Biochimica et Biophysica Acta 1798 (2010) 1348–1356

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

j ourna l homepage: www.e lsev ie r.com/ locate /bbamem

Review

Ceramide: From lateral segregation to mechanical stress

Iván López-Montero a,⁎, Francisco Monroy a, Marisela Vélez b,c, Philippe F. Devaux d

a Universidad Complutense de Madrid, Madrid, Spainb Instituto de Catálisis y Petroleoquímica, CSIC, Madrid, Spainc IMDEA Nanociencias, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spaind Institut de Biologie Physico-Chimique, Paris, France

Abbreviations: Chol, cholesterol; Cer, ceramide; C2CeC16Cer, palmitoyl-Cer; C18Cer, stearoyl-Cer; C20Cer,dimyristoyl-PC; DPPC, dipalmitoyl-PC; POPC, 1-palmitoyC18SM, stearoyl-SM; ESM, Egg-SM; BSM, brain-SM; PE,disordered phase; Lβ, solid phase; Smase, sphingomyefluorescein isothiocyanate; Cy3, cyanine 3⁎ Corresponding author.

E-mail address: ivanlopez@quim.ucm.es (I. López-M

0005-2736/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.bbamem.2009.12.007

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 September 2009Received in revised form 25 November 2009Accepted 9 December 2009Available online 21 December 2009

Keywords:CeramideSphingomyelinaseMechanical stressLipid domains

Ceramide is a sphingolipid present in eukaryotic cells that laterally segregates into solid domains in modellipid membranes. Imaging has provided a wealth of structural information useful to understand some of thephysical properties of these domains. In biological membranes, ceramide is formed on one of the membraneleaflets by enzymatic cleavage of sphyngomyelin. Ceramide, with a smaller head size than its parentcompound sphyngomyelin, induces an asymmetric membrane tension and segregates into highly ordereddomains that have a much high shear viscosity than that of the surrounding lipids. These physical properties,together with the rapid transmembrane flip-flop of the locally produced ceramide, trigger a sequence ofmembrane perturbations that could explain the molecular mechanism by which ceramide mediates differentcell responses. In this review we will try to establish a connection between the physical membranetransformations in model systems known to occur upon ceramide formation and some physiologicallyrelevant process in which ceramide is known to participate.

r, acetoyl-Cer; C6Cer, hexanoyl-Cer; C8Cer, octanoyl-Cer;arachidoyl-Cer; C24Cer, lignoceroyl-Cer; C24:1Cer, nervl-2-oleoyl-PC; DOPC, 1,2-dioleoyl-PC; SOPC, 1-stearoyl-1phosphatidyl ethanolamine; DPPE, dipalmitoyl-PE; DOPElinase; AFM, atomic force microscopy; FCS, fluorescenc

ontero).

ll rights reserved.

© 2009 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13492. Beyond ceramide-domain imaging in model membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1349

2.1. Ceramide-containing membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13492.1.1. Lateral phase behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13492.1.2. Visualising protein sorting in ceramide-rich domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13512.1.3. Measuring flip-flop rates and detergent resistance through optical imaging . . . . . . . . . . . . . . . . . . . . . . . . 13512.1.4. Mechanical properties of ceramide-enriched domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1351

2.2. Ceramide enzymatic conversion in SM-containing membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13512.2.1. Lateral phase behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13512.2.2. Domain clustering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13522.2.3. Mechanical stress: vesicle aggregation, budding, rupture, membrane defects and content efflux . . . . . . . . . . . . . . 1352

3. Ceramide and SMase enzymatic activity: a perfect marriage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13523.1. Compression elasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13533.2. Shear viscoelasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13533.3. Curvature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13533.4. Lamellar to hexagonal phase transition and lipid scrambling promotion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13533.5. Surface tension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1353

4. Effects of ceramide generation on cells: a biophysical viewpoint . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13544.1. Clustering and capping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13544.2. Blebbing and lipid scrambling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1354

C10Cer, decanoyl-Cer; C12Cer, lauroyl-Cer; C14Cer, myristoyl-Cer;onoyl-Cer; ECer, Egg-Cer; PC, phosphatidylcholine; DMPC, 1,2--oleyl-PC; EPC, Egg-PC; SM, sphingomyelin; C16SM, palmitoyl-SM;, 1,2-dioleoyl-PE; EPE, Egg-PE; Lo, liquid-ordered phase; Ld, liquid-e correlation spectroscopy; GUV, giant unilamellar vesicle; FITC,

