Changes in the biophysical properties of the cell membrane are involved in the response of neurospora crassa to staurosporine

ORIGINAL RESEARCH published: 11 Oc obe 2018 doi: 10.3389/ phys.2018.01375 F on ie s in Physiology | www. on ie sin.o g 1Oc obe 2018 | Volume 9 | A icle 1375 Edi ed by: Pushpend a Singh, School o Medicine, Johns Hopkins Uni e si y, Uni ed S a es Re iewed by: Anuj Kuma Sha ma, P ince on Uni e si y, Uni ed S a es Rajeshwe Singh Sankhala, Wal e Reed A my Ins i u e o Resea ch, Uni ed S a es *Co espondence: Rod igo F. M. de Almeida [email p o ec ed] Special y sec ion: This a icle was submi ed o Memb ane Physiology and Memb ane Biophysics, a sec ion o he jou nal F on ie s in Physiology Recei ed: 26 June 2018 Accep ed: 11 Sep embe 2018 Published: 11 Oc obe 2018 Ci a ion: San os FC, Lobo GM, Fe nandes AS, Videi a A and de Almeida RFM (2018) Changes in he Biophysical P ope ies o he Cell Memb ane A e In ol ed in he Response o Neu ospo a c assa o S au ospo ine. F on . Physiol. 9:1375. doi: 10.3389/ phys.2018.01375 Changes in he Biophysical P ope ies o he Cell Memb ane A e In ol ed in he Response o Neu ospo a c assa o S au ospo ine Filipa C. San os1, Ge son M. Lobo1, And eia S. Fe nandes2,3, A naldo Videi a2,3,4 and Rod igo F. M. de Almeida1* 1Depa amen o de Química e Bioquímica, Faculdade de Ciências, Cen o de Química e Bioquímica, Uni e sidade de Lisbon, Campo G ande, Lisbon, Po ugal, 2I3S - Ins i u o de In es igação e Ino ação em Saúde, Uni e sidade do Po o, Po o, Po ugal, 3IBMC-Ins i u o de Biologia Molecula e Celula , Uni e sidade do Po o, Po o, Po ugal, 4ICBAS-Ins i u o de Ciências Biomédicas de Abel Salaza , Uni e sidade do Po o, Po o, Po ugal Neu ospo a c assa is a non-pa hogenic ilamen ous ungus widely used as a mul icellula euka yo ic model. Recen ly, he biophysical p ope ies o he plasma memb ane o N. c assa conidia we e ho oughly cha ac e ized. They e ol e du ing conidial ge mina ion a a speed ha depends on cul u e condi ions, sugges ing an impo an associa ion be ween memb ane emodeling and he in ense memb ane biogenesis ha akes place du ing he ge mina i e p ocess. S au ospo ine (STS) is a d ug used o induce p og ammed cell dea h in a ious o ganisms. In N. c assa, STS up- egula es he exp ession o he ABC anspo e ABC-3, which localizes a he plasma memb ane and pumps STS ou . To unde s and he ole o plasma memb ane biophysical p ope ies in he ungal d ug esponse, N. c assa was subjec ed o STS ea men du ing ea ly and la e conidial de elopmen s ages. Following 1 h ea men wi h STS, he e is an inc ease in he abundance o he mo e o de ed, sphingolipid-en iched, domains in he plasma memb ane o conidia. This leads o highe luidi y in o he memb ane egions. The global o de o he memb ane emains hus p ac ically unchanged. Signi ican changes in sphingolipid-en iched domains we e also obse ed a e 15 min challenge wi h STS, bu hey we e essen ially opposi e o hose e i ied o he 1 h ea men , sugges ing di e en ypes o d ug esponses. STS e ec s on memb ane p ope ies ha a e mo e dependen on e gos e ol le els also depend on he de elopmen al s age. The e we e no al e a ions on 2 h-g own cells, clea ly con as ing o wha happens a longe g ow h imes. In his case, he di e ences we e mo e ma ked o longe STS ea men , and a ionalized conside ing ha he d ug p e en s he inc ease in he e gos e ol/glyce ophospholipid a io ha no mally akes place a he la e conidial s age/ ansi ion o he mycelial s age. This could be pe cei ed as a d ug-induced de elopmen a es a e 5 h g ow h, in ol ing e gos e ol, and poin ing o a ole o lipid a s possibly ela ed wi h an up- egula ed exp ession o he ABC-3 anspo e . O e all, ou esul s sugges he in ol emen o memb ane o de ed domains in he esponse mechanisms o STS in N. c assa. Keywo ds: an i ungal d ug, plasma memb ane, sphingolipid domains, biophysical p ope ies, e gos e ol, liposomes, luo escence spec oscopy, conidial de elopmen San os e al. Memb ane Biophysical Response o an An i ungal Agen INTRODUCTION An i ungal d ug esis ance is a majo socie y conce n, since he mo ali y a e associa ed wi h esis an ungal in ec ions has inc eased d ama ically, pa icula ly in hospi al en i onmen s and in pa ien s wi h dec eased immunological esponse (Gulshan and Moye-Rowley, 2007; Shahi and Moye-Rowley, 2009; P e en ion CFDCA., 2017). The eme ging ole o apop osis as key egula o o ungal de elopmen sugges s ha i migh be possible o de elop new means o con olling ungal in ec ions h ough he manipula ion o some key componen s/o ganelles in ol ed in he apop o ic cascade. In ungi, apop o ic-like cell dea h occu s na u ally du ing de elopmen al p ocesses and ep oduc ion, and can be induced by en i