CHO/LY-B Cell Growth under Limiting Sphingolipid Supply: Correlation between Lipid Composition and Biophysical Properties of Sphingolipid-Restricted Cell Membranes

The FASEB Jou nal. 2021;35:e21657.
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1 o 23
h ps://doi.o g/10.1096/ j.202001879RR
wileyonlinelib a y.com/jou nal/ sb2
Recei ed: 7 Augus 2020
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Re ised: 9 Ap il 2021
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Accep ed: 26 Ap il 2021
DOI: 10.1096/ j.202001879RR
RESEARCH ARTICLE
CHO/LY- B cell g ow h unde limi ing sphingolipid supply:
Co ela ion be ween lipid composi ion and biophysical p ope ies
o sphingolipid- es ic ed cell memb anes
Bingen G.Monas e io1,2
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NoemiJiménez- Rojo3
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A i z B.Ga cía- A ibas1,2
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Howa dRiezman3
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Félix M.Goñi1,2
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AliciaAlonso1,2
This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion- NonComme cial- NoDe i s License, which pe mi s use and dis ibu ion in any medium,
p o ided he o iginal wo k is p ope ly ci ed, he use is non- comme cial and no modi ica ions o adap a ions a e made.
© 2021 The Au ho s. The FASEB Jou nal published by Wiley Pe iodicals LLC on behal o Fede a ion o Ame ican Socie ies o Expe imen al Biology.
In memo iam MJO Wakelam (1955- 2020), colleague and iend.
Abb e ia ions: AFM, a omic o ce mic oscopy; Ce , ce amide; Chol, choles e ol; FBS, e al bo ine se um; GP, gene alized pola iza ion; GPL,
glyce ophospholipids; GPMV, gian plasma memb ane esicles; GUV, gian unilamella esicles; HexCe , hexosyl ce amide; PM, plasma memb ane; SL,
sphingolipids; SM, sphingomyelin; SPT, se ine palmi oyl ans e ase; SUV, small unilamella esicles.
1Ins i u o Bio isika (CSIC, UPV/EHU),
Uni e sidad del País Vasco, Leioa, Spain
2Depa amen o de Bioquímica, Uni e sidad
del País Vasco, Leioa, Spain
3NCCR Chemical Biology, Depa men
o Biochemis y, Uni e si y o Gene a,
Gene a, Swi ze land
Co espondence
Félix M. Goñi, Ins i u o Bio isika (CSIC,
UPV/EHU), Uni e sidad del País Vasco,
Leioa 48940, Spain.
Email: [email p o ec ed]
Funding in o ma ion
Spanish Minis y o Economy, G an /
Awa d Numbe : PGC2018- 099857- B- I00;
Basque Go e nmen , G an /Awa d Numbe :
IT1264- 19 and IT1270- 19; Swiss Na ional
Science Founda ion, G an /Awa d Numbe :
310030- 184949
Abs ac
Sphingolipids (SL) a e ubiqui ous in mammalian cell memb anes, ye he e is li le
da a on he beha io o cells unde SL- es ic ion condi ions. LY- B cells de i e om
a CHO linein whichse ine palmi oyl ans e ase (SPT), hus de no o SL syn hesis, is
supp essed, while main aining he capaci y o aking up and me abolizing exogenous
sphingoid bases om he cul u e medium. In his s udy, LY- B cells we e adap ed o
g ow in a e al bo ine se um (FBS)- de icien medium o a oid ex e nal up ake o li-
pids. The lowes FBS concen a ion ha allowed LY- B cell g ow h, hough a a slow
a e, unde ou condi ions was 0.04%, ha is, 250- old less han he s anda d (10%)
concen a ion. Cells g own unde limi ing SL concen a ions emained iable o
a leas 72hou s. En iching wi h sphingomyelin he SL- de icien medium allowed
he eco e y o g ow h a es analogous o hose o con ol LY- B cells. S udies in-
cluding whole cells, plasma memb ane p epa a ions, and de i ed lipid esicles we e
ca ied ou . Lau dan luo escence was eco ded o measu e memb ane molecula
o de , showing a signi ican dec ease in he igidi y o LY- B cells, no only in plasma
memb ane bu also in whole cell lipid ex ac , as a esul o SL limi a ion in he
g ow h medium. Plasma memb ane p epa a ions and whole cell lipid ex ac s we e
also s udied using a omic o ce mic oscopy in he o ce spec oscopy mode. Fo ce
measu emen s demons a ed ha lowe b eak h ough o ces we e equi ed o pen-
e a e samples ob ained om SL- poo LY- B cells han hose ob ained om con ol
cells. Mass- spec oscopic analysis was also a help ul ool o unde s and he ea -
angemen unde gone by he LY- B cell lipid me abolism. The mos abundan SL in
LY- B cells, sphingomyelin, dec eased by abou 85% as a esul o SL limi a ion in
he medium, he bioac i e lipid ce amide and he ganglioside p ecu so hexosylce a-
mide dec eased simila ly, oge he wi h choles e ol. Quan i a i e SL analysis showed
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MONASTERIO ET Al.
1
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INTRODUCTION
Sphingolipids (SL) a e cha ac e ized by a sphingoid s uc-
u al backbone, sphingosine being he mos abundan base
in mammals.1 In i o s udies o he SL oles a e hampe ed,
among o he easons, by he ac ha hey can be ei he syn-
hesized de no o o aken up om he die . A plausible ap-
p oach would be o in es iga e mu an cells con aining he
smalles possible amoun s o SL, o e en none a all, bu his
is no a s aigh o wa d p ocedu e because SL appea o be
essen ial o cell g ow h and su i al.2- 4 In pa icula , SL a e
conside ed as ins umen al in he a chi ec u e o euka yo ic
cell memb anes. Aside om s abilizing he lamella s uc-
u e and helping o main ain i s asymme y, he endency o
ce ain SL o unde go la e al phase sepa a ion o o m mic o
o nanodomains has been cha ac e ized.5- 8 Lipids and p o-
eins co- localize wi h hese domains, in which sphingomy-
elin (SM) is he mos abundan SL,9 and choles e ol (Chol)
is o en p esen .7,8 The sphingoid base p o ides SL wi h
hyd ogen- bonding accep o s and dono s (amide and ee hy-
d oxyl g oups, espec i ely) ha a e a e in glyce ophospho-
lipids, leading o a dense in e molecula hyd ogen- bonding
ne wo k.10,11 Hyd ogen bonding allows SM o in e ac p e e -
en ially wi h Chol12,13 and wi h ce amide (Ce ),6 and ecen
s udies ha e ound ha sphingomyelin (SM), Ce and Chol a e
able o coexis in a single e na y gel phase (a a 54:23:23mol
a io) wi h in e media e p ope ies be ween SM- Ce en iched
gel domains and Chol- d i en liquid- o de ed phases.14 The
abo e da a ha e been ob ained mos ly om model memb ane
s udies. In es iga ions a he cellula le el ha e allowed o
assign a wide a ie y o unc ions o SL, including apop o-
sis, cell g ow h, cell memb ane unc ion, umo o ma ion,
d ug esis ance, deg anula ion, and phagocy osis, among
o he s.15- 18 Al e a ions in he no mal ac i i ies o SM- cycle
enzymes ha e been linked o many cen al ne ous sys em-
ela ed pa hologies such as Alzheime 's, Pa kinson's, isch-
emia/hypoxia, dep ession, schizoph enia, o Niemann- Pick
diseases.19 Speci ic SL unc ions ha e o en been cha ac-
e ized in cells wi h dec eased amoun s o SL, using ei he
SL- deg ading enzymes (eg, sphingomyelinases o ce ami-
dases),20 o speci ic enzyme inhibi o s,15,21- 23 o which he
SPT inhibi o my iocin is a good example.24- 27
P ocedu es o dec ease he SL con en s o cells con-
s i u e good ools o de e mine hei po en ial unc ions in
i o. De no o SL biosyn hesis is ini ia ed by he conden-
sa ion o L- se ine wi h palmi oyl CoA. This eac ion is
ca alyzed by SPT o gene a e 3- ke odihyd osphingosine.