1349I. López-Montero et al. / Biochimica et Biophysica Acta 1798 (2010) 1348–1356

5. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1355Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1355References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1355

1. Introduction

Ceramides are sphingolipids which can be either synthesised denovo in the endoplasmic reticulum or generated within the plasmamembrane from sphingomyelin (SM) by the action of sphyngomye-linase (SMase). They are constituted by a sphingosine backbone N-acylated with fatty acids of different chain length and degree ofsaturation. They are capable of forming hydrogen bonds that promoteintermolecular lateral interactions, as was first postulated in the earlyworks of Pascher [1,2]. The polar headgroup, the sphingosinehydroxyl groups and the fatty acid chains form hydrogen bonds thatstabilise the segregated lateral domains. Furthermore, the low affinityof ceramides for cholesterol and their atypical high meltingtemperature (∼90 °C) [3] confer the domains a very tight lipidpacking and an extremely solid character, typical of gel/solid phases(Lβ). Within these domains, the lipid molecules form a crystallinetwo-dimensional array and their lateral diffusion is highly inhibited.

The fluid mosaic membrane model proposed by Singer andNicholson in 1972 [4] was widely accepted as the membraneorganization. However, in 1997 Simons and Ikonen [5] proposed theexistence of liquid-ordered (Lo phase) domains (rafts) enriched in SMand sterols, the so called “raft hypothesis”, modifying the prevailingpicture of biological membranes as a homogeneous fluid lipid bilayerwhere proteins are embedded [6]. Although the functional role ofliquid-ordered domains in vivo remains controversial [7,8] there is noquestion about their existence in vitro lipid systems. The presence ofthe more ordered gel/solid phases in biological membranes howeverhas not been considered as relevant. The physicochemical propertiesof ceramide indicate its solid character at physiological temperatures,thus its biological relevance could rely on their highly ordered domainforming capacity.

Work in model lipid bilayers provides relevant biophysicalinformation to understand the segregation of lipid mixtures and itsinfluence on the mechanical properties of the membranes. Ceramide,was first described as a main constituent of the stratum corneum, andits physiological role was associated to the creation of a hydrophobicbarrier necessary to prevent water evaporation through the skin.Since the stratum corneum is mainly composed of a mixture ofceramide, cholesterol and fatty acids, the first lateral segregationstudies of ceramides into crystalline ordered domains were made inmodel systems including cholesterol–ceramide lipid mixtures [9].More recently, ceramide generation has been associated to manyother relevant biological processes, for example cellular stress andapoptosis [10–12] and has also been postulated to act as a secondmessenger and as a metabolic signalling molecule. The ascription ofthese physiologically relevant roles has generated a huge amount ofexperimental work on their lateral segregation and interactions withother phospholipids present in eukaryotic plasma membrane.Ceramide-enriched solid domains in phospholipids were firstfound in a PC fluid matrix [13,14]. The lack of understanding of themechanism behind many of its signalling functions [15,16] togetherwith the fact that the formed solid domains are highly ordered,suggests that the strong impact on the plasma membrane mechanicalproperties that this ordered ceramide domains have could explain themechanism by which ceramide exerts its signalling functions.

Different biophysical, structural and biological aspects of ceramideand ceramide-enriched domains have been reviewed extensively inthe last years [17–19]. A very recent and thorough review by Goñi andAlonso has addressed the effect of ceramides on membrane lateralstructure [20]. However, the physical characteristics of ceramidedomains as well as the mechanical perturbations undergone by the

lipid membrane upon SMase enzymatic SM to ceramide conversionhave not been as widely discussed. In this review we first summarizethe relevant information obtained by different imaging techniques onthe structural and mechanical properties of ceramide-enricheddomains and their formation. We then present a comparative analysisof the physicochemical properties of SM and ceramide, emphasizingthe relevant role played by the SM to ceramide conversion inmodulatingmechanical and physical properties of model membranes.The final section of this review will attempt to correlate somephysiologically relevant cell processes triggered by the presence ofceramide (protein aggregation, blebbing and lipid scrambling) withthe membrane mechanical perturbations associated to enzymatic SMto ceramide conversion described experimentally in model lipidmembranes.