onmen al ac o s and exposu e o oxic me aboli es o abio ic ac o s. The co e apop o ic p ocesses in ungi a e simila o hose in mammals, howe e , he apop o ic ne wo k is less complex (Sha on e al., 2009). The alkaloid s au ospo ine (STS) is well-known o i s an i ungal (Omu a e al., 1977; Pa k e al., 2006) and an i umo al cha ac e is ics (Co ea e al., 2011), bu also o being he mos po en p o ein kinase inhibi o , wi h a hal maximal inhibi o y concen a ion in i o in he nanomola ange (Tamaoki e al., 1986), and an induce o p og ammed cell dea h in neu onal cells (e.g., Wiesne and Dawson, 1996), p o ozoans (e.g., Yin e al., 2010) human mac ophages (e.g., Dunai e al., 2012) and in he ilamen ous ungus Neu ospo a c assa (Gesche , 2000; Cas o e al., 2010; Fe nandes e al., 2011, 2013). STS-induced p og ammed cell dea h in ilamen ous ungus (Fe nandes e al., 2011) is hus one o he many examples o impo an ungal physiological ac i i ies, such as cell p oli e a ion o di e en ia ion, sensing and signaling, ha a e usually ound o be closely ela ed o memb ane composi ion and biophysical ea u es, h ough hei in ol emen wi h memb ane mic odomain o ganiza ion and lipid homeos asis (Malinsky and Ope ako a, 2016). Indeed, a gene exp ession (mic oa ay) s udy (Fe nandes e al., 2011) shows ha he e a e impo an changes in he le els o mRNA coding o se e al enzymes o lipid me abolism and also o (signaling) p o eins ha in e ac wi h he memb ane and may a ec domain o ma ion and p ope ies (Table S1). The plasma memb ane p o ein o which mRNA le els de e mined in he ansc ip ional p o iling s udy a e inc eased by a la ge amoun , a aining a 30- old inc ease is ABC- 3 (Fe nandes e al., 2011). Mo eo e , his p o ein is esponsible o mos o he ene gy-dependen e lux o STS and a null-mu an o his ABC anspo e (abc3) is ex emely sensi i e o STS and accumula es mo e STS han he wild ype s ain (Fe nandes e al., 2011). In he p esen s udy, we use he biological model N. c assa conidial cells and STS o biochemically cha ac e ize ungal plasma memb anes when challenged wi h an an i ungal d ug. We Abb e ia ions: ABC, ATP Binding Casse e; Di-4-ANEPPS, 4-(2-(6- (dibu ylamino)-2-naph halenyl)e henyl)-1-(3-sul op opyl)-py idinium; DOPC, 1,2-dipalmi oyl-sn-glyce o-3-phosphocholine; DPH, 1,6-diphenyl-1,3,5- hexa iene; FGSC, Fungal Gene ics S ock Cen e ; POPC, 1-palmi oyl-2-oleoyl-sn- glyce o-3-phosphocholine; STS, s au ospo ine; Tm, main ansi ion empe a u e; -PnA, ans-pa ina ic acid. asked whe he he e could be a biophysical esponse a he plasma memb ane le el ha could be consequence o STS challenge. This d ug does no di ec ly a ge he cellula en elope o any p o ein ac i i y in ol ed in memb ane lipid syn hesis and ca abolism, con a y o many an i ungal d ugs, such as polyenes, azoles, and sphingoid base analogs, bu migh signi ican ly change he plasma memb ane composi ion, as indica ed by ansc ip omic analysis. The use o STS will disclose i e en in such a case memb ane lipid composi ion and biophysical p ope ies should be conside ed o unde s and he physiological esponse o ilamen ous ungi o he d ug. Un il ecen ly, he biophysical p ope ies o conidial cell memb ane, namely o N. c assa, we e p ac ically unknown, bu a ho ough biophysical cha ac e iza ion o N. c assa plasma memb ane was ca ied ou and impo an biophysical p ope ies o he plasma memb ane o N. c assa conidia a e now well cha ac e ized, as well as hei dynamic beha io along conidial ge mina ion (San os e al., 2017). The cell memb ane becomes globally mo e luid wi h g ow h ime, i con ains o de ed sphingolipid-en iched domains ha di e om he ones known as lipid a s since hey ha e no e gos e ol, and he gel-like na u e o hose domains, despi e being much less igid han hose ound in he yeas Saccha omyces ce e isiae (A es a- B anco e al., 2011), leads o a highe global memb ane o de han in he budding yeas (San os e al., 2017). In he p esen wo k, we s udied he biophysical e ec s o STS on he plasma memb ane lipid domains o N. c assa and also e alua ed i he d ug in e ac s wi h lipids o he plasma memb ane. We show, in ac , ha he mechanism o STS esponse in ol es changes in memb ane biophysical p ope ies dependen on lipid o ganiza ion and composi ion, and hus hese should be aken in o accoun when s udying he mechanism o STS ac ion and he physiological esponses o ilamen ous ungi o d ugs. Using his d ug also p o ides an oppo uni y o disclose i he e could be a as esponse in ol ing eo ganiza ion o lipids and lipid domains a he plasma memb ane be o e he gene ic esponse has ully eme ged adding o knowledge on an i ungal ac ion and p o iding clues o a be e unde s anding o an i ungal d ug esponse by ilamen ous ungi. MATERIALS AND METHODS