ihscmne.Bt upr Ca support Both manner. biphasic hrb oetrssi [Ca in rises modest whereby oehrwt h yndn eetr(y) r h principal the are (RyR), receptor ryanodine Ca intracellular the with together ylcAPrbs)adaergltdb yooi [Ca cytosolic by regulated are and ribose) ADP cyclic odnW1 B,UK. 6BT, WC1E London w-oechannel NAADP, Two-pore modelling, Computational site, contact Membrane hitpe .Penny J. Ca Christopher lysosome–ER of model computational A ARTICLE RESEARCH ß 2934 2014 March 26 Accepted 2014; January 3 Received 1 not Ca of (ER) mobilisation reticulum controlled by endoplasmic achieved only be can which signals, Ca cytosolic in Changes INTRODUCTION Ca WORDS: KEY Ca global driving of lysosome–ER capable that are demonstrated microdomains NAADP messenger mobilising Ca global described qualitatively model The ‘trigger’. Ca intracellular important an form Acidic ABSTRACT Ato o orsodne([email protected]) correspondence for *Author Zealand. New 1142, Auckland hs hnescnb ciae yscn esnes(IP 2007). messengers (Clapham, second ER by activated and be (SR) can channels reticulum These sarcoplasmic the to noprtdapeiu oe fteioio rshsht (IP trisphosphate inositol the that of coupling model lysosome–ER previous of a model incorporated computational a generated aapoieacmuainlfaeokfrpoiglysosome–ER probing for Our framework Ca computational signals. a global provide modulated data or triggered microdomains dictated whether leaks lysosomal the of density overall and distribution relative hsmdlt hsooia yooa ek nue yteCa the by induced leaks but lysosomal Adapting controlled physiological lysosome. to a the model from caused solutes this small which of GPN, depletion substantial agent lysosomotropic the h bqiosioio ,, rshsht eetr(IP receptor trisphosphate 1,4,5 2010). inositol Docampo, and ubiquitous (Patel lysosomes The as such organelles acidic elsdtruhtmoa n pta opeiisi h Ca the is in Specificity for complexities 2000). spatial responsible al., and et temporal from pathway (Berridge through ranging death realised events signalling cellular to disparate ubiquitous fertilisation of multitude a a regulating of basis Ca of formation allow might sites contact E)Ca (ER) neetnl,orsmltossgetta h irdmi [Ca microdomain the that suggest simulations our Interestingly, hi iernesosraino Ca of observation renders size their rv lblCa global drive acigterltvl ihafnt fIP of affinity high thus relatively occur, to responses the for matching the in that than higher be not need eetra h RCa ER the as receptor eateto eladDvlpetlBooy nvriyCleeLondon, College University , Developmental and Cell of Department 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,23–93doi:10.1242/jcs.149047 2934–2943 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 2+ dynamics. 2+ trs eetyietfe yooeE membrane lysosome–ER identified Recently stores. 2+ 2+ inl hog opigwt nolsi reticulum endoplasmic with coupling through signals 2+ ees hnes hc oaiepredominantly localise which channels, release yooe,Edpamcreticulum, Endoplasmic Lysosomes, , 2 eateto ahmtc,Uiest fAuckland, of University Mathematics, of Department 2+ apiir n yooa ek steCa the as leaks lysosomal and ‘amplifier’ 2+ 1 ehnS Kilpatrick S. Bethan , o ocnrto ([Ca concentration ion 2+ eeaeaCa a generate ] 2+ 2+ idcdCa -induced yaisipatcl ee we Here, impractical. dynamics 2+ 2+ 3 trs u lotoein those also but stores, eetr o Ca for receptors irdmis although microdomains, 2+ 2+ pk (Roderick spike 2+ 2+ ees (CICR) release 2+ 2+ olta can that pool epne to responses 1 )fr the form ]) oscillations. ugMnHan Min Jung , 2+ 2+ ]ina 3 .The 3 and R), 2+ 2+ 2+ 2+ 3 ) ] 0) hs bevtoshv e oa‘rge yohss of hypothesis’ IP ‘trigger ER-resident a to whereby led action have NAADP observations These 20%). ln slmtd hsi wn obt h nestvt fNAADP- of insensitivity the Ca both to induced owing organelles is acidic This through limited. responses is such alone evoke to ability its types, eeeaieCa regenerative fCa of ( apposition 2000). n h Raaoost hs ewe h Radother and ER the between Ca local those support to to known analogous are which ER organelles, the lysosomes between 2013). and sites Patel, contact and/or and membrane identified Marchant modest recently 1999; We al., are et signals (Cancela localised NAADP-induced highly the that suggesting 03.Teedt ute nesoetelclsdntr of al., putative et the (Brailoiu nature (TPCs) that channels localised notable two-pore is the the NAADP, it for context, targets underscore this In further signalling. NAADP data These 2013). n os,19;GnzaiadGloe 96,adtelow the and 1996), Galione, and ( volume Genazzani lysosomal 1996; Dousa, and eodmsegrta oiie Ca mobilises that messenger second other system. endolysosomal with the associations within those ER membrane as for such SR/ER–plasma organelles, precedent and a ER–mitochondria provide Schendelassociations 2012). 1999; al., al., et et Franzini-Armstrong excitation–contraction 2002; for (Bers, crucial coupling is SR the on receptors ryanodine ye loigmtcodilutk fE-eesdCa ER-released of uptake mitochondrial allowing types mitochondria ( example, tight For form 2013). al., et (Helle network signalling Ca 2013). al., et Helle 2006; (Berridge, microdomains’ -yeCa ( junction L-type cleft dyadic the in microdomain acigmtcodilAPpouto oclua demand cellular to production ATP mitochondrial matching 02.Frhroe ncricmsl,asrnl ope Ca coupled strongly a muscle, cardiac in Furthermore, 2012). (Csorda AD-nue Ca NAADP-induced loigcerneb of ehnsssc stesarco/ the as such mechanisms Ca ‘off’ reticulum spike, by endoplasmic the terminates clearance feedback negative allowing Subsequent 2003). al., et ntmclbssfrteecdn n eoigo ucinl‘Ca functional of decoding and encoding the for basis anatomical iohnra sawl-hrceie osiun fteCa the of constituent as well-characterised such organelles, a or membrane is plasma the mitochondria, and ER the between loso htlclNAPrsoss(esrdindirectly a (measured EGTA, of presence responses Ca the in slow NAADP persist pH) luminal local in changes through that studies eggs, show recent Moreover, urchin also 2002). sea al., upon et disrupted in (Churchill is homogenisation ER Indeed the and 2013). NAADP between al., coupling functional et (Kilpatrick signalling cdcognle Cucile l,20) lhuhNAPis NAADP Although Ca 2002). global producing al., of et capable (Churchill organelles acidic hs uaieNAPtigreet antb eovdi the in resolved IP be cannot of events presence trigger NAADP putative these ebaecnatstsaergoso ls membrane close of regions are sites contact Membrane ioii cdaeiedncetd hsht NAP sa is (NAADP) phosphate dinucleotide adenine acid Nicotinic 2 ae Sneyd James , 2+ ´ ta. 06 zbda ta. 06 aao tal., et Tarasov 2006; al., et Szabadkai 2006; al., et s 2+ inligpoen nrsrce pcs omn the forming spaces, restricted in proteins signalling 2+ ufrta rvnsgoa epne Mra tal., et (Morgan responses global prevents that buffer , 2+ , hneso h lsammrn n lsesof clusters and membrane plasma the on channels 0n)pyia ucin ihteE nmn cell many in ER the with junctions physical nm) 10 0n)btenognle,alwn congregation allowing organelles, between nm) 30 ees octslcCa cytosolic to release 2+ 3 n y niiosi eti eltypes, cell certain in inhibitors RyR and R pksta hrceieIP characterise that spikes , 2+ 2+ –%o el oprdt h R( ER the to compared cell) a of 1–2% 2 inl Cneae l,19) Importantly, 1999). al., et (Cancela signals n adpPatel Sandip and microdomains 2+ Tae(EC)(erdee al., et (Berridge (SERCA) ATPase 2+ inl nawd ag fcell of range wide a in signals 2+ 2+ rmlssmsadother and lysosomes from hc rcue direct precludes which , 3 , sadRR amplify RyRs and Rs 5n ie between wide) 15-nm 3 sadRR (Chini RyRs and Rs 1, * 2+ microdomain 2+ 2+ signalling thereby , , 10– 2+ 2+ 2+

