for ER associations with other organelles, such as those within the endolysosomal system.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that mobilises Ca2+ from lysosomes and other acidic organelles (Churchill et al., 2002). Although NAADP is capable of producing global Ca2+ signals in a wide range of cell types, its ability to evoke such responses through acidic organelles alone is limited. This is owing to both the insensitivity of NAADP-induced Ca2+ release to cytosolic Ca2+, which precludes direct regenerative Ca2+ spikes that characterise IP3Rs and RyRs (Chini and Dousa, 1996; Genazzani and Galione, 1996), and the low lysosomal volume (∼1–2% of a cell) compared to the ER (∼10–20%). These observations have led to a ‘trigger hypothesis’ of NAADP action whereby ER-resident IP3Rs and RyRs amplify NAADP-induced Ca2+ signals (Cancela et al., 1999). Importantly, these putative NAADP trigger events cannot be resolved in the presence of IP3R and RyR inhibitors in certain cell types, suggesting that the NAADP-induced signals are modest and/or highly localised (Cancela et al., 1999; Marchant and Patel, 2013). We recently identified membrane contact sites between lysosomes and the ER analogous to those between the ER and other organelles, which are known to support local Ca2+ microdomain signalling (Kilpatrick et al., 2013). Indeed in sea urchin eggs, functional coupling between NAADP and the ER is disrupted upon homogenisation (Churchill et al., 2002). Moreover, recent studies also show that local NAADP responses (measured indirectly through changes in luminal pH) persist in the presence of EGTA, a slow Ca2+ buffer that prevents global responses (Morgan et al., 2013). These data further underscore the localised nature of NAADP signalling. In this context, it is notable that the putative targets for NAADP, the two-pore channels (TPCs) (Brailoiu et al., 2009; Calcraft et al., 2009; Zong et al., 2009), have a low maximal open probability (Pitt et al., 2010) and potentially poor selectivity for Ca2+ over Na+ (∼0.1) (Wang et al., 2012, but see Schieder et al., 2010).

The spatial and temporal scales involved in studying microdomains impede full experimental characterisation owing to the relatively low spatial resolution of light microscopy (∼200 nm) and the static nature of electron microscopy. However, these techniques, in tandem with mathematical modelling, can capture essential features of Ca2+ microdomain signalling. Computational modelling of Ca2+ microdomains in interstitial cells of Cajal has helped rationalise our understanding of slow wave generation in gastrointestinal smooth muscle (Means and Sneyd, 2010), and models of the cardiac muscle dyadic cleft have been constructed to accurately describe the behaviour of Ca2+ during excitation–contraction coupling (Cannell et al., 2013; Greenstein et al., 2006; Jafri et al., 1998; Rice et al., 1999). Such approaches allow prediction of the Ca2+ dynamics within microdomains, and of the behaviour of both microdomain and non-microdomain targets in response to applied signals.

In this study, we built a simple two-compartment model of a lysosome–ER microdomain and the contiguous bulk cytosol that is capable of qualitatively describing experimentally observed Ca2+ signalling behaviours induced by the lysosomotropic compound glycyl-L-phenylalanine 2-naphthylamide (GPN). We then adapted this model to a more physiological setting by generating the first computational model of NAADP action at TPCs. We demonstrate that, depending on their relative distribution and density, small NAADP-induced Ca2+ leaks couple with the IP3R at lysosome–ER microdomains to either drive or modulate global Ca2+ oscillations. Interestingly, these microdomains do not require high Ca2+ concentrations for their activity.