S ains, G ow h Techniques and Chemicals N. c assa wild ype s ain (FGSC 2489) and he null-mu an o he ABC anspo e ABC-3 (abc3) (FGSC 14572) we e ob ained om he Fungal Gene ics S ock Cen e (Mccluskey, 2003). S anda d p ocedu es we e employed o g ow h and handling o N. c assa in Vogel’s Minimal Medium (Da is e al., 1970). STS was ob ained om LC-Labo a o ies, DPH (1,6-diphenyl- 1,3,5-hexa iene) and di-4-ANEPPS (4-(2-(6-(dibu ylamino)- 2-naph halenyl)e henyl)-1-(3-sul op opyl)-py idinium) we e ob ained om In i ogen (Mad id, Spain). The p obe -PnA ( ans-pa ina ic acid) was pu chased om San a C uz Bio ech. (San a C uz, C.A.). Ludox R (colloidal silica dilu ed o 50% in wa e ) was pu chased om Sigma-Ald ich (S . Louis, MO). Sol en s/co-sol en s such as e hanol, me hanol, and glyce ol we e spec oscopic g ade and pu chased om Me ck and Scha lau. All o he eagen s we e o he highes pu i y F on ie s in Physiology | www. on ie sin.o g 2Oc obe 2018 | Volume 9 | A icle 1375 San os e al. Memb ane Biophysical Response o an An i ungal Agen a ailable. S ock solu ions o STS (13.2 mM STS in DMSO) and o luo escen p obes we e quan i ied spec opho ome ically (de Almeida e al., 2005; Bas os e al., 2012a). Fo luo escence spec oscopy s udies, STS was dilu ed in PBS a 700 µM be o e use. All he esul s ob ained a e p esen ed as he mean ± s anda d de ia ion (S.D.) ob ained om independen liposomal suspensions/ biological eplica es, and s a is ical signi icance was de e mined using S uden ’s - es . Mean alues we e conside ed signi ican ly di e en o p alues below 0.05. Fluo escence Spec oscopy o STS in Liposomes Liposome (mul ilamella esicles) suspensions o 3 mM lipid we e used. This high lipid concen a ion was used o ensu e ha e en a weak in e ac ion wi h he memb ane could be pe cei ed. Th ee di e en lipid composi ions we e p epa ed: DPPC (1,2- dipalmi oyl-sn-glyce o-3-phosphocholine), POPC (1-palmi oyl- 2-oleoyl-sn-glyce o-3-phosphocholine), and a bina y mix u e o DPPC/ Choles e ol (1:1 mol:mol). Choles e ol was chosen ins ead o e gos e ol because e gos e ol abso bs ligh in he same ange as STS and, as i co esponds o 50 mol% o he lipid, is p esen in a much highe concen a ion (1.5 mM s. 12.5 µM), p ecluding eliable measu emen s o STS luo escence. To p epa e he liposome suspensions, lipid s ock solu ions we e added o a glass ube and he sol en was slowly apo ized by a mild low o ni ogen, o ming a hin laye o lipid. The lipid was hyd a ed by he addi ion o 1 mL o PBS p e iously hea ed abo e he main ansi ion empe a u e (Tm) o he lipids. The samples we e hen p og essi ely o ex-s i ed and submi ed o a leas 5 eeze/ haw cycles. A e wa ds, STS was added o he p epa ed mul ilamella esicles suspensions o a inal concen a ion o 12.5 µM, and he suspension was incuba ed a oom empe a u e (23 ±2◦C). A e 1 h, he liposome suspensions we e analyzed by s eady-s a e and ime- esol ed luo escence spec oscopy, aking ad an age o he in insic luo escence o he d ug, using exci a ion and emission wa eleng hs o 290 and 377 nm, espec i ely, and bandwid h o 1.5 nm in spec al acquisi ion and 3 nm in aniso opy measu emen s. STS in PBS was used as con ol. Liposome suspensions wi hou STS we e used as blanks. Bo h s eady-s a e and ime- esol ed luo escence measu emen s we e pe o med wi h a Ho iba Jobin-Y on Spex Fluo olog 3.22, a 30◦C in a empe a u e-con olled sample compa men unde magne ic s i ing. Fo ime- esol ed measu emen s by he single pho on coun ing echnique, a nanoLED N-280 o exci a ion a 279 nm; emission was collec ed a 377 nm wi h a bandwid h be ween 5 and 7 nm. The o he expe imen al condi ions and da a analysis ollow he p ocedu e desc ibed below o he s udies wi h cell suspensions. STS Challenge and Fluo escence Spec oscopy in Cells N. c assa conidia we e g own in liquid minimal medium a a s a ing concen a ion o 107cell/mL, a 30◦C and 150 pm. A e 2 o 5 h o g ow h, each cul u e was submi ed o a 12.5 µM STS challenge and cells we e u he incuba ed o 1 h. In pa allel, 3 o 6 h g own cul u es we e challenged wi h 12.5 µM STS du ing 15 min, unde he same condi ions. The same olume o PBS was added o con ol cul u es. This 1 h delay ensu ed ha he o al g ow h ime o he con ols was simila in bo h se s o expe imen s, i.e., 3 and 6 h o 3 h 15 min and 6 h 15 min, espec i ely. Cells we e washed by cen i uga ion a 10,000 g o 2 min and esuspended in PBS. The luo escence spec oscopy p ocedu es used in he p esen wo k we e p e iously op imized (San os e al., 2017). As be o e, he luo escen p obes -PnA (2 µM), DPH (2 µM), and di- 4-ANEPPS (1 µM) we e added o he cells and incuba ed o 10 min a 30◦C unless o he wise s a ed. Fo s eady-s a e aniso opy measu emen s, exci a ion and