Journal of Cell Science iee l,19) uhapoce lo rdcino h Ca 1998; the of al., prediction et allow Jafri approaches 2006; Such al., 1999). et al., Greenstein et Rice 2013; al., et (Cannell ydccethv encntutdt cuaeydsrb the describe muscle accurately Ca cardiac to muscle the of constructed of smooth been models behaviour have gastrointestinal and cleft in 2010), dyadic Sneyd, generation and wave understanding (Means our slow rationalise helped has of Cajal of cells interstitial inlig opttoa oeln fCa of modelling Computational signalling. pnpoaiiy(ite l,21)adptnilypo selectivity poor potentially and Ca maximal 2010) low for al., a have et 2009), (Pitt al., probability et open Zong 2009; al., et Calcraft 2009; ARTICLE RESEARCH scpbeo ulttvl eciigeprmnal observed experimentally describing qualitatively that of cytosol capable bulk contiguous is the and microdomain lysosome–ER both of behaviour applied to the response signals. in of targets non-microdomain and and microdomain microdomains, within dynamics oee,teetcnqe,i admwt mathematical with Ca of tandem features microscopy in essential light capture techniques, can of modelling, resolution these spatial However, low relatively ( owing the characterisation experimental to full impede microdomains 2010). al., et , nti td,w ul ipetocmatetmdlo a of model two-compartment simple a built we study, this In h pta n eprlsae novdi studying in involved scales temporal and spatial The 0 m n h ttcntr feeto microscopy. electron of nature static the and nm) 200 2+ vrNa over + 2+ ( , .)(age l,21,btseSchieder see but 2012, al., et (Wang 0.1) uigectto–otato coupling excitation–contraction during 2+ irdmisin microdomains 2+ microdomain 2+ irbat hrfr rvd rcal ytmfrstudying for system tractable a Ca lysosomal provide therefore Fibroblasts irdmisd o eur ihCa high require not do microdomains Ca ntae n anandi Ca in maintained and initiated rv rmdlt lblCa global modulate or drive rvoswr,teeGNeoe Ca GPN-evoked these work, previous ellrhmottcmcaim aal fmaintaining of capable mechanisms C. removing homeostatic potentially employed therefore cellular an have preparations, to studies lysosomal owing Previous lysosomes isolated osmolarity. rupture luminal intralysosomal to in thought increase is the GPN of Hydrolysis for substrate ua irbat ihGNeiiscmlx IP cultured complex, primary elicits of GPN stimulation with that fibroblasts shown human recently have We RESULTS opewt h IP the with couple activity. Ca Ps edmntaeta,dpnigo hi relative their Ca on NAADP-induced depending small by density, that, at setting action and physiological demonstrate NAADP more distribution of We model a computational to TPCs. first model the this generating adapted We (GPN). then 2-naphthylamide glycyl-L-phenylalanine compound 2+ 2+ inl Klarc ta. 03.Cnitn ihour with Consistent 2013). al., et (Kilpatrick signals inligbhvor nue ytelysosomotropic the by induced behaviours signalling ora fCl cec 21)17 9424 doi:10.1242/jcs.149047 2934–2943 127, (2014) Science Cell of Journal 2+ inlig P safel ifsbedipeptide diffusible freely a is GPN signalling. 3 tlssm–Rmcooan oeither to microdomains lysosome–ER at R P ntepeec A rasne()of (B) absence or Ca (A) extracellular presence the 200 in with GPN stimulated fibroblasts of responses Ca complex GPN-evoked 1. Fig. yorce-oddclsfo he rfour experiments. or independent 43 three and from dextran- cells 54 Lysotracker-loaded GPN from s) are (540–600 Results after addition. and s) (0–60 before channels) 200 individual of right; addition (middle, or after overlay) s (left, 152 before (red) Red and Lysotracker (green) fluorescein–dextran fibroblast with individual co-loaded representative a of images oue rmlysosomes. mass from molecular solutes small of leak with associated aus E olddt peetdas (presented mean data fluorescence Pooled initial (E) to values. normalised are 200 Data with GPN. stimulated and Red or Lysotracker Rhodamine–dextran either with cells loaded from of recorded courses responses Time epifluorescence (D) (blue). DAPI using 6 ... uniyn loecneintensity fluorescence quantifying s.e.m.) 2+ siltos neetnl,these Interestingly, oscillations. 2+ 2+ m fe eim(i.1A,B). (Fig. medium -free P.Nce eestained were Nuclei GPN. M C ofclfluorescence Confocal (C) . 2+ ocnrtosfrtheir for concentrations 2+ inl r readily are signals AB igecl Ca Single-cell (A,B) 3 2+ R-dependent inl are signals 2+ m M 2935 leaks m M 2+

Journal of Cell Science opeerpueo yooe vrtetmcus four evoke of not timecourse data, does the These over GPN 2013). lysosomes that as al., of suggest experiments, rupture 1E, et Fig. of complete parallel (Kilpatrick loss in in complete 1D) summarised in (Fig. induced fluorescence decrease GPN expected increase Red modest contrast, not a noted In Lysotracker cultured did we fluorescence. Indeed, 1D). fluorescence in primary (Fig. GPN that to epifluorescence response rhodamine–dextran-labelled showed live-cell signal, Furthermore, fibroblasts of Lysotracker increased 1C). and (Fig. the imaging punctate of intensity remained labelling ablation in dextran complete the a whereas caused GPN oeua as(9 a cdtoe niaigsimilar indicating 200 with small acidotrope, Treatment lysosomes. kDa) a (10 to Da) Red, probes (399 both fluorescein–dextran Lysotracker of targeting mass of of fluorescence lysosomal molecular fluorescence on with fluorescent GPN 1C, colocalised with images Fig. confocal of lysosomes the in labelled effect shown in As we delivery. the Jadot endosomal cells, by 1984; examine dextrans live al., To in et Jadot 1990). integrity 1994; al., al., et et (Berg integrity lysosomal ARTICLE RESEARCH 2936 m M ouela rmlssmsta sascae ihkinetically with associated controlled data, is induces that These Ca GPN cytosolic lysosomes 2B). that distinct (Fig. from cells) show leak increased all 2C–F, solute further Fig. in in was Lysotracker summarised latency of the loss and complete (despite responded fet farneo P ocnrtoso ohLysotracker both on Ca concentrations cytosolic GPN of and range fluorescence small a of of leak effects limited more ( a solutes induces mass molecular rather but experiments, oetcnetaino P etd(20 tested GPN of concentration lowest Fg B.Smlrrslswr banda P concentration GPN a at obtained 100 were of results Similar 2B). (Fig. i.2,tert fdces fLstakrfursec in lysosomes. of fluorescence are lysis data osmotic Lysotracker 200 causing At These GPN of concentration-dependent. with inconsistent decrease was again GPN of to rate response the 2A, Fig. yooe n h R edvlpdasml closed-cell simple a developed we ER, the and lysosomes ofrhrivsiaeteato fGN eeaie the examined we GPN, of action the investigate further To ogi ehnsi nih nofntoa opigbetween coupling functional into insight mechanistic gain To m m ora fCl cec 21)17 9424 doi:10.1242/jcs.149047 2934–2943 127, (2014) Science Cell of Journal ;hwvr hr a ogrltny(i.2) tthe At 2B). (Fig. latency longer a was there however, M; P,cmlxCa complex GPN, M i.2 yooa ekadctslcCa cytosolic and leak Lysosomal 2. Fig. EF h antd fteCa the of magnitude The (E,F) rm3 (20 37 from ( ecnaeo el htrsoddwt Ca a with responded that cells of percentage yorce e loecne()adctslcCa cytosolic and on (A) GPN fluorescence of Red concentrations Lysotracker different of Effect concentration-dependent. (A,B) are GPN to response loecne()o aia Ca maximal Red Lysotracker or the of (C) loss on fluorescence half-maximal GPN achieve of to effects taken the time summarising data Pooled (C–F) w rtreidpnetexperiments. independent three or two . . ai)aesoni n ,rsetvl.Rslsare Results respectively. F, and E in shown are ratio) 0.4 2+ responses. m , ) 4(100 64 M), 0kDa). 10 2+ 2+ inl eeeoe nms cells most in evoked were signals ocnrto.A hw in shown As concentration. m )ad4 (200 48 and M) 2+ 2+ epne n the and responses epne (D). responses m ) ee cells fewer M), 2+ m )clsfrom cells M) 2+ inl in signals increase 2+ (B).

