Downloaded by guest on September 25, 2021 a ihyngtvl uvddrn uin r nihdi cone- in enriched are fusion, (ii)inCa (5); during 2-aminoethylphosphonolipids shaped curved become which negatively regions, protozoon highly membrane the pore-containing in the mating mophila, For during curved. highly the (i) often which are example, observed in is matching sorting regions of or Membrane regions inhomogeneity toward (4). shapes curvature different membrane of pref- the to due of or (3) erences sterols result and a lipids lipid between as interactions particular formation the domain of of 2), (1, association preferential membrane with to types due be may bilayer igelpdseisi eemndb nitiscpoet fthe of property intrinsic an by determined is species of lipid composed single monolayer a a of curvature The (10). curvatures ferent shape. lipid and curvature by determined membrane be between may coupling lipids the the of the distribution raised the have that observations possibility These (9). lipids pathway vesicles intermediate endosomal (iii) and the tubules and an the in (8); in sorted site of differently are fusion shapes formation the different of at the localizing facilitate by structure to fusion thought are cone-shaped 7), and (6, fusion, important vesicle are synaptic lipids phosphatidylinositol-4,5-bisphosphate regulated in and exocytosis I | vesicle unilamellar small | quenching curvature fluorescence spontaneous lipid | coupling membrane curvature of sensors discriminating curvature spontaneous lipids the are by nor curvature. driven lipids, mem- be the biological to of likely in not found are distributions implies branes preference lipid curvature asymmetric weak the The of that areas. values low molecular the lipid of the because nevertheless preferences they curvature curvatures, weak spontaneous showed high corresponding have lipids the though some that, of show mea- We diffraction. curvatures X-ray earlier using lipids spontaneous nonfluorescent on the based of predictions in surements are with measurements mea- agreement Our to coefficient. quantitative distributions us coupling allowing lipid curvature curvature, the transverse sure membrane the on that linearly observed depended across We distribution bilayer. equilibrium the NBD the and measured lengths egg-PC and chain saturation, of different of (NBD)-labeled mixture lysophospholipids N-nitrobenzoxadiazole or a of phospholipids fraction with mole small sizes small a prepared different and We of several method. vesicles of fluorescence-based unilamellar preferences a curvature using bio- views, the by in different lipids measured observed these directly that have between than we distinguish weaker To is processes. alone, logical shape of preference to curvature pre- due the theoretical lipids, that hand, suggest other simulations the and related On dictions shape. is membrane molecular which lipid’s the curvature, the between spontaneous to lipid’s coupling the been the and has to curvature due sorting be lipid to This regions into hypothesized curvatures. sorted membrane are shapes different different with of has lipids it that trafficking, shown and fusion been fission, as such processes, biological In Kamal M. Marzuk curvature leaflet and between shape coupling lipid weak only reveals phospholipids preference of curvature membrane the of Measurement w.nsog/ci/di/1.03/pnas.0907354106 / 10.1073 / doi / cgi / www.pnas.org dtdb xlT rne,Safr nvriy tnod A n prvdNvme ,20 rcie o eiwJl ,2009) 2, July review for (received 2009 2, November approved and CA, Stanford, University, Stanford Brunger, T. Axel by Edited Germany Main, am Frankfurt 60438 3, Max-von-Laue-Strasse a lnkIsiueo oeua elBooyadGntc,Poehurt 0,037Dedn emn;and Germany; Dresden, 01307 108, Pfotenhauerstr Genetics, and Biology Molecular of Institute Planck Max eedn ntemlclrsae iisfr tutrso dif- of structures form lipids shape, molecular the on Depending eul.Teihmgniyo ii itiuin ihna within distributions lipid homoge- distributed of not inhomogeneity are The lipids neously. membranes, biological n a eykMills Deryck , b ihlGrzybek Michal , erhmn ther- Tetrahymena 2 + -dependent a n oahnHoward Jonathon and , hc eoti h uvtr opigcefiin ftelipids. Model the of coefficient coupling curvature the obtain from we technique, which quenching fluorescence a by determined is bution distri- (see transverse S1 lipid Fig. (NBD)-labeled and of N-nitrobenzoxadiazole 1 The Fig. diagrams in shown schematic are studied The lipids physiological the concentration. at salt studied vesicles are and unilamellar diameters temperature small mean in different of lipids (SUVs) labeled fluorescently quan- trace of lipids technique, tities this different In structurally method. in fluorescence-based several a reside using of to measured preferences directly preference we curvature its curvature, and the particular lipid of leaflet a membrane of a shape the between ship was nanotubes membrane stud- into 23). these (22, lipids with observed of sorting than Consistent significant weaker systems. no is biological ies, lipids in of observed preference that curvature the these that on 21) suggested (20, based simulations studies molecular-dynamic and Theoretical 19) 18). (2, measurements (17, type lipid second the aesaluiaelrvsce SV)wt itr of mixture a with (SUVs) vesicles unilamellar small pare qeu ouin h eidct fteH the of periodicity The solution. aqueous n h edn tfns fteDP iis(6 7.Ntall Not 17). (16, lipids DOPE the H of the form curvature stiffness lipids spontaneous bending the the X-ray both by estimate and measured to been used has and pressures diffraction osmotic different of tions hsatcecnan uprigifrainoln at online information supporting 0907354106/DCSupplemental. contains article This reyaalbeoln hog h NSoe cesoption. access open PNAS the through online available Freely Submission. Direct PNAS a is article This interest. of conflict no declare paper. authors ana- the The M.M.K. wrote per- tools; J.H. M.G. and reagents/analytic M.M.K. and new and D.M., contributed data; J.H. M.M.K., lyzed and research; M.M.K. designed research; J.H. formed and M.M.K. contributions: Author Distribution. Lipid Transverse