emission wa eleng hs o 320 and 404 nm ( -PnA) o 360 and 425 nm (DPH) we e used. Bandwid hs o 4 nm we e used o bo h -PnA and DPH. Fo di-4-ANEPPS spec a acquisi ion, bandwid hs o 4 nm we e used. The memb ane dipole po en ial was measu ed h ough he a io o exci a ion in ensi ies a 420 and 520 nm o di-4-ANEPPS wi h emission a 635 nm. The Rex can be linea ly ela ed wi h he memb ane dipole po en ial unde he condi ions me in his s udy [30–33], hence he exci a ion spec a we e acqui ed wi h emission wa eleng h o 635 nm, which is a he ed edge o he spec um, allowing o ule ou he memb ane luidi y e ec s, and as e e ed abo e he e we e no shi s obse ed in he spec a upon he STS challenge. Thus, he dipole po en ial (ψd) in mV was calcula ed om Rex using he linea ela ionship (Equa ion 1) (Halda e al., 2012): ψd=Rex +0.3 4.3 ×10−3(1) The s eady-s a e luo escence aniso opy ( ) was calcula ed acco ding o, =(IVV −G×IVH) (IVV +2G×IVH)(2) in which Gis he ins umen al co ec ion ac o and he subsc ip s Vand H ep esen he e ical and ho izon al o ien a ions o he pola ize s. The o de o he subsc ip s co esponds o exci a ion and emission. An adequa e blank was sub ac ed om each in ensi y eading. Fo ime- esol ed measu emen s by he single pho on coun ing echnique, a nanoLED N-320 and a nanoLED N- 460 we e used o exci a ion o -PnA and di-4-ANEPPS, espec i ely. Emission was se o 404 and 634 nm, espec i ely. Ludox R was used as he sca e e o ob ain he ins umen al esponse unc ion. The p og am TRFA da a p ocesso e sion 1.4 (Minsk, Bela us) was used o he analysis o he expe imen al luo escence decays. To desc ibe he decays, a sum o exponen ials wi h αi he no malized ampli ude and τi he li e ime o componen i, was used: I( )= n X i=1 αiexp − τi(3) F on ie s in Physiology | www. on ie sin.o g 3Oc obe 2018 | Volume 9 | A icle 1375 San os e al. Memb ane Biophysical Response o an An i ungal Agen The ampli ude-weigh ed mean luo escence li e ime was calcula ed as ollows, τa = n X i=1 αiτi(4) and he in ensi y-weigh ed mean luo escence li e ime was ob ained h ough he ollowing exp ession, < τ >=Pn i=1αiτ2 i Pn i=1αiτi (5) The quali y o he i was judged by andom dis ibu ion o weigh ed esiduals and esiduals au oco ela ion and a educed χ2 alue close o 1. SDS-Page and Wes e n Blo N. c assa a a s a ing concen a ion o 107conidia/mL was g own in 10 mL liquid minimal medium a 30◦C and 150 pm, ollowed by he addi ion o 12.5 µM STS o same olume o DMSO, and incuba ed unde he same condi ions. Cells we e collec ed by as il a ion. To al p o ein ex ac s we e ob ained om dis up ion wi h zi conia beads in a Fas P ep-24 (MP Biomedicals). SDS- PAGE and wes e n blo using an an ibody agains ABC-3 p o ein we e pe o med as desc ibed in Fe nandes e al. (2011). RESULTS STS-Lipid In e ac ions To s udy a possible in e ac ion o STS wi h memb ane lipids, h ee di e en model sys ems we e used, co e ing he h ee lipid bilaye phases hough o ep esen he mos impo an ypes o lipid domains in ungal plasma memb ane (Rose i e al., 2017): gel [composed o DPPC, wi h a Tm alue 41.5◦C (Huang and Li, 1999)], liquid diso de ed [composed o POPC, Tmca. −3◦C (Koyno a and Ca ey, 1998)], and liquid o de ed [composed o a bina y mix u e o DPPC/choles e ol, which is liquid o de ed a bo h oom empe a u e and 30◦C (de Almeida e al., 2007)]. Mo eo e , due o STS in insic luo escence, his s udy could be pe o med wi hou eso ing o ex e nally added labels. Di e en pho ophysical pa ame e s o STS we e measu ed. The s eady-s a e luo escence aniso opy e lec s he o a ional mobili y o he d ug, which should be signi ican ly es ic ed upon memb ane adso p ion o inco po a ion. The luo escence in ensi y decay allows compu ing he ampli ude-weigh ed and in ensi y-weigh ed mean luo escence li e ime, a e analysis using equa ion 3 wi h a bi-exponen ial model (n=2) and equa ions 4 and 5. Those wo pa ame e s e lec he mic oen i onmen o he p obe: he con ibu ion o each o hem o he o al decay can change wi h he sol en pola i y and speci ic in e ac ions, o wi h he pa hways he exci ed luo opho e ollows o e u n o he g ound s a e, which depend on collisions wi h o he molecules, ib a ions and o sions (Be ezin and Achile u, 2010). None o he STS pa ame e s analyzed we e signi ican ly al e ed by he p esence o any o he model sys ems used (Figu e 1). These esul s show ha STS does no in e ac di ec ly wi h he memb ane lipids. The a ia ions de ec ed in he conidial plasma memb ane (nex sec ion) mus he e o e a ise om ac i e memb ane eo ganiza ion and/ o lipid composi ion al e a ions ac i ely pe o med by he cell. ABC-3 Exp ession a he Plasma Memb ane: In luence o Ge mina ion and STS Exposu e Time The exp ession o memb ane p o eins, such as he ones