Journal of Cell Science P n stu otlkl ob oe reat(see artefact model a be to likely of most addition thus rapid the is by caused and an is exhibits GPN which model spike, For mathematical early 3B,C). the additional (Fig. concentrations, the system GPN experimental to some the similar in qualitatively observed responses behaviour oscillatory demonstrates xoeta ucinfte otedt nFg A hnwe When 2A. Fig. in data 200 with the treatment to simulate fitted function exponential opttoa oe fCa of model computational ARTICLE RESEARCH Rfue ae nmvmn fCa and of leaks and movement lysosomal on Materials both based fluxes incorporated (see ER model microdomain This Methods). lysosome–ER contiguous P.Lstakrfursec a sda rx o the for proxy a as Ca used of lysosomal effects was the modelled luminal fluorescence we instance, is Lysotracker first model the the GPN. In summarising 3A. schematic Fig. A in SERCA. shown and 2013) al., et 2+ ocnrto ( concentration 2+ yaisi ohtectslada and cytosol the both in dynamics m r100 or M 2+ hog h IP the through C m L P,temodel the GPN, M ,where ), C 3 L (Cao R san is rcs.Itrsigy ysihl leigtemxmlfu value flux black maximal 3D, the (Fig. altering ( data slightly observed by the Interestingly, to 20 traces). similar is of again which case spike, the In Discussion). hrfr aal fqaiaieydsrbn Ca describing qualitatively of capable therefore ( niaeta h pksaedie hog h IP the through driven are spikes the that indicate stoeidcdb AD,aelkl ob ihyrgltdand regulated highly be to likely are NAADP, by induced those as yorce loecne(i.2)ad yifrne luminal inference, by and, 2A) Ca (Fig. fluorescence al., Lysotracker et (Kilpatrick pharmacology expected 2013). the with fibroblasts ugsigta hscnetaino P sa h hehl for threshold the at is GPN of Ca concentration this that suggesting k k IPR Ls P sasnhtccmon htcue opeels of loss complete causes that compound synthetic a is GPN 2+ 2+ o 20 for ) oee,mr hsooia yooa Ca lysosomal physiological more However, . ~ pk eeain motnl,stigteIP the setting Importantly, generation. spike )aoihdtesie Fg BC e rcs.Teedata These traces). red 3B,C, (Fig. spikes the abolished 0) ora fCl cec 21)17 9424 doi:10.1242/jcs.149047 2934–2943 127, (2014) Science Cell of Journal m P,i spsil opeetteeoscillations, these prevent to possible is it GPN, M h w compartments two the Ca GPN-induced of oscillations. simulations Model 3. Fig. orsodn iuain r at are concentration. simulations GPN Corresponding of this reflective at depicted red) behaviour not, are ‘threshold’ one cells black; two D, responsive, In (one respectively. s, 320 and BD yia Ca Typical (B–D) ocnrto fGN epne ihu IP without Responses GPN. of indicated concentration the at (right) simulations corresponding and ( bak n .09(e) epciey(ieinterval (time s). respectively 390 (red), 0.0019 and (black) irdmi M)rsetvl,wt Ca with respectively, (MD) microdomain .05s 0.0075 ( k J IPR hw nbu n IP and blue in shown ) ~ 0 r nred. in are ) 2 1 nBadC ihtm nevl f20s 200 of intervals time with C, and B in A ceai ftemdldepicting model the of schematic A (A) m P,w bev single a observe we GPN, M 2+ epne ffbolss(left), fibroblasts of responses k Ls C auswr .1 s 0.015 were values 3 and eaoim( metabolism C m 3 o h yoo and cytosol the for k .Ormdlis model Our R. 2+ Ls 2+ auso 0.002 of values 3 epne in responses lxt 0 to flux R lxs such fluxes, V 2+ ngreen. in ) fluxes 2 3 1 flux R 2937 and 2+

Journal of Cell Science Rmcooan r hrfr aal fdiiggoa Ca global driving lysosome– of capable Oscillatory therefore trace). are red microdomains 4C, ER (Fig. ceased oscillations ite l,21) easm htti ekde o as a cause not does turn leak this in that assume in which 4 We figure in 2010). (see probability, change al., leak lysosomal et open the model, Pitt of our magnitude TPC2 the In determines dictates NAADP. for NAADP relationship concentration–effect lxwti h irdmi oprmn o0( 0 to compartment microdomain TPC the the setting to by Nevertheless, within identical 4C,D). almost (Fig. flux cytosol is the stimulation in that NAADP upon microdomain xmndteefc ficesn P lxdniyit the we into clustering, density TPC flux TPC potential ( cytosol increasing those the simulate to of relative as microdomain effect to such the al., order et microdomains, examined (Schendel ER In other or SR the at 2012). and membrane cluster plasma the to between known are hs odtos(i.5) ssoni i.5,tefeunyof frequency the 5B, Fig. in shown Ca As 5A). (Fig. conditions these ta. 09,w eeae oe ae napreviously a (low on lysosomal Ca based the high was mimic dataset model pH, that This conditions 2010). a Zong a al., under 2013; et generated Ca acquired as al., (Pitt acts et we dataset lysosomal biophysical Jha that 2009), published 2009; channel prevent al., al., ion et to et lysosomal (Calcraft NAADP a order for is target in TPC2 As magnitude depletion. in smaller ARTICLE RESEARCH 2938 the scenario, ( microdomain this the both ( In into cytosol area). density bulk NAADP Ca flux of shaded TPC range microdomain relative limited 4A, relatively (Fig. high a concentrations over place of takes NAADP absence the in concentrations. even signals Ca which the 4B) (Fig. Interestingly, observed by microdomain are phase. the in cytosol those the follow within oscillations ag vrwihNAPwscpbeo generating of capable was NAADP Ca which global microdomain-driven over range hta h pia AD ocnrto,pritn Ca persistent concentration, NAADP optimal the at that normdl opigbtenlssmsadteE by ER the and lysosomes between coupling model, our In 2+ siltossoe la eedneo h NAADP the on dependence clear a showed oscillations C L 2+ seMtrasadMtos.Orsmltosshow simulations Our Methods). and Materials (see k n eosrtstecaatrsi bell-shaped characteristic the demonstrates and ) t eeasmdt eeul oee,channels However, equal. be to assumed were ) 2+ 2+ inl a xaddunder expanded was signals mk ocnrto ihnthe within concentration , uigtepacemaker the during s 2 t ~ 10 k t .Ipraty the Importantly, ). mk mk t t and ) ~ 0), 2+ 2+ 2+ 2+ zbda ta. 06 setbihd u tpeetw know we present at but established, Ca is about 2006) little al., et Szabadkai aercnl hw htmblsto flssmlCa lysosomal of mobilisation that shown recently have ih xli iceace nteltrtr eaigt h Ca the to relating literature the in discrepancies explain might P ssfiin oeoecmlxadpritn Ca persistent and complex evoke to sufficient is GPN hteii epne(i.5) oee,goa Ca global However, 5D). to (Fig. effect concentrations response similar NAADP a a of range had elicit the manner that widening uniform is this increasing that in clustering, Interestingly, density TPCs. flux overexpressed the of studies to akin opigbtenaii n RCa ER and acidic between physical and coupling functional intimate indicates evidence Accumulating DISCUSSION vn ta. 08 hne l,21) P,hwvr osnot does however, GPN, 2012). al., et (Lloyd- Ca Shen complex disease evoke 2008; C1 al., type et Niemann–Pick Evans in Ca lysosomes of ER content and lysosomal dissociate pharmacologically ocnrto,gvn iet h igotcbell-shaped diagnostic the these Ca compared to increasing We to effects 5C). rise (Fig. relationship concentration–effect giving concentration, iuain nFg ugs htbt h itiuinand distribution the the both together, that microdomains. IP through regulate suggest Taken can TPCs the 5 uniformly. of of Fig. density magnitude increased in the is when simulations role leak modulatory signals and a lysosomal global of serves generation absence rather for prerequisite but the a Microdomain not therefore 5E,F). in the is (Fig. 5E, of flux frequency effect (Fig. signals stimulatory oscillatory flux a on the revealed microdomain TPC microdomain of the microdomain of Comparison presence of traces). absence red the in persisted ak ato srqie,hwvr neutn GPN-evoked cross equating lysosome–ER in probing however, for required, Ca tool is cytosolic valuable Caution a IP talk. thus ER is with GPN coupling require that n tmtcodi–Rjntos(Csorda junctions