Curvature-Dependent oeohrlpd a eetmtdb nlzn h perturbation H the DOPE analyzing the of by periodicity estimated the in be can lipids other some 1 aueo iisrle ntefraino nivre hexago- inverted an of formation the on (H relies nal lipids of vature preference (13–15). curvature lipids membrane the of in lat- role a undergo is play curvature to lipids spontaneous hypothesized Lipid the redistribution. and/or the transverse and stress, curvature eral packing its the has reduce It changes to (12). membrane order lipids in bilayer the that the hypothesized within been and stress curvature packing spontaneous introduces curvature lipid mis- the The between (10). match respectively curvatures, spontaneous negative positive, zero, and have lipids conical and The inverted-conical, (10). cylindrical, respectively conical monolayers, concave and and Cylindrical convex inverted-conical, shape: form and lipids its monolayers, to heuristically planer related form be lipids can lipid curva- a spontaneous of The ture (11). curvature spontaneous called lipids owo orsodnesol eadesd -al [email protected]. E-mail: addressed. be should correspondence whom To ots hs hoeia rdcin n eemn h relation- the determine and predictions theoretical these test To h nykontcnqefrmauigtesotnoscur- spontaneous the measuring for technique known only The II hs fdoeypopaiyehnlmn DP)in (DOPE) dioleoylphosphatidylethanolamine of phase ) PNAS a,1 II eebr2,2009 29, December hs;hwvr h pnaeu uvtr of curvature spontaneous the however, phase; II b a lnkIsiueo Biophysics, of Institute Planck Max hs asdb h diinof addition the by caused phase o.106 vol. www.pnas.org/cgi/content/full/ II o 52 no. tutrsi solu- in structures IAppendix). SI fw pre- we If 22245–22250

BIOPHYSICS AND APPLIED PHYSICAL COMPUTATIONAL BIOLOGY SCIENCES    −1 Ai uo − ui no = 1 + exp . [3] Ao kBT Eq. 3 relates the NBD-labeled lipid transverse distribution func- tion, no, to the bilayer curvature, H (= 1/R), and the NBD-labeled lipid spontaneous curvature, c0. For the NBD-labeled lipids with positive spontaneous curvature, the energy cost for being in the inner leaflet is higher than that for being in the outer leaflet (ui > uo); therefore, no increases with the increasing bilayer cur- vature as the NBD-labeled lipids have preference for the outer leaflet of the bilayer. On the other hand, for negative spontaneous curvature NBD-labeled lipids, no decreases with increasing bilayer curvature as uo > ui, meaning that the NBD-labeled lipids have preference for the inner leaflet of the bilayer. When the membrane bilayer is flat or the lipid spontaneous curvature is very small, the energy cost for the NBD-labeled lipids being in either of the leaflets is almost equal (uo ≈ ui). In both cases, the NBD-labeled lipids are symmetrically distributed in the two leaflets, and Eq. 3 becomes A 1 D n = o ≈ + H (for R  D/2), [4] s A 2 2 Fig. 1. The schematic diagram of the POPC, NBD-lyso-PPE, NBD-DPPE, NBD- lyso-OPE and NBD-DOPE lipids used in this study. POPC is the major compo- where A is the total area of the two leaflets. Therefore, the symmet- nent of egg-PC. The structures of NBD-lyso-MPE, NBD-DMPE, NBD-lyso-OPS ric distribution function, ns, is the frame of reference with respect and NBD-DOPS lipids are shown in Fig. S1 (see SI Appendix). to which we can estimate the asymmetric transverse distribution of the NBD-labeled lipids in the bilayer leaflets. egg-phosphatidylcholine (egg-PC) and a small mole fraction of NBD-labeled lipids, then the presence of the NBD-labeled lipids Curvature Coupling Coefficient. In biological vesicle systems such is not expected to affect the membrane properties, such as bending as synaptic vesicles or the vesicles in the endosomal pathway, the −1 −1 stiffness and membrane curvature. Rather, the membrane curva- typical membrane curvature ranges from 0.005 nm to 0.05 nm ture may affect the transverse distribution of the NBD-labeled (corresponding to diameters from 400 to 40 nm). In this curvature lipids (i.e., across the bilayer). The NBD-labeled lipids will pre- range, no (Eq. 3) is approximately proportional to membrane cur- ∂no ≈ fer the leaflet that costs less energy. The bending energy cost per vature because, as we show, ∂H constant. Therefore, the Taylor molecule for the NBD-labeled lipid to be in one of the leaflets expansion of Eq. 3 around zero mean curvature is (11)   = 1 + D + Γ 2 no H H, [5] ul = 2κa(Hl − c0) , [1] 2 2

where Hl is the mean curvature of the leaflet in which the NBD- where labeled lipids are located, κ is the leaflet or monolayer bending 2κac0 stiffness and, c0 and a are the mean spontaneous curvature and Γ = , [6] molecular area of the NBD-labeled lipids, respectively. The sign kBT of c0 depends on the shape of the NBD-labeled lipid. The bend- κ is a good approximation for the lipid distribution. The first term ing stiffness, , is assumed equal in both of the bilayer leaflets. in Eq. 5,(1+ DH)/2, expresses the symmetric distribution of the When the system is in equilibrium, the NBD-labeled lipids, along NBD-labeled lipids in a bilayer of curvature H and is equivalent to with the other lipids, translocate between the two leaflets and will Eq. 4 for R  D/2, a valid approximation in our range of bilayer prefer the leaflet that is energetically more favorable. According curvatures. The second term, ΓH, is the measure of fractional lipid to Eq. 1, the energy cost per lipid for the NBD-labeled lipids asymmetry due to the curvature preference of the NBD-labeled = κ − 2 being in the outer and inner leaflets are uo 2 a(Ho c0) lipids. For Γ > 0, the NBD-labeled lipids prefer the outer leaflet = κ − 2 = + and ui 2 a(Hi c0) , respectively, where Ho 1/(R D/2) and for Γ < 0, the lipids prefer the inner leaflet. As Γ encapsu- and Hi =−1/(R−D/2) are the mean curvatures of the outer and lates all the necessary mechanical parameters, including c0, that inner leaflets, assuming the SUVs are spherical, R is the radius of may contribute to the curvature dependent asymmetric transverse the SUV bilayer midplane, and D is the membrane bilayer thick- distribution of the lipid of interest, we call Γ the curvature coupling ness. Note that the