uigpopoiaeCmdae eldeath cell phospholipase-C-mediated during eria28 0033Pro Portugal. Porto, 4050-313 228, Ferreira Biome ap lge83 1010Pro Portugal. Porto, 4150-180 823, Alegre Campo inln aha n eeino L-,wihw hwt be the to trigger to show inability we Ca an C staurosporine-induced in which characteristic phospholipase results PLC-2, development, the hyphal of in of deletion involved player and modulation pivotal pathway because a signaling death, as identified cell was during C Phospholipase approaches. .PdoGonc Pedro A. Ca intracellular and channel TRP-like a of Activation ARTICLE RESEARCH eevd2 eray21;Acpe 3Jn 2014 June 23 Accepted 2014; February 24 attributed. properly Received is work original the Attribution that Commons provided Creative medium the any of distribution in terms use, reproduction the unrestricted and under permits distributed which article (http://creativecommons.org/licenses/by/3.0), Access License Open an is This ` 1 (Ca Calcium INTRODUCTION potential channel receptor Transient Staurosporine, C, Phospholipase Inositol-1,4,5-trisphosphate, death, Cell WORDS: KEY Ca free cytosolic that in show we changes Here, defined ([Ca staurosporine. causes to exposed staurosporine when death cell organism model The ABSTRACT Teeatoscnrbtdeulyt hswork this to of equally University contributed Street, authors UK. Grafton *These 9NT, Building, M13 CTF Manchester Repair, Manchester, and Inflammation of Institute ihteetaellrCa extracellular the with ciiyadcnrbt oacsaeo inln vnsthat events signaling of electrostatic cascade a their Ca and a to modulate defines ultimately interactions contribute conformational These and a proteins. activity these to promotes in binds EF-hand-like alterations and ion or EF-hand The at commonly domains, humans. proteins, of to range diverse prokaryotes from organisms, [Ca Ca lcaeo h epnet tuoprn fe niiino a of inhibition after properties staurosporine (IP Partial to with family. inositol-1,4,5-trisphosphate response (TRP) channels putative the potential of membrane receptor blockage transient plasma the of resembling opening the ecie ih n o-fiiyCa low-affinity and high- described netgtdtemlclrbsso hsCa this of basis molecular the investigated Ca D xrclua Ca extracellular ikD Read D. Nick uhr o orsodne([email protected] [email protected]) ([email protected]; correspondence for Authors BCIsiuod ilgaMlclreCllr-Uiesdd oPro u do Rua Porto, do Universidade - Celular e Molecular Biologia de IBMC-Instituto fig-1 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,31–89doi:10.1242/jcs.152058 3817–3829 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 2+ 2+ 2+ 2+ ´ ia eAe aaa,Uiesdd oPro u eJreViterbo Jorge de Rua Porto, do Universidade Salazar, Abel de dicas ] nlxfo h xenlmdu n nenlCa internal and medium external the from influx ees rmitra trsfloigIP following stores internal from release c ] and c esrmnscmie ihpamclgcladgenetic and pharmacological with combined measurements yaisadadsic Ca distinct a and dynamics ) D yvc-1 2+ sa seta nrclua esne nall in messenger intracellular essential an is ) 3 2+ ¸alves n rad Videira Arnaldo and uat n ag fihbtr,w hwthat show we inhibitors, of range a and mutants nr osntocrtruhtehitherto the through occur not does entry ersoacrassa Neurospora 2+ 2+ 1,2, mdae ellrrsos oastimulus a to response cellular -mediated influx. * , ` .Mge Cordeiro Miguel J. , 3 acetrFna neto Group, Infection Fungal Manchester 2+ paesses u through but systems, uptake 2 CA-nttt eCie de ICBAS-Instituto 2+ 2+ 3 eetrsget that suggests receptor ) intr.Using signature. nege programmed undergoes intr htinvolves that signature 1,2, 2+ 3 ` omto combines formation epneb using by response 2+ trs We stores. 2, ˆ ,Joa *, ncias D cch-1 oMonteiro ˜o 2+ , ees fCa of release h xrclua iiucnocrb ifrn mechanisms, different by Ca high-affinity occur The stimulus. can the on milieu depending extracellular the Ca mobilize Cells 2007). (Clapham, eosrtdta [Ca that demonstrated IP 2003). Lew, and Silverman-Gavrila 2002; Lew, htata nrclua trs nfni Ca fungi, In organelles stores. of intracellular membranes the as in act or medium) that external the from SlemnGviaadLw 02 orlae l,2001). al., IP et recognizable Torralba 2002; a Lew, However, and (Silverman-Gavrila ytm Cvne ta. 01 azk ta. 99 e tal., et Lew 1999; al., et Kanzaki some 2011; in al., protein et as channel (Cavinder behave to systems cation of reported role stretch-activated been exact also non-selective has The protein a 2001). the al., as et unclear, Muller is Mid1 2011; and al., Mid1 proteins et regulatory (Martin the Ecm7 and Cch1 channel the comprises 08.TeHC em ob atclryatv nminimal in active particularly Ca be low-affinity the to whereas seems medium, HACS The 2008). HC)i h etdsrbdfna Ca fungal described best the is (HACS) nti td,teiaigo [Ca of imaging 2012b). the al., study, et (Kim this challenged In been recently has hyphae growing hc sivle nmtn Cvne n ri,21;Muller 2012; channel, Trail, Ca Fig1 glucose-induced and the A (Cavinder 2003). is mating al., the system in et far, LACS involved So the is 2001). of which al., member et known (Muller only medium nutrient-rich in active cu ngoighpa is ihtersl httetip-focused the that result the with tips, hyphal growing in occur ob novdi h eeaino h i-ih[Ca tip-high the of generation the in believed hyphal is involved the latter be the channel, near Only to accumulate 2002). conductance Lew, to and proposed (Silverman-Gavrila small tip been Golgi-derived have a and that (ER)- and vesicles reticulum endoplasmic 2002), Silverman- with 1989; Lew, associated al., and et (Cornelius Gavrila membrane vacuolar the with h xsec facniuu i-ih[Ca tip-high 2004). continuous al., a et Zelter of 2004; al., existence et The (Borkovich fungi in identified pnn fcanl ntepam ebae(o Ca (for membrane plasma the in channels of opening i-ih[Ca tip-high in growth Hyphal growth. Ca fioio-,,-rshsht (IP inositol-1,4,5-trisphosphate of crassa are components molecular its but 2011) unknown. al., et (Groppi proposed e,20;SlemnGviaadLw 03.Evidence 2003). Lew, and IP and Silverman-Gavrila that Silverman-Gavrila 2001; indicates 2002; Lew, Lew, and (Silverman-Gavrila Ca oaie hshlps htsne eso u ohyphal to phosphatidylinositol-4,5-bisphosphate due tension converts (PIP senses and that expansion C phospholipase localized ntpgoigognsssc stefungus the as such organisms tip-growing In 2+ 2+ 2 )toIP nlx(e,19) u sitral eie ymeans by derived internally is but 1999), (Lew, influx 1 yooi reCa free cytosolic , eotrepesdin expressed reporter let Mun Alberto , 3 2+ 2+ n icllcrl(A)(ivra-arl and (Silverman-Gavrila (DAG) diacylglycerol and ] c hog ag odcac hne,associated channel, conductance large a through rdetta sntmitie yextracellular by maintained not is that gradient 3 sgnrtdb tec-ciae tip- stretch-activated a by generated is ˜oz 2+ ] 2+ c .crassa N. 3 al Correia-de-Sa Paulo , pkswt nirglrfrequency irregular an with spikes ([Ca 3 2+ 2+ Fusarium ] eetrhsntytbeen yet not has receptor ] c 2+ c a enrpre orl na on rely to reported been has sn eeial encoded genetically a using , 3 sipratfrpolarized for important is ) -ciae Ca )-activated 2+ nlxsse a recently was system influx 2+ 2+ ytm(AS smore is (LACS) system otectslb the by cytosol the to 2+ dynamics and paesse.It system. uptake 2+ 2+ 2+ ] 3 paesystem uptake c Magnaporthe paefrom uptake rmtsthe promotes 2+ 2+ ´ Neurospora rdetin gradient 2 ] c , 2+ channels gradient influx 3817 ,

Journal of Cell Science oo-piie,bouiecn Ca bioluminescent 2013; Gonc codon-optimized, 2014; al., al., et et Fernandes Goncalves 2014). 2010; 2011; Videira, al., al., et et Fernandes (Castro staurosporine to yooi erimn fognlesoe Ca organelle-stored of recruitment cytosolic oaie ntevcoa ebaeadi novdi h ees of release the in involved Ca is and membrane vacuolar the in localized In In 2004). 2013). (Rohacs, (TRPA) crassa ankyrin and (TRPML) mucolipin classical (TRPP), subfamilies: polycystin (TRPM), several melastatin (TRPV), comprises vanilloid (TRPC), family channel TRP The rmtswl-eie hne n[Ca staurosporine that in demonstrate changes We well-defined staurosporine. promotes by death cell ta. 00 esne l,20;Topn ta. 03 nodrto order in 2013) Ca al., of et role Troppens 2004; the al., analyze et Nelson 2010; al., et S ta. 09 n PIP and 2009) al., et (Su EERHARTICLE RESEARCH 3818 includes staurosporine to response the during The Ca player death. both pivotal cell a of is C induction phospholipase that and signature Ca to permeable are which channels, (TRP) potential receptor constant at extending hyphae in rates. disappears and appears gradient Ca well-defined a induces Staurosporine 1. Fig. nlxsse eebigaTPcanlta em obe IP to an seems and HACS that the channel of TRP absence the a in resembling upregulated system influx n nh 00 ece,20) cetaigtene for need We drug. the of type accentuating this that of recently 2000), action showed of (Gani Gescher, mechanisms trials the clinical 2010; understanding in evaluated Engh, being CGP41251 and currently UCN-01, are as PKC412 such or analogs profiles, staurosporine selectivity Some for improved agent. anticancer toxic with inducer an too archetypal and of an death it as cell lack remains of makes drug the the Although effects However, therapy. side models. activity, drug concomitant mammalian largely anticancer and is in potent selectivity It death 2008). exhibits cell al., behave staurosporine et trigger to (Karaman shown to inhibitor later known kinase was broad and 1977) a al., as et (Omura inhibitors C staurosporeus Streptomyces 2012). Taylor, and (Prole homologs TRPchannel additional identified pathogens fungal of comparison genomic tuoprn SS r20 or (STS) staurosporine i.S.