RESULTS

We have recently shown that stimulation of primary cultured human fibroblasts with GPN elicits complex, IP3R-dependent Ca2+ signals (Kilpatrick et al., 2013). Consistent with our previous work, these GPN-evoked Ca2+ signals are readily initiated and maintained in Ca2+-free medium (Fig. 1A,B). Fibroblasts therefore provide a tractable system for studying lysosomal Ca2+ signalling. GPN is a freely diffusible dipeptide substrate for the intralysosomal hydrolase cathepsin C. Hydrolysis of GPN is thought to rupture lysosomes owing to an increase in luminal osmolarity. Previous studies have employed isolated lysosomal preparations, therefore potentially removing cellular homeostatic mechanisms capable of maintaining lysosomal integrity (Berg et al., 1994; Jadot et al., 1984; Jadot et al., 1990). To examine the effect of GPN on lysosomal integrity in live cells, we labelled lysosomes with fluorescent dextrans by endosomal delivery. As shown in the confocal images in Fig. 1C, fluorescence of fluorescein–dextran (10 kDa) colocalised with fluorescence of Lysotracker Red, a small molecular mass (399 Da) acidotrope, indicating similar targeting of both probes to lysosomes. Treatment with 200 µM GPN caused a complete ablation of the Lysotracker signal, whereas the dextran labelling remained punctate and increased in intensity (Fig. 1C). Furthermore, live-cell epifluorescence imaging of rhodamine–dextran-labelled primary cultured fibroblasts showed that fluorescence did not decrease in response to GPN (Fig. 1D). Indeed, we noted a modest increase in fluorescence. In contrast, GPN induced complete loss of Lysotracker Red fluorescence in parallel experiments, as expected (Fig. 1D) (Kilpatrick et al., 2013). These data, summarised in Fig. 1E, suggest that GPN does not evoke complete rupture of lysosomes over the timecourse of our experiments, but rather induces a more limited leak of small molecular mass solutes (<10 kDa).

Fig. 1.
Fig. 1.

GPN-evoked complex Ca2+ signals are associated with leak of small molecular mass solutes from lysosomes. (A,B) Single-cell Ca2+ responses of fibroblasts stimulated with 200 µM GPN in the presence (A) or absence (B) of extracellular Ca2+. (C) Confocal fluorescence images of a representative individual fibroblast co-loaded with fluorescein–dextran (green) and Lysotracker Red (red) before (left, overlay) or 152 s after (middle, right; individual channels) addition of 200 µM GPN. Nuclei were stained using DAPI (blue). (D) Time courses of epifluorescence responses recorded from cells loaded with either Rhodamine–dextran or Lysotracker Red and stimulated with 200 µM GPN. Data are normalised to initial fluorescence values. (E) Pooled data (presented as mean±s.e.m.) quantifying fluorescence intensity before (0–60 s) and after (540–600 s) GPN addition. Results are from 54 dextran- and 43 Lysotracker-loaded cells from three or four independent experiments.

To further investigate the action of GPN, we examined the effects of a range of GPN concentrations on both Lysotracker fluorescence and cytosolic Ca2+ concentration. As shown in Fig. 2A, the rate of decrease of Lysotracker fluorescence in response to GPN was concentration-dependent. These data are again inconsistent with GPN causing osmotic lysis of lysosomes. At 200 µM GPN, complex Ca2+ signals were evoked in most cells (Fig. 2B). Similar results were obtained at a GPN concentration of 100 µM; however, there was a longer latency (Fig. 2B). At the lowest concentration of GPN tested (20 µM), fewer cells responded (despite complete loss of Lysotracker in all cells) and the latency was further increased (Fig. 2B). These data, summarised in Fig. 2C–F, show that GPN induces controlled solute leak from lysosomes that is associated with kinetically distinct cytosolic Ca2+ responses.

Fig. 2.
Fig. 2.

Lysosomal leak and cytosolic Ca2+ signals in response to GPN are concentration-dependent. (A,B) Effect of different concentrations of GPN on Lysotracker Red fluorescence (A) and cytosolic Ca2+ (B). (C–F) Pooled data summarising the effects of GPN on the time taken to achieve half-maximal loss of Lysotracker Red fluorescence (C) or maximal Ca2+ responses (D). (E,F) The magnitude of the Ca2+ responses and the percentage of cells that responded with a Ca2+ increase (>0.4 ratio) are shown in E and F, respectively. Results are from 37 (20 µM), 64 (100 µM) and 48 (200 µM) cells from two or three independent experiments.