mitochondria–ER 1999) al., et at (Franzini-Armstrong between ER junctions and and at membrane Signalling plasma 2012). the Brailoiu, and Patel 2013; ora fCl cec 21)17 9424 doi:10.1242/jcs.149047 2934–2943 127, (2014) Science Cell of Journal 2+ 2+ inl n yooa Ca lysosomal and signals yaisbtenlssmsadteE.We ER. the and lysosomes between dynamics 2+ inl nalcl ye freape MDCK example, [for types cell all in signals irdmi yooa ek( without leak Responses lysosomal cytosol (D). microdomain bulk microdomain the the in and nM) (C) (15 NAADP Model to (C,D) NAADP. responses from of resulting addition artefact, fast simulation the a The is nM). spike (23 first concentration to NAADP response optimal Model the (B) non- (arc). the region and microdomain area) hatched (MD, (Lyso) TPCs microdomain of ER–lysosome density the in even the (cyclinders) depicts inset The in grey. shown is produce oscillations that global values microdomain-driven of range The 2010). probability open ( and NAADP of concentration i.4 oe iuain fNAADP-induced of Ca simulations Model 4. Fig. nred. in P 2+ )o P2bsdo aai ite l Pt tal., et (Pitt al. et Pitt in data on based TPC2 of o) 2+ lxi ohcmatet scenario a – compartments both in flux oscillations. 3 -eedn lblCa global R-dependent 3 s(iptike l,2013). al., et (Kilpatrick Rs 2+ A eainhpbtenthe between Relationship (A) trs(iptike al., et (Kilpatrick stores 2+ otn.Fiueto Failure content. ´ ta. 2006; al., et s mk t ~ 0 r shown are ) 2+ 2+ 2+ 2+ 2+ signals signals signals stores using 2+

Journal of Cell Science oe iuae h Ca the The behaviour. simulates microdomain potential the model understand computational to a model developed we intractable, experimentally largely EERHARTICLE RESEARCH inligaprtsit ptal etitdzn ocreate to zone Ca restricted together spatially bringing a Ca into potentially apparatus membranes, signalling lysosomal SR/ER between sites contact and membrane identified have and 2013) or lysosomes volume between cell-specific coupling lysosomal of reflect total extent ER. might levels, the which C and/or number, 1996)], cathepsin al., in et differences (Haller cells oeta h antd ftefue notemcooanis microdomain the into fluxes the of magnitude the that note oprmns h irdmi n h ukctsl in cytosol, bulk the Ca intracellular and various microdomain to response the compartments, e(iptike l,21)adohr vnBemne al., et Breemen (van others and 2013) al., et (Kilpatrick We 2+ irdmis stesz fteergosrnesthem renders regions these of size the As microdomains. 2+ ocnrto ihntwo within concentration 2+ lxs ti motn to important is It fluxes. 2+ rvosyt xedtepro fIP of period the extend to previously ta. 06.Pam ebaeCa membrane Plasma 2006). al., et r cldb ohsraeae n ouerto seMaterials IP (see the of ratios model volume a fluxes and employed these We area Methods). because surface and both cytosol by the scaled into are than smaller substantially h hne ae nIP on of probability based open the channel define the to dataset biophysical extensive an ocnrto,Ca concentration, 03.Ti ipyia aaidctsta tagvnIP given a at that indicates data biophysical This 2013). oteaiiyo yooeE rstl opoedi h absence the in proceed Ca to extracellular crosstalk of lysosome–ER of ability the to iptike l,21) etu nlddi u model our in included thus We 2013). ( Ca al., dynamic longer et however, much cells, Kilpatrick within typically signals crosstalk are lysosome–ER of period ora fCl cec 21)17 9424 doi:10.1242/jcs.149047 2934–2943 127, (2014) Science Cell of Journal 2+ sniieIP -sensitive 2+ 2+ rvssotpro siltos( oscillations short-period drives Fg A oti ta. 02 acl tal., et Cancela 2002; al., et Boittin 1A; (Fig. est a ihrslcieyicesdi the in [ increased microdomain selectively either was density oscillations. flux TPC Ca of NAADP-induced density modulates and Distribution 5. Fig. [ compartments both in increased rsne nCadF. and C in are presented frequency oscillation and NAADP for relationships red. concentration–effect in Resulting are leak lysosomal microdomain nM without Responses 50 (C,F) and NAADP. nM middle) bottom) 9 (B,E, (B,E, top), nM (B, 23 nM top), 7 (E, to responses show traces Example NAADP. of at concentrations simulations increasing Model (B,E) (grey). highlighted AD eeae lblCa global generates NAADP probability open ( and NAADP for of 4 concentration the Fig. between see Relationship (insets; (A,D) D abbreviations). and A in depicted mk P 3 )o P2 ihterneoe which over range the with TPC2, of o) 3 t n Ca and ~ eaoim hc a enshown been has which metabolism, k t ~ . 0 0s Cneae l,1999; al., et (Cancela s) 50 : 2+ 0364 2+ iuain hr P flux TPC where Simulations 3 mk lxswr xlddowing excluded were fluxes -rvnoclain (Politi oscillations R-driven ocnrto Coe al., et (Cao concentration s { t 5 1 10 DF]a schematically as (D–F)] k t AC]o uniformly or (A–C)] 3 htdasfrom draws that R 2+ oscillations 2+ , ) The s). 5 2939 3

Journal of Cell Science eutn oe scpbeo eciig tlatqualitatively, the least Nevertheless, at describing, cells. of capable intact IP is model in resulting compartments endosomal ifrne nincrcrigcniin ih explain Ca might 2013; modest although al., conditions 2013), et al., (Churamani and recording et sensitivity constructs Jha NAADP TPC ionic tagged in N-terminally discrepancies in of Use differences 2012). al., et hsooial eeatoigt mlfcto hog RCa ER through amplification to owing relevant physiologically P sadppiesbtaefrctesnC(ao ta. 1984) Ca al., investigate et to (Jadot used C widely cathepsin been for has substrate and dipeptide a is GPN orse l,20) eteeoemdle hsmore TPCs this implicating accumulated Ca has modelled Zong NAADP-activated 2009; evidence as al., much therefore et 2009), Calcraft al., 2009; likely We al., et as et identification (Brailoiu 2007). targets initial NAADP their Since al., scenario. physiological et Soares Fg CE.UigLstakrfursec sapoyfrthe for dextrans proxy a pH-sensitive as Ca of fluorescence lysosomal Lysotracker GPN fluorescence Using 1C–E). during the in explains (Fig. thus also Lysotracker increases pH active, lysosomal of in observed remain increase loss will the Indeed, it complete treatment. pH 1969), explaining broad al., relatively likely a et has (McDonald C cathepsin optimum As pH. luminal increasing xtfo h og ope Gohe l,20) hsrie the Ca raises This mobilises 2003). , GPN al., et that upon (Ghosh lysosomal complex possibility compartments Golgi other pre-lysosomal the from through exit Like traffics C to preparations. thought cathepsin on based is cell again GPN is assumption concentrations. broken this of but lysosomes range target selectively a at leak lysosomal P2 wn otesalsz ftela n ieypeec of presence likely and of Ca leak model functional the our of unidentified size In as-yet small 2010). an the al., to et owing (Pitt TPC2, physiologically observed be 99 iptike l,21;PrzTri ta. 95.The of leak 1995). a by al., perturbed et is model Ca Perez-Terzic simple lysosomal this 2013; of al., steady-state stable et Kilpatrick 1999; ARTICLE RESEARCH 2940 first the These Ca 4A). artificial luminal generate (Fig. in high probability measurements with to single-channel bilayers, previous open concentration from channel derived given 2010) used were the a data sets al., whereby We NAADP TPCs, et of 2013). of model (Pitt Patel, computational data and biophysical (Marchant channels NAADP- are TPCs that suggested has Na insensitive data recent However, ftersos t20 nature at threshold response the the including of experimentally, observed those to Hle ta. 96.W ocueta npiaycultured primary mass in molecular low that GPN of Ca conclude leak with including graded We treatment solutes, a produces upon 1996). GPN cells larger al., fibroblasts, of MDCK et retention in the Our (Haller kDa) showing 2006). (70 al., work et previous dextrans Gerasimenko with 1994; agrees of al., data release et in result (Berg to preparations unlikely ( therefore hydrolases is GPN lysosomal 