Coupling acidic organelles with the ER through Ca2+ microdomains at membrane contact sites

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Review

Coupling acidic organelles with the ER through Ca2+ microdomains at membrane contact sites Christopher J. Penny a , Bethan S. Kilpatrick a , Emily R. Eden b , Sandip Patel a,∗ a b

Department of Cell and Developmental Biology, University College London, London WC1E 6BT, UK Department of Cell Biology, Institute of Ophthalmology, University College London, London EC1V 9EL, UK

a r t i c l e

i n f o

Article history: Received 18 December 2014 Received in revised form 13 March 2015 Accepted 14 March 2015 Available online xxx Keywords: Acidic Ca2+ stores Lysosomes Vacuole Endoplasmic reticulum NAADP Inositol trisphosphate Cyclic ADP-ribose Two-pore channels TPCN1 TPCN2 IP3 receptors Ryanodine receptors Membrane contact sites CICR Computational modelling Ca2+ microdomain

a b s t r a c t Acidic organelles such as lysosomes serve as non-canonical Ca2+ stores. The Ca2+ mobilising messenger NAADP is thought to trigger local Ca2+ release from such stores. These events are then amplified by Ca2+ channels on canonical ER Ca2+ stores to generate physiologically relevant global Ca2+ signals. Coupling likely occurs at microdomains formed at membrane contact sites between acidic organelles and the ER. Molecular analyses and computational modelling suggest heterogeneity in the composition of these contacts and predicted Ca2+ microdomain behaviour. Conversely, acidic organelles might also locally amplify and temper ER-evoked Ca2+ signals. Ca2+ microdomains between distinct Ca2+ stores are thus likely to be integral to the genesis of complex Ca2+ signals. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction: canonical and non-canonical Ca2+ stores Calcium ions perform a ubiquitous signalling role, controlling a vast array of physiological processes [1]. Ca2+ signals must be tightly controlled so that only appropriate processes are affected. At rest, the Ca2+ concentration is kept low (∼100 nM), and is elevated either through Ca2+ influx or through release from intracellular stores. Cells achieve signal specificity by controlling both the location and timing of this increase in Ca2+ , with responsive proteins being tuned to specific Ca2+ signatures [1]. Cellular Ca2+ buffers limit free diffusion of the ion. Targeted Ca2+ release from intracellular stores represents one way to ‘globalise’ Ca2+ signals. The best characterised of these stores is the endoplasmic reticulum (ER), or sarcoplasmic reticulum (SR) in muscle. Ca2+

∗ Corresponding author. Tel.: +44 207 679 4437. E-mail address: [email protected] (S. Patel).

can be released from the ER/SR by intracellular second messengers such as inositol trisphosphate (IP3 ) or cyclic ADP-ribose (cADPR), which act on Ca2+ release channels such as IP3 receptors (IP3 Rs) or ryanodine receptors (RyRs), respectively. Sarco/Endoplasmic Reticulum Ca2+ ATPases (SERCA) refill the stores. Cells regulate these processes to further enhance spatiotemporal signal diversity. These mechanisms have been extensively studied since the early 1980s and are relatively well understood [1,2]. In the mid-1990s, a third Ca2+ -mobilising messenger was discovered [3]. Nicotinic acid adenine dinucleotide phosphate (NAADP), a contaminant in commercial stock of NADP, was shown to mobilise Ca2+ from cellular preparations desensitised to both IP3 and cADPR, suggesting the existence of a novel Ca2+ release channel [4,5]. Moreover, the target Ca2+ stores also appeared novel and were later identified as a lysosome-like acidic vesicle [6,7]. Whilst the endocytic and hydrolytic functions of the endolysosomal system are well established [8], the Ca2+ signalling function of these organelles remained obscure until relatively recently [9]. A