2. Beyond ceramide-domain imaging in model membranes

There are two alternative ways of enriching the ceramide contentof a lipid bilayer: incorporating increasing amounts of ceramide in thelipid mixture used to prepare the bilayers, or increasing the contentlocally by the enzymatic transformation of SM into ceramide inducedby the catalytic activity of sphingomyelinases. In the first case themodel systems formed reach equilibrium whereas in the second casethe local perturbations induced by the sudden interconversion of thephospholipid to ceramide is a non equilibrium process that can triggerdifferent dynamic effects. In this section we will first recall the mainobservations obtained by microscopy techniques concerning physi-cochemical properties of ceramides (the lateral phase separation,protein sorting, flip-flop, and mechanical properties) (Section 2.1) tofollow with a description of some of the membrane alterations(domain clustering and mechanical stress) induced when ceramideconcentration is increased locally through SMase enzymatic activity(Section 2.2).

2.1. Ceramide-containing membranes

2.1.1. Lateral phase behaviorIn general, long chain-ceramides segregate into lateral domains

when mixed with phosphatidylcholines (DMPC and POPC) in a widerange of temperatures and ceramide molar ratios [14,21,22]. Forexample, only ∼5 mol% of C16Cer is needed to form gel domains inPOPC bilayers. In contrast, a very recent systematic study carried outon Langmuir monolayers by Kartunnen et al. [23] has shown thatshorter ceramides (from C2Cer, C6Cer and C8Cer) are miscible inDMPC at 1:2 Cer:DMPC molar ratio at all surface pressures. For thesame molar ratio, the morphology of the domains was found todepend on the acyl chain length. For C10Cer, C12Cer and C14 Cer,domains have a flower-like shape. And for longer ceramides (fromC16Cer to C24:1) domains present however a circular shape. Avery similar behavior was found in BCer/POPC mixtures in GUVs,as revealed by confocal fluorescent microscopy [24]. The presence of5 to 23 mol% of ceramide is sufficient to segregate into solid ribbon-like domains. In that work, AFM images of POPC:C16Cer (5:1 molarratio) supported bilayers confirmed the coexistence of solid flower-like domains in a fluid continuous phase. Similar flower-likedomains were seen by fluorescent microscopy in EPC:ECer GUVs(at 90:10 and 80:20 molar ratio) within the temperature range 5–30 °C [25]. From these results it can be deduced that ceramides, atphysiologically relevant molar concentrations, form solid domains ina fluid matrix.

Fig. 1. Supported bilayer (EPC:EPE:BSM:Chol 1:1:1:1 mol%) incubated with B. cereus sphingomyelinase. The ceramide generation by SM enzymatic degradation leads to a surfacetension mismatch between both membrane leaflets which promote the apparition of nanoscale membrane defects (signalled with white arrows in figure b). The new formedceramide is segregated in higher domains (see the AFM profiles in figure d) enriched in ceramide molecules (signalled with white arrows in figure c). The dashed square in (c)delimits the same zone scanned in a) and b). Taken from [44].

1350 I. López-Montero et al. / Biochimica et Biophysica Acta 1798 (2010) 1348–1356

Immiscibility is also observed by imaging techniques in mono-layers made of ceramide and cholesterol mixtures [26–28]. In thiscase, both C16cer and C24cer segregated into rectangular, sticklikedomains for molar ratios of 1:1 and 1:2 (Cer:chol respectively). Thedomain size depends on the ceramide chain length and thecholesterol molar ratio [28]. Edge-shaped domains suggest thepresence of two-dimensional crystals as shown before in Ref. [27].At higher cholesterol ratio, C16Cer mixed well with cholesterol, anddomains were not visible.

To our knowledge only one paper has reported images of phasecoexistence in SM-ceramide mixtures by confocal imaging [29]. Inthat work, Sot et al. found that ceramide-rich domains segregatedfrom the gel SM matrix for ceramide molar ration ranging from 5 to30 mol%. This observation represents a direct proof of the differentnature of SM and ceramide domains. The partitioning of thefluorescent probe used in that study in less ordered phases indicatesthe more ordered character of ceramide domains compared to thoseformed by SM. Further, the shape of ceramide domains changed fromcircular to ribbon-like domains at increasing ceramide concentrations.

A very interesting result reported by the group of Prieto was foundon ternary mixtures made of POPC, cholesterol and ceramide. For alow cholesterol content (up to 20 mol%, for a 5:1 POPC:Cer molar

ratio), ceramides form solid domains within a more fluid Lo phase[24]. Increasing the cholesterol molar concentration the ceramidesolid domains disappeared and a single phase was observed throughconfocal fluorescence microscopy in GUVs [30]. The cholesterol-driven ceramide solubilisation by a high molar concentration ofcholesterol was also observed in the 5–37 °C temperature range [25].