2A). molecular into Fig. small 1; of water (Fig leak ( controlled of solutes a causes We ingress mass GPN gradient. Lo that the osmotic here 1996; the show in al., by increase down an et vesicles followed to cathepsin-C-containing Haller leads GPN osmolarity, 2006; of Churchill al., Hydrolysis luminal 1994; 2013). et al., al., et Duman et (Berg Sanjurjo 2002; contexts of al., range et a in stores acidic ER. the and lysosomes between 3 ieGN AD a eri h IP the recruit can NAADP GPN, Like P a sdt ntaealssmlCa lysosomal a initiate to used was GPN -rvnCa R-driven 2+ 2+ + , 2+ nodrt anisgtit ucinlcoupling functional into insight gain to order in hneswt o Ca low a with channels 0ka nacnetaindpnetmanner concentration-dependent a in kDa) 10 ocnrto,w oeldteGPN-induced the modelled we concentration, pksi h yoo hthv iia period similar a have that cytosol the in spikes 2+ m .H . P Fg 3B–D). (Fig. GPN M 2+ 5ka,a a enrpre nother in reported been has as kDa), 35 + rsmbyas qiirtsthereby equilibrates also presumably hnes(opradPtl 2012). Patel, and (Hooper channels 2+ n o uia H swould as pH, luminal low a and 2+ 2+ rmbt yooa and lysosomal both from eutk ehns,we mechanism, re-uptake 2+ 3 Clrf ta. 2009; al., et (Calcraft R 2+ lxsmgtsilbe still might fluxes 2+ 2+ emaiiy(Wang permeability eki h model. the in leak inligthrough signalling ´ pez- 2+ ope ml AD-nue yooa Ca lysosomal NAADP-induced small couples ugssta irdmi eaiu shgl eedn nthe on result dependent non-intuitive highly Ca of This distribution is spatial proceed. behaviour microdomain to that coupling suggests for prerequisite a o edt ec ihlvl nodrt ntaeCC.I u model, our In CICR. initiate to order C in levels high reach to need not etrn agt ihlwrafnt o Ca for affinity lower with targets featuring ekit h irdmi.Cnrr oorepcain,our expectations, our Ca to higher substantially achieve Contrary not do microdomain. microdomains that indicate simulations the into leak pta oe sn iieeeet n ata differential computational partial Detailed 2010). and Ca full Sneyd, elements of a and models to finite model (Means this using equations extend not to model (computations required function spatial is early work arbitrary its Further these to an shown). flux Indeed, using lysosomal value ramping simplification. gradually simulations maximum this by our eliminated in of are spikes spikes artefacts Early model. likely to are difficult NAADP time-dependent are several of that include steps actions however, GPN, biological particular, The in stimulus and, rapid. models, our are In increases 3–5). (Figs intensity action GPN and NAADP 4B,C). both (Fig. cytosol the in spikes regular causing o eesr o h irdmi odiewoecl responses whole-cell drive to microdomain IP the through for are concentrations high necessary such not but 1998), al., et (Jafri concentrations Ca signals. stemcooan hspolmhsbe atal solved detail partially of been level has same problem the This at cell at cell microdomain. the feasible whole the of not the is rest as 2013); compute It the al., to problem. to multi-scale present et – challenging which Breemen a – intensive microdomains remains van computationally such couple 1999; highly to how and are al., of Means issue 1998; et the al., however, et Rice Jafri 2006; 2010; al., et Sneyd, Greenstein 2013; al., et fiiyfrCa for affinity Ca of concentrations that principle Ca the demonstrates NAADP-induced model small our is simplicity, Nevertheless, It model. its whole-cell despite 2007). the a incorporating al., in for necessary microdomain et be lysosome–ER will (Higgins methods similar approach that likely homogenisation a using ag fcnetain vrwihNAPpoue global produced The NAADP which 5). (Fig. biphasic over recapitulated concentrations NAADP of model for range our relationships frequency, assessing By concentration–effect spike 2010). al., at global et described (Pitt decreases the TPC2 been for level recently level has single-channel optimal the behaviour an biphasic increasing This beyond 2002); activity. Misler, concentration and NAADP Johnson 2000; the al., et (Berg shaped hog yooeE irdmis(i.4) hsglobal Thus 4C). (Fig. microdomains Ca lysosome–ER through oprdwt h ndrcinltasotosre nteGPN the in whereby observed transport data, unidirectional the with compared motn ont htIP that note to important up ihntemcooan hc srqie obalance to required is which microdomain, IP the within pumps oSRApmsmgtvr elaheehg Ca high achieve well very might pumps SERCA no iohnra npre,weehg Ca high where uniporter, mitochondrial sueta uia yooa Ca lysosomal luminal that assume Ca m 3 ent h rsneo ntnaeu pksi u oesof models our in spikes instantaneous of presence the note We eas eosrt h rnil htteNAADP-induced the that principle the demonstrate also We h ocnrto–fetrltosi o NAADP-evoked for relationship concentration–effect The 2+ 2+ 2+ ciiya aa IP basal at activity R ausaelwoigt h fiin ciiyo SERCA of activity efficient the to owing low are values ekfo yooe scpbeo rvn lbloscillations global driving of capable is lysosomes from leak inl eealtdb eetvl lmntn h lysosomal the eliminating selectively by ablated were signals inligi amla el suuuli hti sbell- is it that in unusual is cells mammalian in signalling ora fCl cec 21)17 9424 doi:10.1242/jcs.149047 2934–2943 127, (2014) Science Cell of Journal 3 2+ s h iuto slkl eydfeeta microdomains at different very likely is situation The Rs. 2+ 2+ ocnrtosta h yoo Fg D.I is It 4D). (Fig. cytosol the than concentrations . C irdmisaerltvl omn(Cannell common relatively are microdomains L elts.W eosrt httemodel the that demonstrate We depletes). 2+ 2+ n hrfr irdmi Ca microdomain therefore and ees n eutk ahasadtheir and pathways re-uptake and release 3 2+ ocnrtos irdmiswith Microdomains concentrations. 3 saeatvtdb nanomolar by activated are Rs ek a eeaegoa Ca global generate can leaks 2+ 2+ ocnrtosaelikely are concentrations ( C L 2+ scntn (as constant is ) ek oteIP the to leaks 2+ ,suchasthe 2+ does 3 R, 2+ 2+

Journal of Cell Science hog h IP the through ulttvl eciigteCa the describing qualitatively otoldslt ekfo h yooe rbn more Probing Ca lysosome. but the the substantial through from induces leaks leak demonstrate physiological we solute which controlled GPN, agent hst hne lseig ohRR n IP and RyRs Both clustering. channel to this bevdclua epne.Coeaierglto fIP to of relative regulation model Cooperative our TPC responses. in of cellular narrow induces Regardless NAADP remained observed which 2000). over responses range al., global the et density, and endogenous (Ishibashi distribution the for at level expression also in transcript differences but tissue-specific studies show TPCs for which between only TPCs, recombinant not coupling implications overexpressed a of has of oscillation This mode of levels ER. the setting the expression reveals and alter that lysosomes in could possibility channels microdomain manipulation the lysosomal raises relationship the This this for concentration–effect frequency. role Additionally, the modulatory for on a 5D–F). important during has (Fig. also effect density be RyR TPC in broadening increase to al., for uniform et a Interestingly, Schendel thought particularly 2012). 