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body of research has identified key physiological roles for NAADP. These include several events associated with fertilisation in invertebrate gametes [10–12], neurotransmitter release in amphibians [13] through to numerous processes in mammals such as muscle contraction [14], neurite extension [15], differentiation [16] and autophagy [17] (reviewed in [18–20]). More generally, the endolysosomal system is one of several acidic Ca2+ stores – a collection of morphologically distinct Ca2+ - and H+ -rich organelles found in all major kingdoms of life [21,22]. A number of candidates have been proposed as the molecular target for NAADP (reviewed in [23]). In 2009, papers from three independent groups converged on the two-pore channels (TPCs) as the molecular targets for NAADP [24–26]. TPCs possess a duplicated domain structure in which two voltage-gated ion channel-like domains are concatenated [27]. TPCs likely assemble as dimers [28,29], thus generating the (pseudo-) tetrameric arrangement that is characteristic of the voltage-gated ion channel superfamily, to which TPCs belong [30]. Indeed, TPCs are likely to be evolutionary intermediates between one-domain channels (such as voltagegated K+ channels and TRP channels) and four-domain channels (such as voltage-gated Ca2+ and Na+ channels) of this superfamily [31]. Consistent with their identity as NAADP targets, TPCs localise to the endolysosomal system (reviewed in [19]). Sea urchins and most other animals possess three TPC isoforms (TPC1-3), although TPC3 appears to have undergone striking lineage-specific loss in humans and certain rodents such as mice [32,33]. NAADP function is ablated by knocking-down, knocking-out or mutating TPCs, whilst NAADP signalling is augmented by overexpressing TPCs (reviewed in [19]). Extensive subsequent research including the use of transgenic animals [25,34–37] and direct measurements of NAADP-induced TPC currents [37–43] supports the conclusion that TPCs are the targets for NAADP (reviewed in [19]). A recent report highlights a potentially important role for TPCs in Parkinson Disease, whereby chemical or molecular inhibition of TPC2 corrects lysosomal morphology defects in patient fibroblasts [44]. But controversy was never far away; a 2012 report [45] and subsequent follow-up studies [46–48] suggested that TPCs are Na+ selective channels that may be insensitive to NAADP and instead activated by PI(3,5)P2 and/or voltage. These conclusions have been questioned [49,50] and conflicting data reconciled, at least in part, by the demonstration of TPC co-regulation by NAADP and PI(3,5)P2 [37,41,44]. Nonetheless, the mechanism of NAADP action on TPCs is decidedly complex. Photo-affinity labelling studies have found no evidence for a direct association between NAADP and TPCs, suggesting instead that NAADP binds low-molecular weight protein(s) in complex with TPCs [34,51,52]. These NAADP binding proteins are yet to be identified at the molecular level [53]. On balance, TPCs are likely Ca2+ - and Na+ -permeable channels subject to multimodal regulation.

2. The trigger hypothesis: NAADP connects Ca2+ stores In the early reports of NAADP action in homogenised sea urchin eggs, NAADP activated a Ca2+ release mechanism distinct from either IP3 Rs or RyRs [4,54,55]. It was therefore surprising to find that NAADP-induced Ca2+ signals in intact mammalian cells were dependent upon functioning IP3 Rs and RyRs [6,56]. These observations were rationalised in the NAADP trigger hypothesis [20,57]. A key property of both IP3 Rs and RyRs is their biphasic regulation by Ca2+ . Slight elevations in Ca2+ open the channels and permit ‘Ca2+ -induced Ca2+ -release’, whereby small Ca2+ signals amplify themselves [58]. Conversely, high Ca2+ concentrations are inhibitory, thus terminating Ca2+ release. In contrast, NAADPinduced Ca2+ release is insensitive to cytosolic Ca2+ [59], indicating that NAADP-induced signals cannot amplify themselves. Instead,

NAADP may provide a so-called ‘trigger’ release of Ca2+ that is then amplified indirectly via ER Ca2+ channels. Whilst inhibition of IP3 Rs and RyRs prevents NAADP-induced Ca2+ signalling in many cell types (reviewed in [60]), in general NAADP inhibition does not affect IP3 or cADPR induced signals [56] (see Section 6). This indicates that NAADP acts ‘upstream’ of IP3 Rs and RyRs, consistent with a ‘triggering’ role. Indeed, the need for NAADP can be circumvented in vitro by using a lysosomotropic agent that directly mobilises Ca2+ from the acidic stores and generates ER-dependent Ca2+ signals [61]. Such data further underline an upstream role for acidic organelles and highlight that this role is independent of NAADP, avoiding complications from any potential promiscuity of NAADP action [23]. The NAADP trigger hypothesis therefore posits that small and often unresolvable NAADP-induced Ca2+ release events are amplified by ER receptors through Ca2+ -induced Ca2+ release (Fig. 1). The trigger hypothesis necessitates questions about functional coupling between acidic organelles and the ER. The inability to observe NAADP-induced Ca2+ signals in isolation [56] would suggest a tight coupling. A close anatomical association between trigger and amplifier would explain why brief/small Ca2+ signals cannot be resolved by standard Ca2+ imaging approaches upon ER blockade in some cells. Some groups have recorded relatively low conductances for TPCs in biophysical experiments [38,39,42,43] and reports of poor Ca2+ -permeability [37,41,45] further indicate that the Ca2+ flux may be modest. If correct, such properties would probably require an amplification mechanism to produce global signals. Trigger and amplifier events can be dissociated upon cell homogenisation, as evidenced by the insensitivity of NAADPinduced Ca2+ signals in sea urchin egg homogenates to ER blockade [4,5]. Such uncoupling may also be achieved in live cells. Deleting or mutating an endolysosomal trafficking motif in the N-terminus of TPC2 redirects the channel to the plasma membrane [38]. Cells expressing the wild-type channel display fast signals that can be blocked either by inhibiting RyRs with ryanodine or by inhibiting acidification of acidic organelles with bafilomycin A1 . Cells expressing the mutant, redirected channel instead show slow, sluggish responses that are insensitive to both ryanodine and bafilomycin [38]. Close functional coupling of the acidic and ER stores therefore appears key to NAADP action.