3- Ke odihyd osphingosine is hen con e ed o dihyd osphin-
gosine, which is N- acyla ed and (mos o i ) dehyd ogena ed
a he endoplasmic e iculum o o m Ce . A e mo ing o
he Golgi appa a us, Ce is con e ed o SM o glycosphin-
golipids.6,28 Finally, hese complex SL a e ansloca ed o
he PM.29,30 Thus, SPT is a key enzyme o he egula ion o
cellula SL con en .9,29 Using a gene ic selec ion me hod in
CHO cells,31 he Hanada lab isola ed he de ec i e LY- B cell
line, which had a loss o unc ion o se ine palmi oyl ans-
e ase (SPT) enzyme ac i i y h ough a de ec i e SPTLC1
subuni . The mu an cells main ained he abili y o ake up
and me abolize exogenous sphingoid bases om he cul u e
medium.31 Mu an LY- B and wild- ype CHO cells could be
compa a i ely s udied o de e mine he e ec o SL deple ion
on he biophysical p ope ies o cell memb anes. LY- B cells
ha e been used in mul iple s udies explo ing SL e ec s, and
hei in e ac ion wi h glyce ophospholipid me abolism.32- 35
SM syn hases, which use Ce and phospha idylcholine as
subs a es o p oduce SM and diacylglyce ide, a e used in
he de no o syn hesis pa hway, some imes also in ol ed in
eu iliza ion o ce amide30,36 om exogenous o endogenous
sou ces. In a p e ious wo k,37 plasma memb ane (PM) p epa-
a ions om CHO cells and model memb anes p epa ed om
hei lipid ex ac s had been cha ac e ized. In he p esen
s udy, we ha e applied hose me hods o he s udy o SL-
syn hesis- de icien LY- B cells o de e mine he ole o SL on
cell g ow h, memb ane physical p ope ies, and composi ion.
ha a 250- old educ ion in sphingolipid supply o LY- B cells led only o a six old
dec ease in memb ane sphingolipids, unde lining he esis ance o changes in com-
posi ion o hese cells. Plasma memb ane composi ions exhibi ed simila changes,
a leas quali a i ely, as he whole cells wi h SL es ic ion. A linea co ela ion was
obse ed be ween he sphingomyelin concen a ion in he memb anes, he deg ee o
lipid o de as measu ed by lau dan luo escence, and memb ane b eak h ough o ces
assessed by a omic o ce mic oscopy. Smalle , hough signi ican , changes we e also
de ec ed in glyce ophospholipids unde SL- es ic ion condi ions.
KEYWORDS
AFM, CHO, Lau dan, lipidomics, LY- B, mass- spec oscopy, memb ane luidi y, plasma
memb ane, sphingolipids, sphingomyelin
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2
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MATERIALS AND METHODS
2.1
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Cell g ow h
Wild- ype CHO (ATCC, Manassas, Vi ginia, US) and
a se ine- SPT- de icien CHO cell line, known as LY- B31
(RIKEN BioResou ce Resea ch Cen e , Koyadai, Japan),
we e used in his s udy. Unless o he wise men ioned, cells
we e g own on DMEM:F12 (Dulbecco's Modi ied Eagle
Medium: Nu ien Mix u e F- 12) medium con aining 10%
FBS (Fe al Bo ine Se um), 100 U/mL penicillin, 100 U/
mL s ep omycin, and 6mM glu amine (Glu aMax supple-
men ed) a 37°C and 5%CO2 humidi ied a mosphe e. All
cell cul u e p oduc s we e pu chased om The mo ishe
(Wal ham, MA).
2.1.1
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Cell adap a ion: S anda d s. de icien
(low- FBS) medium
CHO and LY- B cells we e adap ed o g ow h in de icien
(low- FBS) medium. Fo his pu pose, cells we e i s seeded in
DMEM:F12 medium con aining 10% FBS, 100 U/ml penicillin
and 100 U/mL s ep omycin, and 6mM glu amine ( his me-
dium will be e e ed o as ”s anda d medium”). A e 24- hou s
cell g ow h, when a 15%- 25% con luence was eached, he
s anda d medium was disca ded, cells we e washed wi h PBS
bu e (137mM NaCl, 3mM KCl, 80mM Na2HPO4, 7mM
KH2PO4), and DMEM:F12 medium con aining 0.04% FBS,
100 U/mL penicillin, 100 U/mL s ep omycin and 6mM glu-
amine was added ( his medium will be named “FBS- de icien ”
o ”SL- de icien medium”). Cells we e g own in he app op i-
a e medium o 24, 48, 72, o 96hou s. O he low- FBS media
(5%, 2.5%, o 1.25%) we e also used in some speci ic cases.
2.2
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G ow h a e and iabili y es s
2.2.1
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Cell g ow h
2.65×105 cells we e seeded in 25- cm2 lasks in s anda d me-
dium and g own o 24hou s un il 15- 25% con luence. Then,
he s anda d medium was disca ded, cells we e washed wice
wi h PBS, and he app op ia e medium (s anda d o de icien )
was added. Cells we e g own o 24, 48, 72, o 96hou s. Cell
g ow h quan i ica ion was pe o med by cell coun ing wi h
a hemocy ome e (BioRad TC20 Au oma ed Cell Coun e ,
He cules, CA). P o ein was assayed wi h he colo ime ic
Pie ce BCA P o ein Assay Ki (The mo ishe , Wal ham,
MA). I was es ed whe he LY- B cells we e able o each he
ull g ow h a es when he SL- de icien medium was supple-
men ed wi h SL. SM (0.2mg/5mL; ≈80µM SM), sphinga-
nine (0.0125mg/5mL ≈8µM), ce eb oside (a 0.2mg/5mL,
≈55 µM ), o sphingosine (0.02 mg/5 mL, ≈13 µM) we e
used o his pu pose.
2.2.2
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Viabili y es
Flow cy ome y was pe o med o e alua e how he dec eased
FBS concen a ion in he medium a ec ed cell iabili y.38
Cells we e s ained wi h Annexin- V- FITC and p opidium
iodide as indica ed in he manual o he annexin V- FITC
de ec ion ki (CalbioChem, Da ms ad , Ge many), and luo-
escence was measu ed using a FACS Calibu low cy ome e
(Bec on- Dickinson, F anklin Lakes, NJ) as in Ahyayauch
e al.39 Annexin V- FITC luo escence in ensi y was meas-
u ed in luo escence channel FL- 1 wi h λex = 488nm and λem
= 530nm, while FL- 3 was used o p opidium iodide de ec-
ion, wi h λex = 532nm and λem = 561nm. All measu emen s
we e pe o med in iplica e. Da a analysis was pe o med
using Flowing So wa e 2.
2.3
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Sample p epa a ion
In ac cells (whole cells), wo di e en PM p epa a ions
(gian plasma memb ane esicles, known as GPMV o blebs,
and PM pa ches) and SUV o GUV o med wi h whole- cell
o PM lipid ex ac s we e used.
2.3.1
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PM p epa a ions
PM p epa a ions we e ob ained as desc ibed in Monas e io
e al.37 B ie ly, GPMV o ma ion was induced adding he
GPMV o ma ion eagen [ eshly p epa ed 2 mM di hi-
o h ei ol, 25mM pa a o maldehyde in GPMV bu e (2mM
CaCl2, 10mM HEPES, 150mM NaCl, pH 7.4)] o T25 lasks
wi h cells a con luence. Cells we e incuba ed o 1hou a
37°C. A e incuba ion, he GPMV- con aining GPMV ea-
gen was collec ed om he lasks and cen i uged a 14000g
o 20minu es. The supe na an was disca ded, he pelle was
e- suspended in GPMV bu e , and he sample was cen i-
uged a 14000g o 20minu es. The p ocedu e was epea ed
wice o emo e aces o di hio h ei ol and pa a o mal-
dehyde. Finally, he GPMV we e esuspended in 500 µL
GMPV bu e .40
PM pa ches we e isola ed by a modi ica ion37 o he p o-
ocol desc ibed by Bez uko e al.41 In summa y, cells we e
seeded a app oxima ely 50% con luence and incuba ed o
2 hso ha hey adhe ed o he suppo . A e incuba ion, 2
washing s eps we e pe o med using cold TBS (T is Bu e
Saline: 150 mM NaCl, 25 mM T is- HCl, 2 mM KCl) o
disca d non- a ached cells. Then, cold dis illed wa e was
added o 2 minu es o induce cell swelling. Mechanical
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MONASTERIO ET Al.
cell dis up ion was achie ed using a p essu e s eam om
a 20- mL sy inge coupled o a 19X1- 1/2(TW)A needle. In
he p ocess, in acellula con en s we e eleased, while PM
s ayed a ached o he suppo . Se e al washing s eps we e
pe o med o disca d he eleased in acellula con en s.
Pu i ica ion quali y was checked using Di- 4- ANEPPDHQ
(λex = 465 nm, λem = 635 nm) as a gene al luo escen
s aining, oge he wi h o ganelle- speci ic luo opho es as
desc ibed in Monas e io e al.37 Images we e aken in a
Leica TCS SP5 II mic oscope (Leica Mic osys ems GmbH,
We zla , Ge many) a oom empe a u e wi h ImageJ so -
wa e. The luo escence in ensi ies o he a ious ma ke s
we e compa a i ely measu ed in PM pa ches and in ac cells,
so ha speci ic o ganelle con amina ion could be es ima ed.
2.3.2
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Whole cell lipid ex ac
Lipid ex ac ion was pe o med ollowing he me hod used
in Ahyayauch e al.39 B ie ly, cell pelle s we e i s dispe sed
in aqueous pe chlo ic acid (60% / ), hen cen i uged a
14000g o 15minu es, and he supe na an was disca ded.