()Cl et saraoto ebaepreblzto a xmndatrsann ihpoiimidd P) aaso h mean the show Data (PI). iodide propidium with staining after examined was permeabilization membrane of readout a as death * Cell (B) S1. Fig. ihtret i elcts h sarsoieidcdapiueo epne a acltdb utatn h ovn MOcnrlcreshown curve control DMSO solvent the [Ca subtracting 2–6). by Figs calculated following was the response’ for of performed amplitude also ‘staurosporine-induced was The (this replicates. figure six to three with otie w ao Ca major two contained P , nti ae,w aeused have we paper, this In h hshlps aha stemi ouao ftransient of modulator main the is pathway C phospholipase The h laodsarsoiewsiiilyioae from isolated initially was staurosporine alkaloid The 2+ 0.05. notectsl(amre l,20)atratvto ystretch by activation after 2001) al., et (Palmer cytosol the into h nykonTPtp hne sYC1(etre al., et (Zelter YVC-1 is channel TRP-type known only the , 2+ acaoye cerevisiae Saccharomyces paefo h xrclua iiub oe fungal novel a by milieu extracellular the from uptake 2+ .crassa N. rnins(hss‘’ad‘’ n hr ra [Ca broad third a and ‘B’) and ‘A’ (phases transients 2+ m 2 C-1 n h ieoreeiso flmnsec a oioe vr5hus h T-nue Ca STS-induced The hours. 5 over monitored was luminescence of emission timecourse the and UCN-01, M Dn ta. 00.Rcnl,an Recently, 2010). al., et (Dong inln uigteiiito ffungal of initiation the during signaling uigasre o rti kinase protein for screen a during n ahgncfniaesensitive are fungi pathogenic and .crassa N. h ooo fYC1is YVC-1 of homolog the , 2+ eotraqoi (Binder aequorin reporter 2+ ] c 2+ ihadsic Ca distinct a with 2+ el xrsigthe expressing cells . signature. 2+ 3 avsand ¸alves -mediated ] c A eurnepesn idtp el rw o or eeicbtdwt 20 with incubated were hours 6 for grown cells wild-type Aequorin-expressing (A) nsilico in esrmnsi is16aeas rsne iherr asi upeetr material supplementary in bars errors with presented also are 1–6 Figs in measurements 2+ N. 2+ . 2+ ] c nrae(C)adrpeet naeaeo 0idpneteprmns each experiments, independent 30 of average an represents and (‘C’) increase yrxsarsoiecretyi lncltil o cancer immediate an for provoked also trials [Ca 2010), of Engh, peak clinical and (Gani in treatment currently hydroxystaurosporine onie ihasgiiatices ntepercentage the available in Total increase Ca 1B). (Fig. for significant cells aequorin propidium-iodide-positive a of with coincides hogotteetr egho h tuoprn-nue Ca staurosporine-induced the of length entire the throughout h intr nlddtomjrCa major two included signature The tuoprn,crepn oseii Ca specific with treatment to of hours 2 correspond first that the within view of staurosporine, occur the that B, supports images presented and This A are S2A). peaks representative Fig. time-points material 1B; different supplementary (Fig. at in staining drug iodide the propidium membrane with of plasma hours incubation of 3 after reporter increased significantly a only were permeabilization, iodide, propidium with staining 1A). (Fig. staurosporine by caused that from h oo-piie ilmnset[Ca wild-type bioluminescent culture, codon-optimized of the hours 6 Ca After well-defined a induces Staurosporine RESULTS In Ca staurosporine-induced The S2B). Fig. material (supplementary signature Ca staurosporine-induced the The disruption. of membrane (C) plasma to phase due cytosol third the into entry Nlo ta. 04 eeicbtdwt 20 with incubated were 2004) al., et (Nelson nue eldfndsgaueo [Ca Staurosporine of time. signature over well-defined monitored a was induced luminescence the and n auh oa,21;Trab ta. 01.W ie to aimed We 2001). Ca al., of et source(s) on Torralba Latha the depends Lavanya identify 2011; distribution Naveena Mohan, 2011; its Maruthi al., and et and wall, (Bowman phase cell growth the the in and organelles raetapiue curdatr3–0mntsadlasted and minutes 35–40 after staurosporine occurred for of for amplitude, addition greatest upon lasted immediately and occurred A Peak ptemo eldahadd o nov o-pcfcCa non-specific involve not do and death cell of upstream Ca cytosolic in increase broad third a and ‘B’, and ‘A’ nue [Ca induced otiuino xrclua Ca extracellular of contribution xrclua n nenlCa internal and extracellular h eeso tuoprn-nue eldah smaue by measured as death, cell staurosporine-induced of levels The .crassa N. , 0mnts C-1 aua trosmro 7- of stereoisomer natural a UCN-01, minutes. 80 ora fCl cec 21)17 8732 doi:10.1242/jcs.152058 3817–3829 127, (2014) Science Cell of Journal 2+ 2+ ] reCa free , c ] lhuhteoealCa overall the although , c 2+ intr,adw tre yloiga the at looking by started we and signature, , eeto a o fetdb h drug the by affected not was detection 0mnts n ekB aigthe having B, peak and minutes, 20 2+ 2+ ssoe ndfeetintracellular different in stored is htgv iet h staurosporine- the to rise give that 2+ 2+ stores 2+ intr sdrvdfrom derived is signature 2+ r-nuainwt the with Pre-incubation . .crassa N. ek htw dniidas identified we that peaks 2+ 2+ 2+ intr a different was signature ] 2+ c 2+ ] c hne Fg 1A). (Fig. changes signature m eotraequorin reporter inln events signaling staurosporine, M 2+ el expressing cells signature 2+ 6 s.e.m.; signature 2+ m M nthis in (‘C’). 2+ 2+

Journal of Cell Science nue Ca induced opeeaoiino h [Ca the of abolition complete 1,4 r10mnts blse usqeteeain in elevations subsequent abolished minutes) 180 staurosporine or [Ca with incubation 40 the after (15, time-points later at added Ca ebaeiprebeCa membrane-impermeable ARTICLE RESEARCH i.2 h tuoprn-nue Ca stores. staurosporine-induced The 2. Fig. o cu uigpae n fteCa the of B and A phases during occur not tuoprn-nue Ca staurosporine-induced oto.(iv MBPAwsapida h niae iepit arw) h iue ersn h tuoprn-nue mltd frespons of amplitude staurosporine-induced the represent figures The (arrows). time-points indicated the 10 at of applied influence was BAPTA mM 4 (ii–v) control. mean xrsigwl-yecls EF uniiain(nabtayuis .. fte[Ca the of A.U.) units, arbitrary (in Quantification (E,F) cells. wild-type expressing 2+ 2+ 6 ] nlxocr otnosytruhu h staurosporine- the throughout continuously occurs influx c ... * s.e.m.; A h nlec fBPAo h Ca the on BAPTA of influence The (A) fteCa the of 2+ m P hpiagn() 0.6 (B), thapsigargin M intr.Bcuennseii Ca non-specific Because signature. , 0.05. 2+ intr Fg Ai–) hsidctsthat indicates This 2Aiii–v). (Fig. signature 2+ intr Fg A,iEF.BAPTA 2Ai,ii,E,F). (Fig. signature 2+ 2+ ceao AT eutdin resulted BAPTA -chelator ] c 2+ m nrae soitdwt the with associated increases aioyi 1()ad50 and (C) A1 bafilomycin M intr eut rmCa from results signature 2+ intr fe eurnepesn idtp el eetetdwt 20 with treated were cells wild-type aequorin-expressing after signature 2+ 2+ intr (see signature nlxdoes influx 2+ m u6 D ntesarsoieidcdCa staurosporine-induced the on (D) Ru360 M nlxit h yoo rmteetaellrmdu n rmitra Ca internal from and medium extracellular the from cytosol the into influx 2+ intr a vlae ytetn el ihteER-selective the with cells treating by Ca evaluated was signature rvosscin,orrslspitt h rsneo Ca of presence the to point results our section), previous elto fE Ca ER of depletion emal hnesi h lsammrn htaeivle in involved are [Ca that these membrane generating plasma the in channels permeable hne h intr fteCa the of thapsigargin signature with the Pre-treatment 1990). changed al., et (Thastrup organelle ] c rninsAadB epciey hc r hw nAD aaso the show Data A–D. in shown are which respectively, B, and A transients h otiuino h Rt h tuoprn-nue Ca staurosporine-induced the to ER the of contribution The 2+ APs niio hpiagn hpiagncue the causes Thapsigargin thapsigargin. inhibitor -ATPase ora fCl cec 21)17 8732 doi:10.1242/jcs.152058 3817–3829 127, (2014) Science Cell of Journal 2+ 2+ ] c n rvnsCa prevents and transients. m 2+ 2+ tuoprn SS.()N BAPTA No (i) (STS). staurosporine M intr epnei aequorin- in response signature epnet tuoprn and staurosporine to response 2+ eusrto ythis by sequestration .(–)The (B–D) e. 3819 2+ 2+ 2+ -