om he ATP Binding Casse e (ABC) anspo e amily, is an impo an pa o an i ungal d ug esponse. These a e p ima y ac i e anspo e s in ol ed in he modula ion o abso p ion, me abolism and oxici y o pha maceu ical d ugs (Gla inas e al., 2004). F om his amily, he ABC-3 anspo e o N. c assa, homologous o he human P-glycop o ein 1 (Cannon e al., 2009), is esponsible o he STS e lux (Fe nandes e al., 2011). As men ioned abo e, his is he single p o ein whose le els o mRNA showed by a , o speci ic condi ions, he la ges inc ease (Fe nandes e al., 2011). The e o e, we chose o s udy in mo e de ail he exp ession o his p o ein a he plasma memb ane because i is expec ed o obse e e y clea ends ha can be used in combina ion wi h ou p e ious biophysical s udy along conidial ge mina ion o choose a easible numbe o expe imen al condi ions ha clea ly complemen each o he as a as STS physiological esponse o N. c asssa conidia is conce ned. In Figu e 2, he Wes e n blo s o ABC-3 a e shown o di e en g ow h imes and di e en STS ime exposu e. Howe e , we used only one concen a ion o STS. P e ious s udies om Cas o e al. (2010), show ha he concen a ion and incuba ion imes used he e ensu e high su i al a es and no iceable physiological e ec s. Hence, he concen a ion o STS used in he p e ious wo k by Fe nandes e al. (2011) o mos o he expe imen s was al eady 12.5 µM. Mo eo e , he e we e p ac ical easons o choose his concen a ion. Lowe alues o STS would yield low abso bance o luo escence in ensi y alues, which would in oduce la ge unce ain y in he s udy o STS in acellula accumula ion (Fe nandes e al., 2011). La ge concen a ion alues could in e e e wi h he luo escence s udies using -PnA, since he e is a la ge o e lap be ween he abso p ion and emission spec a o his memb ane p obe and hose o STS. As de ailed below, -PnA is an essen ial p obe in memb ane biophysical s udies because o i s unique sensi i i y o acyl chain packing in he mos o de ed lipid domains, i.e., o s udy he impo an sphingolipid-en iched plasma memb ane domains. STS a a concen a ion o 12.5 µM induces ABC-3 exp ession a e 1 h o incuba ion bu much mo e ma kedly in 5 h-g own cells han in 2 o 3 h-g own cells (Figu e 2A), and he exp ession is only de ec ed a e 45 min (Figu e 2B), which means ha a e 15 min exposu e he e is p ac ically no o e exp ession a he plasma memb ane. Biophysical Changes o Plasma Memb ane Lipids Upon STS Challenge To assess he in luence o ge mina ion ime and du a ion o STS s imulus, compa ison will be a emp ed o he ou F on ie s in Physiology | www. on ie sin.o g 4Oc obe 2018 | Volume 9 | A icle 1375 San os e al. Memb ane Biophysical Response o an An i ungal Agen FIGURE 1 | STS does no in e ac wi h lipid bilaye s: he in insic luo escence p ope ies o STS a e simila in PBS and in he p esence o di e en lipid phases. (A) s eady-s a e luo escence aniso opy; (B) ampli ude-weigh ed (black ba s) and in ensi y-weigh ed (whi e ba s) mean luo escence li e ime. The esul s ep esen he mean ±S. D. o a leas h ee independen expe imen s, n≥3. FIGURE 2 | Wes e n blo s o o al p o ein ex ac s om N. c assa cells, using an ise um agains he 130 kDa ABC-3 and he cons i u i e 30.4 kDa subuni o complex I (NUO30.4), as con ol o loading. (A) N. c assa was g own o he indica ed imes in hou and subsequen ly exposed o 12.5 µM STS o 1 h. (B) N. c assa was g own o 5 h and subsequen ly incuba ed wi h 12.5 µM STS (S) o DMSO as con ol (C) o he indica ed imes in min. S anda d molecula weigh s (kDa) a e indica ed on he le o each blo . combina ions o 2 and 5 h g ow h plus 1 h d ug challenge and 3 and 6 h plus 15 min d ug challenge, and espec i e con ols. To acili a e he analysis o he esul s, hey a e p esen ed as N. c assa cells in he absence ( ull pa e n) and in he p esence o STS (s ipped pa e n) wi h incuba ion ime o 1 h (black colo ed) and o 15 min (g ay colo ed). These wo g ow h imes allow compa ison o he ungal d ug esponses a he ea ly s. la e conidial s ages o de elopmen , ensu ing a he same ime complemen a y in o ma ion ega ding he exp ession o ABC-3 a he plasma memb ane. The STS ea men imes we e chosen aking in o accoun he da a p esen ed in Figu e 2B, whe e i is possible o obse e he highes le els o p o ein ABC-3 exp ession be ween he STS incuba ion imes o 45 min and 60 min, hus we ha e chosen he STS 1 h ea men . Fo a mo e comple e s udy we decided o pe o m a 15 min ea men wi h STS, o ensu e ha he inc ease o ABC-3 exp ession le els a he plasma memb ane we e s ill unde ec able, e en o a g ow h ime o 5 h. This allows assessing i we can dis inguish a as s. a slow esponse o he d ug in e ms o memb ane biophysical p ope ies in condi ions whe e he exp ession o la ge memb ane p o eins such as