The effects of ceramide in the canonical lipid mixture for raftformation (DOPC: C18SM:chol 1:1:1 molar ratio) were studied byAFM imaging in supported bilayers. It was found that the substitutionof C18SM by C18Cer keeping the molar ratio 1:1:1 (DOPC:Chol:C18SM) produced lipid bilayers in which three different phases couldbe distinguished above 8 mol% of ceramide incorporation [31]. Thethinner region were assigned to the DOPC-rich phase (fluid phase),the ∼0.8 nm thick domains (with circular shape) over the fluid phasecorresponded to a SM-rich phase (Lo phase/rafts), and the newhighest domains (circular shape) were attributed to ceramide-richdomains. These domains were located within the SM-rich domains. Inthat work, Chiantia et al. also found that the diffusion coefficientassociated to the fluid phase decreased at higher ceramide contents,which is compatible with a cholesterol enrichment of this fluid phase.This excess of cholesterol could come from the SM-rich domains as itis displaced by the newly formed ceramide [32]. It has been suggested

1351I. López-Montero et al. / Biochimica et Biophysica Acta 1798 (2010) 1348–1356

that ceramide may displace cholesterol from Lo domains due to thestrong interaction with SM in PC:SM:Chol mixtures. The same grouphas performed a similar study with ceramides having different acylchain length [33]. They found that only long ceramides (C16Cer andC18Cer) were segregated from Lo domains to form ceramide-richdomains. Conversely, C2Cer, C6Cer and C12Cerwere not excluded intosolid domains. An original approach, combining AFM imaging withtime-of-flight secondary ionmass spectroscopy (ToF-SIMS), enabled awell defined localisation of SM and ceramide in the ordered domainsobserved for DOPC:ESM:Chol (2:2:1 molar ratio) Langmuir–Blodgettmonolayers transferred at different surface pressures [34]. They foundthat SM is uniformly located within the domains and ceramides areheterogeneously distributed in small clusters within the surroundingdomain. The three phase coexistence (Ld, Lo and Lβ) was alsovisualised by fluorescence microscopy in giant vesicles as darkflower-like and circular domains simultaneously coexisting in thebright fluid phase [25].

All these results indicate that both morphology and biophysicalproperties of ceramide-rich domains are dependent on the initial lipidcomposition, specifically on the cholesterol content. Particularly it hasrevealed that ceramide forms solid domains at physiological tem-peratures and at very low molar concentrations.

2.1.2. Visualising protein sorting in ceramide-rich domainsThe role of ceramide in protein distribution in lipid bilayers has

been also studied by AFM imaging combined with FCS and confocalmicroscopy. Chiantia et al. showed both the partial immobilisation ofplacental alkaline phosphatise protein (GPI-PLAP) by ceramide soliddomains and the partitioning of the glanglioside GM1-cholera toxin(CTx-B) complex (used as a typical marker for raft domains in cell andmodel membranes) into ceramide-rich domains [35]. These experi-ments signal that the physicochemical properties of ceramidedomains could favour protein aggregation. Notice that the samegroup has shown [36] that CTx-B induces a relatively fast redistribu-tion of GM1 from disordered to more ordered domains. Althoughfurther caution must be taken when labelling ordered domains withthe GM1-CTx-B complex, it should be very interesting to extend thistype of studies to proteins involved in cell processes (cell stress orsignalling) which are relevant for determining the functional role ofceramides, as receptor proteins like CD95 or CD40. In fact, ceramidedomains have been suggested to aggregate protein signal receptors inorder to amplify the primary signal (see Section 4.1).

2.1.3. Measuring flip-flop rates and detergent resistance through opticalimaging

We have measured the spontaneous transbilayer movement ofunlabelled ceramides with different acyl (C6, C10 and C16) chainlength in fluid EPC GUVs by optical microscopy. By observing thereversibility of a budding transition produced by the externalincorporation of a very small amount of ceramides into the outerleaflet, a half-time for unlabeled ceramide flip-flop was found to be onthe order of 1 minute at 37 °C [37]. In addition, ceramides have anunusual rapid spontaneous transbilayer movement (flip-flop) in Ld aswell as in Lo phases [37,38], where they are thought to be generated invivo. This capacity to rapidly go through the bilayer implies that thenew asymmetrically formed ceramides (in vivo as well as in modelmembranes) can be equilibrated into both monolayers to formsymmetric domains.

