From zero to six double bonds: phospholipid unsaturation and organelle function

From

zero

to

six

double

bonds:

phospholipid

unsaturation

and

organelle

function

Bruno

Antonny

,

Stefano

Vanni

,

Hideo

Shindou

,

and

Thierry

Ferreira

1_{InstitutdePharmacologieMole´culaireetCellulaire,Universite´deNiceSophiaAntipolisandCentreNationaldelaRecherche}

Scientifique(CNRS),660routedesLucioles,06560Valbonne,France

2_{DepartmentofLipidSignaling,ResearchInstitute,NationalCenterforGlobalHealthandMedicine,1-21-1Toyama,Shinjuku-ku,}

Tokyo162-8655,Japan

3_{CREST,JapanScienceandTechnologyAgency,Kawaguchi,Saitama332-0012,Japan}

4_{LaboratoireSignalisationetTransportsIoniquesMembranaires(STIM),CNRSEquipedeRechercheLabellise´e(ERL)7368,}

Universite´dePoitiers,1rueGeorgesBonnet,86073PoitiersCEDEX9,France

Cellularphospholipids(PLs)differbythenatureoftheir polarheads aswell asbythe length and unsaturation leveloftheirfattyacylchains.Wediscusshowtheratio betweensaturated,monounsaturated,and polyunsatu-ratedPLsimpactsonthefunctionsofsuchorganellesas theendoplasmicreticulum,synapticvesicles,and pho-toreceptor discs. Recent experiments and simulations suggestthatpolyunsaturatedPLsresponddifferentlyto mechanicalstress, including membrane bending, than monounsaturatedPLsowingtotheirunique conforma-tionalplasticity. Thesefindingssuggestarationalefor PLacylchainremodelingbyacyltransferasesanda mo-lecular explanation for the importance of a balanced fattyaciddiet.

Fattyacylchain diversity:thedarkfaceofcellular membranes

Thelipidcompositionofcellularmembranesvaries signifi-cantlyamongorganisms,tissues,andorganelles,andour knowledgeofthisdiversityhasgreatlyincreasedthanksto progressinorganellefractionation,lipidanalysis,notably massspectroscopy,andtheidentificationand characteri-zationofmostenzymesresponsibleforlipidsynthesis[1– 5].However,ourunderstandingoftherolesofthevarious lipids that coexist in biological membranes has not im-provedasfast.Hundredsofdifferentlipidspeciescanbe foundinthemembraneofasingleorganelle, butwecan attributeaclearfunctiontoonlyafewofthem.

With the exception of sterols, the lipids of cellular membranes are made of three building blocks: a polar head, a central group (glycerol or sphingosin), and long hydrocarbonchains (thefattyacids). Thisschemeallows numerouscombinationsandisattherootoflipiddiversity. However,biochemistsandcellbiologistsgenerallydocare moreaboutthepolarheadthanabouttheacylchains.This

biasisreflectedinthelanguage:wenamelipidsbytheir polar head (e.g., phosphatidylcholine, PC; phosphatidyl-serine,PS;phosphatidylethanolamine,PE),butwerarely detail their acyl chaincontent. It alsoacknowledges the fact that lipid polar heads are associated with defined functionsowingtotheirspecificinteractionswithproteins

[6].

Theroleofthevarious fattyacylchainsofPLsisless wellunderstood.Fattyacidsaredividedintothreeclasses accordingtothenumberofdoublecarbonbondsalongthe carbonchain(Figure1A)[7].Saturated fattyacids(SFA) containonlysaturatedcarbonbonds(C C); monounsatu-ratedfattyacids(MUFA)containasinglecisdoublebond (C=C);polyunsaturatedfattyacids(PUFA)contain2–6cis double bonds and are themselves divided into two sub-classes,v3andv6,dependingonthepositionofthedouble bonds. Palmitate, oleate, arachidonate, and docosahexa-noicacid(DHA)arethemostabundantfattyacidsofeach class.Theirabbreviation(C16:0,C18:1-n9,C20:4-n6,and C22:6-n3, respectively) indicatesthe number of carbons, thenumberofdoublebonds,andthepositionofthefirst doublebondwith respecttotheterminalmethyl.

Onaverage,thefirstacylchainofcellularPLsis satu-ratedwhereasthesecondacylchainismonoor polyunsat-urated. However, there are large variations around this canonicalscheme.Whyistheendoplasmicreticulum(ER) richer in monounsaturated PLs than the plasma mem-brane(PM)[8,9]?WhyarepolyunsaturatedPLsso abun-dantinsomespecializedorganelles(e.g.,synapticvesicles) butabsentinsomeorganisms(e.g.,Saccharomyces cerevi-siae) [9–12]? These questions, which echo back to the discoveryintheearly1930softheessentialroleofsome PUFAsinmammaliandevelopment[13],arestilltopical: wefrequentlyhearthatoliveoilisbetterthanbutterand that v3 arebetter than v6lipids forourhealth,but the molecularbasisforthesedifferences remainselusive.