ABC anspo e s a e unchanged o no . Packing and O de o he Acyl Chains The luo escence in ensi y decay o -PnA can be used o iden i y he p esence o o de ed domains. These can be a ibu ed o a gel phase in he p esence o a e y long li e ime componen in he luo escence decay (A es a-B anco e al., 2011; Bas os e al., 2012b; Vece e al., 2014) o o a liquid o de ed phase (de Almeida e al., 2009). In N. c assa conidia, he e y low le els o e gos e ol impede he o ma ion o de ec able amoun s o liquid o de ed domains by -PnA (San os e al., 2017). The li e ime alue o he long componen is ela ed o he packing e iciency o he acyl chains. In u n, he ampli ude o he long componen is ela ed o he ela i e abundance o hose o de ed domains. Commencing wi h he 1 h challenge, STS did no ha e a majo impac on he long li e ime componen o -PnA (Figu e 3A and Table S2). This pa ame e inc eased om ∼20 ns a 2 h o ∼25 ns a 5 h g ow h, and was independen o he p esence o STS, e lec ing he p esence o o de ed domains as epo ed p e iously by us (San os e al., 2017). These a e sphingolipid-en iched domains ha a e no highly igid gel, because hei mel ing empe a u e is e y close o N. c assa g ow h empe a u e. Howe e , signi ican e ec s we e obse ed F on ie s in Physiology | www. on ie sin.o g 5Oc obe 2018 | Volume 9 | A icle 1375 San os e al. Memb ane Biophysical Response o an An i ungal Agen FIGURE 3 | Time- esol ed luo escence spec oscopic pa ame e s o -PnA in he plasma memb ane o N. c assa conidia. (A) The long li e ime componen , τ3, (B) ampli ude-weigh ed mean luo escence li e ime, τa (ampli ude-weigh ed), (C) no malized ampli ude o he long componen , α3, and (D) in ensi y-weigh ed mean luo escence li e ime, τa (in ensi y-weigh ed), o -PnA we e ob ained om he luo escence in ensi y decay o he p obe a 30◦C. The esul s p esen he mean ± S.D. o a leas h ee independen expe imen s, n≥3. *p<0.05, **p<0.01, ***p<0.001. o he ela i e abundance o he sphingolipid-en iched domains, since he ampli ude associa ed wi h he long li e ime componen inc eased in he p esence o STS a 5 h o g ow h (Figu e 3C). Rega ding he mean luo escence li e imes o -PnA, he gene al end is o obse e an inc ease o hei alues induced by STS (Figu es 3B,D). The o de ing o diso de ing e ec s o STS in he memb ane we e also assessed. The s eady-s a e luo escence aniso opy alues o DPH in he con ols we e simila o p e iously epo ed (San os e al., 2017). DPH, no being sensi i e o any pa icula kind o domain, gi es a iew o he plasma memb ane global o de . The alues o s eady-s a e luo escence aniso opy o DPH dec ease om 2 o 5 h o g ow h (Figu e 4), showing a luidiza ion o he plasma memb ane along ge mina ion. Fo wild ype cells, STS did no ha e any signi ican e ec on his pa ame e , sugges ing ha he e ec s sensed by - PnA luo escence in ensi y decays a e localized in o speci ic memb ane domains, since he global p ope ies o he memb ane emain essen ially unchanged. Conce ning he abc3 mu an , o 3 h g ow h, DPH s eady- s a e luo escence aniso opy epo s no s a is ically signi ican e ec o STS. The s eady-s a e luo escence aniso opy o DPH o abc3 cells was ca. 0.24 a 3 h o g ow h independen ly o he p esence o STS. Wi h 6 h o g ow h, he aniso opy alue FIGURE 4 | S eady-s a e luo escence aniso opy o DPH in he plasma memb ane o N. c assa conidia, a 30◦C o he wild ype (w ) o abc3 mu an , a e 1 h incuba ion in he absence o p esence o 12.5 µM STS. The alues a e he mean ±S.D. o a leas h ee independen expe imen s, n≥3. **p<0.01. o he con ol dec eased o ∼0.18, ollowing he gene al end al eady epo ed o he wild ype, wi h he plasma memb ane becoming mo e luid along he ge mina ion p ocess. Howe e , F on ie s in Physiology | www. on ie sin.o g 6Oc obe 2018 | Volume 9 | A icle 1375 San os e al. Memb ane Biophysical Response o an An i ungal Agen o his g ow h ime, upon STS challenge, he DPH aniso opy o abc3 unde goes a ma ked inc ease o ∼0.21. Fo his g ow h ime, abc3 cells p esen a end qui e di e en om he wild ype cells, since he memb ane o he mu an cells becomes much mo e igid wi h he STS challenge, in ac eco e ing om a con ol si ua ion o highe luidi y, a aining a memb ane luidi y ha is be ween he con ol o 3 and 6 h o g ow h. This inc eased igidi y o he memb ane as a whole is a biophysical esponse FIGURE 5 | Time- esol ed luo escence spec oscopic pa ame e s o -PnA in he plasma memb ane o N. c assa conidia. (A) The long li e ime componen , τ3, (B) ampli ude-weigh ed mean luo escence li e ime, τa (ampli ude-weigh ed), (C) no malized ampli ude o he long componen , α3, and (D) in ensi y-weigh ed mean luo escence li e ime, τa (in ensi y-weigh ed), o -PnA we e ob ained om he luo escence in ensi y decay o he p obe a 