Pelle s we e e- suspended in 2.5 mL chlo o o m:me hanol
(2:1, / ) and samples we e mixed o 15 minu es. Then,
5mL cold 0.1mM HCl was added o he mix u e. A e ho-
mogenizing, samples we e cen i uged a 1,700g o 20min-
u es. Supe na an s we e disca ded while he lipid- con aining
o ganic phase emained in he bo om laye . Phospholipid
concen a ion was assayed as ino ganic phospho us a e acid
diges ion.
2.3.3
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PM pa ch lipid ex ac ion
PM pa ches we e o med ollowing he abo e men ioned
p o ocol.37,41 Chlo o o m:me hanol (2:1) o ganic sol en
was used o eco e he lipid ac ion o he a ached PM
pa ches.
2.3.4
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GUV o ma ion
GUV we e o med in a PRETGUV 4 chambe supplied by
Indus ias Técnicas ITC (Bilbao, Spain) using he modi ied
elec o o ma ion me hod42 i s de eloped by Angelo a and
Dimi o .43
2.3.5
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SUV o ma ion
The sample was kep unde acuum o 2 hou s o e-
mo e sol en aces and he lipids we e swollen in PBS
bu e . SUV we e ob ained by sonica ion o he swollen
lipid suspensions wi h a p obe- ype Sonip ep 150 sonica-
o (MSK, London, UK) o 10minu es, in 10- s on, 10- s
o in e als.
2.4
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SM quan i ica ion wi h lysenin
2.4.1
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Lysenin- mChe y exp ession and
pu i ica ion
The non- oxic monome ic C- e minal domain o he SM-
speci ic oxin, NT- lysenin, was exp essed and pu i ied as
desc ibed by Ca quin e al.44 B ie ly, he exp ession plas-
mid pET28/lysenin encoded NT- lysenin as a usion p o ein
wi h an N- e minal 6xHis- ag ollowed by he monome ic
ed luo escen p o ein mChe y. The plasmid was ex-
panded in Esche ichia coli BL21 (DE3) and he ecombi-
nan p o ein was exp essed in lysogeny b o h (LB) medium
a 16°C o 72hou s in he p esence o 0.4mM isop opyl
β- D- hiogalac oside. Bac e ial ex ac s we e p epa ed as de-
sc ibed45 and he ecombinan p o ein was pu i ied using an
Ni- NTA Supe low ca idge (Qiagen, Hilden, Ge many)
and elu ed wi h imidazol.46 F ac ion analysis by SDS- PAGE
e ealed ecombinan NT- lysenin wi h he expec ed size
(45kDa). The mos en iched ac ions we e pooled, concen-
a ed, and desal ed. The aliquo s we e s o ed in 20mM NaCl
and 25mM Hepes pH 7.2 and 5% glyce ol a −80°C. P o ein
concen a ion was calcula ed by measu ing abso bance a
280nm.
2.4.2
|
SM s aining and quan i ica ion wi h
lysenin- mChe y
Whole cells and PM pa ches we e s ained wi h lysenin-
mChe y( o SM) and NBD- PE [(N- (7- ni obenz- 2- oxa-
1,3- diazol- 4- yl)- 1,2- dihexadecanoyl- sn- glyce o- 3- phospho
e hanolamine, ie hylammonium sal ] luo opho e ( he la -
e a gene al memb ane s ain) and samples we e isualized
using a con ocal mic oscopy Nikon D- ECLIPSE C1 (Nikon,
Mel ille, NY). In whole cells, he mChe y signal was also
quan i ied using a FL- 3 FACS Calibu low cy ome e
(Bec on- Dickinson, F anklin Lakes, NJ) wi h λex = 532nm
and λem = 561nm.
Fo sample isualiza ion, cells we e seeded in glass-
bo om dishes and g own as abo e. Cells we e i s s ained
wi h 100 µM NBD- PE as a con ol o gene al memb ane
s aining. A washing s ep was pe o med wi h PBS, and
lysenin- mChe y was added a 100µM. Fo low cy ome y
analysis, cells we e s ained in suspension a a inal concen-
a ion o 100µM lysenin- mChe y.
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MONASTERIO ET Al.
2.5
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Lau dan gene al pola iza ion
Lau dan is a luo escence pola i y p obe whose emission
unde goes a spec al shi due o he eo ien a ion o wa e
molecules in he glyce ol backbone egion o he memb ane,
and his shi can be co ela ed o he lipid phase.47 In he gel
phase, when li le wa e is p esen , lau dan maximum emis-
sion is a ound 440nm, whe eas in he liquid c ys alline phase
he spec um is ed- shi ed o a ound 490 nm. In ac cells,
PM p epa a ions and model memb anes o med wi h lipid
ex ac s ha e been used o compa e he lau dan luo escence
o CHO and LY- B cells g own in s anda d and de icien
media. Bo h luo escence mic oscopy imaging and spec o-
luo ome ic analysis ha e been pe o med o lau dan luo-
escence cha ac e iza ion.
2.5.1
|
Con ocal mic oscopy
In ac cells g own in glass- bo om dishes we e s ained wi h
lau dan (Molecula P obes, Eugene, OR) as ollows: Fi s ,
cell cul u e medium was disca ded and wo PBS washing
s eps we e pe o med. Then, o lau dan s aining, lau dan
dissol ed in DMSO was added o a inal concen a ion o
5µM and cells we e incuba ed a 37°C o 5minu es. Finally,
a washing s ep wi h PBS was conduc ed p io o cell isu-
aliza ion. Simila ly, PM pa ches o med as desc ibed abo e
we e s ained wi h 5µM lau dan.
GPMV we e s ained wi h lau dan by adding 5µM ( inal
concen a ion) o he luo opho e dissol ed in DMSO.
GPMV we e ans e ed o polylysine- coa ed glass- bo om
dishes (Ma Tek, Ashland, OR) and esicles we e le o sed-
imen o 3hou s be o e isualiza ion.40 Fo GUV, 0.2mM
lipid ex ac s in chlo o o m:me hanol (2:1, / ) we e mixed
wi h 0.01 mM lau dan. A quan i y o 3 µL o he lipid
s ocks we e added on o he su ace o P elec odes and
sol en aces we e emo ed unde high acuum o a leas
2hou s. The P elec odes we e hen co e ed wi h 400μL
o 300mM suc ose bu e and he P wi es we e connec ed
o an elec ic wa e gene a o (TG330 unc ion gene a o ,
Thu lby Thanda Ins umen s, Hun ing on, UK) unde al-
e na ing cu en ield condi ions (10 Hz, 2.5 VRMS o
2hou s) a 37°C. A e GUV o ma ion, he chambe was
placed on an in e ed con ocal luo escence mic oscope o
GUV isualiza ion.
2.5.2
|
Image acquisi ion and analysis
A Leica TCS SP5 II mic oscope (Leica Mic osys ems
GmbH, We zla , Ge many) was used o image acquisi ion.
A 63x wa e - imme sion objec i e (nume ical ape u e NA
=1.2) was used and samples we e imaged a 512 × 512 pixel
and 400Hz pe scanning line. Equa o ial planes we e imaged
o a oid pho oselec ion e ec s. A pulsed i anium- sapphi e
(Mai- Tai Deepsee, Spec a- Physics) lase uned a 780 nm
was used o wo- pho on imaging o lau dan- labeled sam-
ples. Fluo escence emission was collec ed by non- descanned
(NDD) hyb id de ec o s, as hey o e highe sensi i i y com-
pa ed o descanned pho omul iplie s. The blue edge o he
emission spec um was collec ed by NDD 1 a 435±20nm
and he ed edge by NDD 2 a 500 ± 10 nm. I adiance
a he sample plane was ≈500 GW·cm– 2 o wo- pho on
exci a ion.48
Gene alized pola iza ion (GP) alue o samples was cal-
cula ed using a MATLAB (Ma hWo ks, Na ick, MA)- based
so wa e. Images we e smoo h in each channel wi h 2 pixel
a e aging, and he GP alue was calcula ed using he ollow-
ing Equa ion49:
whe e IB is he in ensi y collec ed by NDD 1, IR is he in ensi y
collec ed by NDD 2, and Gis he co ec ion ac o . The G ac o
is calcula ed measu ing he GP alue o he same luo opho e
concen a ion used in sample s aining, dissol ed in his case in
pu e DMSO.50 In whole cell images, he egion o in e es , ha
is, he PM, was selec ed when equi ed.