outer leaflet is defined to have positive mean coefficient. curvature (Ho > 0) and, therefore, the inner leaflet has negative Finally, in order to calculate the values of Γ and c0 from the mean curvature (Hi < 0). When the outer and inner leaflets have experimental measurements of the transverse distribution, no, no and ni fractions of NBD-labeled lipids, then the lipid densities κ ρ = ρ = we have used literature values of the parameters , D and a in are o no/Ao and i ni/Ai where Ao and Ai are the outer Eq. 5 (see Relationship Between Curvature Coupling Coefficient and and inner leaflet surface areas at the level of the headgroups. By Spontaneous Curvature). using the Boltzmann distribution, we calculate the lipid density ρ ρ = [− − ] ratio as o/ i exp (uo ui)/kBT . Therefore, the ratio of the Results outer and inner leaflet NBD-labeled lipid fractions is   Properties of the SUVs. We have prepared the SUVs of different n A u − u sizes and lipid compositions by using the protocol described in the o = o exp − o i , [2] n A k T Materials and Methods section. The SUV size distributions were i i B measured by dynamic light scattering (DLS) (Zetasizer Nano ZS). where kB is the Boltzmann constant and T is the temperature. The DLS data (scattered light intensity vs. hydrodynamic diame- Because the lipids are confined within the bilayer leaflets, we have ter) were converted to SUV number (%) vs. hydrodynamic diam- no + ni = 1. Therefore, by using Eq. 2, we can calculate no as eter (Fig. 2A) using the nonnegatively constrained least squared

22246 www.pnas.org / cgi / doi / 10.1073 / pnas.0907354106 Kamal et al. Downloaded by guest on September 25, 2021 Downloaded by guest on September 25, 2021 E eecnitn ihec te;bcuetecryo-TEM the because other; each with consistent were TEM r akdwt ige obe n rpearwed,respectively. arrowheads, triple and double, single, with marked are imtr fteSVdsrbtosmaue yDSadcryo- and DLS by mean measured The distributions 1. prepara- Table SUV in sample the summarized of separate are diameters obtained category three size diameters each from of mean vesicles tions their 200 and analyzing bilayers MLV distribution by third the and second in the present of amount fractional two The trilamel- containing lar. considered MLVs are concentric, The necessarily observe not bilayers. SUVs, not three smaller did than We more distributions. with MLVs all SUV in MLVs 36-nm-diameter trilamellar the and bilamellar but that of show fractions images small cryo-TEM are The MLVsthere 2B). of (Fig. number samples the fractional in the present estimate size to different order of in SUVs distributions unstained of microscopy images electron obtain cryotransmission to (cryo-TEM) used therefore We tech- quenching nique. our confound potentially can DLS, by SUVs. ascertained the by into incorporated micelles log- lipids any the lyso detect the with that not data showing did DLS, the We fitting function. the by distribution of estimated normal deviation were standard 1) and (Table diameter SUVs mean The regularization (24). CONTIN package the in included algorithm fitting (NNLS) and 2. Fig. aa tal. et Kamal different preparation. of after in diameter SUVs hours mean 24 shown NBD-lyso-PPE of about are + are taken SUVs were egg-PC function images of The distribution images distributions. log-normal size Cryo-TEM the (B) 1. with Table fitting mea- by by deviations measured standard sured and sizes diameters Mean pore (DLS). scattering indicated light the dynamic with filters membrane polycarbonate h rsneo utlmla eils(Ls,wihcno be cannot which (MLVs), vesicles multilamellar of presence The (iv ) 83 ie fteSUVs. the of Sizes ± 5n Tbe1.Uiaelr iaelr n rlmla vesicles trilamellar and bilamellar, Unilamellar, 1). (Table nm 25 (A) (i h iedsrbto fSV xrddthrough extruded SUVs of distribution size The ) 31 ± 3nm, 13 (ii ) 50 ± 8nm, 18 (iii ) 70 ± 1nm 21 eodblyrfraction, bilayer Second o hs Lst obtain to MLVs these for endaee,n,cy-E 31 cryo-TEM nm, diameter, Mean ue eflt fteSV.FrteohrSV,teewsasmall a was 1); there (Table the SUVs, MLVs other in with the distributed For contamination SUVs. lipids the NBD-labeled of leaflets of outer fraction average the of hr iae fraction, bilayer Third costeSVblyr eemaue yqecigthe quenching by sodium with SUVs measured the Na of were (SDT), leaflet lipids outer dithionite the bilayers NBD-labeled in lipids SUV the NBD-labeled of the distributions across transverse fractional rn iae 2,2) ro oteemaueet,teSUVs 14 37 the for at incubated hours measurements, and 16 buffer these to NaCl-HEPES to in 10-fold Prior mem- diluted 26). were the through (25, permeability bilayer low brane very has SDT nonfluorescent. NBD Furthermore, makes and NBD the with reaction reduction esrmn fteNDLbldLpdTases Distribution. Transverse Lipid NBD-Labeled the of Measurement eil property Vesicle data cryo-TEM and DLS measured using sizes through by pore extrusion indicated by the prepared with SUVs membranes polycarbonate the of diameters The 1. Table Coeffi- Coupling Curvature the and Lipids cient. of Preference Curvature sago siaino h enfato fteNBD-labeled ensemble. the SUV of the of fraction leaflets mean outer the the This in of distribution 3A). lipid estimation (Fig. gives good buffer drop a the fluorescence us in initial the abundant lipids that still NBD-labeled demonstrates is observation leaflet which to inner SDT, due the remaining zero to the to fluorescence of the exposure in (26). the drop fast X100, Triton rates another mM 20 observed detergent, bleaching we with SUVs photo the dissolved we and When translocation, mem- SDT NBD-dipalmitoylphosphatidylethanolamine (NBD-DPPE) the 3A). than permeation, (Fig. higher much outward lipids brane is the rate NBD-labeled to reaction leaflet NBD-SDT due fol- inner The leaflet decrease the outer fluorescence of the slow in translocation very lipids a NBD-labeled fast by the a lowed with observed SDT We the reaction 10). reduction of mM irreversible (pH the 15 base to Trizma due added fluorescence M then in decrease and 1 seconds in NBD- 200 1-mL prepared the a about SDT of of for fluorescence SUVs distribution the total of the lipid the across solution