Journal of Cell Science fCa of Crer ta. 03.Tepeec fbflmcnA resulted A1 bafilomycin [Ca of presence a The in 2013). al., et (Cordeiro hog h iohnra Ca mitochondrial the through Rcnrbtn opa .Hwvr niiino h RCa ER the of inhibition However, A. peak to contributing ER 99 ivra-arl n e,20) ie h importance the Given phospholipase-C–IP 2002). of Lew, al., and et (Cornelius Silverman-Gavrila vacuole 1989; and 2002) Lew, and (Silverman-Gavrila of addition Ca the with before consistent thapsigargin is by result depleted this staurosporine, likely is ER the eivle ntefna epnet staurosporine. 2002; to response Lew, fungal the could and it in that involved Silverman-Gavrila hypothesized be we 2003), 2001; Lew, and Lew, Silverman-Gavrila and Gavrila igepa n asdteaoiino h xeddices in increase extended the of abolition the [Ca caused and peak single saCa a as heterogeneous in and complex system the vacuolar whether assess to A1 bafilomycin Ca niio -32 Sihe l,19) h [Ca The 1990). al., et (Smith phospholipase-C-selective the U-73122 with inhibitor pre-treated cells in treatment enrpre opooeterlaeo Ca of release the promote to reported been amla el,Ca cells, mammalian u6 Zzeae l,19)bfr h diino staurosporine inhibitor of MCU-specific addition the [Ca the before the 1999) converted with al., et Pre-incubation (Zazueta Ru360 2012). al., et the to homologous putatively period. uigti eodpaeo [Ca of phase second this during Ca Because 2B,E,F). (Fig. A peak of absence the in resulted ARTICLE RESEARCH 3820 In 2009). activates (Berridge, DAG (PKC) whereas C kinase stores, protein intracellular from mobilization PIP converts C Phospholipase IP and phospholipase-C- activates Staurosporine C. phase during not ER, the to contrast in but, response Fg AFG,cnitn ihasgiiatrqieetfor suppressed requirement greatly (LiCl significant Lithium was response. a this timecourse during with C phospholipase 5-hour consistent entire 3A,F,G), (Fig. the over gradient proton the of disruption Ca in for results required which activity, Taersle na nraei h mltd fpa B peak of ER that amplitude suggesting the control, untreated in the Ca increase of that an with compared in resulted ATPase ossetwt h aulrsse lyn oei sequestering in role a playing Ca system vacuolar are the results with These consistent 2C,E,F). (Fig. treatment staurosporine following aioyi 1hspeiul enue oihbtCa inhibit to used been previously has A1 Bafilomycin eodmsegrad ybnigt t eetr eut nCa in results receptor, its to binding by and, messenger second mitochondria. and organelles n yai epnet tuoprn,weenclsmobilize complex cells a wherein to staurosporine, point results to Ca our response together, dynamic Taken and B. and A phases rlne [Ca prolonged yvcoe n mle cdcvsce ybokn H blocking by vesicles acidic smaller and vacuoles by el htwr r-rae ihtasgri,idctn that indicating thapsigargin, with pre-treated Ca were continued that cells iohnrapa oei h eusrto fCa of sequestration the in role a play mitochondria erimn fCa of recruitment emaue [Ca measured We In iohnraaeas novdi Ca in involved also are Mitochondria 2+ 2+ 2+ 2+ 2+ APs ciiypasasgiiatrl nsqetrn Ca sequestering in role significant a plays activity -ATPase .crassa N. 2+ ] rmadt h xrclua eim R vacuoles/acidic ER, medium, extracellular the to and from ntectsl(onlu n aahm,18) eused We 1987). Nakashima, and (Cornelius cytosol the in uigpae n ftesarsoieidcdCa staurosporine-induced the of B and A phases during c 2+ ne tescniin oaodtetxcacmlto of accumulation toxic the avoid to conditions stress under uigpaeC(i.2–) hs eut ugs that suggest results These 2D–F). (Fig. C phase during 2+ tr uigtesarsoieidcdCa staurosporine-induced the during store ] c nraei ek n u o uigpa C peak during not but B and A peaks in increase 2+ 2+ h aulsaersosbefrtesequestration the for responsible are vacuoles the , APs ciiymgtas cu uigthis during occur also might activity -ATPase ] 2+ c 2+ lvto uigpaeCwsas nrae in increased also was C phase during elevation 2+ ] .crassa N. paeb h aulrCa vacuolar the by uptake c 2+ ek soitdwt hssAadBit a into B and A phases with associated peaks 2+ 3 ] c inln o yhlgot (Silverman- growth hyphal for signaling paeb iohnraocr mainly occurs mitochondria by uptake yaisi epnet staurosporine to response in dynamics .crassa N. Bwa ta. 01 ly role a plays 2011) al., et (Bowman 2 2+ 2+ noIP into ] c nrae h mltd fthe of amplitude The increase. npre MU,wihis which (MCU), uniporter 3 rti C016(Bick NCU08166 protein n A.IP DAG. and 2+ 2+ .crassa N. eusrto.In sequestration. 2+ 2+ ees rmthe from release /H rmteER the from 2+ 3 + 2+ ] -mediated 2 c 3 a been has ) exchanger 2+ signature. + 2+ ,IP csa a as acts -ATPase response uptake during 3 2+ has 2+ 2+ 2+ 2+ in - hsihbto eue hshlps ciiyoigt the to owing PIP of activity levels C 1991). adequate of phospholipase (Hanson, absence reduces phosphoinositides inhibition of This synthesis the preventing [Ca and type caused staurosporine with treatment r-rae el.Tu,i em httestaurosporine-induced the that seems YM-58483- it in Ca decreased Thus, also cells. was significantly C pre-treated was Phase peak 3E–G). same the (Fig. of reduced part second the whereas phase, Pte,18) eeautdwehrSC a rgeigthe triggering was SOCE whether Ca evaluated we 1986), (Putney, tuoprn infcnl eue h [Ca the reduced significantly staurosporine sdin used rgee ytedpeino IP of depletion the by triggered hs eut niaeta hshlps srqie for required is C phospholipase 3A). pre- (Fig. that cells U-73122 indicate inhibitor wild-type C results in phospholipase These response the with the treated paralleling 4C–E), (Fig. [Ca the of al., that observed et beginning we (Zitt and the staurosporine, YM-58483 with incubation inhibitor before CRAC-selective 2004) the with cells eas etdteefc f2AB hc a enpreviously been has which 2-APB, IP of an block effect to shown the tested also We rvnigIP preventing LiCl 1989). Irvine, and (Berridge DAG orsodn oteIP the to corresponding oto h [Ca the of most crassa N. hssAadBadams opeeyaoihdte[Ca the abolished completely almost and B and A phases ih xli t ifrn fet ntestaurosporine-induced the of on inhibition effects the different Ca This from 2005). its al., result explain et effects (Clapham might claimed its previously as that channels, likely TRP is it release, [Ca on effects These 3B,F,G). (Fig. C phase of increase infcn nraei h ecnaeo ppoi el nthe in a cells to apoptotic led of the in percentage staurosporine marker not but the with YO-PRO1 wild-type in Treatment the increase 1995). with significant al., apoptosis of et of staurosporine (Idziorek levels to the confirmed We resistance measuring 4A). (Fig. increased strain wild-type the whereas the than Interestingly, resistant more 2004). resistant, al., more et slightly deletion Zelter 1997; the of D al., genes of C et staurosporine phospholipase predicted (Jung to four the sensitivity for strains the examined We growth and hyphal death polarized cell staurosporine-induced regulates PLC-2 Ca of recruitment the that phospholipase and results of activity these C the together, promotes staurosporine Taken that C. indicate xestospongin inhibitor receptor a o enrpre nflmnosfnibefore. fungi filamentous in reported been not has tr-prtdCa store-operated eetrslcieihbtrxsopni Gfie l,1997) al., LiCl that et suggests This (Gafni 3C,F,G). C (Fig. xestospongin inhibitor receptor-selective eurstegnrto fIP of generation the requires togrihbtr fetta etsognC(i.3D,F,G). (Fig. IP C blocking be xestospongin might than 2-APB Although effect inhibitory stronger nenlsoe htse opriiaeeryo u otnet be to continue [Ca but entire on the early throughout participate IP involved to seem the that stores of internal effects the from resulting those to similar very plc-1 eas Ca Because 2+ 2+ 2+ 2+ rlaeatvtdCa -release-activated epnein response ] intr hncmae ihtemr eetv IP selective more the with compared when signature c ora fCl cec 21)17 8732 doi:10.1242/jcs.152058 3817–3829 127, (2014) Science Cell of Journal epnet tuoprn a eryaoihdaltogether abolished nearly was staurosporine to response ( D .crassa N. , C025 and NCU06245) SlemnGviaadLw 02.2ABblocked 2-APB 2002). Lew, and (Silverman-Gavrila .%in 9.9% 3 omto n h osqetrlaeo Ca of release consequent the and formation 2+ .crassa N. 2+ 2+ nlxfo h xrclua pc a be can space extracellular the from influx ] 3 c D oihbtioio oohshts,thus monophosphatase, inositol inhibit to nr SC,o aaiaieCa capacitative or (SOCE, entry rcpo-iecanladhpa rwhin growth hyphal and channel -receptor-like plc-2 epnet tuoprn,ehbtn a exhibiting staurosporine, to response D D 3 plc-2 atvtditaellrsoedepletion store intracellular -activated plc-2 2+ 2+ nldstepoeso OE which SOCE, of process the includes el.I h bec fPC2 the PLC-2, of absence the In cells. D 3 ] hne CA) epre-treated We (CRAC). channel . c plc-3 eeinsri Fg B.A2-hour A 4B). (Fig. strain deletion ( ekAwspeet probably present, was A peak D 3 2 sniieCa -sensitive C026 a substantially was NCU01266) 2+ ob yrlzdit IP into hydrolyzed be to 2+ ( ] , D c 2 n etsognCatby act C xestospongin and epnet staurosporine. to response C065 tan were strains NCU09655) 34 ppoi nwild- in apoptosis 23.4% rmitaellrstores intracellular from 3 rcpo-ciae Ca -receptor-activated 2 ntepeec of presence the in 2+ 2+ ] D c plc-2 trsthrough stores ek during peaks .crassa N. 2+ 2+ el by cells 2+ ] c entry) 3 from were 2+ and 2+ 3 ] 3 c -

Journal of Cell Science EERHARTICLE RESEARCH tuoprn-nue eldahadspottecnlso that conclusion the support Ca and death cell staurosporine-induced or fclue lotalwl-yeclshdgerminated had cells By 4I). wild-type (Fig. After observed all was 4H). elongation (Fig. almost hyphal normal medium and culture, (95.8%) liquid of in growth hours 8 poor of very Deletion in 4F,G). (Fig. resulted delayed severely was medium ailgot of growth Radial 2+ inln sipratdrn h process. the during important is signaling D plc-2 el nsldVglsminimal Vogel’s solid in cells plc-2 also 4huso rwh hra h idtp tanwsfully was strain fused and wild-type after branched the the long evident and hyphae, whereas germination also 99.9% growth; with was developed of difference hours The 24 germination). (39.7% i.3 tuoprn ciae hshlps--adIP and phospholipase-C- activates Staurosporine 3. Fig. emnto,psesdsolnclsadafwelongated few of presence a The evident. and fusion cell little cells with hyphae swollen branched possessed germination, contrast, ramn ihdfeetihbtr nteCa the on inhibitors different with treatment epciey hc r hw nAE aaso the show Data A–E. in mean shown are which respectively, rirr nt,AU)o h [Ca the of A.U.) units, arbitrary 10 and (D) 2-APB 32 A,1 MLiCl mM 10 10 with (A), pre-treated 73122 were 20 Cells with (STS). treated staurosporine were cells wild-type aequorin-expressing eitdrcuteto Ca of recruitment mediated 6 ... * s.e.m.; ora fCl cec 21)17 8732 doi:10.1242/jcs.152058 3817–3829 127, (2014) Science Cell of Journal D plc-2 P D , plc-2 m 0.05. M543() FG uniiain(in Quantification (F,G) (E). YM-58483 M el newn ral eue germination reduced greatly underwent cells 2 B,10 (B), ncotmtn nyudret58.5% underwent only mutant knockout 2+ 2+ . m AE h nlec fpre- of influence The (A–E) ] etsognC() 50 (C), C xestospongin M c rninsAadB, and A transients 2+ intr after signature m MU- m M m M 3 - 3821