2.5.3
|
Fluo escence spec oscopic analysis
PM p epa a ions, and SUV o med wi h whole cell o
PM lipid ex ac s we e measu ed in a spec o luo om-
e e . Samples (82.5µM lipid concen a ion) we e labeled
wi h 0.75µM lau dan. Fo his pu pose, lipid ex ac s in
chlo o o m:me hanol (2:1) we e mixed wi h lau dan and
he sol en was e apo a ed o d yness unde a s eam o
N2. Then, he sample was kep unde acuum o 2hou s
o emo e sol en aces and he lipids we e swollen in
bu e (NaCl 150mM, Hepes 25mM, pH 7.4). Sonica ed
SUV we e ob ained as desc ibed abo e and luo escence
measu emen s we e pe o med using a Quan aMas e 40
spec o luo ome e (Pho on Technology In e na ional,
Law ence ille, NJ).51
2.6
|
AFM
Con ac mode AFM imaging has been used o s udy bilaye
opog aphy, looking a possible la e al seg ega ion e ec s
h ough bilaye hickness analysis. A NanoWiza d II AFM
(JPKIns umen s, Be lin, Ge many) was used o pe o m
opog aphic measu emen s unde con ac mode scanning
(cons an e ical de lec ion). Fo measu emen s, he AFM
GP
=
I
B−
G
⋅
IR
I
B
+G⋅I
R

6 o 23
|
MONASTERIO ET Al.
was coupled o a Leica mic oscope and moun ed on o a
Halcyonics Mic o 40 an i ib a ion able (Halcyonics, Inc,
Menlo Pa k, CA) and inside an acous ic enclosu e (JPK
Ins umen s, Be lin, Ge many).52 V- shaped MLCT Si3N4
can ile e s (B uke , Bille ica, MA) wi h nominal sp ing con-
s an s o 0.1 o 0.5N/m we e used. The sample hickness was
es ima ed by c oss- sec ion heigh analysis.53
2.6.1
|
Fo ce spec oscopy
V- shaped MLCT Si3N4 can ile e s (B uke , Bille ica, MA)
wi h nominal sp ing cons an s o 0.1 o 0.5 N/m we e in-
di idually calib a ed in a lipid- ee mica subs a e in assay
bu e using he he mal noise me hod. A e p ope bilaye
a ea localiza ion by means o AFM opog aphy and di ec
epi luo escence mic oscopy, o ce spec oscopy was pe -
o med a a speed o 1μm/s. Fo ce s eps we e de e mined o
each o he inden a ion cu es as ep oducible jumps wi hin
he ex ended aces. A leas h ee independen sample p epa-
a ions we e scanned o each case and 50- 100 cu es we e
measu ed in each sample.
Topog aphic images and o ce spec oscopy analysis o
PM pa ches and suppo ed plana bilaye s (SPB) o med
om lipid ex ac s, and o ce spec oscopy analysis o
GPMV we e pe o med. GPMV opog aphic obse a-
ions could no be pe o med o expe imen al easons;
hese s uc u es would no la en on he mica o AFM
examina ion.
SPB we e p epa ed on high V- 2 quali y sc a ch- ee mica
subs a es (Ashe ille- Schoonmake Mica Co., Newpo
News, VA). A quan i y o 180 μL assay bu e con aining
3mM CaCl2 was added on o a 1.2- cm2 eshly clea ed mica
subs a e moun ed on o a BioCell (JPK Ins umen s, Be lin,
Ge many). Then, 80μL sonica ed 0.4mM SUV o med wi h
CHO o LY- B lipid ex ac was added on op o he mica.
BioCell empe a u e was g adually inc eased (5°C e e y
5minu es) up o 80°C. Vesicles we e le o adso b and ex-
end o 30minu es keeping he sample empe a u e a 80°C.
Samples we e le o equilib a e o 30minu es a oom em-
pe a u e be o e pe o ming i e washing s eps wi h CaCl2-
ee bu e in o de o disca d non- adso bed esicles and
emo e he emaining Ca2+ ca ions.52 Isola ed PM pa ches
o o ce spec oscopy we e p epa ed as p e iously de-
sc ibed,37,41 his ime using polylysine- coa ed mica slips in-
s ead o glass- bo om dishes. GPMV we e i s s ained using
Di- 4- ANEPPQHD o allow de ec ion on he mica slip. Then,
samples we e le o 3hou s o sedimen o e he polylysine-
coa ed mica slip be o e measu emen s we e pe o med.
2.7
|
Mass spec oscopic analysis
Mass spec oscopic analysis was pe o med essen ially as
desc ibed in Monas e io e al.37 A me hodological summa y
ollows.
2.7.1
|
Sample ea men
Lipid ex ac ion was pe o med using a modi ied me hyl
e - bu yl e he (MTBE) p o ocol.54 B ie ly, cells we e
washed wi h cold PBS and sc aped o in 500μL cold PBS
on ice. The suspension was ans e ed o a 2- ml ube in
which i was spun down a 3200 pm o 5minu es a 4°C.
A e emo ing he PBS, samples we e s o ed a −20°C o
di ec ly used o u he ex ac ion. GPMV and PM pa ch
samples we e p epa ed as p e iously men ioned. Then,
360μL o me hanol was added and o exed. A mix u e o
lipid s anda ds (see Table1) was included and samples we e
o exed o 10minu es a 4 ºC using a Cell Dis up o Genie
(Scien i ic Indus ies, Inc, Bohemia, NY). MTBE (1.2mL)
was hen added and he samples we e incuba ed o 1hou a
oom empe a u e wi h shaking (750 pm). Phase sepa a ion
TABLE 1 MS de ec ion condi ions o he di e en lipid classes
Lipid class S anda d Pola i y Mode m/z ion
Collision
ene gy
Phospha idylcholine [M+H]+DLPC + P oduc ion 184.07 30
Phospha idyle hanolamine [M+H]+PE31:1 + Neu al ion loss 141.02 20
Phospha idylinosi ol [M- H]- PI31:1 − P oduc ion 241.01 44
Phospha idylse ine [M- H]- PS31:1 − Neu al ion loss 87.03 23
Ca diolipin [M- 2H]2- CL56:0 − P oduc ion acyl chain 32
Ce amide [M+H]+C17Ce + P oduc ion 264.34 25
Dihyd oce amide [M+H]+C17Ce + P oduc ion 266.40 25
Hexosylce amide [M+H]+C8GC + P oduc ion 264.34 30
Hexosyldihyd oce amide [M+H]+C8GC + P oduc ion 266.40 30
Sphingomyelin [M+H]+C12SM + P oduc ion 184.07 26
|
7 o 23
MONASTERIO ET Al.
was induced by adding 200μL H2O. A e 10minu es in-
cuba ion a oom empe a u e, he sample was cen i uged
a 1,000 x g o 10minu es. The uppe (o ganic) phase was
ans e ed o a 13- mm sc ew- cap glass ube and he lowe
phase was ex ac ed wi h 400 μL a i icial uppe phase
(MTBE/me hanol/wa e (10:3:1.5, / / )). The wo uppe
phases we e combined and he o al lipid ex ac was di-
ided in o h ee equal aliquo s [one o phospholipids (TL),
one o s e ols (S) in 2- mL ambe ials, and one o sphin-
golipid (SL) de ec ion in a 13- mm glass ube] and d ied
in a Cen i ap a 50°C o unde a ni ogen low. The SL
aliquo was deacyla ed by me hylamine ea men (Cla ke
me hod) o emo e glyce ophospholipids. 0.5 mL mono-
me hylamine eagen [MeOH/H2O/n- bu anol/me hylamine
solu ion (4:3:1:5 / )] was added o he d ied lipid, ol-
lowed by sonica ion (5minu es). Samples we e hen mixed
and incuba ed o 1hou a 53°C and d ied (as abo e). The
monome hylamine- ea ed lipids we e desal ed by n- bu anol
ex ac ion. 300 μL H2O- sa u a ed n- bu anol was added o
he d ied lipids. The sample was o exed, sonica ed o
5minu es, and 150μL o MS- g ade wa e was added. The
mix u e was o exed ho oughly and cen i uged a 3200 x
g o 10minu es. The uppe phase was ans e ed o a 2- mL
ambe ial. The lowe phase was ex ac ed wice mo e wi h
300μL H2O- sa u a ed n- bu anol and he uppe phases we e
combined and d ied (as abo e).
2.7.2
|
Glyce ophospholipid and sphingolipid
de ec ion in a T iple Quad upole Mass
Spec ome e
TL and SL aliquo s we e esuspended in 250μL chlo o o m/
me hanol (1:1 / ) (LC- MS/HPLC g ade) and sonica ed o
5minu es. The samples we e pipe ed in a 96- well pla e ( inal
olume =100μL). The TL we e dilu ed 1:4 in nega i e- mode
sol en (chlo o o m/me hanol (1:2) + 5mM ammonium ac-
e a e) and 1:10 in posi i e- mode sol en (chlo o o m/me ha-
nol/wa e (2:7:1 / ) + 5mM ammonium ace a e). The SL
we e dilu ed 1:10 in posi i e- mode sol en and in used on o
he mass spec ome e . Tandem mass spec ome y o he
iden i ica ion and quan i ica ion o sphingolipid molecula
species was pe o med using Mul iple Reac ion Moni o ing
(MRM) wi h a TSQ Van age T iple S age Quad upole Mass
Spec ome e (The mo ishe Scien i ic, Wal ham, MA)
equipped wi h a obo ic nano low ion sou ce, Nanoma e HD
(Ad ion Biosciences, I haca, NY). The collision ene gy was
op imized o each lipid class. The de ec ion condi ions o
each lipid class a e lis ed in Table1. Ce species we e also
quan i ied wi h a loss o wa e in he i s quad upole. Each
biological eplica was ead in wo echnical eplicas (TR).