3. Ca2+ microdomains at the endolysosome–ER interface: a role for membrane contact sites? NAADP-induced Ca2+ signals stem from acidic organelles but are likely to be ‘local’ in the first instance and then amplified by the ER. This intimate functional coupling may permit the formation of Ca2+ microdomains at the lysosome–ER interface. Indeed, there are precedents for this. The ER is rather promiscuous in forming Ca2+ microdomains with both the plasma membrane and other organelles. In cardiomyocytes, Ca2+ influx through plasma membrane voltage-gated Ca2+ channels (Cav ) generates local Ca2+ signals that are subsequently amplified by RyRs through Ca2+ -induced Ca2+ release (Fig. 1). This is critical for excitation–contraction coupling (see other reviews in this issue). Similarly, Ca2+ microdomains form between IP3 Rs on the ER and Ca2+ uniporters on the mitochondria. The resulting mitochondrial Ca2+ uptake not only tempers IP3 -evoked Ca2+ signals, but matches energy supply (Ca2+ -dependent stimulation of oxidative phosphorylation) to demand (Ca2+ signalling) (see other reviews in this issue) (Fig. 1). In addition, ER Ca2+ depletion leads to store-operated Ca2+ entry and store refilling through Ca2+ microdomains between plasma membrane Orai channels and SERCA transporters on the ER (see other reviews in this issue). Thus, analogous functional microdomains

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Fig. 1. Likening ER Ca2+ microdomains between acidic organelles, plasma membrane and mitochondria. (A, B) Nicotinic acid adenine dinucleotide phosphate (NAADP) activates Ca2+ release from acidic organelles through two-pore channels (TPCs), which initiates Ca2+ -induced Ca2+ release from the neighbouring endoplasmic reticulum (ER) through inositol trisphosphate receptors (IP3 Rs) and/or ryanodine receptors (RyRs) (A). This is similar to Ca2+ signalling during excitation–contraction coupling, whereby Ca2+ influx through voltage-gated Ca2+ channels (Cav ) initiates Ca2+ -induced Ca2+ release from the neighbouring sarcoplasmic reticulum (SR) through RyRs (B). (C) Retrograde NAADP signalling whereby Ca2+ release from ER IP3 Rs or RyRs stimulates local NAADP production and subsequent Ca2+ release from acidic organelles. (D, E) Ca2+ release from the ER might be tempered through Ca2+ uptake by lysosomes (D) similar to tempering of ER-derived Ca2+ signals by mitochondria through the mitochondrial uniporter (E).

between the ER and acidic organelles might support triggering of Ca2+ signalling by NAADP and associated downstream Ca2+ dependent output. Membrane contact sites (MCS) are central to the formation of Ca2+ microdomains. MCS are regions of close membrane apposition (typically <30 nm) [62–64]. Given the restricted diffusion of Ca2+ in the bulk cytosol, this juxtaposition may facilitate inter-organellar communication by bringing Ca2+ signalling proteins together. An extreme example of this is at the triadic cleft of skeletal muscle whereby the opposing proteins are physically linked. As at the dyadic cleft in cardiac muscle, excitation–contraction coupling involves sequential activation of voltage-gated Ca2+ channels and RyRs. However in skeletal muscle, the two channel types are physically coupled and it is the conformational change in the plasma membrane Ca2+ channels that opens the RyRs (i.e. not a flux of Ca2+ ). Close apposition of membranes also creates a restricted volume, thus facilitating generation of ‘hot spots’ of high Ca2+ concentration. Indeed, at mitochondrial-associated membranes (MAMs), such hot spots are thought necessary for mitochondrial Ca2+ uptake due to the relatively low affinity of the mitochondrial Ca2+ uniporter (MCU) for Ca2+ [65]. Thus, MCS between acidic organelles and the ER could serve to position trigger and amplifier channels and facilitate Ca2+ signal transmission through Ca2+ microdomains [60]. Is there any evidence of physical contacts between acidic organelles and the ER? The answer is yes. In smooth muscle cells, type 3 RyRs show a greater degree of colocalisation with lysosomes than type 1 and 2 receptors [66]. Such colocalisation was proposed