2012; (Rahman, is activity, coupling excitation–contraction arrangement channel an coordinating such and n rex 1997). Erneux, and aaeesVlePrmtr Value Parameters V mk k Value m Parameters parameters. Model 1. Table ARTICLE RESEARCH tetmcnad10uism eiiln(l rmIvtoe)a 37 at medium Invitrogen) 100 from Eagle serum, (all bovine penicillin modified units/ml fetal 100 Dulbecco’s v/v and 10% streptomycin in with supplemented maintained (DMEM) healthy from were established fibroblasts females skin human primary Untransformed culture Cell METHODS AND MATERIALS Ca by receptors density the Ca increasing selectively TPC2 by of widened be could responses e d c b k k K K ta. 08 iaaie l,19)adcoe ogv cetbesteady- acceptable give to chosen and 1997) al., solutions. et state Miyawaki 2008; al., et epne pnbec ftrsod[Ca threshold of breach upon responses o yooeE irdmist rv lblCa global potential drive the to demonstrates microdomains channel lysosome–ER target for likely its and NAADP ud eeomn fasml opttoa oe of model computational simple a Ca of lysosome–ER development action. NAADP guide of range concentration architecturally the might enhance of sensitivities to NAADP recruitment act differing progressive with microdomains context distinct but cellular population a microdomain homogeneous in a assumed we model, a k t ausfrteeprmtr r ae rmDpn n rex(Dupont Erneux and Dupont from taken are parameters these for Values ausfrteeprmtr r ae rmrpre ags(Lloyd-Evans 2006). ranges al., et reported (Politi from al. taken et are Politi parameters from these taken for Values are parameters these for Values images. experimental from Ca estimated GPN-induced were experimental Results replicate to fitted Results a deg 3 t IPR a SERCA PLC SERCA a K t ncnlso,w aeue bevdeprmna aato data experimental observed used have we conclusion, In a a a a d c c 2+ 3 lxit h irdmi Fg AC.W liken We 5A–C). (Fig. microdomain the into flux R. 1 s 2.8 0.2 0.3 4.9 s 0.0324 s 0.0324 s 0.21 1s 0.0189 2+ 2+ m M m m m irdmis h oe scpbeof capable is model The microdomains. 2 ieycnrbtst hs ‘switch’-like these to contributes likely M M Ms 2 1 m 1 2 2 2 M 1 1 1 2+ epne otelysosomotropic the to responses A C A V V D D A A C Lm ERm LC ERC m C L ER P C e b b a a 2+ b b e b b 2+ mblsn messenger -mobilising yNAP nour In NAADP. by ] 3 sfr clusters, form Rs 2+ oscillations. 2+ 1 1 50 300 1 2000 s 50 1s 500 300 m m m responses 2 m m m m 2 m m m 1 m 2 2 3 m m M M 1 2 m 2 m 3 g/ml ˚ C 3 Ca yorce e 10n;Ivtoe)fr3 i nHS el were Cells HBS. in min label 30 to for 20–200 Invitrogen) medium with with stimulated nM; fibroblasts culture (100 incubating dextran-free by Red labelled in Lysotracker also h were 3 Lysosomes lysosomes. for chased subsequently ne uiiidamshr ih5 CO 5% with atmosphere humidified a under epciey where respectively, and Coe l,21) Ca 2013). al., et (Cao eunilectto t30n n 8 m yorce e and Red respectively, filter. Lysotracker 590-nm nm, a 570 nm. using and 380 captured was nm fluorescence and 568 emitted at and nm excited 340 were after at Rhodamine–dextran visualised was excitation nm) 440 sequential (emission; inverted fluorescence Fura-2 IX71 source. Olympus coupled light an cooled 20 a to a with fitted attached with microscope fluorescence photonics) s (TILL 3 camera every device captured were images Epifluorescence microscopy Epifluorescence Ca cytosolic of measurement For probes Fluorescent r e yteIP the by set are Ca model, the In Ca Ca the variables, bulk two the containing compartments, each IP contiguous microdomain, and two the with and model cytosol a constructed We Modelling 200M Axiovert a with inverted fitted (Zeiss) an scanner confocal using LSM510 63 a acquired to attached were microscope images Confocal microscopy Confocal smdle yHl functions: Hill by modelled is where nert,fbolsswr oddwt ihrdextran-conjugated either for Invitrogen) molecular from mg/ml; with lysosome both (0.2 Da; of fluorescein 10,000 loaded measurements dextran-conjugated mass For or were B Sigma). Rhodamine from fibroblasts all 7.4; integrity, (pH HEPES 10 rmIvtoe)fr1hi EE-ufrdsln HS opiig(in comprising (HBS) saline KH HEPES-buffered 1.25 in mM) h 1 for Invitrogen) from (2.5 AM Fura-2 with incubated rvosy(a ta. 03.Tefue notectsland cytosol the into fluxes The 2013). al., et are: microdomain (Cao previously oesisbfr xeietto n sdbtenpsae 9–13. passages between used and experimentation before coverslips eecridotuigxpu Emnru,20) oe aaeesare parameters 1. Model Table 2002). in (Ermentrout, given xppaut using out carried were pnpoaiiiswti h yols n microdomain, and the within probabilities open adps itr,rsetvl.ZisLM50sfwr a sdto used was software 510 LSM Zeiss images. captured the respectively. acquire was 560–615-nm filters, and fluorescence 505–530-nm pass or Emitted filter band long-pass respectively. 385-nm a nm, nm, either 364 543 through of and wavelengths using nm excited 488 were Red Lysotracker and dextran aia activation. maximal 6 2+ 2+ dynamics lnAohoa ae-meso betv.DP,fluorescein– DAPI, objective. water-immersion Apochromat Plan nHSwsrpae ih1m GA(Sigma). EGTA mM 1 with replaced was HBS in 3 V concentrations, ora fCl cec 21)17 9424 doi:10.1242/jcs.149047 2934–2943 127, (2014) Science Cell of Journal SERCA 2 srltdt h EC est n is and density SERCA the to related is PO 2+ 3 4 CaCl 2 , srlae rmteE hog h IP the through ER the from released is n Ca and k 2+ IPR J J mIPR eutk noteE hog h EC pumps SERCA the through ER the into re-uptake J C m mSERCA J srltdt h est fteIP the of density the to related is SERCA P SnarzBoeh.Weeindicated, Where Biotech). (SantaCruz GPN M IPR or 2+ 2 ~ MgSO 2 , ~ C ocnrtoswti hs compartments these within concentrations k ~ IPR k ~ m m IPR K )ad005 / lrncai (both acid pluronic v/v 0.005% and M) and mP K V SERCA P 1 V SERCA 1 : SERCA 75 O 2+ : SERCA O 75 4 P ðÞ ðÞ C,16NC,1 lcs and 10 NaCl, 156 KCl, 3 , 6 C C ocnrto,fbolsswere fibroblasts concentration, z or ER z ER betv,adamonochromator a and objective, C C C { 1 P { C , m 1 : 2 1 75 m : m 1 el eepae nglass on plated were Cells . 75 C : 75 C 8hi utr.Clswere Cells culture. in h 18 epciey Simulations respectively. , : 75 m , , , K 3 SERCA ,a modelled as R, 3 s h IP The Rs. P O and h half- the 2941 mP 3 2+ R O

Journal of Cell Science epciey whereby respectively, ocnrto fGNand GPN of concentration areas, by: given oprmnswt netmtdrt constant rate estimated an with compartments EERHARTICLE RESEARCH 2942 the in included we oscillations, Ca observed the the of model period long the to Owing IP in decreases lysosome, as-yet of the presence on the to and mechanisms Owing NAADP value. re-uptake by final induced undefined its flux to of zero magnitude of low value the initial an from linearly increases concentration. where eoe as denoted afmxmldgaaino IP of degradation half-maximal yatm nevldtrie yteGNcnetaini which in concentration GPN the by determined simulated is interval GPN of time Addition a 2A). (Fig. by GPN to response in fluorescence aia rate maximal P hr h ufc fteE htajistectpamhsalre area, larger a has cytoplasm the adjoins that ER the of surface the where whereby A h microdomain, the hr sn lxars h lsammrn.Thus, membrane. plasma the across flux no is there rstl inl ess nteasneo xrclua Ca extracellular of absence the in persist signals crosstalk n rex 97 oiie l,20) rdcino IP of Production 2006). al., et Politi 1997; Erneux, and sdfndi i.4A. Fig. in defined as h irdmi n yoo srpeetdb h ratio: the by represented is cytosol and microdomain the elto flssmlCa lysosomal of depletion fIP of ucino gns concentration, agonist of function to due fluxes are: the respectively, instance, microdomain, first and cytosol the the In into Results). GPN (see organelles acidic sue ob elgbei h AD oe.TeNAADP-induced The model. NAADP the Ca in of negligible fluxes be to assumed ERC oTPC 3 yooa ek fCa of leaks Lysosomal h Ca The Ca dynamics hntesraeta don h irdmi,woeae is area whose microdomain, the adjoins that surface the than , dC 2+ 3 dt .IP steTC pnpoaiiya ie ocnrto fNAADP, of concentration given a at probability open TPC2 the is R K m 2 PLC ifssbtentecniuu yoo n microdomain and cytosol contiguous the between diffuses ~ ~ 2+ k 3 Ls A stecnetaino Ca of concentration the is 2+ R yaisi h irdmi r xrse as, expressed are microdomain the in dynamics srmvdtruhpopoyainb IP by phosphorylation through removed is ERm A A 2 k 2+ ERC ERm srltdt h aiu aeo lxa given a at flux of rate maximum the to related is R rmtelssm otectpamadmcooanare microdomain and cytoplasm the to lysosome the from C 3 K dpnetpouto n erdto fIP of degradation and production -dependent R V . dC L dt V ðÞ R Similarly, . Plt ta. 06 n ocnrto fCa of concentration and 2006) al., et (Politi R JM sfte oteepnnildcesso Lysotracker of decreases exponential the to fitted is ~ 2 3 ~ ~ ersnsteefc ftedfeec ntesurface the in difference the of effect the represents IPR oa irdmi volumes microdomain Total J k J IPR t oa yooi volumes cytosolic Total { A A mLs and J LC Lm V J Ls z 2+ 2+ JM mLs J PLC ~ ~ Ls h eaiedfeec nvlm between volume in difference relative The . J ro h AD-nue Ca NAADP-induced the on or mk r oeldbsdo h GPN-induced the on based modelled are LS mk SERCA R ~ ~ k ~ t 3 3 { P t C , k k t m stesraertoo h yooeto lysosome the of ratio surface the is r eae otedniyo Ps and TPCs, of density the to related are P oTPC Ls Ls K L J m C oTPC ðÞ SERCA ðÞ deg n Ca and , z C C 2 stelmnllssmlCa lysosomal luminal the is 2+ z ðÞ L L emdltedgaainas: degradation the model We . C C R { { ðÞ asn afmxmlproduction half-maximal causing K C 3 L 2 z R { C C L PLC 2 V { m D C JM , 2+ C C , , ðÞ n thus and , , C LS , m z D ~ { C R C slysosome–ER As . V V V m C D 3 : C sa increasing an is 3 ðÞ 2+ C iae with kinase, 2+ eassume we , { 2+ 3 lxfrom flux C (Dupont m causing C , L are k 2+ Ls ok ..adSP ocie h td.CJP n ..woetepprwt input with paper the wrote authors. S.P. and experimental all C.J.P. study. the from the performed conceived S.P. B.S.K. and modelling. J.S. work. the performed J.M.H. and C.J.P. contributions Author interests. competing no declare authors The assistance. interests editorial Competing for UK) London, School, fibroblasts, of Latymer (The provision for Patel of UK) Jasmine Institute and London, Neuroscience, London, Clinical College of University (Department Neurology, Schapira H. Anthony thank We Acknowledgements erde .J. M. Berridge, H. A. Guse, and W. G. Mayr, V., B. Potter, I., Berg, T. Berg, and O. Seglen, T., Løvdal, E., Strømhaug, O., T. Zealand. Berg, New of Society References Royal the of Fund Marsden College the University and Research from B.S.K.); Sciences studentship (to IMPACT Biological London an and C.J.P.); Biotechnology (to a studentship by Council supported was work This Funding oee,teei rnpr fIP of constant rate transport with is microdomain, there However, anl,M . og .H . miz .S n ae,D R. D. Laver, and S. M. Imtiaz, T., H. C. Kong, B., M. Cannell, A. Galione, and C. G. Churchill, M., J. Cancela, M. A. Evans, and A. D. Galione, M. F.-X., Bootman, Boittin, M. and D. P. Bers, Lipp, J., M. Berridge, Csorda P. T. Dousa, and N. J. E. Sneyd, Chini, and M. Falcke, G., Donovan, P., Cao, J., Tang, X., Hao, A., Arredouani, X., Cheng, Z., Pan, M., Ruas, J., X., P. Gao, Calcraft, C., G. Brailoiu, G., M. Schrlau, X., Cai, D., Churamani, E., Brailoiu, lpa,D E. D. Clapham, S. Patel, and E. Brailoiu, T., Rahman, R., Hooper, D., Churamani, hrhl,G . kd,Y,Toa,J . eazn,A . ae,S and S. Patel, A., A. Genazzani, M., J. Thomas, Y., Okada, C., G. Churchill, iuloiepopae(AD() sa seta euao fT-lymphocyte of regulator essential an endocytic is Ca(2+)-signaling. (NAADP(+)) prelysosomal phosphate and dinucleotide lysosomes between C distinguish cathepsin . lysosome-disrupting to a substrate, 2-naphthylamide, glycyl-L-phenylalanine . anla .A n Hajno and A. C. Mannella, T., nue a+sgaln atrsb AD npnrai cnrcells. acinar pancreatic in NAADP 398 by patterns Ca2+-signalling induced signalling. calcium of universality Calcium oe ftecricda n motneo nuto ea o CICR for decay induction of importance 3D a and within dyad gating cardiac RyR stochastic the termination. by of release model Ca2+ reticulum sarcoplasmic channels. two-pore through al. organelles al. et acidic et K.-T. calcium from Chuang, S. L., NAADP-mediated Teboul, J. K., in Rietdorf, Marchant, 1 J., channel N. two-pore Dun, for signaling. J., requirement arterial M. Essential in Boulware, R., contraction Hooper, and mechanism. signals two-pool a Ca2+ via muscle mediates smooth phosphate dinucleotide nue a2)rlaede o eaea a2)idcdCa(2+)-release Ca(2+)-induced a as behave system. not does Ca(2+)-release induced data. single-channel on based puffs calcium eae raels nsauci eggs. urchin sea in organelles, related A. by Galione, activation and trafficking regulates 1 channel NAADP. two-pore of region N-terminal hr sn rdcino erdto fIP of degradation or production no is There 74-76. , s . ekn . Va C., Renken, G., ´s, ora fCl cec 21)17 9424 doi:10.1242/jcs.149047 2934–2943 127, (2014) Science Cell of Journal ice.J. Biochem. ice.J. Biochem. 40 20) ada xiaincnrcincoupling. excitation-contraction Cardiac (2002). .Cl Biol. Cell J. ice.J. Biochem. 405-412. , 20) AD oiie a2)fo eev rnls lysosome- granules, reserve from Ca(2+) mobilizes NAADP (2002). ipy.J. Biophys. 20) acu signaling. Calcium (2007). 20) acu irdmis raiainadfunction. and organization microdomains: Calcium (2006). .Cl Biol. Cell J. dP dt 316 453 186 300 V ~ 104 709-711. , 147-151. , deg dP ra,P,Wle,L,Wae,D,Btl,K . Balla, F., K. Buttle, D., Weaver, L., Walter, P., ´rnai, 201-209. , 229-236. , V dt 19) ioiaeaeiedncetd phosphate- dinucleotide Nicotinate-adenine (1996). 2149-2159. , ~ PLC m 150 ~ ! { zy G. czky, ´ k a.Rv o.Cl Biol. Cell Mol. Rev. Nat. R 581-588. , 3 D V K V deg P D C Thus, . 3 P Cell 2 z ðÞ z ewe h yols n the and cytoplasm the between C Cell P D { 2 K ic Res. Circ. 111 P 20) tutrladfunctional and Structural (2006). ipy.J. Biophys. deg 20) AD oiie calcium mobilizes NAADP (2009). 2 ðÞ P 131 P 19) oriaino agonist- of Coordination (1999). 20) ioii cdadenine acid Nicotinic (2002). 703-708. , m m 21) tcatcmdlof model stochastic A (2013). 20) ioii cdadenine acid Nicotinic (2000). 1047-1058. , { : P 20) h estlt and versatility The (2000). Nature : 3 P 91 ntemicrodomain. the in , 1168-1175. , 105 Nature 459 1 11-21. , 1133-1142. , 21) oto of Control (2013). 596-600. , 19) s of Use (1994). 415 21) The (2013). 198-205. , (2009). Nature Cell

Journal of Cell Science acat .S n ae,S. Patel, and S. J. Marchant, resen .L,Hnh .adWnlw .L. Vo and P. R. Deetjen, P., Winslow, Dietl, T., Haller, and R. Hinch, S. L., Kornfeld, J. and Greenstein, M. and N. H. Dahms, O. P., Petersen, Ghosh, V., A. Tepikin, M., Sherwood, V., J. Gerasimenko, A. Galione, and A. A. Genazzani, V. Ramesh, and F. Protasi, C., Franzini-Armstrong, B. Ermentrout, EERHARTICLE RESEARCH Lo Sillence, E., Elliot-Smith, A., D. Smith, X., He, J., A. Morgan, E., S. Lloyd-Evans, Patel, and E. C. Futter, H., A. Schapira, R., E. Eden, S., B. Kilpatrick, S. Misler, and D. J. Johnson, L. R. Winslow, and J. J. Rice, S., M. Jafri, ao,M,Clat . ataxD oic,S n atax R. Bie Wattiaux, M., and Jadot, S. Coninck, Wattiaux-De C., Colmant, M., Jadot, M. Imai, and M. Suzuki, K., Ishibashi, J. Sneyd, and S. Patel, M. and Cannell, R. Hooper, M., D. Bers, L., J. Puglisi, P., Goel, B. R., Kornmann, E. and Higgins, H. A. Michel, A., Lang, K., Kolar, G., Kanfer, J., C. S. Helle, C. Erneux, B. and Hille, G. and Dupont, E. A. Palmer, L., Chen, G., J. Duman, h,A,Aua . ae,S,Bali,E n ule,S. Muallem, and E. Brailoiu, S., Patel, M., Ahuja, A., Jha, e-ajro .I,Tvy .C,Poe .L n alr .W. C. Taylor, and L. D. Prole, C., S. Tovey, I., C. pez-Sanjurjo, ´ yNAADP. by 289-300. sitaellrclimsoei DKcls osbeivleeti InsP3- in involvement possible a cells: MDCK release. in Ca2+ mediated store calcium intracellular as ventricular cardiac the of model integrative myocyte. an in coupling excitation-contraction tale. the in twists new receptors: area. secretory the in store acidic Sci. an and reticulum endoplasmic V. pool. O. Gerasimenko, thapsigargin-insensitive cardiac a and from skeletal Ca2+ J. in mobilizes couplons phosphate and units release Ca(2+) muscles. of Students distribution and Researchers for XPPAUT to SIAM. PA: Guide A Systems: yooe hp n(,,)3eoe a+sgasb selectively by reticulum. endoplasmic signals the from Ca2+ released Ca2+ Ins(1,4,5)P3-evoked sequestering shape causes that Lysosomes disease storage sphingosine M. calcium. F. a Platt, lysosomal is and of A. C1 deregulation Galione, type H., E. disease Schuchman, Niemann-Pick C., G. Churchill, J., D. signals. Ca2+ complex triggers Ca2+ lysosomal Sci. of mobilisation Direct USA cells. Sci. beta human Acad. in Natl. signaling Proc. initiate stores calcium phosphate-sensitive lysosomes. R. 2-naphthylamide. J. glycyl-L-phenylalanine channels. of hydrolysis calcium Intralysosomal and sodium Commun. voltage-gated Res. to Biophys. Biochem. related protein (two-repeat) 347. homogenisation. using 247 microdomains calcium Modelling (2007). sites. contact membrane Acta of function and Organization (2013). oscillations. Ca2+ on activities Calcium 5-phosphatase and store. 