Each TR comp ised h ee measu emen s o each ansi ion.
Lipid concen a ions we e calcula ed ela i e o he ele an
in e nal s anda ds and hen no malized o he o al lipid con-
en o each lipid ex ac (mol %).
2.8
|
Gas ch oma og aphy- mass
spec ome y o choles e ol assay
Lipid ex ac s we e analyzed by GC- MS as desc ibed p e i-
ously.55 B ie ly, samples we e injec ed in o a VARIAN CP-
3800 gas ch oma og aph equipped wi h a Fac o Fou Capilla y
Column VF- 5ms 15m×0.32mm i.d. DF =0.10, and ana-
lyzed in a Va ian 320 MS iple quad upole wi h elec on
ene gy se o – 70eV a 250°C. Samples we e applied o he
column o en a 45°C, held o 4minu es, hen empe a u e was
aised o 195°C (20°C/min). S e ols we e elu ed wi h a linea
g adien om 195 o 230°C (4°C/min), ollowed by ising o
320°C (10°C/min). Choles e ol was iden i ied by i s e en ion
ime (compa ed wi h an e gos e ol s anda d) and agmen a ion
pa e ns, which we e compa ed wi h he NIST lib a y.
2.9
|
Quan i a ion o lipids pe cell
An es ima e o he amoun s o lipids pe cell, o pe weigh
p o ein, was ob ained as ollows. Szelio a e al56 measu ed
he a e age d y weigh o CHO cells as 264pg/cell. Albe s
e al57 indica ed ha he mammalian cell con ained 10 d y
w % phospholipids and 7 d y w % o he lipids. Mo eo e , he
a e age amoun o p o ein pe cell was measu ed expe imen-
ally wi h he BCA p o ein assay. Cell numbe s we e coun ed
wi h a hemocy ome e . F om he abo e da a, and knowing
om MS analysis he concen a ion o a gi en lipid in a sam-
ple, he amoun o such lipid pe cell and pe w p o ein could
be es ima ed.
3
|
RESULTS
3.1
|
CHO- de i ed mu an cells can g ow
and su i e wi h ex emely low sphingolipid
concen a ions in he cul u e medium
3.1.1
|
G ow h and iabili y
The ex en o which CHO (wild ype) and LY- B (SPT- de ec i e)
cell adap a ion o a SL- de icien medium a ec ed cell di ision
a io and in eg i y was assessed. FBS was he only ex e nal
sou ce o SL o cell g ow h, hus FBS in he g ow h medium
was he only SL sou ce o LY- B cells. SL- de icien g ow h
media we e p epa ed con aining 0.04% FBS, ha is, a 250- old
dec ease wi h espec o he s anda d condi ions (10% FBS).
Cell coun measu emen s we e pe o med using a BioRad
TC20 hemocy ome e . Figu e1A shows a compa ison be ween
8 o 23
|
MONASTERIO ET Al.
cell g ow h in s anda d (con aining 10% FBS) o SL- de icien
(con aining 0.04% FBS) medium. Bo h cell lines (CHO and
LY- B) g ew s eadily o a leas 96hou s in ull medium (10%
FBS), and bo h di ided, e en i slowly, o he i s 72hou s in a
low- SL medium. A e 72hou s in he SL- limi ed medium cell
quan i y dec eased. The di e ence be ween CHO and LY- B
|
9 o 23
MONASTERIO ET Al.
cell- g ow h a ios was no s a is ically signi ican when hey
we e g own in s anda d medium. Ne e heless, in SL- de icien
medium CHO and LY- B cells beha ed di e en ly. A e
72hou s, CHO cell numbe was 47% o con ol (comple e me-
dium), while LY- B g ow h was only 18%. G ow h a es o cells
ea ed wi h he SPT inhibi o my iocin (2.5µM) 58 and g own
in de icien medium we e also measu ed as an addi ional con-
ol. CHO cell g ow h a io was dec eased o he le el o LY- B
cells g own in de icien medium. Mo eo e , my iocin ea men
did no a ec he g ow h o LY- B cells (da a no shown). The
esul s concu in sugges ing ha he di e en beha io o CHO
and LY- B cells in ou s udy is due o he lack o an ac i e SPT
in he la e s ain.
Ancilla y expe imen s we e pe o med in which cell
g ow h a e 72hou s in media wi h di e en deg ees o SL-
limi a ion was measu ed (Figu esS1A,B). Cell coun s we e
pe o med as in Figu e 1 (Figu e S1A) and o al cell p o-
ein was quan i ied wi h a BCA p o ein assay (Figu eS1B).
A e 72hou s, di e ences in g ow h we e al eady signi i-
can be ween CHO and LY- B cells when medium con ained
5% FBS. No LY- B cell g ow h could be eliably measu ed
wi h FBS concen a ions below 0.04%. In a di e en se ies o
expe imen s, cell g ow h was in ended on delipida ed FBS-
con aining medium, bu no cell di ision could be obse ed.
To asce ain ha he di e ence be ween CHO and LY- B cell
di ision a io a low FBS concen a ions was indeed due o a
lack o SL, we es ed whe he LY- B cells we e able o each he
ull g ow h a es when he SL- de icien medium was supple-
men ed wi h SL. Fo his pu pose, equimola mix u es o egg
PC and he sphingolipid unde s udy we e sonica ed in bu e
and added o he cul u e lasks in a ious amoun s. The bes
eco e ies we e achie ed wi h sphingomyelin (SM) o sphin-
ganine (Figu eS1E). Figu e1B shows ha LY- B cells g own in
SL- de icien medium o 72hou s eached ≈80% o he con ol
g ow h when 0.2mg SM was added pe T25 cul u e lask (5mL;
80µM SM), while he e was no di e ence in he case o CHO
cells. Wi h 0.0125mg sphinganine/ lask (8µM), cells g own in
SL- de icien medium unde he same condi ions eco e ed 81%
o he high- FBS con ol alue (Figu eS1C). Supplemen a ion
wi h pu e PC esicles did no ha e any e ec when compa ed
o he g ow h o LY- B cells in he non- supplemen ed, FBS-
de icien medium (Figu eS1E). Ce eb oside (16% eco e y a
0.2mg/ lask, 55µM) and sphingosine (33% eco e y a 0.02mg/
lask, 13µM) supplemen a ions we e also es ed (Figu eS1E).
I was concluded ha i is he lack o SPT ac i i y wha makes
he main di e ence be ween CHO and LY- B cell di ision a ios
in SL- de icien medium.
As a u he con ol o asce ain ha he main e ec s o
lowe ing FBS concen a ion we e due o he low supply o
SL, he ime- cou se o cell g ow h in SL- de icien medium
supplemen ed wi h SM and ea ed wi h sphingomyelinase
inhibi o s was measu ed (Figu e1©). When cells we e ea ed
wi h5 µM acid sphingomyelinase inhibi o luphenazine di-
hyd ochlo ide o 20µM neu al sphingomyelinase inhibi o
GW4869, he e ec ha SM addi ion had on cell g ow h e-
co e y was supp essed. The e was no s a is ically signi ican
di e ence be ween he g ow h o cells ea ed wi h sphin-
gomyelinase inhibi o g own in SM supplemen ed de icien
medium and he non- ea ed LY- B cells g own in de icien
medium (Figu e1C).
The possible e ec o low cell g ow h/ SL- de icien me-
dium on cell iabili y was hen es ed using low cy ome-
y analysis wi h Annexin- V- FITC and p opidium iodide.
Flow cy ome y analyses demons a ed ha , despi e he low
g ow h a e, 86% o LY- B cells g own in SL- de icien me-
dium o 72hou s emained iable (Figu eS2E). E hanol-
ea ed CHO cells (Figu e S2A) we e used as a posi i e
con ol o non- iable cells. 95% CHO cells (Figu eS2B)
and 95% LY- B cells (Figu eS2D) g own in s anda d medium
we e iable. Wi h espec o cells g own in SL- de icien me-
dium, 92% CHO (Figu eS2C) and 86% LY- B (Figu eS2E),
as well as 89% LY- B g own in SM- supplemen ed medium
(Figu e S2F) we e iable. These plo s a e ep esen a i e
da a used o quan i y esul s shown in Figu e1A and S1.