Fattyacids could have effectsontheirownor as pre-cursors ofsignaling molecules [14]. In addition, because fatty acids become in large part esterified in PLs, they should impact onthe functioning of cellular membranes

[10]. We summarize here the origin of the acyl chain Correspondingauthor:Antonny,B. (antonny@ipmc.cnrs.fr).

Keywords:polyunsaturatedphospholipid;membranecurvature;lipid-packingdefect; mechanotransduction;lipidremodeling.

http://doc.rero.ch

Published in "Trends in Cell Biology 25(7): 427–436, 2015"

which should be cited to refer to this work.

diversity in PLs and present a few examples ofmutual adaptationbetweenorganellefunctionandPLacylchain composition.Last,wediscusstheimportanceof biophysi-calapproachescoupledtomoleculardynamicssimulations torevealhowdeceptivelysimplechangesinthe hydrocar-bonstructureofPLfattyacidscanimpartbiological mem-braneswithdifferentphysicochemicalproperties.

BiochemicalpathwayspromotingPLacylchain diversity

The acylchaindiversity ofPLsresultsfrom several pro-cesses,fromdietsourcestocomplexreactionswherefatty acids are elongated,desaturated, transported,and even-tuallyesterifiedintoPLs(Figure1A)[15,16].

Almost all organisms contain a D9 desaturase, which introduces adoublebondinthe middleoftheacylchain, thusproducingmonounsaturatedfattyacidsfrom saturat-edones(e.g.,C18:0>C18:1-n9).Bycontrast,theabilityto addadditionaldoublebondsisnotuniversal.Someplants introducedoublebondsbetweentheterminalmethyland

the central carbon, whereas mammals introduce double bonds between the centralcarbon andthe terminal car-boxyl[7].Ourdietreflectsthisdivisionoflabor:weeatthe essentiallinolenicacidC18:3-n3fromplantoilsto synthe-sizeC22:6-n3,whichthenaccumulatesinourbrain mem-branes.AtransporterallowingC22:6-n3tocrossthebrain– bloodbarrierhasbeenrecentlyidentifiedanditsdeletion stronglyimpairsbraindevelopment[17].

Fatty acids are eventually activated in the form of acyl-CoAintermediatesandesterified intoPLsthrough twoalternativepathways:thedenovopathway(or Ken-nedypathway)correspondstothecompletesynthesisofa PLfromelementarybuildingblocks,whereasthe remo-delingpathway(orLandscycle)correspondstothemere substitution of one acyl chain in a preformed PL

(Figure1A)[18].Theacyltransferasesinvolved inthese

pathwayshavebeenidentifiedandtheirsubstrate spec-ificitydeterminestheacylchaindiversityofPLs(Box1)

[15].StrikingexamplesareLPCAT1,whichis enriched in thelung and incorporates saturated fattyacids into

(A) (B) (C) Monounsaturated Saturated Secretory pathway ER Golgi PM Fay acid diversity C16:0 C18:1 C22:6 PL Glycero-3P LysoPA PA PLs LysoPL PLA2 O O O O O O Remodelling De novo synthesis Body Axon Synapc vesicles C18:2 C20:4 C22:6 Diet, transport, elongases, desaturases Acyltransferase repertoire LPAATs LPLATs

Acyl chain diversity of lipid membranes

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Figure1.Acylchaindiversityofmembranelipids.(A)Summaryofthevariouslipidmetabolicpathwaysthatleadtotheacylchaindiversityofphospholipids(PLs)in biologicalmembranes.Thediversityoffattyacidsarisesfromdietarysources,aswellasfromendogenousenzymaticactivitiesthatallowtheacylchaintobeelongatedor desaturated.Inmanyorganisms,however,theseactivitiesarelimited,hencetheimportanceofadiversediet.Fatty-acyl-CoAintermediatesarethenusedassubstratesby variouslysophospholipidacyltransferasestosynthesizeorremodelPLs[5,15].Theseenzymesformtwolargefamiliesandincludemembersdisplayingdifferentspecificity fortheirlysophospholipidandacyl-CoAsubstrates(Box1).(B,C)TheacylchainprofileofPLsvariesalongtheorganellesofthesecretorypathwayaswellasinsome differentiatedstructuressuchasaxons[9,12].

PCto promote theformationof a protectivesurfactant

[5], and tafazzin, which incorporates polyunsaturated fatty acids into cardiolipin (CL) in a curvature-depen-dentmanner[19].

ExamplesofPLfatty acylchaingradients

In mammalian cells, there is a gradual enrichment of saturatedPLspeciesattheexpenseofmonounsaturated species along the organelles of the secretory pathway (ER>Golgi>plasma membrane) [8]. This subcellular gradient, whichparallels main membrane traffic routes, also exists in yeast and results from a change in the esterifiedacylchainsofPEandPS[9](Figure1B).