Ceramide also promotes a mechanical stabilisation againstdetergent solubilisation below the melting temperature of lipidmixtures. As little as 5 mol% of ECer confers detergent resistance tolipid vesicles made of ESM or DPPC. Confocal fluorescence microscopyimages show the prevention of membrane solubilisation of ceramide-rich domains in ESM GUVs even at very large excess of detergentconditions and long equilibration times [29].

2.1.4. Mechanical properties of ceramide-enriched domainsThe above detailed physicochemical characterization of ceramide

domains has revealed that at physiological temperatures they aremost likely segregated as solid phases This suggests that theseceramide domains can have a strong impact on the mechanicalproperties of membranes. AFM has provided direct experimentalinformation of the effect of ceramide on the mechanical properties ofbilayers. First, an AFM and fluorescence combined study has shownthat the ceramide-rich domains are much more viscous than Lodomains are. Solid domains in DOPC/Chol/SM/Cer (1:1:0.52:0.48(16 mol% Cer)) bilayers were deformed by scanning the sample withthe tip at high force and speed. In contrast to Lo domains in theabsence of ceramide (DOPC:Chol:SM, 1:1:1), the initial shape of soliddomains was not recovered at the AFM timescale [31]. Very recently,Sullan et al. [39] have shown by AFM-force maps that ceramide(10 mol%) increased the breakthrough force and the Young's modulusin both liquid-disordered and liquid-ordered domains in DOPC:ESM:Chol bilayers. Furthermore, the ceramide-rich domains observed inthe bilayer presented a very high breakthrough force (>12 nN) and ahigher packing density than the DPPC gel phase. Thus, ceramide isbelieved to promote a high structural rigidity, mechanical stabilityand compactness on lipid bilayers. Further mechanical experimentson ceramide pure systems may provide useful information in order tocharacterize the mechanical impact of ceramide in lipid membranes.

2.2. Ceramide enzymatic conversion in SM-containing membranes

2.2.1. Lateral phase behaviorAfirst examcomparing ceramide domainmorphology produced by

enzymatic conversion of C16SM with that produced by premixingceramide with the phospholipid mixture was carried out by epifluor-escence microscopy on Langmuir monolayers [40,41]. It was foundthat the premixed interface generally contains significantly larger butfewer domains and percolation occurred at lower content of ceramide.A more detailed image analysis revealed that the SM to ceramideformation was mediated by a spontaneous nucleation of circulardomains that grow in time. This circular shape was transformed intoflower-like domains which finally formed a hexagonal lattice.

The SM to ceramide conversion in SOPC:SM:BodipySM (0.75:0.20:0.05 molar ratio) giant vesicles lead the fluorescent probe (homoge-nously distributedwithin themembrane before SMase) to segregate intovisible (fluorescent) solid domainsmade of ceramide [42]. Giant vesicleswith Ld+Lo phase coexistence have been also treated asymmetricallywith SMase [25,43,44] (Fig. 1). The SM to ceramide conversion in thosesystemsfirst induced the coalescenceof Lodomains. After that, theborderregion between Lo and Ld phases started to undulate and finally, thefluorescent probewas extended to thewhole vesicle. Further exposure toSMase provoked membrane invaginations and rupture of the vesicle. Ifvesicles donot collapse (as in the caseof electrode-attachedvesicles [25])ceramide ribbon-like domains could be seen.

Supported bilayers with a lipid composition originating to Ld+Lophase coexistence (DOPC: SM:chol 1:1:1 molar ratio), were alsoincubated with S. aureus SMase and studied by AFM imaging [31]. Adynamical transformation was observed. It is important to note thatthe time evolution as well as the domain morphology was verydifferent from the one observed in experiments in which theequivalent amount of ceramide was directly incorporated to theinitial lipid composition [31]. The continuous SM transformationtriggered the formation of a third thicker phase rich in ceramidesurrounding the Lo domains, indicating that the enzyme acted directlyon liquid-ordered domains rich in SM. In addition, destabilisation andformation of holes are observed. A similar experiment carried out onDOPC:ESM:Chol (2:2:1molar ratio) bilayers [45] showed amembranereorganization which lead to the visualisation of three differenttopographies: a) larger areas of the fluid phasewith occasional defectsb) areas that have ceramide-rich domains either around or within Lo