Journal of Cell Science eim h ecnaeo emntdclsi niae nteuprrgtcre (mean corner right upper the in of indicated 24-hour-cultures is in cells conidia germinated ungerminated of percentage The medium. eetdb tiigwt OPO,adtepretg fpstv el a esrdb lwctmty * and cytometry. wild-type flow in by apoptosis measured of was levels cells The positive (B) of colonies. percentage compact the produce and to YO-PRO1, sorbose with 2% staining contained by medium detected four GFS the The for (STS). strains staurosporine deletion of profiles sensitivity EERHARTICLE RESEARCH 3822 Ca the for required is PLC-2 4. Fig. hwtemean the show strain); mean and wild-type of Growth (F,G) DE uniiain(nabtayuis .. fte[Ca the of A.U.) units, arbitrary (in Quantification (D,E) 6 # ...() H rwho idtp and wild-type of Growth (H) (G). s.e.m. P , .5(o T-rae idtp versus wild-type STS-treated (for 0.05 6 ...()Rpeettv irgah fwl-yeand wild-type of micrographs Representative (I) s.e.m. D plc-2 2+ tan nsldmdu.Tepnl hwgot t4 otiouain()adhpa xeso ae vrtm sthe as time over rates extension hyphal and (F) post-inoculation h 48 at growth show panels The medium. solid in strains intr n o eldahidcdb tuoprn,adi novdin involved is and staurosporine, by induced death cell for and signature D plc-2 D .crassa N. plc-2 D arw) * (arrows). plc-2 2+ tan nlqi eimoe 4huswsotie ymauigasrac As t40n.Data nm. 450 at (Abs) absorbance measuring by obtained was hours 24 over medium liquid in strains ] c el) C [Ca (C) cells). rninsAadB epciey hc r hw nC aaso h mean the show Data C. in shown are which respectively, B, and A transients hshlps ee eeeautdb ptigcndaot F eimcnann 2.5 containing medium GFS onto conidia spotting by evaluated were genes C phospholipase P , .5 cl as 40 bars: Scale 0.05. 2+ D ] c plc-2 a esrdi aequorin-expressing in measured was tan t8husad2 or fe nclto nlqi oe’ minimal Vogel’s liquid in inoculation after hours 24 and hours 8 at strains m m. 6 ora fCl cec 21)17 8732 doi:10.1242/jcs.152058 3817–3829 127, (2014) Science Cell of Journal ....Nt h rsneo wle oii n several and conidia swollen of presence the Note s.e.m.). P , .5(o 50 (for 0.05 D plc-2 .crassa N. el fe ramn ih20 with treatment after cells m T essDS o each for DMSO versus STS M yhldevelopment. hyphal 6 ... * s.e.m.; D plc-2 el were cells P , m 0.05. STS. M A The (A) m M

Journal of Cell Science -P Fg ACD,wihi nw oihbtteIP the inhibit to known is which 5A,C,D), (Fig. 2-APB oesmlrt hto h idtp el,dsieasih [Ca slight a despite cells, wild-type the of that to similar more eakby opeeaoiino h tuoprn [Ca staurosporine the of CCH-1. abolition of absence complete the was in system Remarkably, especially influx activity or staurosporine unknown system by This high-affinity channel stimulated system. CCH-1–MID-1 low-affinity the FIG-1 involves of the that death from distinct cell staurosporine-induced tanepesn eurna ufcetlvl o [Ca for levels sufficient at previously As aequorin a generate 2005). not expressing al., could we et 2013), strain al., (Clapham et (Troppens channels reported TRP also but [Ca h [Ca The netgtdtemcaimo Ca of mechanism the investigated ypetetetwt h Ca the with pre-treatment by C- n I- hne Mle ta. 01 hra the 2003). whereas al., et the 2001) (Muller al., FIG-1 comprises is et LACS HACS (Muller in counterpart level, channel channel MID-1 molecular and the CCH-1 At respectively). eas OEsest eol atal novdi the in involved partially only be Ca to seems staurosporine-induced SOCE Because involving channel TRP Ca putative a activates 2007). Staurosporine al., et C (Gavric phospholipase previously other the suggested of as activity genes, redundant the by in explained hyphae germinated some ARTICLE RESEARCH euaoyprnro C- Hn ta. 03 ok tal., a et be Locke 2013; to al., et considered (Hong CCH-1 is of partner (NCU06703) regulatory MID-1 measurement. when observed was signature C phase in decrease a and Ca that suggest B strongly data phase These 5B–D). in (Fig. increase amplitude paesseshv enetbihdi ug h ih and high- the – fungi Ca in established been low-affinity have systems uptake n a bevdi aequorin-expressing in observed was B and A anydet xrclua Ca extracellular to due mainly ( D rsi nraei h [Ca the in increase drastic A C072 xoe osarsoie(i.5,,) hswas This 5A,C,D). (Fig. staurosporine to exposed NCU02762) 2+ 2+ nlxfo h xenlmedium external the from influx ] c ek soitdwt hssAadB ofr w Ca two far, So B. and A phases with associated peaks 2+ ] c epnei the in response 2+ paesses(ASadLACS, and (HACS systems uptake 2+ D 2+ 2+ D D intr Fg E,w further we 3E), (Fig. signature plc-2 2+ ceao AT Fg 5A,C,D). (Fig. BAPTA -chelator fig-1 cch-1 pae eas twsprevented was it because uptake, ] c 2+ ek soitdwt phases with associated peaks ncotclue ih be might cultures knockout uat( mutant el eepetetdwith pre-treated were cells nlxrsosbefrthe for responsible influx D C029 was NCU02219) 2+ paeduring uptake D cch-1 3 receptor D mid-1 cells 2+ 2+ 2+ 2+ ] ] ] c c c - 03.Bcueo h motneo hshlps uigthe during be might C channel TRP phospholipase a whether of asked we importance staurosporine, (Rohacs, to the pathway response C of phospholipase Because the by 2013). regulated be to known are novdi h Ca the in involved gemn ihoreiec htpeicbto ih2AB nIP an 2-APB, with pre-incubation in that is evidence This our 6B,E,F). with (Fig. agreement suppressed strongly also was staurosporine to o-eetv niio fteMUadapnihbtro TRP of [Ca the pan-inhibitor that a found and and 2005), MCU al., et the (Clapham of channels inhibitor non-selective a rti Cvne ta. 01 azk ta. 99 e tal., et Lew 1999; channel al., et of cation knockout Kanzaki the 2011; stretch-activated MID-1 Nonetheless, 2008). al., of non-selective et reports (Cavinder are a protein there although as 2001), behaving al., et Muller 2000; efre [Ca performed Fg 3D). (Fig. adpsil I-)i orltdwt nrae eldeath. cell increased with correlated is MID-1) possibly (and n nrsos osarsoie(i.5) easse the assessed we 5A), (Fig. staurosporine to of response susceptibility in B and A 05,ptnl eue h [Ca the reduced al., et (Clapham potently channels TRP of 2005), blocker a also but inhibitor receptor h tuoprn-nue [Ca abolished nearly staurosporine-induced non-selectively, 2013), al., albeit the et that, (Guinamard channels chemical TRP some a blocks acid, flufenamic of presence yeiletninrt splmnaymtra i.S3B), Fig. and together. material acting S3A) proteins (supplementary both Fig. with aerial rate consistent reduced material extension as (supplementary such mycelial defects conidiation showed strains hyphae, both as deletion, tuoprn splmnaymtra i.SA.Tu,the Thus, S4A). Ca Fig. a of material upregulation (supplementary to Deletion staurosporine culture S3D). staining Fig. liquid YO-PRO1 material of and in (supplementary S3C) apoptosis growth estimate of Fig. both effect material inhibitory measuring the (supplementary of after analysis the 5E). staurosporine by (Fig. drug corroborated the was to hypersensitive This are strains both that observed naias Ca animals, In ie h accentuated the Given fig-1 ora fCl cec 21)17 8732 doi:10.1242/jcs.152058 3817–3829 127, (2014) Science Cell of Journal eutdi lgtices nssetblt to susceptibility in increase slight a in resulted 2+ ] 2+ c D esrmnsi h rsneo uhnu Red, Ruthenium of presence the in measurements nlxcnb eitdb R hnes which channels, TRP by mediated be can influx cch-1 2+ 2+ nlxfo h xrclua eim The medium. extracellular the from influx nlxsse nteasneo CCH-1 of absence the in system influx and D cch-1 2+ D ] c mid-1 1 AT n 50 mM and 4 BAPTA with pre-treatment of influence D and wild-type aequorin-expressing in compared was (STS) staurosporine r hw npnl ,.Dt show Data mean A,B. the panels in shown are which respectively, B, and A transients and Ca high- low-affinity the from distinct uptake system an through occurs influx Ca Staurosporine-induced 5. Fig. Ca rirr nt,AU)o h [Ca the of A.U.) (in units, Quantification arbitrary (C,D) A. in shown otiig2.5 medium containing GFS onto conidia spotting D of sensitivity The (E) versus cells pre-treated 2-APB and BAPTA D intr Fg AEF.W also We 6A,E,F). (Fig. signature cch-1 mid-1 cch-1 [Ca esswl-yecells); wild-type versus 2+ 2+ ] mid-1 c intr nrsos o20 to response in signature 2+ T-nue Ca STS-induced el osarsoieand staurosporine to cells A or (A) tan a vlae by evaluated was strains ] epnet staurosporine to response c 6 intr uigphases during signature ... * s.e.m.; hncpe the phenocopied D cch-1 D m 2+ fig-1 m STS. M -P nthe on 2-APB M systems. P ihSSalone). STS with B el.The cells. (B) , D cch-1 .5(for 0.05 2+ 2+ # P ] intr is signature c , AB The (A,B) response and .5(for 0.05 2+ D D cch-1 cch-1 3823 ] c cch- m M 2+ 3 -