To gain mechanistic insight into functional coupling between lysosomes and the ER, we developed a simple closed-cell computational model of Ca2+ dynamics in both the cytosol and a contiguous lysosome–ER microdomain (see Materials and Methods). This model incorporated both lysosomal leaks and ER fluxes based on movement of Ca2+ through the IP3R (Cao et al., 2013) and SERCA. A schematic summarising the model is shown in Fig. 3A. In the first instance, we modelled the effects of GPN. Lysotracker fluorescence was used as a proxy for the luminal lysosomal Ca2+ concentration (CL), where CL is an exponential function fitted to the data in Fig. 2A. When we simulate treatment with 200 µM or 100 µM GPN, the model demonstrates oscillatory behaviour qualitatively similar to the responses observed in the experimental system (Fig. 3B,C). For some GPN concentrations, the mathematical model exhibits an additional early spike, which is caused by the rapid addition of GPN and is thus most likely to be a model artefact (see Discussion). In the case of 20 µM GPN, we observe a single spike, which again is similar to the observed data (Fig. 3D, black traces). Interestingly, by slightly altering the maximal flux value (kLs) for 20 µM GPN, it is possible to prevent these oscillations, suggesting that this concentration of GPN is at the threshold for Ca2+ spike generation. Importantly, setting the IP3R flux to 0 (KIPR=0) abolished the spikes (Fig. 3B,C, red traces). These data indicate that the spikes are driven through the IP3R. Our model is therefore capable of qualitatively describing Ca2+ responses in fibroblasts with the expected pharmacology (Kilpatrick et al., 2013).

Fig. 3.
Fig. 3.

Model simulations of GPN-induced Ca2+ oscillations. (A) A schematic of the model depicting the two compartments C and Cm for the cytosol and microdomain (MD) respectively, with Ca2+ fluxes (J) shown in blue and IP3 metabolism (V) in green. (B–D) Typical Ca2+ responses of fibroblasts (left), and corresponding simulations (right) at the indicated concentration of GPN. Responses without IP3R flux (kIPR=0) are in red. kLs values were 0.015 s−1 and 0.0075 s−1 in B and C, with time intervals of 200 s and 320 s, respectively. In D, two cells are depicted (one responsive, black; one not, red) reflective of ‘threshold’ behaviour at this GPN concentration. Corresponding simulations are at kLs values of 0.002 (black) and 0.0019 (red), respectively (time interval 390 s).

GPN is a synthetic compound that causes complete loss of Lysotracker fluorescence (Fig. 2A) and, by inference, luminal Ca2+. However, more physiological lysosomal Ca2+ fluxes, such as those induced by NAADP, are likely to be highly regulated and smaller in magnitude in order to prevent lysosomal Ca2+ depletion. As TPC2 is a lysosomal ion channel that acts as a target for NAADP (Calcraft et al., 2009; Jha et al., 2013; Zong et al., 2009), we generated a model based on a previously published biophysical dataset (Pitt et al., 2010). This dataset was acquired under conditions that mimic the lysosomal lumen (low pH, high Ca2+) and demonstrates the characteristic bell-shaped concentration–effect relationship for NAADP. In our model, NAADP dictates TPC2 open probability, which in turn determines the magnitude of the lysosomal leak (see figure 4 in Pitt et al., 2010). We assume that this leak does not cause a change in CL (see Materials and Methods). Our simulations show that at the optimal NAADP concentration, persistent Ca2+ oscillations within the cytosol are observed (Fig. 4B) which follow those in the microdomain by ∼2 s during the pacemaker phase. Interestingly, the Ca2+ concentration within the microdomain upon NAADP stimulation is almost identical to that in the cytosol (Fig. 4C,D). Nevertheless, by setting the TPC flux within the microdomain compartment to 0 (mkt=0), oscillations ceased (Fig. 4C, red trace). Oscillatory lysosome–ER microdomains are therefore capable of driving global Ca2+ signals even in the absence of high microdomain Ca2+ concentrations.

Fig. 4.
Fig. 4.

Model simulations of NAADP-induced Ca2+ oscillations. (A) Relationship between the concentration of NAADP and open probability (Po) of TPC2 based on data in Pitt et al. (Pitt et al., 2010). The range of values that produce microdomain-driven global oscillations is shown in grey. The inset depicts the even density of TPCs (cyclinders) in the ER–lysosome (Lyso) microdomain (MD, hatched area) and the non-microdomain region (arc). (B) Model response to the optimal NAADP concentration (23 nM). The first spike is a simulation artefact, resulting from the fast addition of NAADP. (C,D) Model responses to NAADP (15 nM) in the bulk cytosol (C) and the microdomain (D). Responses without microdomain lysosomal leak (mkt=0) are shown in red.