1352 I. López-Montero et al. / Biochimica et Biophysica Acta 1798 (2010) 1348–1356

domains and c) areas that have clusters of heterogeneous domainssurrounded by an intermediate phase. Both B. cereus and S. aureusSMase promoted the same changes in membrane structure. SMasewas suggested to act at the interface between Lo and Ld domains. Iraand L. Johnston concluded that protein is not confined on the domainboundaries. A more recent result [46] localized a fluorescentlylabelled enzyme Alexa-SMase in the liquid expanded SM-enrichedphase in SM:Cer (9:1 molar ratio) using epifluorescence microscopy.It seems reasonable to expect SMase to act on zones enriched in SM,the enzymatic substrate. In similar studies, the group of Maggioreported that the SMase activity drives the system out of equilibriumby producing over-saturation of ceramide in the expanded phase withsubsequent ceramide segregation. This effect was shown to beenzymatic rate dependent. Moreover, the ceramide production ratewas also a determinant in the evolution andmorphology of ceramide-enriched domains [47]. On the contrary, biochemical studies haveshown that the addition of cholesterol to SM monolayer or bilayersenhances the enzymatic activity by fluidizing the SM gel phase to amore disordered Lo phase [48,49]. In fact, it has been shown byspectroscopic techniques that the enzyme activity is stronglydependent on membrane physical properties but not on substratecontent, and is higher in raft-likemixtures [50]. To summarize, it mustbe emphasized that ceramide production within model membranesinvolves some kinetic and dynamic aspects which are absent whendomains are formed by premixing ceramides with the lipid compo-sition: a) different domain morphology b) membrane integrityalteration and c) enzymatic activity regulation by the physical stateof the membrane (surface tension or lipid phase).

2.2.2. Domain clusteringSupported bilayers composed of DOPC:ESM:Chol (5:5:1 molar

ratio) are imaged by AFM as heterogeneous membranes with manysmall nanodomains randomly distributed in the fluid phase that areirregular in shape with the corresponding large interfacial area. Thisdomain morphology is compatible with SM-rich gel domains withinthe fluid DOPC-rich phase; in agreement with previously PC:SM:Cholphase diagrams [51,52]. The SMase (either B. cereus or S. aureusSMase) incubation promoted the clustering of existing condenseddomains into large patches [53]. However, spectroscopic studies onPOPC:C16SM:Chol at different molar ratios, all of them includedwithin the Ld+Lo coexistence region of the phase diagram, did notdetect the coalescence of ceramide-rich domains after SM to ceramideconversion by SMase treatment [50,54]. These data suggest that theSMase activity and Cer-induced changes in lipid bilayers are mainlydependent on the domain lipid composition and physical properties.

2.2.3. Mechanical stress: vesicle aggregation, budding, rupture,membrane defects and content efflux

As described above, SM to ceramide conversion perturbs themechanical integrity of membranes to different extents: inducing

Table 1Comparative table showing the very different physicochemical (thermotropic and mechaniand mechanical modifications in lipid membranes which could be important in biological ceand produced exclusively when SM is asymmetrically converted into ceramide is the memscrambling to membrane rupture.

Molecular property SM

Interbilayer movement –

Melting temperature 39 °C [49]Flip-flop Hours [38]Compressibility modulus ( for palmitoyl at 30 mN/m) 200 mN/m [63]Breakthrough force 3.2 nN (in Lo phaseShear modulus 0 mN/m [65]Curvature 0Hexagonal transition promotion NoMean molecular area (for palmitoyl at 30 mN/m) 47 Å2 [63]

SM to ceramide conversio

vesicle aggregation and shape changes as budding or invaginations, orproducing major perturbations associated to the formation ofmembrane defects that increase the membrane permeability andeventually produce membrane rupture.