to correspond to a trigger zone for NAADP action [66]. However, the presence of lysosome–SR contacts was not assessed in this instance due to the limited resolution of confocal microscopy. Indeed, due to their small size, MCS can only be definitively identified using electron microscopy. It is now clear from recent ultra-structural work that MCS do indeed exist between the endolysosomal system and the ER (Fig. 2). The first of these to be identified were those between endosomes and the ER [67–70] (Fig. 2A). These junctions are thought to regulate endosomal positioning, morphology, and dynamics [69,70], EGF receptor signalling [67] and possibly endosomal fission [71]. MCS have also been recently identified between lysosomes and the ER [61] (Fig. 2B). The SR might also form MCS with lysosomes in smooth muscle cells [72]. The functions of lysosome–ER junctions are yet to be elucidated (see Section 5). Electron microscopy has resolved molecular tethers between the ER and both endosomes [67] and lysosomes [61]. These tethers are similar in appearance to those between mitochondria and the ER [73] and would argue against the proposition that inter-organellar contacts simply result from stochastic collisions. Furthermore, some regions of the endolysosomal system and ER are in juxtaposition but with no apparent separation [61]. Perhaps such regions facilitate inter-organellar protein-protein contacts, analogous to those in the triadic cleft of skeletal muscle. MCS between the endolysosomal system and the ER set the precedent for the close association between other acidic Ca2+ stores and the ER [9]. Indeed, MCS have already been observed between phagosomes and the ER

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Fig. 2. Membrane contact site ultrastructure at the endolysosomal–ER interface. HeLa cells were stimulated with epidermal growth factor (EGF) in the presence of antiEGF receptor 10 nm gold conjugate and prepared for electron microscopy. The endoplasmic reticulum (ER) (chevrons) forms membrane contact sites (MCS) (arrows) with endosomes (endo, A) and lysosomes (B). Scale bar, 200 nm.

[74]. Acidic organelles might also form contacts with organelles other than the ER, as evidenced by contacts between mitochondria and both melanosomes [75] and yeast vacuoles [76,77]. In summary, MCS are strong anatomical candidates for facilitating the functional crosstalk between acidic and canonical Ca2+ stores.

4. Anatomy of membrane contact sites between the endolysosomal system and the ER: making the connection If MCS between the endolysosomal system and the ER allow formation of functional Ca2+ microdomains, then a key goal is to define how these sites are both composed and regulated. This might allow manipulation of MCS in order to probe their role in Ca2+ signal transmission between acidic Ca2+ stores and the ER. MCS between endosomes and the ER are thought to facilitate interaction of the EGF receptor and the ER-localised phosphatase, PTP1B [67] (Fig. 3A). By bringing the two together, MCS allow PTP1B to dephosphorylate the EGF receptor, leading to downregulation of EGF receptor signalling. Overexpressing a substrate-trapping mutant of PTP1B enhanced the extent of these contacts. Conversely, silencing PTP1B modestly reduced the number of contacts. These data suggest that endosome–ER contacts are stabilised by the interaction of the EGF receptor and PTP1B [67]. Contacts between the ER and more ‘mature’ endosomes might be mediated by interaction of Rab7 and RILP on the endosomes and VAP proteins on the ER, in complex with ORP1L [69] (Fig. 3A). This interaction is regulated by cholesterol, likely through conformational changes in ORP1L [69]. Endosome–ER contacts also partner the ER-resident VAPs with the STARD3 and STARD3NL proteins on endosomes [70] (Fig. 3A). Overexpressing either STARD3 or STARD3NL increases the extent of endosomal–ER contacts. By using an elegant in situ proximity ligation assay [78], the authors visualised these proteins interacting with VAP-A using light microscopy. Interestingly, knockdown of PTP1B or ORP1L did not affect this interaction, suggesting that these proteins might belong to distinct classes of MCS [70]. How lysosomes form contacts with the ER is unknown at present. VAP proteins may again be involved, given their general role in forming ER-based MCS [79]. Indeed, in addition to their role in forming endosome–ER junctions, VAPs are also found at MCS between the ER and plasma membrane [80], mitochondria [81] and the Golgi complex [82]. VAPs often partner with lipid binding