measured the outside first gradient we measure and lipids, pH to inside labeled the order maintained by was In distrib- 7.4 affected SUVs. lipid pH is Because (27), bilayer 20. membrane the around in signal-to-noise maintained ution The always Inc). was fluorometer Yvon a ratio using Jobin by HORIBA measured measurements). was (FluoroMax-3, quenching SUVs the the of by Fluorescence (confirmed bilayer the across rmtreidpnetypeae ape rmtecy-E mgsof deviations. images standard cryo-TEM are vesicles errors the The 200 from category. analyzing size samples by each prepared estimated independently were MLVs, three the from in diameters mean tive DLS nm, diameter, Mean hr iae endaee,n 63 37 52 31 46 26 31 — — nm diameter, mean bilayer Third nm diameter, mean bilayer Second ubro ape,w sdteSVsz-itiuindt nour in data size-distribution SUV smaller calculations. the the used we to samples, due of deviation number standard larger have measurements uin oterpdfatoa rpi intensity, in drop distri- SUV fractional the rapid in the present MLVs so no bution, were there For SUVs, subtraction. 36-nm background the after normalized fluorescent were The data distributions. intensity size SUV different in fractions lipid (NBD-lyso-PPE) NBD-palmitoylphosphatidylethanolamine and h rcinlaon ftescn n hr iaes n hi respec- their and bilayers, third and second the of amount fractional The i.3B Fig. emaue h rnvredistribution, transverse the measured We hw esrmnso h ue efltNBD-DPPE leaflet outer the of measurements shows PNAS ◦ oalwteNDlbldlpd oequilibrate to lipids NBD-labeled the allow to C f t 2 (%) f S b eebr2,2009 29, December 2 (%) O n 4 o 2) D nege nirreversible an undergoes SDT (25). (see 36 IAppendix SI 53 0100 80 30 15 ± ± 114 11 8 0 . 0.8 0.6 2 0 350 13 746 I o a hrfr corrected therefore was o.106 vol. oesz,nm size, Pore ± ± o details). for 870 18 967 I o n sameasure a is , o 52 no. o ± ± o l the all for , 183 21 483 14 22247 ± ± The 25 17

BIOPHYSICS AND APPLIED PHYSICAL COMPUTATIONAL BIOLOGY SCIENCES (NBD-lyso-MPE), showed increasing preference for the pos- itive curvature outer leaflet with the increase in SUV mem- brane curvature (Fig. 4A and Fig. S2A of the SI Appendix). On the other hand, the saturated NBD-DPPE and NBD-di- myristoylphosphatidylethanolamine (NBD-DMPE) lipids showed decreasing preference for the outer leaflet with increasing bilayer curvature, which implies that these lipids have preference for the inner leaflet of the bilayer. The unsaturated NBD-DOPE and NBD-dioleoylphosphatidylserine (NBD-DOPS) lipids showed curvature-dependent asymmetric distribution toward the inner leaflets (Fig. 4B and Fig. S2B of the SI Appendix). Taking the symmetric lipid-distribution function, ns (Eq. 4), as the frame of reference, the saturated lyso lipids, NBD-lyso-MPE and NBD-lyso-PPE, show ≈10% enrichment in the outer leaflet, and the saturated NBD-DMPE and NBD-DPPE lipids show ≈15% enrichment in the inner leaflet of the SUVs with mean curvature 0.056 nm−1 (36-nm diameter). For the same SUV curvature, both the unsaturated NBD-DOPE and NBD-DOPS lipids showed ≈24% enrichment in the inner leaflet, whereas the NBD-oleoylphosphatidylethanolamin (NBD-lyso-OPE) and NBD-oleoylphosphatidylserine (NBD-lyso-OPS) lipids did not show any significant preference for either of the leaflets, as indi- cated by the overlap of the transverse distribution data points with the line corresponding to ns. The curvature coupling coefficient, Γ, was estimated (Table 2) by fitting Eq. 5 with the no vs. H data (Fig. 4 and Fig. S2 of the SI Appendix) by chi-squared minimization weighted by the stan- dard deviations along both of the axes. Thus, the smaller SUVs with narrower size distributions give slightly more weight to the Fig. 3. Fluorescent-quenching method for estimating the fractional outer fitting. leaflet NBD-labeled lipid distribution in the SUVs. (A) SUVs of 83 nm mean Relationship Between Curvature Coupling Coefficient and Sponta- diameter were prepared from egg-PC with a small mole fraction (0.01%) Γ of NBD-DPPE. Addition of 15 mM SDT caused a fast decrease in total fluo- neous Curvature. To relate the curvature coupling coefficient, , rescence due to NBD quenching. The following slow decrease is mainly due to the spontaneous curvature, c0, via Eq. 6, we used the following to outward translocation of inner leaflet NBD-DPPE lipids. Addition of 20 parameter values for the different NBD-labeled lipids from the mM detergent, Triton-X100, dissolves the membrane, exposing the rest of literature (where possible). The bending stiffness of a lipid mono- the NBD-DPPE lipids in the inner leaflet to SDT, causing the total fluores- layer has the same order of magnitude for different lipids and cence to drop to zero. Data were normalized after background subtraction. has been estimated as κ = 10 kBT (κ range, 9–11 kBT) (28–30). (B) Fluorescence quenching of the SUV outer leaflet NBD-lyso-PPE (Left) and The thickness of the egg-PC SUV membrane bilayer, D, is 3.6 nm NBD-DPPE (Right) lipids by SDT. The fast drop in fluorescence, Io, represents (31, 32). The NBD fluorophore is attached to the head group of the the fractional amount of NBD-lyso-PPE or NBD-DPPE present in the outer phosphatidylcholine (PE) and phosphatidylserine (PS) lipids, and leaflet of the SUVs. In case of NBD-lyso-PPE, Io increases with decreasing SUV diameter. The opposite effect is observed for NBD-DPPE lipids. the NBD is outside the membrane surface (33); because only a very small proportion of the lipids in the SUVs are labeled with NBD, NBD-labeled lipids (Fig. 4 and Fig. S2 of the SI Appendix). In we assumed that there is no interaction between the NBD-labeled all the measurements, the transverse distribution of the NBD- head groups. Therefore, the NBD molecules do not contribute to the molecular area, a, of the attached lipids. The molecular area of labeled lipids was approximately proportional to the SUV mem- 2 2 2 brane curvature, in agreement with Eq. 5. The saturated lyso lipids, the DMPE is 43 Å (34), DPPE is 48 Å (35), DOPE is 68 Å (36), NBD-lyso-PPE and NBD-myristoylphosphatidylethanolamine DOPS is 66 Å2 (37), lyso-MPE is 31 Å2 and lyso-PPE is 38 Å2 (38).