Journal of Cell Science RC Mle ta. 01 n oovossqec homologs sequence and obvious [Ca TRPC4 no in mammalian and present by against 2011) are explained al., selective be et very might (Miller is this TRPC5 it and as that inhibitors, drastic fact TRP as the not other was the ML204 of of that effect The 6C,E,F). (Fig. reduced RC n RC Mle ta. 01.Wt hsihbtr the inhibitor, this With 2011). of al., antagonist et [Ca specific (Miller a TRPC5 ML204, and of TRPC4 presence the in staurosporine EERHARTICLE RESEARCH 3824 Ca extracellular for responsible channel TRP-like a activates Staurosporine 6. Fig. AT n -32,aogwt h eiin Ca ML204, deficient the 2-APB, with Red, along Ruthenium U-73122, and acid, BAPTA flufenamic of presence ciae ysarsoieadta h osqetdpeinof depletion consequent the that and PIP staurosporine by activated Zage l,21) eaaye h [Ca the analyzed we 2013), al., et (Zhang mean tuoprn SS naqoi-xrsigwl-yeclsatrpetetetwt ifrn Ca different with pre-treatment after cells wild-type aequorin-expressing in (STS) staurosporine expressing lfnmcai A lfai) 100 acid), fluf (A, acid flufenamic eas u aapoie vdneta hshlps is C phospholipase that evidence provides data our Because 2 2+ 2+ 6 sa ciainfco o pcfcTPcanl TRPC4 channel, TRP specific a for factor activation an is ] ] c c ... * s.e.m.; ek soitdwt hssBadCwr significantly were C and B phases with associated peaks epnet tuoprn-nue eldahi the in death cell staurosporine-induced to response D yvc-1 P , .crassa N. 0.05. el.(,)Qatfcto i rirr nt,AU)o h [Ca the of A.U.) units, arbitrary (in Quantification (E,F) cells. ae oehr h ifrne nthe in differences the together, Taken . m uhnu e B uhnrd n 50 and red) ruthen (B, Red Ruthenium M 2+ ] c 2+ epneto response epneof response m L0 C.()TeCa The (D) (C). ML204 M 2+ ] ta. 01.Tu,orcmie hraooia n genetic and Ca a pharmacological of existence combined the to our points Thus, approach 2001). al., et ta. 04.Teiiilpr ftesarsoieidcd[Ca staurosporine-induced the of part initial (Zelter The annotation) 2004). recent al., a et in NCU16725 or C. (NCU07605 phospholipase YVC-1 by regulated channel TRP-like a of opening the n ) u [Ca and but wild-type B), in and similar was signature es ooo slclzdt h aulrmmrn n this and membrane vacuolar the Ca to mobilizes localized channel is homolog yeast tuoprn,smlrt hto h idtp strain wild-type in the protein this of for role that A Ca S4B). extracellular Fig. to of material growth (supplementary similar The staurosporine, 6D–F). (Fig. cells c rninsAadB epciey hc r hw nAD aaso the show Data A–D. in shown are which respectively, B, and A transients ofr h nyTPcanlrpre in reported channel TRP only the far, So D plc-2 2+ ora fCl cec 21)17 8732 doi:10.1242/jcs.152058 3817–3829 127, (2014) Science Cell of Journal influx. 2+ uatsri,sgetta tuoprn nue the induces staurosporine that suggest strain, mutant mdltn rg.Clswr r-rae ih100 with pre-treated were Cells drugs. -modulating AC h Ca The (A–C) 2+ 2+ ] paewudntb xetd eas the because expected, be not would uptake 2+ c uigpaeCwsdcesdin decreased was C phase during 2+ intrsi epnet 20 to response in signatures rmtevcoet h yoo (Palmer cytosol the to vacuole the from 2+ intrsi epnet 20 to response in signatures D 2+ yvc-1 D prebecanlthat channel -permeable yvc-1 .crassa N. a niie by inhibited was m T naequorin- in STS M el pae A (phases cells m M a been has m D M yvc-1 2+ ] c

Journal of Cell Science ne ftersos.A osqec fisactivation, its of consequence a As the from the prompts of response. PIP that alleviation hydrolyses that al., C liberation the the PKC phospholipase Given et is by of it (Omura C 1990). and that onset PKC phospholipase propose al., of of to 2003) tempting phosphorylation inhibitor et is powerful it (Ryu al., 1977), a is PKC et staurosporine G-protein-coupled by membrane with (Werry phosphorylation include interaction 2000), mechanisms receptors Possible (Kinnunen, activation mechanism. stretch unknown C currently a phospholipase by C al., phospholipase et in (Norberg known neurons are and homologs channel cells HCN2 cancer No 2010). lung nucleotide-gated human in HCN2 cyclic hyperpolarization-activated Rhc,21) u eut upr h yohssthat hypothesis the support results PIP depletes Our C phospholipase 2013). (Rohacs, fetteatvt fTPcanl Rhc,21) is,the First, 2013). (Rohacs, channels PIP TRP of of depletion activity the affect [Ca characteristic euaindpnso h pcfciett ftecanl in channel; PIP the of of identity depletion specific the the animals, on depends regulation uigsarsoieidcdcl death. cell staurosporine-induced during fPIP of h eeaino A ih o aeasgiiatrl nour in role significant a have not might DAG of generation the 08 obr ta. 00 e n e,20) u the Ca clinically but Recent its induces 2009), as unknown. PKC412, well Seo, as analog and staurosporine, largely that relevant Seo shown remain has 2010; al., research mechanisms al., et Norberg 1998; et underlying al., et Norberg Kruman 1993; 2008; al., et Himpens 2012; nraei [Ca in increase aebe hw ob ohngtvl n oiieyregulated positively TRPV1 and and negatively TRPC5 PIP both TRPC4 like be by to channels, activates shown Other it been 2013). whereas have al., 2008), et al., (Zhang et (Lemonnier TRPC7 uha N rgetto,acmlto fratv oxygen reactive of accumulation fragmentation, DNA phenotypes, cellular as several such of manifestation the by accompanied organism model The DISCUSSION Ca and of YVC-1 from mediator distinct is the channel, is TRP-like a of properties has ARTICLE RESEARCH poal L-) hc rge Ca C phospholipase a and trigger channel which TRP-like PLC-2), novel promoted of putative is (probably this influx complex a level, molecular the the by a At stores. involving activates intracellular from response interest, Ca intracellular extracellular therapeutic 2011; dynamic cell of al., archetypal and an et inducer staurosporine, that Fernandes show death we Dementhon 2013; Here, 2009). 2008; al., al., al., et et et Fernandes Castro Gonc 2006; 2010; resistance al., multidrug al., of et of et activation (Castro compartmentalization and proteins synthesis, export glutathione ceramide cells, (ROS), species u h xsigdt r ofitn nwehrterdcinin reduction the whether on conflicting are PIP data existing the but tuoprn,PCi niie Ouae l,17) n this it and allowing 1977), channel, PIP TRPC4-like al., open. a to of et inhibition (Omura the relieve inhibited would is PKC staurosporine, htteslcieTP4atgns L0 atal eue the reduced found partially we ML204 and [Ca antagonist 2003), TRPC4 al., to selective et the shown (Venkatachalam that was PKC TRPC4 by Indeed, inhibited TRPC4. be like behaves that channel u aaidct htsarsoietigr h ciainof activation the triggers staurosporine that indicate data Our 2 2+ avsadVdia 04 lsfk ta. 08 Videira 2008; al., et Plesofsky 2014; Videira, and ¸alves ciae riatvtstecanl(oas 03.Second, 2013). (Rohacs, channel the inactivates or activates ] c 2 eeaino A n eeaino IP of generation and DAG of generation , 2 epnet tuoprn.I h rsneof presence the In staurosporine. to response elto,dpnigo h ellrenvironment cellular the on depending depletion, 2 elto loafcsTP1(hage l,2001), al., et (Chuang TRPV1 affects also depletion 2+ 2+ 2 ] 2+ c otosTPpoen,atog h oeof mode the although proteins, TRP controls swl stectslcrcuteto Ca of recruitment cytosolic the as well as ] nrsos osarsoie(eaie al., et (Dezaki staurosporine to response in c .crassa N. intr.Sm eot aesonan shown have reports Some signature. 2+ nlxfo h xrclua medium extracellular the from influx 2 nege rgamdcl death cell programmed undergoes 2 n hscue h pnn fa of opening the causes this and 2 ihtreotoe depletion – outcomes three with niisTP3 RC and TRPC6 TRPC3, inhibits 2+ .crassa N. 2+ nlxtruhthe through influx epnewt a with response 3 l fwhich of all , . 2+ Bne ta. 00 rwt h atra eaoie2,4- metabolite bacterial the to leads with 2013) al., or et Ca 2010) (Troppens (DAPG) al., diacetylphloroglucinol et (Binder eeaino IP of generation nmmeso h aiysc sTP4adTRPC5 and TRPC4 more TRPC4-like is as a it is PIP death, such there that cell likely staurosporine-induced indeed family in If involved occur 2003). the channel not al., of does et of and interaction (Venkatachalam specific direct members protein is PKC-independent in channels this TRP previously that with shown DAG shown Nonetheless, as 2003). was al., by channels et it TRP Venkatachalam blocked 1999; on al., is et directly that (Hofmann exclude acting which cannot We is 1977). PKC, DAG al., et activates (Omura alone DAG staurosporine because system, bevto htdrn ato h epneteE otiue to contributes ER [Ca the in response increase the the of part during that observation ta. 00 atne l,21;Mle ta. 01 ulre al., Ca et Muller putative 2001; a al., describe et Muller we 2011; Here, Locke al., (FIG-1) 2003). 