proteins such as ORP1L and STARD3 (see above) through interaction with their FFAT motifs. Given that the lysosome–ER interface might also serve as a conduit for cholesterol traffic, perhaps such contact sites are formed through VAPs and cholesterol-binding proteins. Other candidates that could support the formation of lysosome–ER junctions include homologues of yeast proteins involved the in the formation of the nucleus–vacuole junction (NVJ) [83] (Fig. 3B). This contact site facilitates autophagic turnover of nuclear material in yeast. The NVJ can be likened to the lysosomal–ER interface; the nuclear membrane is contiguous with the ER whilst the vacuole is an acidic Ca2+ store and therefore functionally similar to the lysosome [60]. The NVJ is formed by interaction of Nvj1p on the outer nuclear membrane and Vac8p on the vacuole [83] (Fig. 3B). Deletion of either protein disrupts NVJ contact formation. Recent work has identified Nvj2p as an additional component of the NVJ [84]. Nvj2 contains a Synaptotagmin-like mitochondrial–lipid-binding protein (SMP) domain [85], which appears to be a conserved component of all MCS in yeast; the seven yeast proteins that contain an SMP domain localise to inter-organellar junctions [84]. These proteins include three of the four components of the ERMES complex at mitochondria–ER junctions (Mmm1p, Mdm12p and Mdm34p) and the tricalbins (Tcb1p, Tcb2p and Tcb3p) at ER–PM junctions [84]. HT008, the human homologue of Nvj2p, also localises to the NVJ upon heterologous expression in yeast [84], suggesting a potential conservation of MCS composition across taxonomic kingdoms. Indeed, human homologues of the yeast tricalbins (the extended synaptotagmins) localise to ER–PM contacts in mammalian cells (see other reviews in this issue). However, the molecular composition of mitochondrial–ER junction is likely to be distinct between animals and yeast [86]. It remains to be seen whether HT008 and/or other homologues of NVJ components localise to lysosome–ER contacts in mammalian cells, if they even exist. Recent proteomic analysis of TPCs identified several proteins of interest in the context of vesicular membrane trafficking (e.g. Rabs, including Rab7) and membrane organisation (e.g. sigma receptors) [87]. Sigma receptors have been localised to MAMs where they interact with ER-resident chaperones. This complex can regulate IP3 receptors and thus facilitate ER-coupled mitochondrial Ca2+ uptake [88]. Perhaps these proteins might play analogous roles at the lysosome–ER interface. In summary, the composition of MCS between the endolysosomal system and the ER is beginning to be defined. This could

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Fig. 3. Composition of membrane contact sites between acidic organelles and the ER. Schematic outlining the molecular components of tethering complexes between endosomes and the endoplasmic reticulum (ER) (A), and at the nuclear–vacuole junction in yeast (B), which may be analogous to lysosome–ER junctions. ER-resident protein tyrosine phosphatase 1B (PTP1B) interacts with epidermal growth factor receptors (EGFR) on the endosomal membranes (A, left). A complex of the small GTPase Rab7 and Rab-Interacting Lysosome Protein (RILP) on the endosomal membrane forms contacts with the ER through a complex of Oxysterol-binding protein Related Protein 1L (ORP1L) and Vesicle-associated membrane protein Associated Protein (VAP) (A, centre). VAPs may also form ER-endosome tethers with StAR-related lipid transfer domain protein 3 N-terminal like protein (STARD3NL) (A, right). In yeast, the ER-related outer nuclear membrane is tethered to the vacuole by an interaction between nucleus–vacuole junction protein1 (Nvj1p) and Vac8p (B).

facilitate future functional analyses. Putative distinct classes of endosome–ER MCS might increase the functional heterogeneity of such sites. 5. Modelling Ca2+ signalling at lysosome–ER membrane contact sites: seeing the invisible By their very nature, MCS are difficult anatomical features to probe experimentally. The span between apposing membranes is <30 nm, which is far beyond the range of standard light microscopy (∼200 nm) and even goes beyond the limit for functional super resolution microscopy (∼50 nm). Although electron microscopy can achieve the required resolution, the structures are static and thus cannot be analysed over the timescales of Ca2+ signalling. It is therefore technically challenging to functionally characterise Ca2+ signalling at these sites. This problem has been recently approached using computational modelling of Ca2+ signalling within these sites [72,89]. In human fibroblasts, it had been previously shown that the lysosomotropic agent, GPN evokes complex global Ca2+ oscillations driven by IP3 Rs [61], similar to the actions of NAADP. GPN is a freely diffusible synthetic dipeptide substrate for the lysosomal hydrolase cathepsin C. When hydrolysed, GPN increases the osmolarity inside the lysosomes, leading to the swelling of the organelles and leak of small molecular weight solutes, including ions. Leakage can be tracked by monitoring loss of fluorescence of the acidotropic dye Lysotracker. This serves as a proxy for Ca2+ depletion from the lysosome and is amenable to computational modelling. Using a previous model of IP3 R activation by IP3 and Ca2+ [90], we modelled lysosomal Ca2+ leaks in the context of global signalling [89]. Assembling the lysosomal (trigger) and ER (amplifier) leak models qualitatively recapitulated the observed data, whereby GPN-induced Ca2+ release from the lysosome coupled to IP3 Rs on the ER and initiated global Ca2+ signals in a concentration dependent manner [89]. This then provided a modular platform for modelling more physiological NAADP-induced lysosomal leaks. TPCs are the likely target for NAADP and have been biophysically characterised (see Section 1). Much of the electrophysiological data is inconsistent, likely owing to the difficulty of directly measuring currents from lysosomal channels in their native state. Nonetheless, consensus is tending towards conclusions that TPCs are both Ca2+ - and Na+ -permeable [91]. An asparagine-based selectivity filter of TPCs – which is not unlike that in Ca2+ and Na+ conducting NMDA receptors – may support this dual permeability [31]. One