3-kinase acidic trisphosphate an implicating dynamics: calcium Traffic to compartments intracellular mitochondria. and ER between Biol. linkage Cell physical J. the of significance and features euaino h yooa w-oecanl2b g+ AD,P(,5)P2 PI(3, NAADP, Mg2+, by load. kinases. channel-2 protein two-pore reticulum multiple lysosomal and the sarcoplasmic of and regulation adaptation receptor J. ryanodine Biophys. of roles 315 219 19) yooiiyadefc fgyy--hnllnn--ahhlmd on glycyl-D-phenylalanine-2-naphthylamide of effect and Cytotoxicity (1990). 623-644. , 119 126 1833 721-725. , 965-970. , 7 226-238. , 60-66. , 859-872. , 22 ipy.J. Biophys. ipy.J. Biophys. 2526-2541. , 321-331. , lne . euoe . ataxD oic,S n Wattiaux, and S. Coninck, Wattiaux-De V., Beauloye, V., ´lande, 74 Messenger ici.Bohs Acta Biophys. Biochim. 174 1149-1168. , 915-921. , (2002). 20) AD,cDRadI3alrlaeC2 rmthe from Ca2+ release all IP3 and cADPR NAADP, (2006). 77 90 21) AD ntarget. on NAADP (2012). 2 1528-1539. , 77-91. , elCalcium Cell 113-119. , iuaig nlzn,adAiaigDynamical Animating and Analyzing, Simulating, 19) iuain fteefcso nstl1,4,5- inositol of effects the of Simulations (1997). 21) usinn euaino w-oechannels two-pore of regulation Questioning (2013). 99 MOJ. EMBO 14566-14571. , a.Rv o.Cl Biol. Cell Mol. Rev. Nat. 20) ioii cdaeiedinucleotide acid-adenine Nicotinic (2002). 19) ioii cdaeiedinucleotide acid-adenine Nicotinic (1996). k,H. lkl, ¨ a.Med. Nat. 270 1027 20) oeua lnn fanvlform novel a of cloning Molecular (2000). 19 370-376. , 33 157-165. , 205-209. , 501-511. , 19) ada a+dnmc:the dynamics: Ca2+ Cardiac (1998). 19) h yooa compartment lysosomal The (1996). 14 20) ans 6-phosphate Mannose (2003). 1247-1255. , d.Ep e.Biol. Med. Exp. Adv. 19) hp,sz,and size, Shape, (1999). 20) ehnssof Mechanisms (2006). 20) otiuin of Contributions (2006). 4 21) Convergent (2013). 202-213. , ici.Biophys. Biochim. .Cl Sci. Cell J. Philadelphia, . .Ter Biol. Theor. J. Biochem. Biochem. 740 (2013). (2008). (2013). (1984). .Cell J. .Cell J. 325- , 126 Cell , aao,A . rfih,E .adRte,G A. G. Rutter, and J. E. Griffiths, I., A. Tarasov, Va K., Bianchi, G., Szabadkai, Y., Prakash, M., Yamasaki, A., Isbell, T., White, M., Thompson, S., Soares, ie .J,Jfi .S n iso,R L. R. Winslow, and S. M. Jafri, J., J. Rice, T. Rahman, Ho and P. A. Thomas, D., L. Gaspers, M., A., Politi, Ruas, K., Rietdorf, E., Venturi, M., Sitsapesan, M., T. Funnell, E. J., D. S. Clapham, and Pitt, P. T. Dousa, S., S. Shen, N., E. Chini, M., C. Perez-Terzic, hn . ag . i . hn,X,Yo . ibe . og .P,Y,T., Yu, P., X. Dong, S., Dibble, Z., Yao, X., Zhang, X., Li, X., Wang, D., Shen, Ro M., Schieder, D. M. Bootman, and J. M. Berridge, L., H. Roderick, en,S .adSed J. Sneyd, and A. S. Means, S. Ellis, and B. B. Zeitman, X., P. Callahan, K., J. McDonald, ae,S n oap,R. Docampo, and S. Patel, E. Brailoiu, and S. Patel, cedl . hl . ny,J n ace M. Falcke, and J. Sneyd, R., Thul, T., Schendel, og . cidr . uy . ese . rnr . Ro C., Gruner, S., Fenske, H., Cuny, M., Schieder, X., A., Zong, Goschka, X., Cheng, X., Li, M., Samie, M. X.-P., Dong, A. X., Zhang, Evans, X., and Wang, N. Fameli, C., Breemen, van ogn .J,Dvs .C,Wge,S .T . ei,A . arntn J., Parrington, M., A. Lewis, Y., T. K. S. Wagner, C., L. Davis, J., A. Morgan, iaai . lps . em . cafr,J . dm,J . kr,M and M. Ikura, A., J. Adams, M., J. McCaffery, R., Heim, J., Llopis, A., Miyawaki, rdcinb iohnra Ca(2+). mitochondrial R. by production Rizzuto, and channels. T. Ca2+ mitochondrial Biol. Balla, and Cell reticulum J. I., endoplasmic of A. coupling Nagy, mediated for D., necessary Cavagna, cells. are myometrial N. reaction in E. NAADP base-exchange of C239. Chini, nor generation and vivo CD38 in A. neither Galione, messenger: E., F. Lund, fC2 ees ntefntoa nto h ada idcspace. diadic cardiac the of unit functional 1871-1884. the in release Ca2+ of underlying of identification Trans. and encoding frequency feedbacks. oscillations: of sensor Ca2+ dual a as Ca2+. al. operating and et channel, X. pH release M. luminal Zhu, Ca2+ C., NAADP-sensitive G. Churchill, novel R., Gosain, A., Ganesan, in phosphate eggs. dinucleotide urchin adenine sea nicotinate by intact triggered release Ca2+ (1995). 277-286. yooa rfikn yihbtn R hne n yooa calcium lysosomal and channel TRP a inhibiting by release. al. trafficking et D. lysosomal Ca2+ H. Showalter, P., (TPCN2)-mediated A. Lieberman, 2 channel lysosomes. two-pore isolated in of currents Characterization (2010). n erdto fguao ydppiy mnppiaeI(ahpi )o rat of C) (cathepsin I aminopeptidase dipeptidyl liver. by glucagon of degradation and xadn h oeta o nrclua a+signaling. Ca2+ intracellular for potential the expanding sites? contact membrane for role 40 a channels: two-pore 805. eetri h etiua yct aeu:b igeo ymlil pnL- open multiple by or single a by up: channels? wake Ca2+ myocyte type ventricular the in receptor release. calcium PN eitsNAPdpnetC(+-ees rmlssmlstores. lysosomal C. from Wahl-Schott, Ca(2+)-release and Arch. Pflugers NAADP-dependent M. mediates Biel, TPCN2 H., Harz, lysosomes. O., and in channels 151 ion sodium-selective al. et activated J. Harlow, Y., Zhou, D., Shen, signalling. transport Ca2+ Ca2+ function-specific nanospace and muscle: site- smooth for vascular in reticulum sarcoplasmic hrhl,G .adGloe A. Galione, and C. G. Churchill, Cajal. of cells interstitial the of units pacemaking ewe h nolsi eiuu n cdcorganelles. acidic and reticulum endoplasmic the between rtisadcalmodulin. and proteins Y. R. Tsien, 152. 153-157. , 372-383. , .Bo.Chem. Biol. J. ora fCl cec 21)17 9424 doi:10.1242/jcs.149047 2934–2943 127, (2014) Science Cell of Journal 40 a.Commun. Nat. 325-330. , 21) yai lseigo P eetr yIP3. by receptors IP3 of clustering Dynamic (2012). ipy.J. Biophys. 19) loecn niaosfrC2 ae ngenfluorescent green on based Ca2+ for indicators Fluorescent (1997). 175 zr . Bru K., tzer, ¨ 458 901-911. , ur Biol. Curr. 891-899. , 244 u.Bohs J. Biophys. Eur. .Bo.Chem. Biol. J. 90 ice.J. Biochem. 3 6199-6208. , Nature 731. , 21) rgeigo a+sgasb NAADP-gated by signals Ca2+ of Triggering (2012). 3120-3133. , gmn,A,Be,M n alSht,C A. C. Wahl-Schott, and M. Biel, A., ggemann, ¨ 13 21) cdcclimsoe pnfrbusiness: for open stores calcium Acidic (2010). ra,P,D tfn,D,Wekwk,M R., M. Wieckowski, D., Stefani, De P., ´rnai, R425. , 21) ptotmoa acu yaisin dynamics calcium Spatio-temporal (2010). .Bo.Chem. Biol. J. 388 21) iietoa Ca Bidirectional (2013). 21) P rtisaephosphoinositide- are proteins TPC (2012). 312 882-887. , elCalcium Cell 285 41 955-959. , 19) oeiggi n gradedness and gain Modeling (1999). 27-39. , 35039-35046. , 21) ii trg iodr block disorders storage (2012). e,T. fer, ¨ .Physiol. J. 285 21) o osteryanodine the does How (2012). 20) h w-oechannel two-pore The (2009). 20) AD sasecond a as NAADP (2007). 52 21219-21222. , m .Physiol. J. Am. 21) euaino ATP of Regulation (2012). .Ter Biol. Theor. J. 28-35. , 20) oeso P and IP3 of Models (2006). 20) Calcium-induced (2003). 21) Pan-junctional (2013). ice.Sc Trans. Soc. Biochem. rnsCl Biol. Cell Trends .Cl Biol. Cell J. 591 zr . Griesbeck, K., tzer, ¨ 20) Chaperone- (2006). 2+ 16) Inactivation (1969). 21) P2i a is TPC2 (2010). 2043-2054. , inln occurs signaling ice.Soc. Biochem. ipy.J. Biophys. 292 267 200 C227- , 2943 137- , 789- , Cell 77 20 , ,

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