2013; et al., Martin LACS et 2000; al., Hong and et 2012; MID-1) Trail, and and (Cavinder mechanisms (CCH-1 HACS characterized Cell- PAF. for tested Ca not was survival-associated this whereas FIG-1, of independent nohrmrhgntcpoess uha appressorium as 2009). al., et such Rho 2011; al., processes, et (Choi of conidiation morphogenetic deletion and is formation that other spikes shown these in have in generating genes studies in C phospholipase Other elongation PLC-2 hyphal unclear. for in currently role requirement their the but and instead, tips hyphal growing hsrpeet oe ehns o Ca for protein. mechanism TRP novel a a of represents This properties pharmacological the with membrane anr AGidcdCa DAPG-induced manner. hne,hsbe hw oocri aallwt Ca with parallel in occur to shown been has channel, oiiaino Ca of mobilization rdeti otnosygoighpa Kme l,2012b). al., [Ca transient et that (Kim shown hyphae have authors growing These continuously in gradient in Ca involved tip-high is the vesicles of Golgi-derived maintenance and the ER intracellular from rsne n hsrqieet facntn i-ih[Ca tip-high constant main a of the requirement, be thus the about might and questions in raised presence, observations have PLC-2 Recent however, fungi, process. that filamentous this other in suggest engaged C results phospholipase Lew, Our and (Silverman-Gavrila elongation 2002). hyphal for required be to emnto n hti ly nipratrl nhpa growth. on hyphal studies from in presented role We previously important been an 2007). has of plays Evidence al., it absence not that et and morphogenesis the germination cell (Gavric that of growth aspects observed hyphal in polarized implicated be and involving characterized, to previously was seems PLC-1 it only four, the Of 2004). ( C phospholipase htspotteie htteIP the that idea the support that h amla R hnesi ahgncfni(rl and (Prole fungi pathogenic of far homologs in putative channels so as TRP proposed channel mammalian recently were the these TRP proteins of few all only a or some The in in described involved responses. be might aforementioned it that conceivable is tesaet(oil ta. 02.Teei hseiec o the for evidence thus ER is an Ca There fungal tunicamycin, 2002). of al., with presence et treated (Bonilla agent yeast stress in Cch1–Mid1 through codnewt u eut,ioio traintigr ER- (Gue triggers yeast starvation fission in inositol death results, cell our mediated with accordance In h eoeof genome The 2+ nlxfo h xenlmdu naCCH-1-independent a in medium external the from influx .crassa N. ora fCl cec 21)17 8732 doi:10.1242/jcs.152058 3817–3829 127, (2014) Science Cell of Journal .crassa N. 2 elto stemjrrgltr ehns.In mechanism. regulatory major the is depletion ramn ihteatfna etd PAF peptide antifungal the with treatment , 3 d fe ramn ihsarsoieldt the to led staurosporine with treatment after 2+ tp)gns(uge l,19;Zle tal., et Zelter 1997; al., et (Jung genes -type) 2+ ] 2+ c . .crassa N. 2+ sYC1(etre l,20) although 2004), al., et (Zelter YVC-1 is rmitra trs nln ihthe with line in stores, internal from paessesi diint h already the to addition in systems uptake pae eitdb nunidentified an by mediated uptake, anprh oryzae Magnaporthe 2+ nlxhsas ensont be to shown been also has influx 3 mdae oiiaino Ca of mobilization -mediated plc-2 2+ nopse orputative four encompasses rdet hc sreported is which gradient, 2+ ed oaern spore aberrant to leads 2+ ´ hne nteplasma the in channel i ta. 09.The 2009). al., et rin nlxi ug,adit and fungi, in influx 2+ ] c eut ndefects in results pksocrin occur spikes .crassa N. 2+ 3825 entry 2+ 2+ ] c

Journal of Cell Science el r estlrn ohg eeso Ca of levels high to tolerant less are cells fpopoiaeC hc ed otegnrto fIP of generation the to leads which activation C, the includes phospholipase staurosporine of of action The mechanisms. edl euse h xesCa vacuoles excess The channel. the TRP-like sequester the of readily regulation the to possibly i.7 epooeta h tuoprn-nue nraei the in increase staurosporine-induced the that [Ca in propose presented We is 7. 2011). death Fig. al., cell et phospholipase-C-mediated Martin and 2010; induced al., drugs et azole Hong or 2002; tunicamycin al., and by caused et CCH-1 stress (Bonilla of ER lack to results, sensitive very our with line renders MID-1 In S3E). Fig. material soitddltrosefcso Ca of effects deleterious associated o lc h hs ftesarsoieidcdCa staurosporine-induced the of phase C the does block A1 bafilomycin because not saturate eventually to seems system 01 odioe l,21) eusrto fCa of Sequestration 2013). al., et Cordeiro 2011; pae el akn C- adMD1 neg oecell more undergo MID-1) (and CCH-1 lacking cells uptake, in knockout channel cells wild-type the respective [Ca from is the differences the of major YVC-1 terms show because that not does plausible staurosporine mutant not by is activated It 2012). Taylor, ARTICLE RESEARCH 3826 the that demonstrate reports H recent vacuolar Interestingly, signature. that staurosporine. observation with the treated with when consistent cells is wild-type This the than death n ees fCa SOCE) of and channel release TRP-like unidentified and an (through medium es v1i oaie ntevcoe(amre l,20) The 2001). al., et (Palmer vacuole the Ca in TRP localized is Yvc1 yeast cu ymaso Ca a of means by occur Ca in upregulation this of consequence a as Possibly CCH-1. of rdetb niiigtevcoa H proton vacuolar the on the A1 inhibiting bafilomycin by of effect gradient disruptive the by blocked oe lutaigtemcaim novdi staurosporine- in involved mechanisms the illustrating model A 2+ ] c 2+ scue ycniuu Ca continuous by caused is paesse em ob oeatv nteabsence the in active more be to seems system uptake + APs sacnrlmdao fcl et in death cell of mediator central a is /ATPase .cerevisiae S. 2+ ] c 2+ epnet tuoprn n,furthermore, and, staurosporine to response rmitra trsb ait of variety a by stores internal from 2+ /H and + niotsse hti indirectly is that system antiport 2+ rpoocsneoformans Cryptococcus na tep oaodthe avoid to attempt an in 2+ 2+ + APs Crer tal., et (Cordeiro -ATPase hssqetaincan sequestration This . nlxfo h external the from influx D 2+ cch-1 (supplementary 2+ hog this through and D mid-1 3 cells and 2+ 2+ eue ntepeec fR30 ugsigta h oeof role Ca the to that restricted suggesting not Ru360, of is presence mitochondria the in reduced tde,i ilb neetn odtrieterl fthe of role the determine to interesting be H will vacuolar it studies, niie ihR30sgetta iohnrapa neryrole early an Ca play mitochondria in that suggest Ru360 with inhibited Ptre n ekrtk,20) xrclua Ca Extracellular 2007). Verkhratsky, and (Petersen hssBadC hs eut a epsil xlie ythe by explained Ca possibly exhibit be ER can and mitochondria results that These hypothesis C. and B phases eas h mltdso h [Ca the Ru360, of with amplitudes treated those the to because thapsigargin-treated response opposite two the Interestingly, generated the cells 2007). between contact (Clapham, of organelles microdomains so-called the through hog nIP an through rmrgoso h Rta r ihi IP in rich are that ER the of regions from iohnraadE,adams ntnaeul ees Ca release instantaneously almost and ER, and mitochondria lutae htteetn fsarsoieidcdcl et is death cell staurosporine-induced of extent the that illustrates aetm epcal uigpaeB e h staurosporine- the see B, phase during (especially time same nue yooi [Ca cytosolic induced h Rcnla ihCa with load which by can mechanism tunneling ER a by explained the be might 2B) Fig. ogprosb rnprigCa transporting by periods long in changes initial The staurosporine. to Ca staurosporine-induced response the the in brief to even sensitive very [Ca are (von in cancer activity and elevations 2010) ATPase In al., cells. et vacuolar 2013) Zhang al., 2012a; et al., Schwarzenberg et (Kim fungal paetprdxclcnlso htteE srlaigCa releasing is ER the that conclusion paradoxical apparent novsete ietrlaeo Ca of release direct either involves tuoprn,a eue rmteeprmnsinvolving experiments to Ca the response the the from with of deduced duration treatment as whole the staurosporine, throughout continues iohnra lxso Ca of fluxes Mitochondrial 2+ ora fCl cec 21)17 8732 doi:10.1242/jcs.152058 3817–3829 127, (2014) Science Cell of Journal eusrto.Hwvr [Ca However, sequestration. + APs in -ATPase 3 atvtdcanlbtsqetrn h o tthe at ion the sequestering but channel -activated 2+ ] c 2+ ] .crassa N. FrtradKn,20) nfuture In 2000). Kane, and (Forster 2+ c ceao AT.Ormdlalso model Our BAPTA. -chelator rnin ntasgri-rae cells, thapsigargin-treated in transient icsinfrdtis lecrls Ca circles, Blue details. for Discussion nr;SS staurosporine. STS, entry; action in the staurosporine for model of proposed A 7. Fig. lttin;SC,soeoeae Ca store-operated SOCE, glutathione; reduced GSH, species; reactive oxygen ROS, vacuoles; V, reticulum; endoplasmic ER, mitochondria; M, 2+ 2+ hog otc pt between spots contact through 2+ 2+ intr hnteMUis MCU the when signature eldeath. cell 2+ 2+ ] lose ob involved be to seem also c .cerevisiae S. rninsicesdduring increased transients notectslo h ER the or cytosol the into osbtente.The them. between ions 2+ 2+ ] c eusrto u also but sequestration ek n are C and B peaks 3 atvtdchannels -activated .crassa N. 2+ el lacking cells , rstl over crosstalk 2+ . See uptake 2+ 2+ 2+ 2+ ;