Conside ing ha e en LY- B cells g own in 0.04 FBS e-
ained a ai iabili y, hese cells we e conside ed as a good
ool o ob ain eliable in o ma ion on he pu a i e e ec s
o a de ec i e SPT ac i i y on hei biophysical p ope ies.
3.1.2
|
Lysenin s aining
Cells we e s ained wi h SM- speci ic NT- lysenin- mChe y
and isualized wi h con ocal mic oscopy. LY- B cells g own
FIGURE 1 LY- B cells can g ow on e y small amoun s o sphingolipids. A, LY- B (emp y symbols) and CHO ( illed symbols) cell g ow h
as a unc ion o ime in s anda d (10% FBS) ( iangles) and sphingolipid- de icien (0.04% FBS) (ci cles) medium. Inse : LY- B cell g ow h in
sphingolipid- de icien medium, Y- axis expanded (B) LY- B and CHO cell g ow h a e 72hou s in sphingolipid- de icien medium supplemen ed
wi h SM (seeded cells: 0.25 × 106; medium olume =5mL)). C, LY- B cell g ow h as a unc ion o ime in sphingolipid- de icien (0.04% FBS)
medium supplemen ed wi h SM (0.2mg ≈ 80µM) (ci cles) and ea ed wi h sphingomyelinase inhibi o s, luphenazine o GW4869 (squa es)
(seeded cells: 0.25 × 106). In A- C, da a co espond o a e age alues ± SD (n=3). In A, e o ba s a e smalle han he symbols. Fluo escence
images and low cy ome y- media ed mChe y- lysenin ( ed) quan i ica ion. CHO (D) and LY- B (E) cells g own in s anda d medium. CHO (F) and
LY- B (G) cells g own in SL- de icien medium. NBD- PE (g een) was used in luo escence images o gene al memb ane s aining. Measu emen s
a e shown a e 72- h g ow h. Geome ic mean ± SD (n=3). H, Flow cy ome y: ime- dependen mChe y- lysenin quan i ica ion in LY- B cells
g own ( om le o igh ) in s anda d medium, o de icien medium o 24, 48hou s, and 72hou s. His og ams in black co espond o con ol cells
(wi hou mChe y- lysenin s aining) and hose in ed, o he sample o in e es (mChe y- lysenin signal). Geome ic mean ± SD (n=3)
16 o 23
|
MONASTERIO ET Al.
in medium con aining di e en FBS concen a ions. These
h ee speci ic SL we e selec ed among he lipidomic da a e-
spec i ely because SM is he mos abundan SL, Ce is pa ic-
ula ly impo an in cell signaling, and HexCe is a he o igin
o he biosyn he ic pa hway leading o he complex glyco-
sphingolipids. Signi ican di e ences we e seen be ween SM
amoun s in CHO and LY- B cells (Figu e 6A). When cells
we e g own in s anda d medium, he SPT- de icien LY- B
cells con ained 43% less SM han CHO cells.
When he amoun o FBS in he medium was dec eased,
o al SM was also lowe in bo h wild ype and mu an cells.
Ne e heless, he dec ease was g ea e in LY- B han in CHO
cells, 90% e sus 25% wi h he lowes FBS concen a ion
(0.04%) (Figu e 6A). In cells g own in s anda d medium,
FIGURE 6 Lipidomic analysis o CHO and LY- B cells. To al SM (A), Ce (B) and HexCe (C) om CHO (black ba s) o LY- B (g ay ba s)
whole cells g own in media con aining a ious concen a ions o FBS. D, E, A compa ison o lipid composi ions o whole cell and PM pa ches
o LY- B cells g own in s anda d o sphingolipid- de icien (0.04% FBS) media. Only selec ed lipids a e included in he igu e. A comp ehensi e
desc ip ion o he a ious lipid composi ions can be seen in he Supplemen a y Ma e ial TableS1 and Figu e7. (F) Fully sa u a ed (DB0 = no
double bonds) and (G) sho - chain (30- 32C) GPL o whole cells ea ed o GPMV p epa a ion, GPMV, cells ea ed o PM pa ch p epa a ion,
and PM pa ches. n=3. S a is ical signi icance was calcula ed wi h ANOVA o S uden ´s - es , wi h simila esul s. Signi icance: (*) P<.05; (**)
P<.01; (***) P<.001. Lipid pe cen ages we e compu ed o e he o al lipid amoun measu ed wi h he mass spec ome e

|
17 o 23
MONASTERIO ET Al.
LY- B con ained 70% less Ce han CHO cells. When FBS
concen a ion was dec eased, he o al Ce amoun was also
diminished in bo h cell lines. As wi h SM, he educ ion was
la ge in LY- B han in CHO cells, 66% s. 38% (in 0.04%
FBS- con aining medium). Figu e6C shows he co espond-
ing alues o HexCe . LY- B g own in 10%- FBS medium
con ained 70% less HexCe han CHO, in ag eemen wi h
he Ce da a (Figu e 6B). When FBS in he medium was
dec eased, HexCe also dec eased by 50% in LY- B cells
g own in 0.04% FBS- con aining medium, bu i inc eased
by 85% in CHO (Figu e6C). Conside ing he PM pa ches,
SM, Ce and HexCe we e all lowe in LY- B 0.04 PM pa ches
han in LY- B 10 ones. In summa y, in LY- B cells he h ee
SL unde s udy exhibi ed a simila dec ease (55%- 70%) wi h
he educ ion o FBS, a a iance wi h CHO cells, sugges ing
ha , in he la e , an ac i e de no o SL syn hesis could occu .
FIGURE 7 Lipidomic analysis o whole cells and plasma memb ane p epa a ions. SM (A), Ce (B), Hex Ce (C), Chol (D), PC (E), PE (F),
PI (G), PS (H), CL (I). J- L: GPL sa u a ion dis ibu ion (numbe o double bonds pe GPL molecule). No double bonds (DB 0) (J), one double bond
pe molecule (DB 1) (K), wo – six double bonds pe molecule (DB 2- 6) (L). M- O: GPL leng h dis ibu ion (numbe o C a oms in he wo acyl
chains). 30- 32 C (M), 34- 40 C (N), 42- 44 C (O). Mean alues ± SD (n=3). S a is ical signi icance was calcula ed wi h ANOVA and S uden ´s
- es : (*) P<.05; (**) P<.01; (***) P<.001. Lipid pe cen ages we e compu ed o e he o al lipid amoun measu ed wi h he mass spec ome e
18 o 23
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MONASTERIO ET Al.
The si ua ion wi h GPMV was, howe e , di e en , mainly in
ha Ce and HexCe inc eased when he cells (ei he CHO o
LY- B) we e g own in 0.04 FBS medium (Figu e7B,C).
Wi h low- FBS medium, Chol le els we e dec eased in bo h
cell lines (Figu e7D). Since FBS is a majo sou ce o lipids and
p o eins o cell g ow h unde ou condi ions, he d as ic educ-
ion om 10% o 0.04% in he SL- de icien medium induces
pa ial cell s a a ion. Chol syn hesis is known o dec ease in
as ing condi ions.67- 69 Mo eo e , LY- B PM p epa a ions had
la ge amoun s o Chol han he whole cell a e age, o cells
g own in high- and low- FBS (Figu e6D). This had been ob-
se ed by Monas e io e al37 o he case o CHOcells.
Glyce ophospholipid (GPL) acyl chain also changed
along wi h SL dep i a ion. In Figu e6E we can see ha e he
PC was inc eased in LY- B 0.04 cells. GPL acyl chain sa u-
a ion and leng h also play an impo an ole in he physical
p ope ies o he memb ane bilaye , speci ically on i s diso -
de / luidi y. Speci ically, unsa u a ed and sho e acyl- chain-
con aining GPL inc ease memb ane luidi y.70 In Figu e6F,
he dis ibu ion o ully sa u a ed GPL o con ol and wo PM
p epa a ions (pa ches and GPMV) is shown. PM p epa a-
ions had mo e ully sa u a ed and less polyunsa u a ed (2- 6
double bounds) GPL (Figu e7J- L) han hei espec i e con-
ols in all measu ed samples.37 The di e ences we e la ge
in he case o GPMV. Compa ing CHO and LY- B g own in
s anda d medium, LY- B had mo e ully sa u a ed GPL in all
cases. In addi ion, when FBS in he medium was dec eased,
he sa u a ed GPL inc eased in LY- B, while in CHO cells
hey emained almos cons an (Figu e6F).
As o he GPL chain leng h dis ibu ion, PM p epa a-
ions we e iche in 30- 32 C chains han whole cells, he
di e ence being la ge o he GPMV (Figu e 6G).37 GPL
o LY- B g own in s anda d medium con ained mo e 30- 32
C acyl chains han CHO 10 in all measu ed samples. This
di e ence was inc eased when LY- B we e g own in 0.04%
FBS medium. Con e sely, CHO 0.04 chain- leng h alues e-
mained cons an (Figu e6G). In summa y LY- B cells syn he-
sized sho e and mo e sa u a ed GPL in hei homeos a ic
esponse o SM deple ion. A comp ehensi e desc ip ion o
he a ious lipid composi ions can be seen in he TableS1
and Figu e7.