Recentadvancesinlipidimagingbymassspectrometry revealanotherstrikingacylchaingradient(Figure1C).In neuronalcellcultures,whereaxonsradiatearounda clus-ter ofcell bodies, a gradient ofpolyunsaturated PChas beenimagedatmmresolution[12].PCmolecules contain-ingC20:4orC22:6PUFAsaccumulateattheaxontipbut arerareatthecellbody,whereasless-unsaturatedspecies, notablyC18:2-PC,showanoppositedistribution. Sperma-tozoaprovideanotherexampleoftheunevendistribution ofpolyunsaturatedPLs atthesubcellularlevel[20].

CellsalsochangetheirPLacylchainprofileovertime. Epitheliumdifferentiationisaccompaniedbythe appear-anceofmoresaturatedlipidspecies,probablytomakethe apicalPMa betterprotective barrier[1].The amount of C20:4-containing PC oscillates during the cell cycle

[21]. During cytokinesis, lipid species with well-defined acylchaincompositionappearinthemidbody[22].Last, theratiobetweenmonounsaturatedPLsand polyunsatu-ratedPLsincreasesinseveralcancercelllinesandtissues

[23,24].

Themetabolicpathwaysattheoriginofthe spatiotem-poralcontrolofPLacylchainunsaturationremaintobe investigated,notablytheinvolvementofspecific acyltrans-ferases [5]. However, the fact that this control exists, whereasthe multiplemembranetrafficpathwaysshould leadtoPLhomogenization[4],suggeststhatthe balance betweensaturated,monounsaturated,and polyunsaturat-edPLspecieshasdecisivefunctions.

InfluenceofPLacylchainsonproteinsynthesisand foldingattheER

TheERistheorganelleforthebiosynthesisandfolding of transmembrane and luminal proteins. To maintain thecorrectbalance betweenERclientproteinload and foldingcapacity,cellshavedevelopedapathwayknown astheunfoldedproteinresponse(UPR)[25,26].TheUPR iscontrolledbyintegralproteinsensors,suchas inositol-requiring enzyme 1 (IRE1) and protein kinase-like ER kinase (PERK) [25,26], which are maintained in an inactive monomeric form by the interaction of their luminal domainwith thechaperoneBiP.Accumulation of unfolded proteins correlates with the disruption of this repressive complex, oligomerization, and down-streamactivationoftheUPRcascade[27,28].Induction oftheUPRlimitsproteinoverloadviaageneraldecrease intranslationinitiationandcoordinatedupregulationof genesencodingproteinsinvolvedinfolding(e.g., chaper-ones)andER-associated degradation(ERAD). Interest-ingly,thesepathwaysareactivateduponcellexposureto

SFAs[29–34].

Becauseexogenouslysuppliedfattyacidsareefficiently incorporated into PLs [35–37], several studies point at saturatedPLsasthedirectcauseofER-stressasaresult of their impact on membrane properties. This includes modification ofcalcium permeation, perturbation of pro-teinfolding,andchangesintheoligomericstate ofIRE1. Forexample,palmitatepromotesdepletionofERcalcium stores[30,38] inaprocessthatcould berelated todirect effects of saturated PLs on sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) pump activity [30]. In vitro studies demonstrate that SERCA-2b activity is inhibitedwhen reconstitutedintomicrosomescontaining saturated PLs [39].Saturated PLs alsoinduce the accu-mulationofmisfoldedproteins.TreatingSFA-intoxicated cellswith pharmacologicalchaperonesthat stabilize pro-tein conformation alleviates SFA-induced UPR [37,40– 42]. Various causes could account for misfolded protein accumulationunderSFAtreatment.First,thetranslocon, which catalyzes the translocation of newly translated proteins towards the lumen, is sensitive to membrane

Box1.AcyltransferasesandPLdiversity

The fattyacidcomposition atboth sn-1and sn-2positions ofPLs differsinvariouscelltypesandtissues.Asymmetryanddiversityof membranePLsaregeneratedthroughthedenovopathwayandthe remodelingpathway[18].PLsareformedfromglycerol-3-phosphate (G3P)bytheKennedypathway.Next,usingacyl-CoAsasdonors,the sn-2 acylmoietyofPLsisremodeledintheLands’cycle,whichis conductedbytheconcertedactionsofphospholipaseA2(PLA2)and lysophospholipid acyltransferase (LPLAT) enzymes. The Kennedy pathwayandtheLands’cyclewereproposedinthe1950s.However thecorrespondingLPLATswerediscoveredonlyrecently[15].LPLATs are divided into two families: the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family and the membrane-bound O-acyl-transferase(MBOAT)family.