Fig. 4. The transverse distribution, no, of the NBD-labeled lipids as a function of SUV bilayer curvature, H. The dotted lines correspond to symmetric distribu- tion, ns (Eq. 4). (A) The NBD-lyso-PPE () lipids show preference for the positive curvature outer leaflet, whereas the NBD-DPPE () lipids show preference for the inner leaflet. (B) The unsaturated NBD-lyso-OPE () lipids do not show any significant preference for any of the leaflets as it overlaps with ns. The unsatu- rated NBD-DOPE () lipids prefer the negative curvature inner leaflet. By fitting the data with Eq. 5 (the red and blue lines for the single- and double-chain lipids, respectively) we estimated the curvature coupling coefficient, Γ, and spontaneous curvature, c0, of the lipids (Table 2).

22248 www.pnas.org / cgi / doi / 10.1073 / pnas.0907354106 Kamal et al. Downloaded by guest on September 25, 2021 Downloaded by guest on September 25, 2021 B-yoOE0.10 1.32 1.67 NBD-lyso-OPE NBD-lyso-PPE NBD-lyso-MPE NBD-DOPS NBD-DOPE NBD-DPPE n B-yoOElpd are lipids NBD-lyso-OPE and 48 yolpd,NDls-P n B-yoPE r inverted- are NBD-lyso-PPE, and (c lipids conical NBD-lyso-MPE lipids, saturated The lyso 2). (Table curvatures spontaneous the estimate to us 19). (2, studies SUVs theoretical in and 21) simulations (20, molecular-dynamic coarse-grained predic- of support the observations tions These nm. all 100 than of larger diameter ( a preference low very curvature become would the lipids NBD-labeled 2, Table From respectively. rnvredsrbto aa(i.4adFg 2o the of S2 Fig. and 4 (Fig. data distribution transverse o B-OE ecnetmt that estimate can size we SUV NBD-DOPE, given for a Eq. for using lipid the by of preference curvature fractional h uvtr opigcoefficient, coupling by quantified curvature are the membrane of egg-PC amounts the trace in the lipids of NBD-labeled the preferences curvature the of strength The Discussion lipids. lyso unsaturated the of cases the ngo gemn ihteeXrydfrcinmeasurements: diffraction curvature, X-ray of radius these spontaneous with The are curvatures agreement spontaneous good of values in inferred Our 30). (17, lyso-OPE tively and DOPE be the to measured of were lipids curvature of radius spontaneous the NBD-lyso-OPS rosin errors emaue h ra fls-P n yoOSt e47 be to lyso-OPS and lyso-OPE of areas the measured We limits. confidence 95% are errors Eq. The category. using calculated are values coupling, curvature The 2. Table aa tal. et Kamal ae rmtesadr ro) epciey Our respectively. error), standard the from mated NBD-DMPE Lipid anyo h acltdvle of values calculated the of tainty r oia iis(c lipids conical are eflto h Uso encraue00 nm 0.01 inner curvature curvature mean negative of the SUVs the in of enriched leaflet be would lipids DOPE lipids. unsaturated corresponding NBD-labeled the with compared leaflet outer the for respec- with lipids, NBD- tive NBD-lyso-PPE NBD-DMPE, and asymmet- saturated NBD-lyso-MPE, curvature-dependent The DPPE, display distributions. not transverse do ric lipids these that and nm (0.01 lipids NBD-lyso-OPS small The respectively. nm, rfrnefrteinrlae with leaflet inner higher the comparatively for show preference lipids NBD-DOPS and NBD-DOPE P n B-yoOS r prxmtl yidia sthey values. as curvature spontaneous cylindrical low approximately very are have NBD-lyso-OPS, and OPE fteNDlbldlpd negP membranes egg-PC in lipids NBD-labeled the of h eni bu 0.Terltv netite in uncertainties relative The 10%. about by is divided mean deviation standard the The uncertainty: relative large a has B-yoOS hrfr,terltv netite forcal- our of uncertainties culated relative the Therefore, NBD-lyso-OPS. in those and (<5%) h ausof values The u esrmnso h uvtr opigcefiin allow coefficient coupling curvature the of measurements Our nwn the Knowing nteXrydfrcinmaueeto h H the of measurement diffraction X-ray the In Å Γ 2 epciey(e i.S fthe of S3 Fig. (see respectively , values c Γ 0 ausaeapoiaeyeult htof that to equal approximately are values swl so h netite in uncertainties the on as well as −2.7, o xml,b sn h value the using by example, For 5. Γ 0 r bandb tigEq. fitting by obtained are Γ > −5.49 −4.19 −2.13 −0.08 −2.65 .,17 n . m aeahge preference higher a have nm, 1.3 and 1.7, −2.1, au falpd n a edl siaethe estimate readily can one lipid, a of value 0 )adaltedul-hi B-aee lipids NBD-labeled double-chain the all and 0) Γ,nm < Γ ± ± ± ± ± ± ± ± ) h nauae yolpd,NBD-lyso- lipids, lyso unsaturated The 0). are .066 0.20 .068 0.10 .348 0.13 .648 0.16 .343 0.13 .53 .75.8 3.7 0.01 0.17 0.27 47 38 0.08 31 0.15 0.13 madlre hn4 m respec- nm, 40 than larger and nm ≈−3 hr r bu 0dt onsprlipid per points data 90 about are There 6. %ecp o D-yoOEand NDB-lyso-OPE for except ≈5% Γ auso h B-yoOEand NBD-lyso-OPE the of values n pnaeu curvature, spontaneous and Γ, . mand nm −3.2 c a, 0 Å eed ntemeasurement the on depends .8n,rsetvl)imply respectively) nm, −0.08 Γ 2 Γ r Tbe2.Teunsaturated The 2). (Table 0 values .Teuncer- The Appendix). SI .