30◦C. The esul s p esen he mean ± S.D. o a leas h ee independen expe imen s, n≥3. *p<0.05, **p<0.01, ***p<0.001. FIGURE 6 | Di-4-ANEPPS luo escence p ope ies in N. c assa cells, a 30◦C, (A) luo escence exci a ion (λem =635 nm) and emission spec a (λexc =450 nm), (B) ampli ude-weigh ed mean luo escence li e ime o di-4-ANEPPS. The esul s p esen he mean ±S.D. o a leas h ee independen expe imen s, n≥3. ***p<0.001. F on ie s in Physiology | www. on ie sin.o g 7Oc obe 2018 | Volume 9 | A icle 1375 San os e al. Memb ane Biophysical Response o an An i ungal Agen obse ed only o he mu an ha is unable o e icien ly expo he d ug. Wild ype cells we e also subjec ed o a 15 min challenge. In hese condi ions, he long li e ime componen o -PnA (Figu e 5A and Table S3) showed only a change on he 3 h- g own cells, inc easing ca. 8% om ∼24 ns in he con ol o ∼26 ns in he p esence o STS, i.e., he d ug is inducing igh e packing o he acyl chains in he mo e o de ed domains. The ampli ude (Figu e 5C) associa ed wi h his long li e ime componen p esen s he opposi e beha io , dec easing wi h he addi ion o STS. This is also e lec ed in he ampli ude-weigh ed mean luo escence li e ime (Figu e 5B) bu no on he in ensi y- weigh ed mean luo escence li e ime (Figu e 5D). In he case o 6 h-g own cells, he e we e no signi ican changes, bu in his case he long li e ime is al eady e y high, e en in he con ol si ua ion. Wha is clea is ha a e 3 h g ow h a 15 min challenge FIGURE 7 | Ra io o di-4-ANEPPS luo escence in ensi y p oduced by exci a ion a 420 nm o ha p oduced by exci a ion a 520 nm, Rex (420 nm/520 nm) cells, a 30◦C. The esul s p esen he mean ±S.D. o a leas h ee independen expe imen s, n≥3. *p<0.05. wi h STS induces a ea angemen o sphingolipid-en iched domains. Conside ing he s eady-s a e luo escence aniso opy o DPH (no shown), no signi ican changes induced by STS could be pe cei ed, as obse ed o he 1 h s imulus. Pola i y Changes a he Lipid/Wa e In e ace To e alua e i STS can a ec N. c assa plasma memb ane pola i y p ope ies, conidia we e labeled wi h di-4-ANEPPS, a p obe om he class o po en ial sensi i e naph yls y yl dyes, ex emely esponsi e o ei he e gos e ol- o choles e ol-en iched lipid domains (Loew, 1996; Bas os e al., 2012a; Ama o e al., 2017). STS did no shi he emission o he exci a ion spec a o di-4-ANEPPS (Figu e 6A, see also Figu e S1,Tables S4–S6). Howe e , he in ensi ies o emission and exci a ion bands p esen ed consis en a ia ions ha ma ched he end o he ampli ude-weigh ed mean luo escence li e ime (Figu e 6B). A 5 h g ow h, a signi ican dec ease in he ampli ude-weigh ed mean luo escence li e ime wi h he addi ion o STS was obse ed. This dec ease a 5 h o g ow h is con a y o he beha io o -PnA luo escence in ensi y decays (Figu e 3), implying ha di-4-ANEPPS is p obably epo ing he plasma memb ane su ace beha io o di e en domains han -PnA. As a measu e o he memb ane dipole po en ial, he a io o he di-4-ANEPPS luo escence in ensi y by exci a ion a 420 nm o ha p oduced by exci a ion a 520 nm (Rex =IF420/IF520) was calcula ed (Figu e 7). On 5 h-g own cells, STS signi ican ly dec eased Rex, om ∼0.65 o ∼0.61. This beha io co obo a es he esul s o Figu e 6, whe e bo h he s eady-s a e luo escence in ensi y and ampli ude-weigh ed mean luo escence li e ime o di-4-ANEPPS show simila ends. We can now sugges ha hese di e ences may be due o la ge e gos e ol con en , since despi e he absence o shi s o he spec a whe he in he absence o in he p esence o STS, his s e ol is known o inc ease all he o he pho ophysical pa ame e s o di-4-ANEPPS showed in Figu es 6, 7(Bas os e al., 2012a). Rega ding he 15 min s imulus (Figu e 8), he e ec o STS on he s eady-s a e luo escence in ensi y and ampli ude-weigh ed FIGURE 8 | Di-4-ANEPPS luo escence p ope ies in N. c assa cells, a 30◦C, (A) luo escence exci a ion (λem =635 nm) and emission spec a (λexc =450 nm), (B) ampli ude-weigh ed mean luo escence li e ime o di-4-ANEPPS. The esul s p esen he mean ±S.D. o a leas h ee independen expe imen s, n≥3. *p< 0.05, ***p<0.001. F on ie s in Physiology | www. on ie sin.o g 8Oc obe 2018 | Volume 9 | A icle 1375 San os e al. Memb ane Biophysical Response o an An i ungal Agen FIGURE 9 | Ra io o di-4-ANEPPS luo escence in ensi y p oduced by exci a ion a 420 nm o ha p oduced by exci a ion a 520 nm, Rex (420 nm/520 nm) in cells, a 30◦C. The esul s p esen he mean ±S.D. o a leas h ee independen expe imen s, n≥3. ***p<0.001. mean luo escence li e ime o di-4-ANEPPS is simila o he one ob ained o 1 h s imulus (Figu e 6), howe e , less p onounced a bo h g ow h imes. Conside ing he a io, Rex =IF420/IF520, o he 15 min STS challenge (Figu e 9), he e we e no signi ican changes