In the AGPATfamily, GPAT1-4 and LPAAT1-4 produce lysopho-sphatidic acid (LPA) and phosphatidic acid (PA) in the Kennedy pathway,respectively,whereastheothermembersareLands’cycle enzymes.LPCAT1ismainlyexpressedinthelungandgeneratesPAF and dipalmitoyl-PC, which is a main component of pulmonary surfactantessentialforrespiration [5].LPCAT2functions in inflam-matory cells to synthesize platelet-activating factor (PAF) and PC. LPCAT2isphosphorylatedand isactivatedbyextracellular stimuli

suchasATP,PAF,andlipopolysaccharide[103].LPEAT2isexpressed in the brain and produces PE. LPGAT1 and LCLAT1 generate phosphatidylglycerol(PG)andCL,respectively.IntheMBOATfamily, LPCAT3,LPCAT4,LPEAT1,andLPIAT1havebeenidentified.LPIAT1 synthesizes phosphatidylinositides(PI) containingarachidonicacid, the main forms of PI [94]. LPCAT3 (PC, PE, and PS production), LPCAT4 (PC and PE), and LPEAT1 (PE and PS) possess several enzymaticactivitiesandshowdifferentexpressionpatterns, suggest-ingspecificrolesindifferenttissues[15].

PCisthemajorPLanddisplaysahighly-diversefattyacidprofile depending on the tissue [5]. Fatty acidenrichment of PC can be regulatedbytworeactions:theLPAATstepintheKennedypathway andtheLPCATstepintheLands’cycle.Themolecularmechanismof PCfattyaciddeterminationwasexaminedusingLPAAT1,2,and3,as wellasLPCAT1,2,3,and4.FromenzymaticassayandPCcomposition ofseveraltissues,theLPCATstepseemstocontroltheformationofPC species containing16:0 and 18:1 atsn-2, whereas the LPAATstep seemstocontroltheincorporationof18:2and22:6inPCatthesn-2 position.Furtherinvestigationswillbenecessarytounderstandthe diversityofotherPLs,suchasPS,PE,PI,PG,andCL.Regulationoffatty aciddiversityatthesn-1positionshouldbealsoclarified.

organization[43].Second,manyERchaperonesare depen-dent oncalcium[44],thehomeostasisofwhichisaltered underpalmitateaccumulation.

Intriguingly,recentstudiessuggestthat lipid disequi-librium couldinduceUPRby adirecteffectonIRE1and

PERK[45,46].Mutantsoftheseproteinslackingthe

lumi-nal unfolded protein-sensing domain still respond to in-creased lipid saturation [45]. Moreover, increasing PL saturation in reconstituted liposomes that are devoid of unfolded ER client proteins enhanced the activity of a PERK variant devoid of its luminal domain [45]. Even though the mechanism for saturated PL-induced PERK clustering is a matter ofdebate [47], these observations suggest that misfoldedprotein accumulationmaynot be the onlysignal forUPR induction underSFA accumula-tion.DimerizationofIRE1andPERKuponoverloadofthe ERmembranewithsaturated-PLsmightallowthecellto anticipate futureproteinmisfoldingproblemsinducedby theselipids.Conversely,thefactthatacomponentofthe ERAD machinery, Ubx2p, has been identified as a key activatorofyeastD9desaturase[48]suggestsafeedback loop whereby ER stress induced by misfolded proteins promotescorrectionoftheERunsaturationlevel. PLmonounsaturation,membranecurvature,and proteinadsorption

How the ratio between saturated andmonounsaturated PLs controls transmembrane helices oligomerization at the ER remains elusive, although several mechanisms havebeenproposed[49].Bycontrast,the monounsaturat-ed/saturated PL ratio has another impact that is more

straightforward to rationalize. Introducing monounsatu-ratedPLsattheexpenseofsaturatedonesfacilitatesthe membraneadsorptionofseveralcytosolicproteinsacting on the ER or ER-derivedorganelles such as autophago-somesorGolgi[2].Ingeneral,theseproteinscontainlarge amphipathicmembraneanchors,andtheirpreferencefor monounsaturatedPLscorrelateswithanacutesensitivity tomembranecurvature[2,50,51].

Themotifsthatallowperipheral proteinstosensethe combinatory effects of monounsaturated PLs ratio and membrane curvature have been reviewed elsewhere

[50]. We recall here the underlying mechanism, which revolvesaroundtheideaof‘voids’orlipid-packingdefects

(Figure2A).In thissimple geometricalmodel, lipidsare

consideredasratherstiffobjects.Whenmembrane curva-tureincreases,orwhenmonounsaturatedPLsare substi-tuted for saturated ones, the lipid geometrical arrangementis perturbedandpackingdefects gradually appear, whichcould hosthydrophobic anchorsfrom pro-teins[52,53].

Combiningtheeffectofcurvatureandlipid monounsa-turation in vitro can change protein adsorption by two ordersofmagnitude,suggestingapotentregulatory mech-anism[50–52,54].Testingtherelevanceofthismechanism invivoischallenging becauseitrequiresmasteringboth thegeometryandlipidcompositionofcellularmembranes. However, recent data are in agreement with the model. ExcesslevelsofsaturatedPLshamperprocessesasdiverse as ER-to-Golgi trafficking or autophagosome formation, andpromotethemembranedissociationofsomekey asso-ciatedproteins[54–57]. (A) Bending Saturated acyl chain Monounsaturated acyl chain Polar head

Side view Top view

10 nm

Packing defect map

Packing defects

Large and deep packing defect for hydrophobic inseron

Lipid-packing defect Topographic analysis Bending C=C C=C (B)