biophysical study incorporated purified human TPC2 preparations into lipid bilayers, and recorded currents using physiologically relevant conditions (luminal high Ca2+ , low pH) [39]. In this setting, NAADP evoked Ca2+ currents through TPCs, with the characteristic biphasic concentration–effect relationship, whereby increasing concentrations of NAADP activated and then inactivated the channel. Such behaviour displays many of the hallmarks of NAADP and TPC action in intact cells. Indeed, simulated NAADP treatment using lysosomal leaks modelled on this data produced small Ca2+ signals from lysosomes that were amplified to global signals by IP3 Rs on the ER [89], consistent with previous physiological data [25,92]. Crucially, ‘turning off’ TPCs within the microdomain but not on the remainder of the lysosome ablated the global signals (Fig. 4A). Thus, the model demonstrated the principle that lysosome–ER microdomains are capable of driving global Ca2+ signals [89]. To probe the properties of the microdomain, the ‘density’ of the lysosomal Ca2+ leak was altered. In the first instance, TPC flux was selectively increased within the microdomain, a scenario akin to TPC ‘clustering’ at MCS. Although there is no direct evidence for TPC clustering, other MCS such as MAMs [93] and the dyadic cleft [94,95] are enriched with Ca2+ signalling proteins when compared to the surrounding ER/SR. Clustered TPCs broadened the range of NAADP concentrations that could produce a response in the model [89], and increased the frequency of the oscillations (Fig. 4B). This indicates that the extent of TPC clustering within microdomains may increase the functional diversity of microdomain signals [89]. Increasing the ‘density’ of TPCs both within and outside the microdomain – akin to an increased level of expression – also broadened the concentration range of NAADP action [89]. However, in contrast to the clustering simulations, microdomains were no longer absolutely required to initiate global signals. Instead, microdomain activity served to increase the frequency of global oscillations (Fig. 4C). Lysosome–ER microdomains are therefore capable of both driving and modulating global Ca2+ signals depending on the expression levels both inside and outside the microdomain [89]. Interestingly, in all of the simulations, the concentration of Ca2+ within the microdomain was almost identical to that within the cytosol, even though the signals were kinetically distinct [89]. This was surprising as Ca2+ in other microdomains are thought to reach high concentrations. Previous models of the dyadic cleft, which bring together L-type Ca2+ channels on the ‘source’ plasma membrane and RyRs on the ‘target’ SR membrane, predicted a higher Ca2+ concentration within the microdomain than within the bulk

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Fig. 4. Modelling Ca2+ microdomains at the lysosome–ER interface. (A–C, top) Schematic of models used to stimulate Ca2+ signalling between lysosomal two-pore channels (TPCs) and endoplasmic reticulum (ER) inositol trisphosphate receptors (IP3 Rs) [89], where the density of TPCs is equal in both the microdomain and non-microdomain area (‘dispersed’) (A), increased in the microdomain only (‘clustered’) (B), or ‘increased’ in both the microdomain and non-microdomain area (C). Middle and bottom, example model traces of cytosolic Ca2+ signals, where TPC flux into the microdomain was manipulated as indicated.

cytosol [96]. Similarly in MAMs, IP3 Rs on the ‘source’ membrane neighbour the very low affinity MCUs on the ‘target’ mitochondrial membranes [97,98]. In this case, a high Ca2+ microdomain is necessary to overcome the barrier to Ca2+ uptake into the mitochondria. So why might these microdomains differ? First, the ‘target’ protein in the model lysosome–ER junction is the IP3 R, which has a much higher affinity for Ca2+ (∼0.3 ␮M) [99] than does the MCU (>10 ␮M) [97,98], or the even the RyR (∼1 ␮M) [100]. This means that even a trickle of Ca2+ through TPCs is enough to tickle the IP3 R into action; Ca2+ -induced Ca2+ release by the IP3 Rs amplifies the small NAADP-induced signal and generates the global responses. Thus, the affinity of the protein on the ‘target’ membrane may be matched to the ‘source’ of the Ca2+ . Second, unlike RyRs and MCUs, which can be activated by Ca2+ alone, IP3 Rs are co-activated by IP3 and Ca2+ . This required a basal IP3 concentration in the model, which induced a leak of ER Ca2+ [89]. To generate a steady state at rest, this basal Ca2+ leak was balanced through Ca2+ uptake by SERCA pumps. SERCA is a highly efficient uptake mechanism and thus prevents the microdomain from reaching a high Ca2+ concentration. Microdomain models utilising RyRs do not need SERCA pumps in their microdomains as there is no basal flux to balance, thus allowing high Ca2+ concentrations to accumulate [96]. Similarly, structural isolation of MAMs has shown that both IP3 Rs and MCUs are enriched at these sites, whereas SERCA is not [93]. This potential absence of SERCA in MAMs may permit the generation of high Ca2+ microdomains. Indeed the absence of SERCA could permit IP3 Rs to be basally active, as is required to maintain mitochondrial bioenergetics [101]. Thus, in addition to matching the affinity of the ‘target’ protein to the ‘source’ release mechanism, different Ca2+ transport processes may be mixed together to generate a functionally heterogenous microdomain population [102]. Others have employed a stochastic modelling approach that simulated the trajectories of individual ions within lysosome–SR microdomains in pulmonary artery smooth muscle cells [72], again using the same TPC2 biophysical dataset to model the action of NAADP [39]. In these cells, NAADP has been shown to selectively