In our model, coupling between lysosomes and the ER by NAADP takes place over a relatively limited range of NAADP concentrations (Fig. 4A, shaded area). In this scenario, the relative TPC flux density into both the microdomain (mkt) and bulk cytosol (kt) were assumed to be equal. However, channels are known to cluster at other microdomains, such as those between the plasma membrane and the SR or ER (Schendel et al., 2012). In order to simulate potential TPC clustering, we examined the effect of increasing TPC flux density into the microdomain relative to the cytosol (mkt = 10kt). Importantly, the range over which NAADP was capable of generating microdomain-driven global Ca2+ signals was expanded under these conditions (Fig. 5A). As shown in Fig. 5B, the frequency of Ca2+ oscillations showed a clear dependence on the NAADP concentration, giving rise to the diagnostic bell-shaped concentration–effect relationship (Fig. 5C). We compared these effects to increasing Ca2+ flux in both compartments – a scenario akin to studies of overexpressed TPCs. Interestingly, increasing the flux density in this uniform manner had a similar effect to clustering, that is widening the range of NAADP concentrations that elicit a response (Fig. 5D). However, global Ca2+ signals persisted in the absence of microdomain TPC flux (Fig. 5E, red traces). Comparison of the signals in the absence and presence of the microdomain revealed a stimulatory effect of the microdomain on oscillatory frequency (Fig. 5E,F). Microdomain flux is therefore not a prerequisite for generation of global signals but rather serves a modulatory role when the magnitude of the lysosomal leak is increased uniformly. Taken together, the simulations in Fig. 5 suggest that both the distribution and density of TPCs can regulate IP3R-dependent global Ca2+ signals through microdomains.

Fig. 5.
Fig. 5.

Distribution and density of TPC flux modulates NAADP-induced Ca2+ oscillations. Simulations where TPC flux density was either selectively increased in the microdomain [mkt = 10kt (A–C)] or uniformly increased in both compartments Embedded Image as schematically depicted in A and D (insets; see Fig. 4 for abbreviations). (A,D) Relationship between the concentration of NAADP and open probability (Po) of TPC2, with the range over which NAADP generates global Ca2+ oscillations highlighted (grey). (B,E) Model simulations at increasing concentrations of NAADP. Example traces show responses to 7 nM (B, top), 9 nM (E, top), 23 nM (B,E, middle) and 50 nM (B,E, bottom) NAADP. (C,F) Responses without microdomain lysosomal leak are in red. Resulting concentration–effect relationships for NAADP and oscillation frequency are presented in C and F.

DISCUSSION

Accumulating evidence indicates intimate functional and physical coupling between acidic and ER Ca2+ stores (Kilpatrick et al., 2013; Patel and Brailoiu, 2012). Signalling at junctions between the plasma membrane and ER (Franzini-Armstrong et al., 1999) and at mitochondria–ER junctions (Csordás et al., 2006; Szabadkai et al., 2006) is established, but at present we know little about Ca2+ dynamics between lysosomes and the ER. We have recently shown that mobilisation of lysosomal Ca2+ using GPN is sufficient to evoke complex and persistent Ca2+ signals that require coupling with ER IP3Rs (Kilpatrick et al., 2013). GPN is thus a valuable tool for probing lysosome–ER cross talk. Caution is required, however, in equating GPN-evoked cytosolic Ca2+ signals and lysosomal Ca2+ content. Failure to pharmacologically dissociate lysosomal and ER Ca2+ stores might explain discrepancies in the literature relating to the Ca2+ content of lysosomes in Niemann–Pick type C1 disease (Lloyd-Evans et al., 2008; Shen et al., 2012). GPN, however, does not evoke complex Ca2+ signals in all cell types [for example, MDCK cells (Haller et al., 1996)], which might reflect cell-specific differences in cathepsin C levels, total lysosomal volume or number, and/or the extent of coupling between lysosomes and ER.

