ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 2934–2943 doi:10.1242/jcs.149047 RESEARCH ARTICLE A computational model of lysosome–ER Ca2+ microdomains Christopher J. Penny1, Bethan S. Kilpatrick1, Jung Min Han2, James Sneyd2 and Sandip Patel1,* ABSTRACT et al., 2003). Subsequent negative feedback terminates the spike, allowing clearance by ‘off’ mechanisms such as the sarco/ Acidic organelles form an important intracellular Ca2+ pool that can endoplasmic reticulum Ca2+ ATPase (SERCA) (Berridge et al., drive global Ca2+ signals through coupling with endoplasmic reticulum 2000). (ER) Ca2+ stores. Recently identified lysosome–ER membrane Membrane contact sites are regions of close membrane contact sites might allow formation of Ca2+ microdomains, although 2+ apposition (,30 nm) between organelles, allowing congregation their size renders observation of Ca dynamics impractical. Here, we of Ca2+ signalling proteins in restricted spaces, forming the generated a computational model of lysosome–ER coupling that anatomical basis for the encoding and decoding of functional ‘Ca2+ incorporated a previous model of the inositol trisphosphate (IP3) microdomains’ (Berridge, 2006; Helle et al., 2013). Ca2+ signalling 2+ 2+ receptor as the ER Ca ‘amplifier’ and lysosomal leaks as the Ca between the ER and the plasma membrane or organelles, such as 2+ ‘trigger’. The model qualitatively described global Ca responses to mitochondria, is a well-characterised constituent of the Ca2+ the lysosomotropic agent GPN, which caused a controlled but signalling network (Helle et al., 2013). For example, mitochondria substantial depletion of small solutes from the lysosome. Adapting form tight (,10 nm) physical junctions with the ER in many cell this model to physiological lysosomal leaks induced by the Ca2+ types allowing mitochondrial uptake of ER-released Ca2+, thereby mobilising messenger NAADP demonstrated that lysosome–ER matching mitochondrial ATP production to cellular demand microdomains are capable of driving global Ca2+ oscillations. (Csorda´s et al., 2006; Szabadkai et al., 2006; Tarasov et al., 2+ Interestingly, our simulations suggest that the microdomain [Ca2+] 2012). Furthermore, in cardiac muscle, a strongly coupled Ca need not be higher than that in the cytosol for responses to occur, thus microdomain in the dyadic cleft junction (,15-nm wide) between 2+ 2+ matching the relatively high affinity of IP3 receptors for Ca .The L-type Ca channels on the plasma membrane and clusters of relative distribution and overall density of the lysosomal leaks dictated ryanodine receptors on the SR is crucial for excitation–contraction whether microdomains triggered or modulated global signals. Our coupling (Bers, 2002; Franzini-Armstrong et al., 1999; Schendel data provide a computational framework for probing lysosome–ER et al., 2012). ER–mitochondria and SR/ER–plasma membrane Ca2+ dynamics. associations provide a precedent for ER associations with other organelles, such as those within the endolysosomal system. KEY WORDS: Ca2+, Lysosomes, Endoplasmic reticulum, Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Membrane contact site, Computational modelling, NAADP, second messenger that mobilises Ca2+ from lysosomes and other Two-pore channel 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 INTRODUCTION alone is limited. This is owing to both the insensitivity of NAADP- Changes in cytosolic Ca2+ ion concentration ([Ca2+]) form the induced Ca2+ release to cytosolic Ca2+, which precludes direct 2+ basis of a ubiquitous signalling pathway responsible for regenerative Ca spikes that characterise IP3Rs and RyRs (Chini regulating a multitude of disparate cellular events ranging from and Dousa, 1996; Genazzani and Galione, 1996), and the low fertilisation to cell death (Berridge et al., 2000). Specificity is lysosomal volume (,1–2% of a cell) compared to the ER (,10– realised through temporal and spatial complexities in the Ca2+ 20%). These observations have led to a ‘trigger hypothesis’ of signals, which can be achieved by controlled mobilisation of not NAADP action whereby ER-resident IP3Rs and RyRs amplify only endoplasmic reticulum (ER) Ca2+ stores, but also those in NAADP-induced Ca2+ signals (Cancela et al., 1999). Importantly, acidic organelles such as lysosomes (Patel and Docampo, 2010). these putative NAADP trigger events cannot be resolved in the The ubiquitous inositol 1,4,5 trisphosphate receptor (IP3R), presence of IP3R and RyR inhibitors in certain