recruit RyRs [14]. Unlike the simpler, deterministic modelling approach used for modelling MCS in fibroblasts as outlined above, this stochastic approach is computationally intensive. It therefore assumed, rather than demonstrated, functional coupling between the modelled microdomains and the rest of the cell. As in previous models utilising RyRs [96], a high Ca2+ concentration was found in the lysosome–SR microdomains of smooth muscle [72]. Again, this supports the argument that source and target proteins are ‘mixed and matched’ to generate a functionally heterogeneous microdomain population [102]. Somewhat intuitively, increasing the separation of membranes at the MCS (and thus the MCS volume) reduced the peak Ca2+ concentration achieved upon simulated TPC activation. Such analysis suggested that a distance of 50 nm or less was sufficient to generate Ca2+ signals (>8 ␮M) capable of activating type 3 RyRs [72]. Thus, modelling predicts that lysosome–SR MCS may be necessary to allow Ca2+ -induced Ca2+ release in pulmonary artery smooth muscle cells. Modelling has therefore provided insight into Ca2+ microdomain behaviour within MCS [72,89,102]. Although particular functions of the lysosome–ER MCS are yet to be demonstrated, there is some evidence that they may be involved in inter-organellar crosstalk as modelled [72,89]. Equivalent junctions between endosomes and the ER are better understood at a functional level, though again the role of Ca2+ signalling within these structures is poorly understood. 6. Other functions of lysosome–ER Ca2+ microdomains: beyond the trigger hypothesis. All discussion of lysosome–ER Ca2+ microdomains and MCS has thus far focused on so-called ‘anterograde’ signalling, from the acidic organelles to the ER. Communication might also occur in the reverse ‘retrograde’ direction [103]. Some early observations of crosstalk between Ca2+ mobilising messengers (IP3 , cADPR and NAADP) remain mechanistically unexplained [10,104]. In ascidian oocytes, IP3 evoked robust Ca2+ oscillations that were completely eliminated following pre-treatment with NAADP [10].

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The Ca2+ oscillations that accompany fertilisation were also sensitive to NAADP blockade [10]. Furthermore, NAADP co-injection also blocked the actions of IP3 and cADPR in Jurkat T cells [104]. Using a similar logic to that used to formulate the ‘trigger’ hypothesis (Section 2), these experiments place NAADP-sensitive Ca2+ channels ‘downstream’ of IP3 receptors [57]. Recent studies by Morgan and colleagues have revisited this concept [103]. These authors used acridine orange to indirectly monitor NAADP activity in sea urchin eggs. Acridine orange is a weakly basic fluorescent dye that accumulates in acidic organelles and undergoes selfquenching in a pH-dependent manner. Previous studies in sea urchin egg homogenates have shown that among the Ca2+ mobilising messengers, only NAADP can evoke increases in acridine orange fluorescence [105]. Such increases are consistent with an alkalinisation of the organelles accompanying Ca2+ release. A similar NAADP-evoked alkalinisation can be observed in intact cells [106]. However, unlike in sea urchin egg homogenates, IP3 and cADPR were also able to evoke pH responses in intact cells [103,105]. Blocking NAADP action by a range of manoeuvres also blocked the IP3 and cADPR pH responses [103]. Thus, functional NAADP receptors are required for some aspects of IP3 and cADPR action. These data again place NAADP-sensitive Ca2+ channels downstream of IP3 and cADPR. Given that this ‘retrograde’ coupling is lost in the homogenate system, the data also suggest that – like anterograde signalling – functional coupling requires the stores to be closely linked. So, how might acidic organelles and the ER be coupled in retrograde mode? One key finding is that pH responses by cADPR and IP3 in eggs were blocked by buffering cytosolic Ca2+ with the slow Ca2+ buffer EGTA [103]. This suggests that Ca2+ mediates retrograde coupling. This Ca2+ sensitivity is unlikely to be at the level of the NAADP target itself since it was in the egg homogenate system that the insensitivity of NAADP-induced Ca2+ release to cytosolic Ca2+ was first demonstrated [59]. Moreover, although plant TPCs are regulated by cytosolic Ca2+ through EF hands in the cytosolic linker that connects the two channel domains [107], sea urchin and other deuterostome animal TPCs lack these – or any other – Ca2+ binding motifs [108] and are thought to be insensitive to cytosolic Ca2+ (although see [43]). Instead, retrograde coupling of the ER to lysosomes might occur through Ca2+ -sensitive NAADP production. Like NAADP degradation by dephosphorylation [109], NAADP synthesis can be stimulated by Ca2+ [103]. Thus, localised production of NAADP, stimulated through ER Ca2+ release, could potentially initiate further Ca2+ release from acidic organelles (Fig. 1C). Such bidirectional coupling adds a further layer of regulation for finetuning inter-organellar crosstalk [103] and may serve to increase the functional heterogeneity of MCS. Although the mechanisms of Ca2+ release from acidic organelles are being extensively studied, much less attention has focussed on the mechanisms of Ca2+ uptake. In plants and yeast, Ca2+ –H+ exchangers and Ca2+ ATPases drive vacuolar Ca2+ uptake [110]. In animals however, analogous genes for Ca2+ /H+ exchangers appear to be absent from most genomes, whilst Ca2+ pumps are found at the plasma membrane, the ER and the secretory pathway. Nevertheless, isolated reports of both Ca2+ /H+ exchange and Ca2+ ATPase activity on acidic organelles are present in the literature [21]. Indeed, a proton gradient is required for lysosomal Ca2+ uptake [111] and many studies have demonstrated blockade of NAADP action by preventing acidification [6] (reviewed in [21]). Recent work has highlighted the potential importance of lysosomal Ca2+ uptake in the shaping of cytosolic Ca2+ signals [112,113]. In HEK cells, several independent manoeuvres that interfere with acidic organelles potentiate global Ca2+ signals evoked by the IP3 forming agonist carbachol, by IP3 itself, or by the SERCA inhibitor (and ER-depleting agent) thapsigargin. These data were interpreted as suggesting that lysosomal Ca2+ uptake dampens cytosolic