We (Kilpatrick et al., 2013) and others (van Breemen et al., 2013) have identified membrane contact sites between lysosomal and SR/ER membranes, potentially bringing together Ca2+ signalling apparatus into a spatially restricted zone to create Ca2+ microdomains. As the size of these regions renders them largely experimentally intractable, we developed a computational model to understand the potential microdomain behaviour. The model simulates the Ca2+ concentration within two compartments, the microdomain and the bulk cytosol, in response to various intracellular Ca2+ fluxes. It is important to note that the magnitude of the fluxes into the microdomain is substantially smaller than into the cytosol because these fluxes are scaled by both surface area and volume ratios (see Materials and Methods). We employed a model of the IP3R that draws from an extensive biophysical dataset to define the open probability of the channel based on IP3 and Ca2+ concentration (Cao et al., 2013). This biophysical data indicates that at a given IP3 concentration, Ca2+ drives short-period oscillations (<5 s). The period of lysosome–ER crosstalk signals within cells, however, are typically much longer (>50 s) (Cancela et al., 1999; Kilpatrick et al., 2013). We thus included in our model dynamic Ca2+-sensitive IP3 metabolism, which has been shown previously to extend the period of IP3R-driven oscillations (Politi et al., 2006). Plasma membrane Ca2+ fluxes were excluded owing to the ability of lysosome–ER crosstalk to proceed in the absence of extracellular Ca2+ (Fig. 1A; Boittin et al., 2002; Cancela et al., 1999; Kilpatrick et al., 2013; Perez-Terzic et al., 1995). The stable steady-state of this simple model is perturbed by a leak of lysosomal Ca2+ in order to gain insight into functional coupling between lysosomes and the ER.

GPN was used to initiate a lysosomal Ca2+ leak in the model. GPN is a dipeptide substrate for cathepsin C (Jadot et al., 1984) and has been widely used to investigate Ca2+ signalling through acidic stores in a range of contexts (Berg et al., 1994; Churchill et al., 2002; Duman et al., 2006; Haller et al., 1996; López-Sanjurjo et al., 2013). Hydrolysis of GPN leads to an increase in luminal osmolarity, followed by the ingress of water into cathepsin-C-containing vesicles down the osmotic gradient. We show here that GPN causes a controlled leak of small molecular mass solutes (<10 kDa) in a concentration-dependent manner (Fig 1; Fig. 2A). GPN is therefore unlikely to result in release of lysosomal hydrolases (>35 kDa), as has been reported in other preparations (Berg et al., 1994; Gerasimenko et al., 2006). Our data agrees with previous work showing the retention of larger dextrans (70 kDa) in MDCK cells upon treatment with GPN (Haller et al., 1996). We conclude that in primary cultured fibroblasts, GPN produces a graded leak of low molecular mass solutes, including Ca2+. H+ presumably also equilibrates thereby increasing luminal pH. As cathepsin C has a relatively broad pH optimum (McDonald et al., 1969), it will remain active, thus likely explaining complete loss of Lysotracker during GPN treatment. Indeed, the increase in lysosomal pH also explains the observed increases in fluorescence of pH-sensitive dextrans (Fig. 1C–E). Using Lysotracker fluorescence as a proxy for the lysosomal Ca2+ concentration, we modelled the GPN-induced lysosomal leak at a range of concentrations. GPN is thought to selectively target lysosomes but this assumption is again based on broken cell preparations. Like other lysosomal hydrolases, cathepsin C traffics through pre-lysosomal compartments upon exit from the Golgi complex (Ghosh et al., 2003). This raises the possibility that GPN mobilises Ca2+ from both lysosomal and endosomal compartments in intact cells. Nevertheless, the resulting model is capable of describing, at least qualitatively, IP3R-driven Ca2+ spikes in the cytosol that have a similar period to those observed experimentally, including the threshold nature of the response at 20 µM GPN (Fig. 3B–D).

Like GPN, NAADP can recruit the IP3R (Calcraft et al., 2009; Soares et al., 2007). We therefore modelled this more physiological scenario. Since their initial identification as likely NAADP targets (Brailoiu et al., 2009; Calcraft et al., 2009; Zong et al., 2009), much evidence has accumulated implicating TPCs as NAADP-activated Ca2+ channels (Hooper and Patel, 2012). However, recent data has suggested that TPCs are NAADP-insensitive Na+ channels with a low Ca2+ permeability (Wang et al., 2012). Use of N-terminally tagged TPC constructs and differences in ionic recording conditions might explain discrepancies in NAADP sensitivity (Churamani et al., 2013; Jha et al., 2013), although modest Ca2+ fluxes might still be physiologically relevant owing to amplification through ER Ca2+ channels (Marchant and Patel, 2013). We used previous biophysical data (Pitt et al., 2010) to generate the first computational model of TPCs, whereby a given concentration of NAADP sets the channel open probability (Fig. 4A). These data were derived from single-channel measurements in artificial bilayers, with high luminal Ca2+ and a low luminal pH, as would be observed physiologically (Pitt et al., 2010). In our model of TPC2, owing to the small size of the leak and likely presence of an as-yet unidentified functional Ca2+ re-uptake mechanism, we assume that luminal lysosomal Ca2+ (CL) is constant (as compared with the unidirectional transport observed in the GPN data, whereby CL depletes). We demonstrate that the model couples small NAADP-induced lysosomal Ca2+ leaks to the IP3R, causing regular spikes in the cytosol (Fig. 4B,C).