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Figure2.Cooperationbetweenmonounsaturatedphospholipids(PLs)andcurvatureforproteininsertion.(A)Elementaryviewoftheeffectsofmembranecurvatureand lipidmonounsaturationontheformationoflipid-packingdefects.Thisnaivemodelconsidersthegrossshapeoflipidsaswellastheirrelativetiltingduetocurvature.It accounts,atleastqualitatively,forthecumulativeeffectsofmembranecurvatureandlipidmonounsaturationontheadsorptionofmodelamphipathicmotifsatthe membrane-waterinterface[50,52,53].(B)Moleculardynamicssimulationsgiveamorerealisticviewoftheeffectoflipidcompositionandmembraneshapeonthe formationoflipid-packingdefects[54,58–60].Theexampleshownhereconsidersamembranetubemadeofasinglelipid(C16:0-C18:1-PC)thathasbeen‘coarse-grained’to minimizecalculations.Scanningthemembranesurfaceallowsidentifying‘voids’(i.e.,lipid-packingdefects).Thestatisticaldistributionofthesevoidsconfirmsthe cumulativeeffectsofmembranecurvatureandmonounsaturationonlipid-packingdefects.

Moleculardynamicssimulationsoflipid-packing defects

Although the scheme of Figure 2A is obviously naı¨ve becauselipids arenot stiff, addressingthe molecular or-ganization oflipids in bilayers of different compositions andgeometries isexperimentally very difficult. To over-come these limitations, and to investigate microscopic propertiesoflipidassemblies,a powerfulmethodologyis moleculardynamicssimulations(Box2),acomputational approach that allows investigating the behavior of lipid membraneswithatomic-levelresolution.Usingthis tech-nique it has been possible to characterize lipid-packing defectsoccurringatthemembrane–waterinterface, there-by providing a topographic description of lipid bilayers

[54,58,59](Figure2B).Despitebeingtransient(with

life-timesinthepicosecondregime),lipid-packingdefectsare ubiquitousandcanbepreciselyquantifiedforalltypesof lipidassemblies.Theirsizedisplaysacharacteristic expo-nentialdistribution(largedefectsaremuchlessfrequent thansmallones).

Molecular dynamics simulation of varying membrane geometries and compositions show that lipid-packing defects increase with positive curvature, and also with theintroductionofmonounsaturatedPLs attheexpense ofsaturatedones[54].Mostimportantly,curvatureandPL monounsaturation have a cumulative effect towards the promotion of lipid-packing defects, whereas curvature alone,in the presenceofsaturated PLs, is notsufficient topromotemembraneadsorptionofperipheralproteins,in agreementwithexperimentaldata[54,58–60].This exam-ple suggests that molecular dynamics simulations can provideanatomicviewofan intuitivebutelusivemodel of PL organization, and complements other approaches that consider membranes as continuum material

[61].The simulations show that lipid-packing defects do notnecessarilylocalizeatthepositionofthemostconical lipids[59]andneedtocoalescetoprovideenoughvolumeto host large hydrophobic insertions [58]. Nevertheless,

saturatedandmonounsaturatedPLsarerelativelysimple lipidsforwhichalargebodyofexperimentaldataexists, makingtheirsimulationsanottooriskytask.

PLpolyunsaturationinphototransduction

Phototransduction, one ofthe best-characterized trans-ductioncascades,occursinamembranerichinv3lipids. In thephotoreceptor discs, DHA (22:6-n3) accounts for 50%ofthePLacylchains[62].Theproteinsinvolvedin phototransduction,namelythelightreceptorrhodopsin, theGproteintransducin,anditseffector,acGMP phos-phodiesterase,havebeenreconstitutedintoartificial lipo-somes. This reductionist approach revealed that replacing C16:0-C18:1 by C18:0-C22:6 PLs increases the rates at which rhodopsin switches to the active state and subsequently activates GDP/GTP exchange ontransducin[57,63,64].Becausethefirstreactionisa monomolecularprocessthatoccurswithinthe hydropho-bicmatrix,whereasthesecondreactionisabimolecular processthatoccursatthesurfaceofphotoreceptordiscs, these observations suggest that polyunsaturated PLs provide two advantages: they facilitate the movements ofthehelicalsegmentsofrhodopsinandtheyaccelerate in-planediffusionofperipheralproteins.

Biophysicalmeasurementsofthebehaviorof polyunsaturatedPLsinbilayers

Studies on phototransduction have stimulated detailed biophysical studies on the behavior of polyunsaturated PLs in model membranes [65,66]. Neutron and X-ray diffractionaswellas NMRmeasurementsrevealed that, in bilayerscontainingmixedacyl chainPLs (e.g., C16:0-C18:1vsC18:0-C22:6),polyunsaturatedacylchainsoccupy morespaceatthewaterinterfacethansaturatedor mono-unsaturated chains, despitepolyunsaturated acyl chains beinggenerallylonger(e.g.,C22:6vsC18:1).This counter-intuitivefindingsuggeststhatpolyunsaturatedacylchains are not extended but are curled (Figure 3A). However,

Box2.Moleculardynamics(MD)simulations

MD isa computational methodology that isused tosimulatethe physicalmovementofsystemscomposedofseveralatoms. Origin-allydevelopedinthecontextoftheoreticalphysics,thistechniqueis nowusedinseveraldomains, fromphysicalchemistrytomaterial science, toinvestigate with high-resolution the behavior of multi-atom systemsthataredifficultorexpensive tocharacterize experi-mentally.

