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
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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)
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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
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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
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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 ,
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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.
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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.
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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