We note the presence of instantaneous spikes in our models of both NAADP and GPN action (Figs 3–⇑5). In our models, stimulus intensity increases are rapid. The biological actions of NAADP and, in particular, GPN, however, include several time-dependent steps that are difficult to model. Early spikes in our simulations are likely artefacts of this simplification. Indeed, these early spikes are eliminated by gradually ramping lysosomal flux to its maximum value using an arbitrary function (computations not shown). Further work is required to extend this model to a full spatial model using finite elements and partial differential equations (Means and Sneyd, 2010). Detailed computational models of Ca2+ microdomains are relatively common (Cannell et al., 2013; Greenstein et al., 2006; Jafri et al., 1998; Means and Sneyd, 2010; Rice et al., 1999; van Breemen et al., 2013); however, the issue of how to couple such microdomains – which are highly computationally intensive – to the rest of the cell remains a challenging multi-scale problem. It is not feasible at present to compute the whole cell at the same level of detail as the microdomain. This problem has been partially solved using a homogenisation approach (Higgins et al., 2007). It is likely that similar methods will be necessary for incorporating the lysosome–ER microdomain in a whole-cell model. Nevertheless, despite its simplicity, our model demonstrates the principle that small NAADP-induced Ca2+ leaks can generate global Ca2+ signals.

We also demonstrate the principle that the NAADP-induced Ca2+ leak from lysosomes is capable of driving global oscillations through lysosome–ER microdomains (Fig. 4C). Thus global Ca2+ signals were ablated by selectively eliminating the lysosomal leak into the microdomain. Contrary to our expectations, our simulations indicate that microdomains do not achieve substantially higher Ca2+ concentrations than the cytosol (Fig. 4D). It is important to note that IP3Rs are activated by nanomolar concentrations of Ca2+ and therefore microdomain Ca2+ does not need to reach high levels in order to initiate CICR. In our model, Cm values are low owing to the efficient activity of SERCA pumps within the microdomain, which is required to balance IP3R activity at basal IP3 concentrations. Microdomains with no SERCA pumps might very well achieve high Ca2+ concentrations (Jafri et al., 1998), but such high concentrations are not necessary for the microdomain to drive whole-cell responses through IP3Rs. The situation is likely very different at microdomains featuring targets with lower affinity for Ca2+, such as the mitochondrial uniporter, where high Ca2+ concentrations are likely a prerequisite for coupling to proceed. This non-intuitive result suggests that microdomain behaviour is highly dependent on the spatial distribution of Ca2+ release and re-uptake pathways and their affinity for Ca2+.

The concentration–effect relationship for NAADP-evoked Ca2+ signalling in mammalian cells is unusual in that it is bell-shaped (Berg et al., 2000; Johnson and Misler, 2002); increasing the NAADP concentration beyond an optimal level decreases activity. This biphasic behaviour has recently been described at the single-channel level for TPC2 (Pitt et al., 2010). By assessing the global spike frequency, our model recapitulated biphasic concentration–effect relationships for NAADP (Fig. 5). The range of concentrations over which NAADP produced global responses could be widened by selectively increasing the density of TPC2 Ca2+ flux into the microdomain (Fig. 5A–C). We liken this to channel clustering. Both RyRs and IP3Rs form clusters, and such an arrangement is thought to be important for coordinating channel activity, particularly for RyR during excitation–contraction coupling (Rahman, 2012; Schendel et al., 2012). Interestingly, a uniform increase in TPC density also has a broadening effect on the concentration–effect relationship (Fig. 5D–F). Additionally, this manipulation reveals a modulatory role for the microdomain in setting oscillation frequency. This raises the possibility that expression levels of lysosomal channels could alter the mode of coupling between lysosomes and the ER. This has implications not only for studies of overexpressed recombinant TPCs but also for endogenous TPCs, which show tissue-specific differences in expression at the transcript level (Ishibashi et al., 2000). Regardless of TPC distribution and density, the range over which NAADP induces global responses remained narrow in our model relative to observed cellular responses. Cooperative regulation of IP3 receptors by Ca2+ likely contributes to these ‘switch’-like responses upon breach of threshold [Ca2+] by NAADP. In our model, we assumed a homogeneous microdomain population but in a cellular context progressive recruitment of architecturally distinct microdomains with differing NAADP sensitivities might act to enhance the concentration range of NAADP action.