InaMDsimulation,allatomsinthesystemaretreatedasclassical particles(i.e.,quantumeffectsareneglected),andforcesandenergies betweenatomsaredescribedusingaso-calledmolecularmechanics force-field –in other wordsa simplified potentialenergy function wheretheinteractions betweenatomsaresubdividedintobonded (bonds,angles,anddihedrals)andnon-bonded(electrostaticandVan der Waals) terms. Specifying these interactions requires a large numberofparameters(e.g.,equilibriumvaluesandforceconstants forbonds,angles,anddihedrals;electrostaticcharges;vanderWaals parameters) that are usually obtained fromeither experiments or high-levelquantummechanicalcalculations.Oncetheseparameters havebeenset,thedynamicsofthesystemisstudiedbynumerically integratingtheequationsofmotion,uptothestatisticalconvergence ofthepropertiesofinterest.

Because ofthenumerous approximationsthatarecarriedout to maketheproblemtractable,theresultsfromMDneedtobevalidated

byexperimentaldata.Whentheagreementwithexperimentaldatais satisfactory, the simulations areused toexplain the experimental results and to study phenomena that could not be otherwise investigatedwithexperimentaltechniques.

In the past few decades,MD hasemerged asone of themost powerful tools to investigate the atomistic properties of lipid membranes becausethestructuralcharacterizationoflipidbilayers withatomic-levelresolutionishinderedbythehighdisorderoftheir physiologicalliquid-crystallinephase.

Several atomistic force-fields able to simulatelipid bilayers are currentlyavailableandunderconstantdevelopment;theyhavebeen abletosatisfactorilyreproduceseveralpropertiesoflipidbilayersthat can be measured experimentally using different diffraction or biophysicaltechniques,suchasareaperlipid,hydrophobicthickness, deuteriumorderparameters,ormembraneelasticity(see[104]and http://nmrlipids.blogspot.fr).

Coarse-grainmodels,whereseveralatomsaregroupedtogether andrepresented byasinglebead, andwherethepotentialenergy functionisfurthersimplified,arealsounderheavydevelopmentin thefieldoflipidmembranes.They havebeensuccessfullyusedto interpret and predictlarge-scale membraneremodeling processes, includingvesicleformation,membranefusion,andmembranefission [105–107].

becauseacylchainsundergofastmotionsinbilayers(inthe nsscale),biophysicalapproachescannotgivesnapshotsof theirconformationsbutinsteadrevealtrendsasdeduced from meanorientationanddensitymeasurementsacross thebilayer.

PolyunsaturatedPLsascontortionists

Polyunsaturatedacylchainswereinitiallyconsideredtobe morerigidthansaturatedacylchainsbecauseaC=Cbond cannot rotate about its axis. In polyunsaturated acyl chains,however,theC=Cbondsaresystematicallyflanked by two saturated bonds. Calculations and simulations revealed that this regular pattern of one non-rotating and two rotatingbonds decreases the energyof rotation about the saturated carbons [32,67,68] (Figure 3A). The exceptionalflexibilityofpolyunsaturatedacylchainsgives a straightforward explanation for their benefit on the activation of rhodopsin and, more generally, on many membrane-embedded proteins as well as onthe orienta-tionoffluorescentprobes[69–71](Figure3B). Polyunsat-uratedPLsquicklyadapttheirconformationtofollowthe rapidstructuraltransitionsoftheruggedsurfaceof trans-membranehelixbundles[66,72].

PLpolyunsaturationinsynapticfunctions

Synaptic vesicles (SV) deliver neurotransmitters in the synaptic cleft in response to action potentials.Although the abundance of polyunsaturated PLs is a remarkable featureofSVs[11](Figure1C),studiesontheroleoflipids in the cycling of theseorganelles have focused on other aspects[73].Inthissectionwepresentahypothesisforthe roleofpolyunsaturatedPLsinSVs,whichwasinspiredby observations made years ago but was formulated only recently[74].

In 1986, electron microscopy studies revealed that, amongallexogenous factors that promotethe formation ofsynapticvesiclesinculturesoffetalhypothalamiccells, thepolyunsaturatedfattyacidsC20:4andC22:6werethe mostefficient[75].Theauthorsproposedthatthefeatures ofSVsinPUFA-supplementedmedia,notablytheir regu-larshapeandsmalldiameter,aswellastheirabundance, reflectanenhancementofmembranefluidityinagreement with the model of Singer and Nicolson. Despite its pre-science, this study was largely ignored. Later, other researchers reported that deficiency in polyunsaturated fatty acid synthesis in Caenorhabditis elegans leads to abnormally low levelsofsynaptic vesiclesanddefectsin neurotransmitterrelease[76].