The homeos a ic esponse unde gone by LY- B cells g own
in de icien medium and supplemen ed wi h SM (Figu e5E)
was also obse ed. Da a a e shown as pe cen inc ease o de-
c ease when compa ed o he amoun s ound in CHO cells
g own in de icien medium. SM and Ce le els we e o ally
eco e ed, becoming e en highe han he con ol alues.
Howe e , o easons ha emain unexplained, he HexCe
CHO amoun s we e no eached. PC e he s inc eased when
LY- B cells we e g own in de icien medium; his inc ease
was supp essed wi h SM supplemen a ion. Fully sa u a ed
GPL le els we e sligh ly dec eased wi h SM supplemen a-
ion (Figu e5E).
A quan i a i e es ima e o he amoun o SM and Chol,
wo ep esen a i e lipids in his con ex , was ca ied ou as de-
sc ibed unde Me hods. The esul s can be seen in Figu eS8.
Wi h espec o Chol, he concen a ion in CHO cells g own
in SL- medium was 33% o hose g own in s anda d medium
(da a in pg Chol/cell), and he co esponding igu e o LY- B
was 45% (Figu eS8B). As o SM da a, CHO cells g own on
medium wi h 0.04% FBS con ained 68% o he SM ound in
cells g own on 10% FBS (da a in pg SM/cell) (Figu eS8B),
o 53% (in pg SM/ng p o ein) (Figu eS8A). Fo he SPT-
de ec i e LY- B cells, he co esponding igu es a e 15%
(Figu eS8B), and 16% (Figu eS8A). Thus, a 250- old e-
duc ion in sphingolipid supply o LY- B cells leads o a 6- old
dec ease in memb ane sphingolipids.
As a summa y o he lipidomic esul s, SM, Ce , and
HexCe concen a ions we e lowe in LY- B PM pa ches han
in CHO ones when g own in s anda d medium. All h ee SL
we e simila ly dec eased (55%- 70%) wi h he educ ion in
FBS. LY- B cells con ained la ge amoun s o Chol han CHO
ones in bo h s anda d and SL- de icien media. Wi h espec
o he GPL a y acyl dis ibu ion, LY- B had mo e sa u a ed
and sho e GPL a y acids han CHO cells. These g oups o
a y acids, oge he wi h PC e he s, we e inc eased in LY- B
and main ained in CHO when FBS concen a ion was de-
c eased. Fo LY- B in SL- de icien medium, SM dec eased
bo h in GPMV and in PM pa ches bu Ce and HexCe we e
inc eased wi h lowe FBS concen a ions (Figu e7A- C).
4
|
DISCUSSION
LY- B cells g own in SL- de icien medium we e used o
unde s and he e ec s ha a de ec i e SPT ac i i y migh
ha e on he biophysical p ope ies o he cell. SPT- de ec i e
cells g own wi h e y low SL concen a ions we e iable
(Figu eS2E) and hey we e able o eco e he con ol g ow h
a es when he SL- de icien medium was supplemen ed wi h
SM (Figu e1B) o sphinganine (Figu eS1C).
4.1
|
CHO and LY- B cells g own in
s anda d medium
4.1.1
|
Whole cells
Compa ing SL le els in CHO and LY- B cells g own in s and-
a d medium, hey happened o be ma kedly lowe in he mu-
an cells. Conside ing he h ee mos abundan SL, SM was
43% lowe in LY- B cells (Figu e 6A), Ce was 66% lowe
(Figu e 6B), and HexCe was 70% lowe (Figu e 6C). This
indica ed ha he de no o pa hway could be a majo SL syn-
hesis sou ce. This esul is in ag eemen wi h he one pub-
lished by Ziulkoski e al30 whe e hey used umonisin B1 and
|
19 o 23
MONASTERIO ET Al.
β- chlo oalanine o de e mine he con ibu ion o he di e en
pa hways o he syn hesis o SM in Se oli cells. They ound
ha 40% o 16:0 and 61% o 18:0, 18:1 and 18:3 SM was syn-
hesized by he de no o pa hway. They also obse ed ha hese
alues could be inc eased when he equi emen o cell mem-
b anes was g ea e , as in apidly di iding cells.30 In con as
wi h he SL esul s, Chol concen a ions we e simila in CHO
and LY- B cells g own in s anda d medium (Figu e7D). In ad-
di ion, compa ison o bo h kinds o cells in s anda d medium
showed small changes in GPL, LY- B con ained mo e ully sa -
u a ed, and less monounsa u a ed GPL han CHO, chain leng h
dis ibu ions in GPL being i ually he same (Figu e6F,G).
4.1.2
|
PM p epa a ions
Impo an di e ences we e ound be ween he whole cell and
PM lipid composi ions, as an icipa ed om he s udies in CHO
by Monas e io e al37 Bo h o CHO and LY- B p epa a ions,
PM pa ches con ained less SM (abou one hal ), mo e HexCe
and mo e Chol han he whole cells. GPMV had also less SM
bu con ained highe amoun s o HexCe , and pa icula ly o
Ce and Chol. Changes in GPL we e mode a e o low, excep
o ca diolipin, ha was almos absen in he PM p epa a ions
(Figu e7A- D,I). The highe amoun s o Chol in PM pa ches
(Figu e7D) could be a majo ac o esponsible o compensa -
ing PM molecula o de e en wi h lowe SM le els. Nei he
lau dan GP o GPMV no lau dan GP o b eak h ough o ces o
PM pa ches showed any s a is ically signi ican di e ence be-
ween CHO and LY- B g own in 10% FBS (Table2, Figu e3C).
GPMV cons i u e a equen ly used PM p epa a-
ion.37,40,71,72 Howe e , he lipidomic da a showed ha hei
lipid composi ion depa ed om hose o he whole cells and
om o he PM p epa a ions (pa ches). In pa icula , GPMV
we e en iched in Ce (Figu e7B) and HexCe (Figu e7C), and
hey also exhibi ed an unusual en ichmen in PI (Figu e7G).
Fu he mo e, hei GPL we e en iched in sa u a ed a y acids
(Figu e7J), and con ained co espondingly less unsa u a ed
chains, speci ically wi h 2- 6 double bonds pe GPL mole-
cule (Figu e7L). Also, he p opo ion o medium- leng h a y
acids (C30- 32 pe GPL molecule) inc eased a he expense
o he longe ones (C34- 40) (Figu e7M,N). All hese a e pe-
culia i ies o GPMV, in which hey di e ed om all o he
cell and memb ane p epa a ions, wi h ei he 10% o 0.04%
FBS. The ac ha hese changes we e no modi ied by SL
deple ion, and ha some o hem a ec ed mainly GPL, makes
GPMV a less use ul memb ane p epa a ion in he con ex o
ou s udy. GPMV pene a ion equi ed consis en ly highe
b eak h ough o ces han PM pa ches (Figu eS7). This could
be ela ed o he en ichmen in Ce and HexCe ound in
GPMV wi h espec o whole cells (Figu e7B,C). Bo h Ce
and HexCe a e known o inc ease memb ane lipid o de 73- 75
GPMV ha e been shown o be pe meable o hyd ophilic
mac omolecules,72 and his could again be ela ed o he in-
c eased Ce , and pa ly HexCe , since hese SL happen o
inc ease memb ane pe meabili y.73,74 The obse ed inc eases
in GP and b eak h ough o ces could be seconda y o he
use o di hio h ei ol in GPMV o ma ion. As seen in Eps ein
e al76 di hio h ei ol can be esponsible o inc easing Ce
concen a ions e en in SPT- supp essed cell lines, wi hou
al e ing SM alues. Those au ho s concluded ha di hio h-
ei ol could induce he “un olded p o ein esponse” and his
would lead o an o e - exp ession o he SPT LCB1 subuni
mRNA, pa ially eco e ing i s ac i i y.76 Di hio h ei ol was
also shown o a ec lipid- lipid and lipid– p o ein in e ac ions
and o in eg a e di ec ly in o lipid memb anes.77
4.2
|
CHO and LY- B cells g own in SL-
de icien medium
When LY- B cells we e g own in SL- de icien medium, SM
and Chol pe cen le els we e ma kedly dec eased, espec-
i ely by abou i e old and wo old, wi h no compa able
changes in Ce o HexCe , and he de i ed PM pa ches ol-
lowed pa allel ends (Figu es6A,D, 7A,D). In CHO cells, he
dec ease in SM concen a ion was less clea , and HexCe le -
els ac ually inc eased somewha , o he SL a ying as in LY- B
(Figu e6A,C), wi h he co esponding PM pa ches showing
simila ends (Figu e7A- D). G ow h in SL- de icien medium
did no cause any ema kable changes in GPL, no in hei as-
socia ed a y acids (Figu e7E- N), wi h he excep ion ha he
e y long a y acids (C42- 44 pe GPL molecule) whose con-
cen a ion was in any case e y low, we e u he dec eased
wi h he low FBS medium. No e ha he la ges dec ease in
SM, he mos abundan SL, occu ed in LY- B cells dep i ed
o SL in he g ow h medium, hus he wo ac o s appea o
be equi ed, lack o SL in he nu ien s and lack o capaci y o
syn hesize he sphingosine p ecu so , o ob ain low- SL cells.