Journal of Cell Science u6,LiCl Ru360, a ig,C)adbflmcnA Wk hmcl,Rcmn,VA). Richmond, Chemicals, (Wako A1 bafilomycin and CA) Diego, Biochemicals, 1-[6-((17San (Alexis (U-73122) yl)amino)hexyl]-1H-pyrrole-2,5-dione Germany); Darmstadt, o iaetu ug sn h ilmnsetCa bioluminescent the using fungi filamentous for 1 measurements medium, growth minimal For liquid medium. in minimal solid containing diameter) lttin GH Csr ta. 00 enne ta. 2013). reduced al., of et efflux Fernandes 2010; the al., by et CZT-1 facilitated (Castro is by (GSH) turn, glutathione controlled in staurosporine-induced which, also for required death, is is cell and accumulation 2014) al., (Goncalves ROS et CZT-1 (Goncalves 2014). factor transcription al., the of et control which the 2011), ATP- under al., staurosporine-exporting et is (Fernandes the ABC3 transporter of cassette activity binding the by determined ARTICLE RESEARCH eeial noe Ca encoded Genetically Ca Intracellular 1,2-bis(ortho- UK); Bristol, Bioscience, (Tocris aminophenoxy)ethane-N,N,N (2-APB) borate St 2-aminoethoxydiphenyl and (Sigma-Aldrich, (YM-58483/BTP2) following C 4-methyl-4 thiadiazole-5-carboxanilide MA); xestospongin The MO); and Woburn, S1. acid Laboratories, Louis, Table flufenamic (LC ML204, material staurosporine this DMSO, in supplementary used: used were in experiments strains deletion chemicals all listed and in are Wild-type used 1970). study was Serres, sucrose de (w/v) and 1.5% (Davis plus medium minimal crassa chemicals N. and media culture Strains, METHODS AND MATERIALS Ca manipulate experimentally to used commonly h niiosepoe norsuy eea aebeen have several study, our in these in used employed Of of detail. previously inhibitor in targets inhibitors characterized the been precise not from the have cells the results fungal in of because inhibitors interpretation especially the experiments, taken to be to regard needs caution with against some specificity Therefore, lack targets. drugs purported these their of some and cells, mammalian td fitaellrCa intracellular of study als X nmnmlmdu.Aiut f100 of Aliquots medium. minimal in TX) Dallas, el n bobnewsmntrda 5 moe 4hours. 24 over nm 450 at monitored (200 was plates 96-well absorbance in and light well) constant under rpm, 100 otiig1 containing utprtr(abr,Gray t10 o s Aequorin- of concentration ms. 5 a at for incubated V and 1800 obtained 2 were at conidia Germany) expressing was (Hamburg, aequorin synthetic Multiporator the transform encoding to gene used the containing vector for pAB19 codon-optimized been has that et n ntedvlpeto hrpui ramnsfrfungal on for cancer. treatments and therapeutic impact cell of infection during development might signaling the Thus, of on action and aspects 2010). death fundamental of the Engh, on mechanism studies and future its (Gani of drugs our characterization anticancer limitations, for prototype these al., Despite et using obtained Matlib 2011). approach. those 2003; genetic with a al., al., consistent are et et results Ishikawa pharmacological with Miller 1997; previously al., 1998; used et been (Gafni cells not C, U-73122 have xestospongin and crassa N. ML204 Ru360, 2004) and 2002). al., YM-58483 Lew, et and (Bowman (Silverman-Gavrila A1 bafilomycin 2012), 6 yeiletninrtsa 26 at rates extension Mycelial hssuyhsmd xesv s fihbtr htare that inhibitors of use extensive made has study This tuoprn sawdl sdto ncl et eerhada and research death cell in tool used widely a is Staurosporine 10 6 el/lwt 5 with cells/ml a ade codn osadr rcdrs Vogel’s procedures. standard to according handled was 6 el u r nw ob eyslciei animal in selective very be to known are but cells 2 10 n -yrxsarsoie(C-1 MrkMillipore, (Merck (UCN-01) 7-hydroxystaurosporine and 3 2+ oii notecnr flrePtidse (14.2-cm dishes Petri large of centre the onto conidia .crassa N. esrmn ihaequorin with measurement .crassa N. m 2+ 2+ 9 olneaie(at rzBiotechnology, Cruz (Santa coelenterazine M -[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]-1,2,3- eotr r h otrlal pinfrthe for option reliable most the are reporters yais n uhamto a developed was method a such and dynamics, 9 el yeetooainwt nEppendorf an with electroporation by cells 6 ,N 10 9 ˚ ttaoim(AT) thapsigargin, (BAPTA), -tetrasodium eemaue fe ncltn 5 inoculating after measured were C hpiagn(aa n Lew, and (Hamam thapsigargin – 4 .crassa N. oii/lwr nuae t26 at incubated were conidia/ml b -3-methoxyestra-1,3,5(10)-trien-17- Nlo ta. 04.The 2004). al., et (Nelson m eeaddt each to added were l 2+ m oa oueper volume total l eotraequorin reporter 2+ inln in signaling ˚ C, m l lto h auso eurnlmnsec,a edu f[Ca of readout a as luminescence, aequorin of values the of plot h tuoprn-rae ape ooti h ‘staurosporine-induced the obtain of values to RLU Ca the samples from staurosporine-treated subtracted were the samples control DMSO solvent olwdb h diino tuoprn n rwhfrafurther a with for twice 0.1 growth washed with centrifugation, and incubated by staurosporine and harvested PBS were of Samples addition hours. 2 the by followed ocnrto f2 of concentration germinated MD). a of MetaMorph Bethesda, percentage (NIH, the ImageJ The by using acquisition. calculated driven was image cells UK) for City, software NX1.1 Garden Welwyn (Hamamatsu, 5 containing medium Vogel’s a with microscope inverted TE2000E Nikon 60 a microscopy, routine For Microscopy for prepared were 6.6 dilutions with threefold starting successive strain nine each assay, spot the For assays death Cell lt rsne nti ae orsodt h vrg fa es three least The at experiment. of per average replicates six the to to three luminescence correspond with aequorin experiments paper of independent this The volume in homogenization. 10 presented the good plots was instances, ensure wells all to the before In solution), to agents added. added pharmacological was chemical(s) various DMSO) with (or minutes staurosporine 15 for incubated yia tuoprn-nue Ca staurosporine-induced typical ld utr hmes(agNn,Rcetr Y n nuae at incubated and NY) Rochester, Nunc, (Nalg 26 chambers culture slide iia eima 26 at medium minimal hmesa 26 at chambers uiecnerslswr omlzdfrec tant lo direct strain, allow each to For follows. strain as each 100 experiments for different between normalized comparisons were results luminescence elo ht pqe9-elpae n nuae o or t26 at hours 6 for incubated and plates 96-well opaque white of well units) oa eurndshremaueet eeue onraiethe normalize to Ca These used [cytosolic experiment. strain were aequorin each each for measurements in total values maximum RLU strain discharge the experimental the each to aequorin of for corresponds expressed total measurement and aequorin emitted a measured of be level was provided could luminescence that This luminescence aequorin minutes. 3 discharged for and plates 96-well aus n aaaeepesda h mean the as expressed experimental are normalized data the and summing values, by performed was quantification I pis loecneiae eeaqie sn lnfur20 fluor plan a with using at acquired equipped obtained were Japan) images were Fluorescence (Tokyo, Olympus optics. microscope images an DIC fluorescence 20 using and inverted obtained growth, applied were IX81 of Micrographs was time-points. hours DMSO appropriate 6 or After staurosporine (Sigma-Aldrich). iodide propidium bandfo 0idpnetexperiments. independent 30 from obtained ihaa upeetdwt h niae hmcl el were Cells chemical. indicated the with 26 at supplemented medium incubated (GFS) agar glucose-fructose-sorbose containing with plates onto spotted OPO Lf ehoois albd A a sd oii ta at Conidia used. 10 was CA) of Carlsbad, concentration Technologies, (Life YO-PRO1 ape eera naBcmnCutrEISX-C Be,CA). (Brea, XL-MCL EPICS mean the Beckman-Coulter represent a Results in read were samples hms K n ifrnilitreec otat(I)otc was optics (DIC) 200- contrast analyses, interference these For differential used. and UK) Thames, ovr L ausit rcs [Ca precise to possible into not VT) values was (Winooski, it RLU HT constraints, convert Synergy in equipment to Bio-Tek measured Owing a was reader. using microplate emitted (RLU) luminescence units The light relative agitation. without dark the n P5050ectto itr loecneeiso a filtered were was Images Japan). emission Tokyo, fluorescence (Olympus, DM570 filter; mirror dichroic excitation a source 510–550 through light fluorescence BP mercury a 100-W a and using lens, objective NA 0.45 6 o h vlaino rpdu oieutk yda el,cndaat conidia cells, dead by uptake iodide propidium of evaluation the For o h eeto faottcclsb lwctmty h fluorophore the cytometry, flow by cells apoptotic of detection the For ˚ 2+ .Iae eecpue iha RAE C camera CCD ORCA-ER an with captured were Images C. 12N ae-meso lnaoojcie(io,Kingston-upon- (Nikon, objective apo plan water-immersion NA /1.2 m 5 f3MCaCl M 3 of l mltd frsos’ hr pcfe,smlswr pre- were samples specified, Where response’. of amplitude xeietlRUvle/oa mte uiecne.Freach For luminescence]. emitted values/total RLU experimental ora fCl cec 21)17 8732 doi:10.1242/jcs.152058 3817–3829 127, (2014) Science Cell of Journal ˚ ˚ ntedr ihu gtto n tie ih5 with stained and agitation without dark the in C n mgswr band7 or later. hours 72 obtained were images and C 2 6 6 n2%ehnlwsppte noetawlso the of wells extra into pipetted was ethanol 20% in el/lwr utrdfr4husi Vogel’s in hours 4 for cultured were cells/ml 10 ˚ ,10rm ne osatlgtconditions, light constant under rpm, 140 C, 6 6 el/lwr nuae negtwl slide eight-well in incubated were cells/ml 6 s.e.m. 6 10 m m OPO1 fe 0mntso ice, on minutes 20 After 1. YO-PRO M 10 7 rp fcnda upnini liquid in suspension conidial of drops l el/l rmec iuin 5 dilution, each From cells/ml. 5 2+ oii/lwr lcdi eight-well in placed were conidia/ml 2+ intr fwl-yeclswas cells wild-type of signature ] c m ocnrtos hrfr,the Therefore, concentrations. fo naporaestock appropriate an (from l 6 ...TeRUvle of values RLU The s.e.m. 2+ ees(arbitrary levels m m 3827 was l ˚ g/ml Cin 2+ m 6 ] M c / ,