In 2000, physicists reported a striking effect of acyl chain polyunsaturation on the mechanical properties of giantliposomesmadeofsyntheticPC.WhenPCcontained atleastonepolyunsaturatedacylchain,theresistanceof thebilayeragainstbendingdroppedabruptlybytwofold, irrespectiveoftheexactchemistryoftheacylchain[77].

Together,theseseeminglydisparateobservationsoffer astraightforwardexplanationforthebenefitof polyunsat-uratedPLsinSVs:theselipidswouldmaketheneuronal membranemoreflexibleandthusmorepronetorespondto themechanicalworkexertedbyproteins(Figure3C).This would explain why SVs are the smallest membrane-enclosed organelles (diameter = 40nm) and why their formation is so fast [78]. Recent experiments conducted withmodellipidbilayersandwithproteinsinvolvedinSV retrieval support this hypothesis [74]. First, the fission activityofthedynamin–endophilincomplex,whichformsa transientspiralattheneckofendocyticstructuresinthe presenceofGTP,increaseddramaticallyuponthe incorpo-rationofC18:0-C22:6-n3PLsattheexpenseofC16:0-C18:1

Bending Light C C C C C C C Rhodopsin C16:0-C18:1 C18:0-C22:6 Energy Conformaonal adaptaon Super-flexible Conformaonal adaptaon α-helix movement (A) (C) (B)

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Figure 3.The conformational flexibility of polyunsaturated phospholipids (PLs) facilitates fast protein movements and membrane bending. (A) In contrast to monounsaturatedacylchains,whichhaveasingleoptimalconformation,polyunsaturatedacylchainsswitcheasilybetweendifferentconformationsofequivalentenergy butdrasticallydifferentgeometry.(B)TheflexibilityofpolyunsaturatedPLsallowsthemtoeasilyaccommodatethesuddenconformationalchangeofproteins,suchasthe movementsofthetransmembranehelicesfromtheGPCRrhodopsindepictedhere.(C)Similarly,polyunsaturatedPLsadapttheirconformationtomembraneshape,hence facilitatingthemechanicalactionofproteinsthatbendandfissionmembranes.

PLs [74]. Second,the PM ofnon-neuronal cells that are forced to incorporate C22:6 in their PLs becomes more deformablebyanexternalpullingforceandmore permis-sivetoendophilin-drivenendocytosis[74].

The shape and function of SVs and of photoreceptor discs are anything but similar. However, the benefits provided by polyunsaturated PLs in both membranes might originate from the same property: the ability of polyunsaturated chains to change their conformation

(Figure3Band3C).Biochemicalexperimentsand

molecu-lar dynamics simulations suggest that polyunsaturated PLscorrectthelipid-packingdefectsinducedbymembrane curvaturebyfillingthedefectsthroughtheirhyperflexible

tail[74,79,80].Thisfindinggivesamolecularexplanation

forthedecreaseinmembranebendingrigidity[74,77],and mayalsoexplainsomepuzzlingeffectsofpolyunsaturated PLsonthetargetingoflipidatedproteins[81].Moreover,it underlinesakeyqualitativedifferencebetween polyunsat-uratedPLsandotherlipids.Whereasmostlipidsdisplay an intrinsic shape (e.g., conical, cylindrical, or inversely conical), polyunsaturated PLs escape this classification. Likecontortionists,theyadoptdifferentshapesofsimilar energy,therebysoftening variousmembrane mechanical stresses(Figure3A–C).

TheproteinsinvolvedinSVsretrievalarewellknown, buttheorderinwhichtheyshapethesynapticmembrane remainsunderintenseinvestigation[82,83].Recent stud-ies suggest a two-step process in which endophilin and dynaminactfirsttopromotetheproductionofatransient endosome,whichisthentransformed intoauthentic SVs throughtheactionoftheAP2–clathrincoat[84–86].The firststepissuper-fast[78]and,giventheprominentroleof endophilin and dynamin at this stage, it is tempting to associatethisfeaturewiththe abundanceof polyunsatu-ratedPLsatthesynapse[74].

Tworecentstudiesnicelyillustratetheeffectof polyun-saturatedPLsonmembranemechanicsinotherneuronal contexts.InC.elegans,mutatingtheenzymesinvolvedin thesynthesisofC20:4-n6orinitsincorporation intoPLs causesadefectintouchsensation[87].However,thedefect is not complete, suggesting that polyunsaturated PLs merely make the membrane of sensory neurons more permissive tomechanotransduction.Thiseffect could re-sultfromareductionintheforcethresholdthat leadsto membrane deformation and/or from a facilitation of the conformationalchangeofthechannel.InDrosophila, ma-nipulatingthediettodecreasetheamountof polyunsatu-rated PLs in the rhabdomere causes a decrease in the speed and gain of phototransduction [88]. Because this defectcorrelateswithastiffermembrane,thisobservation suggeststhatthemembranephysicalpropertiesaffectthe gatingofamechanosensitivechanneldownstreamofthe phototransduction cascade. More generally, many chan-nelsexhibit strong sensitivity toboth mechanical stress and lipid composition, and are excellent candidates to detectsubtlechanges inthehydrophobicmembrane

ma-trix[89–91].