induced by STS a bo h g ow h imes. None heless, no e ha he a io a 6 h g ow h is ca. 0.9. Rega ding he con ol, his alue is conside ably highe han ha obse ed o any o he sho e g ow h imes (Figu e 7 and San os e al., 2017), again e lec ing he change in memb ane composi ion. This ab up change coincides wi h he ansi ion om he conidial o mycelial s age, whe e a ma ked inc ease o e gos e ol con en occu s. STS does no in e ac di ec ly wi h memb ane lipids (Sec ion STS-Lipid In e ac ions). Thus, he di e ences obse ed in he memb ane dipole po en ial, namely he inc ease wi h g ow h ime and he dec ease a 5 h g ow h upon incuba ion wi h STS ela i e o he con ol, can be also explained by he s e ol con en (Renaud e al., 1978). The 1 h ea men wi h he d ug p obably hampe s he de elopmen o he ungus, and he change o g ow h phase om la ency (lag) o exponen ial (log), when mycelium o ms, p e en ing he ise in e gos e ol con en and memb ane dipole po en ial. DISCUSSION P e ious s udies in N. c assa conidia showed ha i is possible o es ablish ela ionships be ween lipid composi ion and dynamics du ing conidial g ow h (San os e al., 2017). In his wo k, his ela ionship was u he exploi ed by s udying he in luence o an STS challenge. The e ec s o STS we e pa icula ly no iceable o he 1 h ea men o conidia ge mina ed o 5 h. These a e he condi ions o which ABC-3 p o ein eaches i s maximum le els a he plasma memb ane. A ha ime poin , he p ocess o STS- d i en p og ammed cell dea h is a i s ea ly s age: eac i e oxygen species had o med, glu a hione e lux is a i s maximum, bu he in eg i y o he memb ane is no ye a ec ed (Fe nandes e al., 2013). The in acellula accumula ion o STS o e ime was ho oughly s udied (Fe nandes e al., 2011). The e is a highe accumula ion o STS a 15 min han a 1 h p obably due o STS expo h ough ABC-3. Howe e , we obse e much s onge changes in memb ane p ope ies ( ela ed o e gos e ol con en ) epo ed by di-4-ANEPPS o he 1 h ea men han o he 15 min, in e es ingly, when we con on he esul s o -PnA luo escence in ensi y decay o he wo STS challenge imes, i is possible o obse e an opposi e beha io o mos o he changing pa ame e s. Al oge he , hese esul s sugges a di e en esponse o he sho e and longe STS ea men s. Upon 15 min ea men wi h STS he signi ican changes epo ed by -PnA a e obse ed only in 3 h-g own cells. The e is an inc ease in he acyl chain packing o he mo e o de ed domains, possibly associa ed wi h he inc emen o he le els o sphingolipids wi h smalle headg oups (e.g., ce amides, which a e known o inc ease in ce ain apop o ic ou es), which induce igh e packing o he lipid acyl chains (Les e e al., 1974). Al hough he inc ease in he alue o τ3seems modes (ca. 2 ns), and he signi icance is p< 0.05, once his alue is con e ed o he a e cons an o non- adia i e p ocesses, kn , conce ning he long-li ed exci ed- s a e -PnA molecules, hese alues become (2.86 ±0.08) ×107s−1and (3.19 ±0.06) ×107s−1, espec i ely o he con ol and ea ed cells, i.e., he e ec su passes 10%, and he s a is ical signi icance is p<0.01). The cons an kn is di ec ly ela ed o he molecula mo ions and collisions esponsible o he non-emissi e elaxa ion o he exci ed p obe molecules, and hus o he mo ions o he su ounding lipid acyl chains, e lec ing he acyl chain packing o he lipids unde physiological condi ions, p o ided ha p obe molecules a e highly dilu ed in he memb ane, which is he case unde ou expe imen al condi ions. This s onge seg ega ion o sphingolipids con ibu es o a dec ease in hei abundance (α3). Bo h hese al e a ions sugges an inc eased sphingolipid ca abolism, which migh be associa ed wi h he ini ia ion o p o- apop o ic signaling. Fo he 1 h STS challenge, an inc ease in he sphingolipid-en iched domain abundance (α3) is obse ed ins ead o bo h g ow h imes, wi hou any change in he acyl chain packing. On ano he hand, he e we e no changes in he pho ophysical p ope ies o di-4-ANEPPS a he ea ly conidial s age (sho e g ow h imes), in clea con as o wha happens o longe g ow h imes, a which he ungus has la ge abili y o exp ess he STS expo e ABC-3. Being a e y la ge ansmemb ane p o ein, om a majo amily ha has been associa ed o lipid me abolism and anspo (Li and P inz, 2004), and o sphingolipid-en iched domains and/ o lipid a s (Hin ichs e al., 2004; Modok e al., 2004), i is concei able ha ABC- 3 exp ession induces changes on he memb ane o ganiza ion, especially in o de ed domains, i.e., hose epo ed, on one hand, by -PnA (abundance inc ease) and on he o he hand by di-4-ANEPPS, which abundance seems o be dec easing, since he inc ease o e gos e ol le els is being limi ed by F on ie s in Physiology | www. on ie sin.o g 9Oc obe 2018 | Volume 9 | A icle 1375