In conclusion, we have used observed experimental data to guide development of a simple computational model of lysosome–ER Ca2+ microdomains. The model is capable of qualitatively describing the Ca2+ responses to the lysosomotropic agent GPN, which we demonstrate induces substantial but controlled solute leak from the lysosome. Probing more physiological leaks through the Ca2+-mobilising messenger NAADP and its likely target channel demonstrates the potential for lysosome–ER microdomains to drive global Ca2+ responses through the IP3R.

MATERIALS AND METHODS

Cell culture

Untransformed primary human skin fibroblasts established from healthy females were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% v/v fetal bovine serum, 100 µg/ml streptomycin and 100 units/ml penicillin (all from Invitrogen) at 37°C under a humidified atmosphere with 5% CO2. Cells were plated on glass coverslips before experimentation and used between passages 9–13.

Fluorescent probes

For measurement of cytosolic Ca2+ concentration, fibroblasts were incubated with Fura-2 AM (2.5 µM) and 0.005% v/v pluronic acid (both from Invitrogen) for 1 h in HEPES-buffered saline (HBS) comprising (in mM) 1.25 KH2PO4, 2 CaCl2, 2 MgSO4, 3 KCl, 156 NaCl, 10 glucose and 10 HEPES (pH 7.4; all from Sigma). For measurements of lysosome integrity, fibroblasts were loaded with either dextran-conjugated Rhodamine B or dextran-conjugated fluorescein (0.2 mg/ml; molecular mass 10,000 Da; both from Invitrogen) for ∼18 h in culture. Cells were subsequently chased for 3 h in dextran-free culture medium to label lysosomes. Lysosomes were also labelled by incubating fibroblasts with Lysotracker Red (100 nM; Invitrogen) for 30 min in HBS. Cells were stimulated with 20–200 µM GPN (SantaCruz Biotech). Where indicated, Ca2+ in HBS was replaced with 1 mM EGTA (Sigma).

Epifluorescence microscopy

Epifluorescence images were captured every 3 s with a cooled coupled device camera (TILL photonics) attached to an Olympus IX71 inverted fluorescence microscope fitted with a 20× objective, and a monochromator light source. Fura-2 fluorescence (emission; 440 nm) was visualised after sequential excitation at 340 nm and 380 nm. Lysotracker Red and Rhodamine–dextran were excited at 568 nm and 570 nm, respectively, and emitted fluorescence was captured using a 590-nm filter.

Confocal microscopy

Confocal images were acquired using an inverted Axiovert 200M microscope attached to a LSM510 confocal scanner (Zeiss) fitted with a 63× Plan Apochromat water-immersion objective. DAPI, fluorescein–dextran and Lysotracker Red were excited using wavelengths of 364 nm, 488 nm and 543 nm, respectively. Emitted fluorescence was captured through either a 385-nm long-pass filter or 505–530-nm and 560–615-nm band pass filters, respectively. Zeiss LSM 510 software was used to acquire the images.

Modelling

We constructed a model with two contiguous compartments, the bulk cytosol and the microdomain, each containing two variables, the Ca2+ and IP3 concentrations, C or Cm and P or Pm, respectively. Simulations were carried out using xppaut (Ermentrout, 2002). Model parameters are given in Table 1.

View this table:
Table 1. Model parameters.