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Ca2+ signals; interfering with lysosomal Ca2+ uptake resulted in exaggerated global Ca2+ responses. Analogous roles for vacuolar Ca2+ uptake have been established in tempering Ca2+ signals that are generated during the response to environmental stressors in plants and yeast [110]. In the HEK cells, Ca2+ influx through storeoperated Ca2+ entry was not affected by interfering with lysosomal Ca2+ uptake [112]. Here we draw an analogy with anterograde coupling of TPCs to ER Ca2+ release channels. As discussed previously, inhibiting either acidic organelles or ER Ca2+ release reduced NAADP-evoked Ca2+ signals in cells expressing TPC2 but not a mutant re-routed to the PM [38] (see Section 2). In the mutantexpressing cells, NAADP-evoked Ca2+ signals result from Ca2+ influx across the plasma membrane. Thus, Ca2+ influx is neither tempered by lysosomal Ca2+ uptake [112] nor amplified by the ER [38]. Selective dampening of ER-evoked Ca2+ signals but not store operated Ca2+ entry further suggests an intimate relationship between ER and lysosome Ca2+ stores [112] (Fig. 1D). In summary, lysosomes may serve as targets as well as sources of Ca2+ signals. 7. Outlook: a series of questions in a section that features ‘might’ a lot! Much evidence has accrued indicating that the endolysosomal system and likely other acidic organelles are intimately connected to the ER, both functionally and physically. At the functional level, a critical player in this process is the Ca2+ mobilising messenger NAADP. Its molecular target is still disputed, although evidence that implicates the TPCs continues to mount. If this is the case, might TPCs localise to MCS? Might ER Ca2+ channels also localise to MCS? In other words, is there juxtaposition between the trigger and amplifier channels? Might differential targeting of IP3 Rs and RyRs to MCS explain why NAADP selectively couples to a particular ER Ca2+ release channel in different cells [14,92]? Is it possible to measure putative local signals at these junctions as has been done at MAMs [114,115]? Might such measurements reveal differences in microdomain Ca2+ concentrations as has been predicted through computational modelling? As ER-evoked Ca2+ signals might also be amplified by Ca2+ -stimulated NAADP production and/or curbed by lysosomal Ca2+ uptake, it is important to identify both the synthetic and transporter machinery. At the physical level, progress towards defining tethering complexes at MCS continues to suggest that distinct classes of contacts might exist, at least between endosomes and the ER. What is the physiological significance of this? Might distinct junctions perform different functions? Emerging evidence links local NAADPdependent Ca2+ fluxes to vesicular trafficking events, likely through regulating vesicle fusion [37,44,87,116]. Might Ca2+ signals also regulate non-vesicular traffic through formation of these junctions? It is clear that MCS regulate lipid traffic [79]. As both lipid transfer and Ca2+ signalling occur at MCS, might these processes interact, or be regulated by similar mechanisms [117]? The convergence of interests of Ca2+ ‘signallers’ and membrane ‘contacters’ might provide answers to these questions in the near future. Conflict of interests The authors declare no conflict of interests. Acknowledgements The authors would like to thank Tim Levine, Shmuel Muallem and Aldebaran Hofer for their helpful comments. This was work was supported by studentship BB/J014567/1 (to C.J.P.) and grants

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