Concludingremarks

Simpleorganismscontainonlysaturatedand monounsat-urated lipids, highlighting a fundamental role of the

monounsaturated/saturated ratio for elementary func-tions. This ratio ranges from low valuesin membranes with a protective barrier function (apical membrane of epithelial cells, lung surfactant) [1], to high values in membranes withabiosyntheticfunction, asexemplified

bytheER(Figure1B).Theevolutionarypressureforthe

conservationofD9desaturaseandtheresultingselection ofoleate(C18:1-n9)mightthenreflectaneconomicalway to synthesize an acyl chain whose structure creates enough packingdefects inmembranes to hostreactions associated with a huge transfer of material, such as the foldingof transmembranehelices. Not surprisingly, this featureis hijacked bysome pathogens, which take advantageoflooselipid-packingattheER fortheirown replication[92].

The concepts that are operational to understand the differencesbetweensaturatedandmonounsaturatedPLs (cylindricalvsconicalshape,tightvsloosepacking,barrier vsbiogenicfunction)arenotadaptedtounderstandingthe propertiesofpolyunsaturatedPLs.Functionally,the hall-markofthesePLsistheirabundanceinmembranesthat hostsuper-fastandefficientreactions(e.g., phototransduc-tion, ATP synthesis, neurotransmission). Molecularly, theirdistinguishablefeatureistheirfastadaptation:like contortionists, theyare alternatively cones, cylinders, or hairpinstominimizethepackingstressthatresultsfrom thefastswitchoftransmembraneproteinsorthe mechan-icalworkofmembrane-bendingproteins. Inotherwords, polyunsaturatedPLsarenotatypeof‘super’ monounsat-uratedPLs;theyarejustdifferent.

Theexamplesevokedheremighthelpinunderstanding the traits imparted by saturated, monounsaturated and polyunsaturated lipidsin other organisms,cells, or orga-nelles(Box3).Suchstudieswillrequirecarefullipid analy-sis, notably at the subcellular level, as wellas sensitive functionalassaysand tediousreconstitutionexperiments, notablyofstructuralfissionandfusionintermediates[93], because the effects are likely to be subtler than those observedbythedeletionofessentialproteins.Thenematode C.elegans,whichisamenabletogeneticmanipulationand displaysalargesetofenzymesforlipidremodeling,provides ausefulmodel[7,76,78,94–96].Inaddition,recentadvances ingene-editingtechnologyopenthepossibilityof compre-hensivestudiesoftheimpactoflipidremodelingincultured cells. Another challenge is to determine the spatial and temporalcontroloflipidremodelingatthesubcellularlevel: most lipid remodeling enzymes have been reported to

Box3.Outstandingissues

Lysophospholipid acyltransferase specificity, regulation and structure

SpatiotemporalcontrolofPLacylchainremodeling

Control of the monounsaturated/saturated PL ratio along the secretorypathway

OriginofthegradientofpolyunsaturatedPLsalongaxons StructureofpolyunsaturatedPLsinfissionandfusion

intermedi-ates

Correlation between fastendocytosisat thesynapseand poly-unsaturatedPLs

Differencesbetweenv3andv6PLsinsynapticfunctions Impact of the acyl chain remodeling of phosphoinositides on

cancercells

localizeattheER,buttheirlipidproductsclearly concen-trateatspecificlocations.Last,wearefarfrom understand-ingallsubtlechangesthatdistinguishfattyacylchainsin PLs.Forexample,thedifferencebetweenv3andv6PLsis not clear-cut: both seem prone to rapid conformational changes,butthecurrentexperimentaldataandsimulations arefragmentary[65,66,97,98].

Counterexamples of the general trends of acyl chain distributionwillalsodeservespecialattention.TheERis notmadeexclusivelyofmonounsaturatedlipids:long sat-uratedlipidsmightparticipateinthecreationofdiffusion barriersimportantforthesegregationofproteins[99]. Con-versely,monounsaturatedlipidsintheexoplasmicleaflet ofthePMmembraneareprivilegedtargetsfortoxinsthat drivemembraneinvagination[100].Overall,lipidspecies whoseacylchainsdifferinlengthandunsaturationfrom bulklipidsaremorelikelytohavespecificroles,a promi-nentexamplebeingphosphoinositides(PI)[94,101].During nuclearenvelopeformation,polyunsaturatedPIsare cru-cialformembranefusionevents[102].Incancercelllines,a marked shiftin the lengthand unsaturation of PIs has beenreportedrecently,butitsimpactoncellularfunctions isunknown[24].

Acknowledgments

WorkinthelaboratoryofB.A.issupportedbytheCNRS,theEuropean ResearchCouncil(advancedgrant268888),andtheAgenceNationalede laRecherche(ANR-11-LABX-0028-01).

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