Journal of Cell Science hag .H,Pect,E . og . hed,S,Jrt .E,Bsam .I., A. Basbaum, E., S. Jordt, S., Shields, H., Kong, D., E. Prescott, H., H. Chuang, ttsial significant. statistically hi . i,K . h,H .adLe .H. Y. Lee, and S. H. Rho, S., K. Kim, J., Choi, F. Trail, and R. R. Lew, A., Hamam, B., Cavinder, F. Trail, and B. Cavinder, Falca C., Lemos, A., Castro, Falca C., Lemos, A., Castro, Bowman, and M. Draskovic, E., Margolles-Clark, S., Abreu, J., B. Bowman, J. B. Bowman, and H. T. Stevens, A., L. Graham, J., E. Bowman, D., N. Read, E., G. Turner, M., Freitag, O., Yarden, A., L. Alex, A., K. Borkovich, hcg,I) h o-aaercMn–hte etwsue for used (SPSS, was 20 test SPSS Mann–Whitney using non-parametric performed was The data IL). the Chicago, of analysis Statistical mean analysis ms the Statistical 50 as expressed to and was adjusted (NIH) cells ImageJ propidium-iodide-positive was using of time quantified percentage exposure The binning. The without Japan). Tokyo, System (Olympus, Imaging Soft II, Mu (ColorView camera GmbH, CCD cooled a with acquired ARTICLE RESEARCH 3828 W. K. Cunningham, and K. K. Nastase, M., Bonilla, F. Marx, and D. N. Read, M., Chu, K. V. U., Mootha, Binder, and E. S. Calvo, G., A. Bick, F. R. Irvine, and J. M. Berridge, J. M. Berridge, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.152058/-/DC1 online available material Supplementary material Supplementary Fundac from fellowship a of recipient was A.P.G. Funding revised A.V. was and and A.V., N.D.R. and P.C.S., N.D.R. A.M., A.P.G., J.M.C., A.P.G., J.M.C., by A.P.G., by written analyzed by was article were The and were A.V. A.M., Data and and A.V. J.M. J.M. and J.M.C., N.D.R. A.P.G., by J.M.C., A.P.G., collected by designed were Experiments contributions Author interests. competing no declare authors The interests Competing groups. two between comparisons eloeTutgat[rn ubrW039M]t ...Dpstdi PMC in Deposited a N.D.R. release. and to A.V.; immediate WT093596MA] to for number PP_IJUP2011-20] [grant number grant [grant Trust Porto Wellcome of University the a from NORTE-07-0124-FEDER-000003]; QCAIII grant number of [grant program FEDER) POCI (co-participant, European the P.C.S.]; numbers and to [grant A.V. PEst-OE/SAU/UI0215/2014 Portugal to FCT FCOMP-01-0124-FEDER-037277 by and supported PEst-C/SAU/LA0002/2013 was Fundac work of This scope FSE-POPH-QREN]. the under hired was Cie J.M.C. and 104210]; number asii eetrfo tIs45P-eitdinhibition. PtdIns(4,5)P2-mediated from receptor capsaicin D. Julius, and V. M. Chao, hshlps ee nfna eeomn n ahgnct of pathogenicity and development fungal in oryzae. genes Magnaporthe C phospholipase zeae. the Gibberella in fungus discharge ascospore filamentous and development, growth, affects channel, ion calcium fungi. filamentous of development sexual Cell and growth Eukaryot. in system, uptake calcium Cell Eukaryot. A. cell programmed phytosphingosine-induced to mutants death. I complex of resistance in levels calcium Cell intracellular Eukaryot. maintaining in cax, and nca-3, J. from E. V-ATPases the of c cerevisiae. 33138. subunit Saccharomyces and in crassa Neurospora site binding bafilomycin/concanamycin of organism. multicellular sequence to al. blueprint genome et genomic N. Plesofsky, the J., Paietta, from D., Bell-Pedersen, S., Seiler, h eiilu hyoeu rti A irpsclimhmotssin stress. reticulum endoplasmic homeostasis to response in calcium calcineurin disrupts PAF protein crassa. chrysogenum Neurospora Penicillium the uniporter. calcium mitochondrial Nature Acta Biophys. Biochim. ni enlga(C)Prua CIE Portugal (FCT) Tecnologia a e ˆncia 21) oeoeehne h niuglpoete fstaurosporine. of properties antifungal the enhances Rotenone (2010). 21) oeo orclimtasotpoen,ecddb c-,nca-2, nca-1, by encoded proteins, transport calcium four of Role (2011). .Bo.Chem. Biol. J. 341 ntr emn)cnetdt optrrnigCl F Cell running computer a to connected Germany) ¨nster, 197-205. , 20) nstltipopaeadclimsgaln mechanisms. signalling calcium and trisphosphate Inositol (2009). 11 9 10 906-914. , 978-988. , 654-661. , uayt Cell Eukaryot. 283 uglGnt Biol. Genet. Fungal 1793 21) oeo i1 opnn ftelow-affinity the of component a Fig1, of Role (2012). 19314-19321. , o . ls,N .adVdia A. Videira, and L. N. Glass, A., ˜o, 20) rdknnadnregot atrrlaethe release factor growth nerve and Bradykinin (2001). o . enne,A . ls,N .adVideira, and L. N. Glass, S., A. Fernandes, A., ˜o, 933-940. , 18) nstlpopae n elsignalling. cell and phosphates Inositol (1989). Science ersoacrassa Neurospora 9 uayt Cell Eukaryot. 1374-1382. , P ˆ vle of -values CA20 rgam gatnumber [grant Programme 2008 NCIA 336 48 21) vltoaydvriyo the of diversity Evolutionary (2012). irbo.Ml il Rev. Biol. Mol. Microbiol. 445-455. , 886. , ¸ 21) h niuglatvt of activity antifungal The (2010). 21) i1 mechanosensitive a Mid1, (2011). a oClut ubnin[grant Gulbenkian Calouste ˜o 21) ifrnilrlso the of roles Differential (2011). 10 # .Bo.Chem. Biol. J. 832-841. , rcn h ahfrom path the tracing : 20) seta oeof role Essential (2002). .5wr considered were 0.05 MOJ. EMBO Nature ersoacrassa. Neurospora 20) Increased (2008). 20) Lessons (2004). 6 21 ¸ a 411 opr a para ˜o s.e.m. 2343-2353. , 279 20) The (2004). 68 957-962. , 33131- , 1-108. , ipn,B,D md,H n ates R. Casteels, and H. Smedt, De B., Himpens, A. B. Hanson, R. R. Lew, and A. Hamam, C. Negro, Del and C. Simard, R., Guinamard, Gue rpi . eot,F,Branda F., Belotti, S., Groppi, ocle,A . al . obl .J,Gas .L n ier,A. Videira, and L. N. Glass, J., D. Kowbel, C., Hall, P., A. Goncalves, Gonc lpa,D . uis . otl,C n cut,G. Schultz, and C. Montell, D., Julius, E., D. Clapham, ug .J,Le .J,Km .W,Cug .R n e,C W. C. Lee, and R. Y. Chung, W., J. Kim, J., E. Lee, J., O. Jung, ece,A. Gescher, lpa,D E. D. Clapham, eai . an,E,St,K,Aia .adOaa Y. Okada, and T. Akita, K., Sato, E., Maeno, K., Dezaki, L. N. Glass, and G. Iyer, K., J. Dementhon, F. Serres, de and H. R. Davis, D. Techel, and G. Gebauer, G., Cornelius, odio . Gonc J., Cordeiro, og .P,V,K,Buo,J n el,A. Gelli, and J. Bautos, K., Vu, P., M. Hong, and T. Gudermann, C., Harteneck, M., Schaefer, G., A. Obukhov, T., Hofmann, siaa . ha . ohn,T,Tkzw,R,Ihkw,A,Kbt,H and H. Kubota, A., Ichikawa, R., Takezawa, T., Yoshino, K., Ohga, C. J., J. Ishikawa, Ameisen, and F. Bels, De J., and Estaquier, P. T., J. Idziorek, Uhrig, M., Jamklang, R., Tham, M., J. Bautos, K., Vu, P., M. Hong, arc . o ats .B n rfih,A. Griffiths, and B. D. Santos, dos O., Gavric, F. A. T. R. Molinski, Engh, and G., A. L. O. Costa, Gani, C., M. Catlin, H., T. Lam, A., J. Munsch, J., Gafni, og .P,Se,D,Wn,X,Dwo,T,L,X,Zag . hn,X., Cheng, Q., Zhang, X., Li, T., Dawson, X., Wang, D., Shen, P., X. Dong, odio .M,Bd,B,Gonc B., Boda, M., J. Cordeiro, onlu,G n aahm,H. Nakashima, and G. Cornelius, ose,C n ae .M. P. Kane, and C. Forster, A. Videira, and A. Castro, S., A. Fernandes, Gonc S., A. Fernandes, hshtso ersoacrassa. neurospora of phosphates 694-702. e6244. uatsriso iaetu ugs ersoacrassa. Neurospora fungus, filamentous a of strains mutant modulator. channel yeast. fission in starvation inositol by induced apoptosis regulates lcs-nue acu nlxi udn es novsanvlcalcium novel a involves yeast calcineurin. activate budding can and in system influx transport calcium Glucose-induced Z- sanvltasrpinfco otoln eldahadntrldrug natural and death cell controlling factor crassa. transcription Neurospora in novel resistance a Is CZT-1 crassa. niuoragents? antitumour 242-249. curneo K of occurrence crassa. Neurospora in death cell Cell programmed for necessary genes of crassa. Neurospora for techniques vacuoles. crassa Neurospora Commun. from release calcium h iecus fnuornmte erto i Ca a via secretion neurotransmitter of course time the Ca diacylglycerol. G. Schultz, fpttv hshioiieseii hshlps ee nflmnosfungi. Cells filamentous Mol. in genes C phosphoinositide-specific phospholipase putative of lymphocytes. T in 170 production IL-2 and influx Ca2+ sustained operated T. Yamada, without (apoptosis) viability. death cell cell with programmed interfering of analysis cytofluorometric permits neoformans. Cryptococcus in Mid1 subunit Cell the of region modulatory A. Gelli, 10951-10958. ebaetafcigb ietatvto fmclpnCa( mucolipin of activation direct by trafficking membrane ucino h -Taei acaoye cerevisiae. Saccharomyces in V-ATPase the of function ftepopoiaeCgn nNuopr crassa. Neurospora in gene C phospholipase the of receptor. 1,4,5-trisphosphate inositol N. the of I. blockers Pessah, and 38245-38253. hn,Y,Wimn .S,Dlig .e al. et M. Delling, S., L. Weisman, Y., Zhang, cells. HeLa in apoptosis to srqie o eiua Ca vesicular for required is 1 589 acu oesai nNuopr crassa. Neurospora in homeostasis calcium fPamclg.XI.Nmnltr n tutr-ucinrltosisof relationships structure-function channels. and potential receptor Nomenclature transient XLIX. Pharmacology. of nraei D1F2sot ucecells. muscle smooth DDT1MF-2 in increase uigporme eldaho ersoacrassa. Neurospora of death cell programmed during profiling. A. transcriptional by Biol. Videira, identified Genet. proteins and targeting by L. staurosporine N. Glass, endolysosome. the tuoprn analogues. staurosporine rn . euead .B,Lru,A n oec,L A. L. Rokeach, and A. Leroux, B., P. Beauregard, R., ´rin, avs .P n ier,A. Videira, and P. A. ¸alves, 2+ 4441-4449. , 149-167. , 5 12 nfna el uigedpamcrtclmstress. reticulum endoplasmic during cells fungal in 2161-2173. , 142-150. , ora fCl cec 21)17 8732 doi:10.1242/jcs.152058 3817–3829 127, (2014) Science Cell of Journal e .Sci. J. 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