%and ≈4.2% =1/c (= 5 c ihteNDlbldlipid NBD-labeled the with 0 −0.42 −0.31 −0.22 −0.01 −0.31 a ,nm 0)i Uswith SUVs in <10%) and . mand nm −4.2 0 r −1 −1 0 ,o NBD-DOPE of ), .The Appendix). SI II h au of value The κ. hs tp 7.0, pH at phase ≥ n .5nm 0.05 and Γ r 1 NBD- ≈21% 0 5n (esti- nm 55 κ =− au (−2.4 value r a 0 xetfor except |r |r = r small are 0 0 −2.4 −3.2 −4.5 −3.3 |≥40 |≥55 c . nm 4.2 Å 0 −1 2 −5.5 ,nm and −1 c c 0 κ 0 , , neo ii eed ieryo h ii pnaeu curvature, spontaneous lipid c the on linearly depends lipid a of ence rvn h ii smer 2) ntecs fsnpi vesicles synaptic of case the In (27). asymmetry lipid the be driving flippases—must lipid as curvature—such membrane than other inbtenteNDDP sngiil n,hne eregard we hence, measured and, negligible repul- our is coulomb of NBD-DOPS effect the the between NBD-DOPS, sion of the fraction decreased small very experiments a X-ray to the the curvature of in Neutralizing radius 2.0 spontaneous positive. pH curvature at charge of repulsion make DOPS mutual to radius the expected spontaneous is and lipids assay, the DOPS our charged negatively in the that between assay than X-ray diffrac- higher the in much X-ray However, DOPS is the (18)]. charged 7.0 negatively from pH of at concentration differs nm the [14.4 7.4 DOPS pH for measurements at tion NBD-DOPS for nm) The are NBD-lyso-MPE ro ftemaueet,t be to measurements, the of error B-OElpd,NDDP iishave lipids NBD-DOPE lipids, NBD-DOPE h -a ifato esrmnsa o H nadto,we addition, In pH. the low that at estimated measurements diffraction X-ray the u siain ept h similar to the According despite lipids. estimation, of our preference curvature promotes area cular area, molecular nauae.I rtrcts(imtr8 (diameter mainly erythrocytes are which In lipids, unsaturated. PS and PE containing systems membrane data. these for account realistic to required not are is explanations (see sorting alone other preference lipid curvature fivefold the on a based that (see work shows own calculation our our as observations well as other 23) contradict of (22, observations diameters These with (42). tubes reported membrane into types lipid area molecular small lipids. the the of because of weak is lipids pref- these lipids curvature of erences membrane some the although curvatures, that, spontaneous demixing high suggest near have observations lipids our of proteins, Thus, heterogeneity point. transmembrane or with clusters, lipid very compared have peptides, area curva- general, in to molecular lipids, be sensitivity individual small can that amplified observations noting These to by 41). (40, understood lead ways various can in gradients point ture demixing a proximity and to curvature- separation lateral Phase (22). clustering, demixing mixtures any the lipid–sterol near of of only point show occur to absence not shown was the sorting the did lipid dependent in lipids In cases, the preference. both clustering, curvature In curva- lipid 23). high of (22, the absence region of out nanotube stay membrane systems, to ture tend vesicle–nanotube low clusters the In lipid in (39). -assisted localize clusters region the curvature clus- where lipid membrane separate systems, phase to bilayer tend supported ters curved periodically could in assembly, shown been that molecular has It of lipids. or of area preference sterols curvature stronger larger by promote a assisted in formation, resulting cluster proteins, mol- lipid larger Also, the areas. of ecular because preference curvature stronger predict the we peptides, monosialotetrahex- in and proteins cardiolipin, transmembrane Therefore, as (GM1), osylganglioside magnitude. such molecules in larger equal of cases almost cur- are spontaneous their values although vature lipids, preference NBD-lyso-MPE curvature the higher than same have the lipids For NBD-DMPE lipids. the NBD-DOPE reason, of the of because area lipids molecular NBD-DMPE larger the of that than preference curvature itiue nteblyrlaesa h ebaecraueof nm curvature [<0.001 membrane small the very as is leaflets erythrocyte bilayer (27). leaflet the inner in the distributed in distributed Eq. are to lipids According PS the of 95% and 0 swl so h ooae edn stiffness, bending monolayer the on as well as , codn otedrvto of derivation the to According e snwcmaeorosrain ihsm biological some with observations our compare now us Let two of sorting relative fivefold a work, published recently a In r 0 au fNDls-P setmtd rmtestandard the from estimated, is NBD-lyso-OPS of value r PNAS 0 ihrbnigsifesa ela agrmole- larger as well as stiffness bending Higher a. . ma H74 ncoeareetwith agreement close in 7.4) pH at nm (−2.4 ohteP n Slpd hudb equally be should lipids PS and PE the both 5, r 0 . nm, −3.3 auso B-ME B-PE and NBD-DPPE, NBD-DMPE, of values eebr2,2009 29, December . m n . m respectively. nm, 3.7 and nm, −4.5 Γ |r c 0 . m(8.A eaeusing are we As (18). nm −2.3 (Eq. 0nm. |≥40 0 −1 .Furthermore, Appendix). SI auso B-MEand NBD-DMPE of values 4).Teeoe factors Therefore, (43)]. ,tecraueprefer- curvature the 6), o.106 vol. μm), .Thus, Appendix). SI . ie higher times ≈1.6 0 ftePE the of ≈80% o 52 no. 0n was nm ≈70 n lipid and κ, 22249