Ca2+ dynamics

In the model, Ca2+ is released from the ER through the IP3R, as modelled previously (Cao et al., 2013). The fluxes into the cytosol and microdomain are:Embedded ImageandEmbedded Imagerespectively, where KIPR is related to the density of the IP3Rs. The IP3R open probabilities within the cytoplasm and microdomain, PO and mPO are set by the IP3 and Ca2+ concentrations within these compartments (Cao et al., 2013). Ca2+ re-uptake into the ER through the SERCA pumps is modelled by Hill functions:Embedded ImageEmbedded Imagewhere VSERCA is related to the SERCA density and is KSERCA the half-maximal activation.

Lysosomal leaks of Ca2+ are modelled based on the GPN-induced depletion of lysosomal Ca2+ or on the NAADP-induced Ca2+ flux from acidic organelles (see Results). In the first instance, the fluxes due to GPN into the cytosol and microdomain, respectively, are:Embedded ImageEmbedded Imagewhereby KLs is related to the maximum rate of flux at a given concentration of GPN and CL is the luminal lysosomal Ca2+ concentration. CL is fitted to the exponential decreases of Lysotracker fluorescence in response to GPN (Fig. 2A). Addition of GPN is simulated by a time interval determined by the GPN concentration in which kLs increases linearly from an initial value of zero to its final value. Owing to the low magnitude of flux induced by NAADP and the presence of as-yet undefined re-uptake mechanisms on the lysosome, decreases in CL are assumed to be negligible in the NAADP model. The NAADP-induced fluxes of Ca2+ from the lysosome to the cytoplasm and microdomain are given by:Embedded ImageEmbedded Imagerespectively, whereby kt and mkt are related to the density of TPCs, and PoTPC is the TPC2 open probability at a given concentration of NAADP, as defined in Fig. 4A.

Ca2+ diffuses between the contiguous cytosol and microdomain compartments with an estimated rate constant DC. As lysosome–ER crosstalk signals persist in the absence of extracellular Ca2+, we assume there is no flux across the plasma membrane. Thus,Embedded Image

The Ca2+ dynamics in the microdomain are expressed as,Embedded Imagewhere the surface of the ER that adjoins the cytoplasm has a larger area, AERC than the surface that adjoins the microdomain, whose area is denoted as AERm. R2 represents the effect of the difference in the surface areas, Embedded Image. Similarly, R3 is the surface ratio of the lysosome to the microdomain, Embedded Image. The relative difference in volume between the microdomain and cytosol is represented by the ratio:Embedded Image

IP3 dynamics

Owing to the long period of the observed oscillations, we included in the model the Ca2+-dependent production and degradation of IP3 (Dupont and Erneux, 1997; Politi et al., 2006). Production of IP3 is an increasing function of agonist concentration, Embedded Image, and Ca2+, and thusEmbedded Imagewhere KPLC is the concentration of Ca2+ causing half-maximal production of IP3. IP3 is removed through phosphorylation by IP3 kinase, with maximal rate k3K (Politi et al., 2006) and concentration of Ca2+ causing half-maximal degradation of IP3, Kdeg. We model the degradation as:Embedded Image

There is no production or degradation of IP3 in the microdomain. However, there is transport of IP3 between the cytoplasm and the microdomain, with rate constant DP. Thus,Embedded ImageEmbedded Image

Acknowledgments

We thank Anthony H. Schapira (Department of Clinical Neuroscience, Institute of Neurology, University College London, London, UK) for provision of fibroblasts, and Jasmine Patel (The Latymer School, London, UK) for editorial assistance.

Footnotes

  • Competing interests

    The authors declare no competing interests.

  • Author contributions

    C.J.P. and J.M.H. performed the modelling. B.S.K. performed the experimental work. J.S. and S.P. conceived the study. C.J.P. and S.P. wrote the paper with input from all authors.

  • Funding

    This work was supported by a Biotechnology and Biological Sciences Research Council studentship (to C.J.P.); an IMPACT studentship from University College London (to B.S.K.); and the Marsden Fund of the Royal Society of New Zealand.

  • Received January 3, 2014.
  • Accepted March 26, 2014.

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Research Article
A computational model of lysosome–ER Ca2+ microdomains
Christopher J. Penny, Bethan S. Kilpatrick, Jung Min Han, James Sneyd, Sandip Patel
Journal of Cell Science 2014 127: 2934-2943; doi: 10.1242/jcs.149047
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