cell types, together with the ryanodine receptor (RyR), are the principal suggesting that the NAADP-induced signals are modest and/or intracellular Ca2+ release channels, which localise predominantly highly localised (Cancela et al., 1999; Marchant and Patel, 2013). to the sarcoplasmic reticulum (SR) and ER (Clapham, 2007). We recently identified membrane contact sites between lysosomes These channels can be activated by second messengers (IP3 and and the ER analogous to those between the ER and other cyclic ADP ribose) and are regulated by cytosolic [Ca2+]ina organelles, which are known to support local Ca2+ microdomain biphasic manner. Both support Ca2+-induced Ca2+ release (CICR) signalling (Kilpatrick et al., 2013). Indeed in sea urchin eggs, whereby modest rises in [Ca2+] generate a Ca2+ spike (Roderick functional coupling between NAADP and the ER is disrupted upon homogenisation (Churchill et al., 2002). Moreover, recent studies 1Department of Cell and Developmental Biology, University College London, also show that local NAADP responses (measured indirectly London WC1E 6BT, UK. 2Department of Mathematics, University of Auckland, through changes in luminal pH) persist in the presence of EGTA, a Auckland 1142, New Zealand. slow Ca2+ buffer that prevents global responses (Morgan et al., *Author for correspondence ([email protected]) 2013). These data further underscore the localised nature of NAADP signalling. In this context, it is notable that the putative Received 3 January 2014; Accepted 26 March 2014 targets for NAADP, the two-pore channels (TPCs) (Brailoiu et al., Journal of Cell Science 2934 RESEARCH ARTICLE Journal of Cell Science (2014) 127, 2934–2943 doi:10.1242/jcs.149047 2009; Calcraft et al., 2009; Zong et al., 2009), have a low maximal Ca2+ signalling behaviours induced by the lysosomotropic open probability (Pitt et al., 2010) and potentially poor selectivity compound glycyl-L-phenylalanine 2-naphthylamide (GPN). We for Ca2+ over Na+ (,0.1) (Wang et al., 2012, but see Schieder then adapted this model to a more physiological setting by et al., 2010). generating the first computational model of NAADP action at The spatial and temporal scales involved in studying TPCs. We demonstrate that, depending on their relative microdomains impede full experimental characterisation owing distribution and density, small NAADP-induced Ca2+ leaks to the relatively low spatial resolution of light microscopy couple with the IP3R at lysosome–ER microdomains to either (,200 nm) and the static nature of electron microscopy. drive or modulate global Ca2+ oscillations. Interestingly, these However, these techniques, in tandem with mathematical microdomains do not require high Ca2+ concentrations for their modelling, can capture essential features of Ca2+ microdomain activity. signalling. Computational modelling of Ca2+ microdomains in interstitial cells of Cajal has helped rationalise our understanding RESULTS of slow wave generation in gastrointestinal smooth muscle We have recently shown that stimulation of primary cultured (Means and Sneyd, 2010), and models of the cardiac muscle human fibroblasts with GPN elicits complex, IP3R-dependent dyadic cleft have been constructed to accurately describe the Ca2+ signals (Kilpatrick et al., 2013). Consistent with our behaviour of Ca2+ during excitation–contraction coupling previous work, these GPN-evoked Ca2+ signals are readily (Cannell et al., 2013; Greenstein et al., 2006; Jafri et al., 1998; initiated and maintained in Ca2+-free medium (Fig. 1A,B). Rice et al., 1999). Such approaches allow prediction of the Ca2+ Fibroblasts therefore provide a tractable system for studying dynamics within microdomains, and of the behaviour of both lysosomal Ca2+ signalling. GPN is a freely diffusible dipeptide microdomain and non-microdomain targets in response to applied substrate for the intralysosomal hydrolase cathepsin C. signals. Hydrolysis of GPN is thought to rupture lysosomes owing to an In this study, we built a simple two-compartment model of a increase in luminal osmolarity. Previous studies have employed lysosome–ER microdomain and the contiguous bulk cytosol that isolated lysosomal preparations, therefore potentially removing is capable of qualitatively describing experimentally observed cellular homeostatic mechanisms capable of maintaining 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 mM 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 mM GPN. Nuclei were stained using DAPI (blue). (D)