BIOPHYSICS AND APPLIED PHYSICAL COMPUTATIONAL BIOLOGY SCIENCES (diameter 40–100 nm), 40–50% of PE and ≈60% of the PS lipids NBD-DMPE, NBD-lyso-MPE, NBD-DPPE, NBD-lyso-PPE, NBD-DOPE, NBD-lyso- are distributed in the inner leaflet (44, 45). According to our mea- OPE, NBD-DOPS, and NBD-lyso-OPS (Fig. 1 and Fig. S1). The major com- surements, both NBD-DOPE and NBD-DOPS lipids asymmetri- ponent of egg-PC lipid mixture is (POPC) 1-palmitory-2-olely-sn-glycero-3- ≈ phosphocholine (Fig. 1). The lipid films were made by drying the lipid mixture, cally distributed with 60% NBD-DOPE and 65% NBD-DOPS which was desolved in chloroform-methanol (2:1). The concentration of the lipids in the inner leaflet of the SUVs in the similar diameter range lipid mixture was 1 mg/mL. The lipid films were kept in a vacuum oven for (36–83 nm). Although the asymmetric distribution of the PS lipids 4 hours at 45◦C, well above the melting point (−5◦C) of the lipid mixture, in synaptic vesicles is similar to that of the NBD-DOPS lipids in then rehydrated with NaCl-HEPES buffer (150 mM NaCl, 50 mM HEPES, 0.2 ≈ ◦ the SUVs of similar size range, the PE lipids in the synaptic vesicles mM EDTA, pH 7.4) for 2 hours with 2 Hz agitation at 45 C. Rehydration causes formation of MLVs of different diameters. SUVs of different diameter show a different trend than the NBD-DOPE lipids in the SUVs. distributions were prepared by extrusion of the MLVs with 50 passes through Thus, in biological systems the curvature preference of the lipids polycarbonate membrane filters of pore sizes 15 nm, 30 nm, 80 nm, and 100 must be influenced by factors other than a spontaneous curvature. nm (Whatman). Prior to extrusion, we applied the freeze-thaw technique to In this work, we measured the curvature coupling coefficient of assure that the extruded SUVs were mostly unilamellar. The double-chain NBD-labeled lipids were purchased from Avanti polar NBD-labeled lipids in SUVs composed of a natural lipid mixture, lipid. The NBD-labeled lyso lipids were prepared by phospholypase A2 hydrol- egg-PC, in physiological conditions. Our observations suggest that ysis of the NBD-labeled double-chain lipids and separated by thin-layer the coupling between membrane curvature and lipid shape is weak chromatography to have single chain NBD-lyso-MPE, NBD-lyso-PPE, NBD- mainly because of small molecular area of the lipids. This implies lyso-OPE and NBD-lyso-OPS. The quality of the prepared NBD-labeled lipids was checked by mass spectrometry. The molecular areas of the nonfluores- that the asymmetric lipid distributions within the biological mem- cent lyso-OPE and lyso-OPS lipids were measured using a Langmuir–Blodgett branes are not likely to be driven by the spontaneous curvature trough as shown in Fig. S3 of the SI Appendix, and the molecular areas of of the lipids, nor are lipids discriminating sensors of membrane other nonfluorescent lipids were taken from the literature. curvature. ACKNOWLEDGMENTS. The authors would like to thank Kai Simons, Sol M. Gruner, and the Howard lab members for their helpful comments on the Materials and Methods manuscript. M.M.K. would like to thank Werner Kühlbrandt for the sugges- Preparation of the SUVs. We first prepared lipid films from 104:1 (mol:mol) tions and support regarding the cryo-TEM imaging, Christoph Thiele, Jacques mixture of egg-PC (99% pure from chicken egg, product ID: 840051, Avanti Pecreaux, and Ünal Coskun for helpful discussions, and Julio Sampaio for his polar lipids) and one of the NBD headgroup-labeled phospholipid derivatives help with the quality control of the lipids using mass spectrometry.

1. Dumas F, Sperotto MM, Lebrun MC, Tocanne JF, Mouritsen OG (1997) Mol- 25. McIntyre JC, Sleight RG (1991) Fluorescence assay for phospholipid membrane ecular sorting of lipids by bacteriorhodopsin in dilauroylphosphatidylcholine/ asymmetry. Biochemistry 30:11819–11827. distearoylphosphatidylcholine lipid bilayers. Biophys J 73:1940–1953. 26. Moreno MJ, Estronca LM, Vaz WL (2006) Translocation of phospholipids and dithion- 2. Zimmerberg J, Kozlov MM (2006) How proteins produce cellular membrane curvature. ite permeability in liquid-ordered and liquid-disordered membranes. Biophys J Nat Rev Mol Cell Biol 7:9–19. 91:873–881. 3. Huttner WB, Zimmerberg J (2001) Implications of lipid microdomains for membrane 27. Devaux PF (1991) Static and dynamic lipid asymmetry in cell membranes. Biochemistry curvature, budding and fission. Curr Opin Cell Biol 13:478–484. 30:1163–1173. 4. McMahon HT, Gallop JL (2005) Membrane curvature and mechanisms of dynamic cell 28. Niggemann G, Kummrow M, Helfrich W (1995) The bending rigidity of phosphatidyl- membrane remodelling. Nature 438:590–596. choline bilayers—Dependences on experimental-method, sample cell Sealing and 5. Ostrowski SG, Van Bell CT, Winograd N, Ewing AG (2004) Mass spectrometric imaging temperature. J Physique II 5:413–425. of highly curved membranes during Tetrahymena mating. Science 305:71–73. 29. Chen Z, Rand RP (1997) The influence of on phospholipid membrane 6. Muller M, Zschornig O, Ohki S, Arnold K (2003) Fusion, leakage and surface hydropho- curvature and bending elasticity. Biophys J 73:267–276. bicity of vesicles containing phosphoinositides: Influence of steric and electrostatic 30. Leikin S, Kozlov MM, Fuller NL, Rand RP (1996) Measured effects of diacylglycerol effects. J Membr Biol 192:33–43. on structural and elastic properties of phospholipid membranes. Biophys J 71:2623– 7. Di Paolo G, et al. (2004) Impaired PtdIns(4,5)P2 synthesis in nerve terminals produces 2632. defects in synaptic vesicle trafficking. Nature 431:415–422. 