Wi h espec o he PM p epa a ion, lau dan GP indica ed
a dec eased lipid o de (inc eased bilaye luidi y) in all sam-
ples unde s udy (Table2) and b eak h ough o ces dec eased
acco dingly, mo e in LY- B han in CHO cells and memb anes
(Figu e3B,C). As a esul , PM pa ches om LY- B cells we e
less o de ed and mo e easily pene able (Table2, Figu e3C).
The close co ela ion be ween dec ease in SM concen a ion
in cell memb anes, as a esul o SL dep i a ion in he nu-
ien s, dec ease in GP alues and dec ease in b eak h ough
o ces can be seen in Figu e3C,D. (Only da a om whole
cell lipid ex ac s a e included in Figu e3D, o simplici y.)
4.3
|
Homeos a ic adap a ions
A leas some o he obse ed changes in memb ane lipid
composi ion as a esul o gene supp ession o o changes in
20 o 23
|
MONASTERIO ET Al.
nu ien media could be explained in e ms o homeos a ic e-
sponses o he no el si ua ions. Pe haps he main obse a ion
in e ms o adap a ion is he ema kable esilience o LY- B
cells ha , when g own unde ex emely low SL concen a-
ions (250- old below s anda d condi ions), a e s ill able o
di ide while keeping SL concen a ions jus six old lowe
han he s anda d alue, and memb ane physical p ope ies
no a away om he wild- ype cells. Examining he da a in
mo e de ail, and speci ically compa ing CHO and LY- B cells
g own in 10% FBS medium, hin s on adap a ion o lack o
de no o SL syn hesis could be e ie ed. In pa icula , as de-
sc ibed abo e, he only no able change be ween he lipidomes
o hose wo cell lines, g own unde s anda d condi ions, is
he clea dec ease in SL as pe cen o al lipids (one- hal on
a e age) in he LY- B cells (Figu e7), while Chol le els did
no a y. The pe cen concen a ion o SM, he mos abun-
dan sphingolipid, wen om 6.7% o 3.8% (Figu e 7A).
Pa allel changes we e eco ded in PM pa ches de i ed om
hose cells. This was no accompanied by any changes in he
measu ed physical p ope ies o he memb anes, lau dan GP
(Table2) o AFM b eak h ough o ces (Figu e3C). Pe haps
he obse ed a ia ion in SL concen a ion was no enough o
cause any obse able physical changes, and a e y mino , o
no adap a ion was equi ed.
The si ua ion was di e en when cells we e g own in SL-
de icien medium. In LY- B cells SM concen a ion d opped
by one o de o magni ude when cells we e g own in 0.04%
ins ead o 10% FBS. O he SL, as well as Chol, we e de-
c eased in pa allel. PM pa ches unde wen simila changes
as he whole cell lipids. Pe haps as a consequence o hese
changes, he memb anes became mo e easily pene able, and
lipids became less o de ed (Table2 and Figu e3) when FBS
concen a ion was lowe ed. Simul aneously, a y acyl un-
sa u a ion was dec eased (Figu e7J- L), a phenomenon ha
could ha e he e ec o inc easing lipid o de , hus ending
o compensa e he dec ease in bilaye - o de ing SM. Ou e-
sul s ega ding he inc ease in e he lipids in sphingolipid-
deple ed cells i p e ious obse a ions 78 and con i m he
co- egula ion be ween hese wo lipid classes.
When CHO cells we e g own in SL- de icien medium he
p opo ion o HexCe was conside ably inc eased, by abou
wo old. This inc eased HexCe syn hesis ( ha could no
occu in LY- B cells because o hei low sphingosine a ail-
abili y, due o he lack o SPT ac i i y), may be one o he
homeos a ic esponses ha wild- ype cells ca y ou unde
s a a ion. HexCe is a he o igin o he complex glyco-
sphingolipid biosyn he ic pa hway.1 Glycosphingolipids a e
equi ed o cellula di e en ia ion and he e a e human dis-
eases esul ing om de ec s in hei syn hesis.79 This may be
one o he easons o he di e en di iding a ios o CHO
and LY- B in SL- de icien medium (Figu e 1A). CHO, bu
no LY- B cells, may o e - exp ess he HexCe syn hesis o
con inue cell di ision in o de o bu e he nu ien deple ion
condi ion. As discussed abo e, Chol le els in CHO and LY- B
g own in s anda d medium emained in a ian , and his could
help in main aining memb ane igidi y unde condi ions o
low SM (Figu e7D, J). Ne e heless, as Chol syn hesis de-
c eases unde cell s a a ion condi ions, igidi y canno be
main ained in his manne unde SL- de icien condi ions.
When FBS in he medium was dec eased, sa u a ed GPL
we e inc eased in LY- B (Figu e6F), while in CHO cells hey
emained almos cons an .
4.4
|
CONCLUDING REMARKS
The p esen s udy has demons a ed ha in cells lacking he
SPT ac i i y, SM, Ce and HexCe a e ma kedly dec eased in
all measu ed samples (con ols and PM p epa a ions).
Fully sa u a ed GPL a e inc eased and polyunsa u a ed
ones a e dec eased. Syn hesizing mo e sa u a ed GPL can
be he way ha LY- B cells ha e o compensa e he low SM.
Choles e ol may also ha e some in luence in ha esponse
bu i s e ec is minimized when i s le els a e dec eased be-
cause o s a a ion. The SM- deple ed cells y o main ain
memb ane o de by unde going a homeos a ic esponse, al-
hough hey achie e i only pa ially as hei PM a e mo e
agile when g own in SL- de icien medium.
These changes in lipid o de and memb ane igidi y
caused by low SL could be linked o a a ie y o phe-
nomena in cell physiology and pa hology. Al e a ions in
he no mal ac i i ies o he SM- cycle enzymes ha e been
associa ed o many cen al ne ous sys em and neu ode-
gene a i e diseases.19 Speci ic SM species ha e been ound
o bind memb ane p o eins he eby modi ying hei unc-
ions.80 The capaci y shown by ce ain cells in his pape o
g ow unde ex emely demanding low concen a ions o SL
opens he way o a a ie y o unc ional s udies on he ole
o SL in memb anes.
ACKNOWLEDGMENTS
We hank D Al ed Me ill (Geo gia Tech) o sug-
ges ing ha we examine he p ope ies o LYB cells in
sphingolipid- deple ed medium. The au ho s a e also g a e-
ul o D Dona ienne Ty eca o he kind gi o plasmid
pET28/D onpa- NT- lysenin and o Ms Ma ina I iondo o
he help in pu i ying lysenin. This wo k was suppo ed
in pa by g an s om he Spanish Minis y o Economy
(g an FEDER MINECO PGC2018- 099857- B- I00) and he
Basque Go e nmen (g an s No. IT1264- 19 and IT1270- 19),
as well as Fundación Bio ísica Bizkaia and he Basque
Excellence Resea ch Cen e (BERC) p og am o he Basque
Go e nmen , and by he Swiss Na ional Science Founda ion
(310030- 184949).
|
21 o 23
MONASTERIO ET Al.
CONFLICT OF INTEREST
The au ho s ha e s a ed explici ly ha he e a e no con lic s
o in e es in connec ion wi h his a icle.
AUTHOR CONTRIBUTIONS
FM Goni and A. Alonso designed he esea ch; BG Monas e io,
N. Jimenez- Rojo, and A. Ga cia- A ibas pe o med he e-
sea ch; H. Riezman con ibu ed analy ic ools (mass spec-
ome y); all au ho s analyzed he da a; BG Monas e io and
N. Jimenez- Rojo w o e he manusc ip , which was edi ed by
FM Goni, H. Riezman, and A. Alonso.
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SUPPORTING INFORMATION
Addi ional Suppo ing In o ma ion may be ound online in
he Suppo ing In o ma ion sec ion.
How o ci e his a icle: Monas e io BG, Jiménez-
Rojo N, Ga cía- A ibas AB, Riezman H, Goñi FM,
Alonso A. CHO/LY- B cell g ow h unde limi ing
sphingolipid supply: Co ela ion be ween lipid
composi ion and biophysical p ope ies o
sphingolipid- es ic ed cell memb anes. The FASEB
Jou nal. 2021;35:e21657. h ps://doi.o g/10.1096/
j.20200 1879RR