31. Wilkins MH, Blaurock AE, Engelman DM (1971) Bilayer structure in membranes. Nat 8. Salaun C, James DJ, Chamberlain LH (2004) Lipid rafts and the regulation of exocytosis. New Biol 230:72–76. Traffic 5:255–264. 32. Tristram–Nagle S, Nagle JF (2004) Lipid bilayers: Thermodynamics, structure, fluctua- 9. Mukherjee S, Soe TT, Maxfield FR (1999) Endocytic sorting of lipid analogues differing tions, and interactions. Chem Phys Lipids 127:3–14. solely in the chemistry of their hydrophobic tails. J Cell Biol 144:1271–1284. 33. Abrams FS, London E (1993) Extension of the parallax analysis of membrane penetra- 10. Israelachvili J (1991) Intermolecular & Surface Forces (Academic, London) 2nd Ed. tion depth to the polar region of model membranes: Use of fluorescence quench- 11. Helfrich W (1973) Elastic properties of lipid bilayers: Theory and possible experiments. ing by a spin-label attached to the phospholipid polar headgroup. Biochemistry Z Naturforsch C 28:693–703. 32:10826–10831. 12. Gruner SM (1989) Stability of lyotropic phases with curved interfaces. J Phys Chem 34. Graf K, Riegler H (1998) Molecular adhesion interactions between Langmuir mono- 93:7562–7570. layers and solid substrates. Colloids Surfaces A 131:215–224. 13. Leibler S (1986) Curvature instability in membranes. J Physique 47:507–516. 35. McQuaw CM, Sostarecz AG, Zheng L, Ewing AG, Winograd N (2005) Lateral het- 14. Seifert U (1993) Curvature-induced lateral phase segregation in two-component erogeneity of dipalmitoylphosphatidylethanolamine-cholesterol Langmuir–Blodgett vesicles. Phys Rev Lett 70:1335–1338. films investigated with imaging time-of-flight secondary ion mass spectrometry and 15. Carnie S, Israelachvili JN, Pailthorpe BA (1979) Lipid packing and transbilayer asym- atomic force microscopy. Langmuir 21:807–813. metries of mixed lipid vesicles. Biochim Biophys Acta 554:340–357. 36. Dufrene YF, Barger WR, Green JBD, Lee GU (1997) Nanometer-scale surface 16. Rand RP, Fuller NL, Gruner SM, Parsegian VA (1990) Membrane curvature, lipid properties of mixed phospholipid monolayers and bilayers. Langmuir 13:4779– segregation, and structural transitions for phospholipids under dual-solvent stress. 4784. Biochemistry 29:76–87. 37. Petrache HI, et al. (2004) Structure and fluctuations of charged phosphatidylserine 17. Fuller N, Rand RP (2001) The influence of lysolipids on the spontaneous curvature and bilayers in the absence of salt. Biophys J 86:1574–1586. bending elasticity of phospholipid membranes. Biophys J 81:243–254. 38. Yamanaka T, Ogihara N, Ohhori T, Hayashi H, Muramatsu T (1997) Surface chem- 18. Fuller N, Benatti CR, Rand RP (2003) Curvature and bending constants for ical properties of homologs and analogs of lysophosphatidylcholine and lysophos- phosphatidylserine-containing membranes. Biophys J 85:1667–1674. phatidylethanolamine in water. Chem Phys Lipids 90:97–107. 19. Derganc J (2007) Curvature-driven lateral segregation of membrane constituents in 39. Parthasarathy R, Yu CH, Groves JT (2006) Curvature-modulated phase separation in Golgi cisternae. Phys Biol 4:317–324. membranes. Langmuir 22:5095–5099. 20. Cooke IR, Deserno M (2006) Coupling between lipid shape and membrane curvature. 40. Groves JT, Boxer SG, McConnell HM (1998) Electric field-induced critical demixing in Biophys J 91:487–495. lipid bilayer membranes. Proc Natl Acad Sci USA 95:935–938. 21. Risselada HJ, Marrink SJ (2009) Curvature effects on lipid packing and dynamics in 41. Veatch SL, et al. (2008) Critical fluctuations in plasma membrane vesicles. Am Chem liposomes revealed by coarse grained molecular dynamics simulations. Phys Chem Soc Chem Biol 3:287–293. Chem Phys 11:2056–2067. 42. Hatzakis NS, et al. (2009) How curved membranes recruit amphipathic helices and 22. Sorre B, et al. (2009) Curvature-driven lipid sorting needs proximity to a demixing protein anchoring motifs. Nat Chem Biol 5:835–841. point and is aided by proteins. Proc Natl Acad Sci USA 106:5622–5626. 43. Khairy K, Foo J, Howard J (2008) Shapes of red blood cells: Comparison of 3D confocal 23. Tian A, Baumgart T (2009) Sorting of lipids and proteins in membrane curvature images with the bilayer-couple model. Cell Mol Bioeng 1:173–181. gradients. Biophys J 96:2676–2688. 44. Deutsch JW, Kelly RB (1981) Lipids of synaptic vesicles: Relevance to the mechanism 24. Provencher SW (1982) CONTIN—A general-purpose constrained regularization pro- of membrane fusion. Biochemistry 20:378–385. gram for inverting noisy linear algebraic and integral-equations. Comput Phys 45. Michaelson DM, Barkai G, Barenholz Y (1983) Asymmetry of lipid organization in Commun 27:229–242. cholinergic synaptic vesicle membranes. Biochem J 211:155–162.

22250 www.pnas.org / cgi / doi / 10.1073 / pnas.0907354106 Kamal et al. Downloaded by guest on September 25, 2021