Direct experimental evidence of membrane perturbations causedby SMase activity on lipid vesicles was first reported in largeunilamelar vesicles made of ESM:EPE:Chol (2:1:1 molar ratio) [55].Electron micrographs showed that large unilamellar vesicles with awell defined diameter (nanometer sized) fused into large membra-nous bags (micrometre sized) after SMase treatment. These observa-tions confirmed the previously described vesicle aggregationobserved with biochemical assays reported by the same group [56].Holopainen et al. carried out a very original experimental approachthat showed vectorial budding in SOPC:SM (3:1 molar ratio) giantvesicles produced by the SMase action [42]. Asymmetrical addition ofSMase by a micropipette in the vicinity of vesicles producedinvaginations or budding if SMase was externally or internallyadded respectively. Invaginations were also observed in EPC GUVscontaining as low as 2 mol% of BSM when treated with SMase [44].GUVs with Ld and Lo phase coexistence (DOPC:C16SM:Chol, 1:1:1molar ratio) were also treated with external SMase in similarexperiments [43]. Taniguchi et al. reported that some vesiclescollapsed after several minutes of incubation with SMase and someof them decreased in size. Although the SM content in GUVs wassimilar in [42] and [43], Holopainen et al. did not report vesiclerupture. In fact, their giant vesicles were attached to the platinumelectrode and therefore, the lipidmixture spread on thewire providednew lipid surface which was transferred to the vesicle, not onlypreventing membrane collapse but also allowing an increasing invesicle size. Membrane holes or defects (a previous stage for rupture)were directly visualised by AFM on supported bilayers DOPC:C18SM:Chol (1:1:1molar ratio) [31], DOPC:ESM:Chol (2:2:1molar ratio) [45],EPC:EPE:BSM:Chol (1:1:1:1 molar ratio) [44] and DOPC:ESM:Chol(5:5:1 molar ratio) [53] incubated with SMase. Finally, the action ofSMase on lipid vesicles leads also to content release as a consequenceof membrane destabilisation. This effect was initially detected withbiochemical methods [57] andmore recently fluorescencemicroscopydirectly showed the efflux, through transiently formed membranepores, of large vesicles enclosed within a giant one [44].

3. Ceramide and SMase enzymatic activity: a perfect marriage

All the effects produced by the replacement of SM by ceramide onmembrane properties described in the above section could have theirorigin on the different physicochemical properties of ceramides andtheir parent compound, SM (Table 1). Ceramides are lipids extremelyinsoluble in water (C6-Cer precipitates at ∼100 μM in water [58]) andtheir intermembrane exchange time is therefore very high [59]. Forthis reason, the potential action of ceramides must be restricted to themembrane interior. Although ceramide is synthesised de novo in the

cal) properties of SM and ceramide. The unique ceramide features promotes structuralll membranes (last column). In addition, a very striking effect observed in lipid bilayersbrane mechanical stress, responsible of many destabilising effects which go from lipid

Ceramide Biological relevance

Hours [59] Membrane localization∼90 °C [3] Lateral segregationSeconds [37,38] Symmetric domain formation600 mN/m [42] Rigid domains

s) [39] >12 nN [39] Structural stability90 mN/m [65] Protein cappingNegative Budding promotionYes [70] Defects promotion40 Å2 [42] Tightly packing

n→mechanical stress

1353I. López-Montero et al. / Biochimica et Biophysica Acta 1798 (2010) 1348–1356

endoplasmic reticulum, the local production of ceramide by sphingo-myelin degradation represents a large local perturbation of themembrane where it is generated (i.e. plasma membrane).

Much emphasis has been made characterizing the behavior ofceramides inside the membrane on the short range scale and theirability to form in-plane lateral domains and their modulation by thepresence of other lipids. As we have already mentioned, ceramideshave also very distinctive mechanical properties compared to othercommon sphingolipids and phospholipids. In this context, bymechanical behavior we refer to the stress response of the mem-brane against compression and shear deformations. The compres-sion response is mainly driven by bilayer compactness, i.e. a highcompression modulus describes the ability of lipids to densely packin such a way that molecular hard cores resist against furthercompression. The shear response, however, arises from bondtightness between neighbor molecules, i.e. the shear modulusmeasures the resistance of a solid arrangement of molecules to bedistorted against a lateral shear deformation not changing the areaper molecule but affecting the relative orientation between them.Consequently, fluid membranes are characterized by more or lesshigh values of the compression modulus (depending of their relativecondensation) but necessarily by a zero shear modulus, whichdefines a system able to flow under an applied force. On the otherside, solid membranes exhibit structural rigidity characterized by afinite shear modulus, independent of compression elasticity, whichwill be higher or lower depending on the compactness of the solidlipid arrangement.

3.1. Compression elasticity

The resistance to compression, parameterized by the compress-ibility modulus, is higher for ceramides than for SM or SM:Cholmixtures (Lo phases) or othermore fluid lipids as POPC, or prokaryoticlipid extracts. Very high values of the compressionmodulus have beenreported for bovine brain ceramide (300 mN/m) [60–62] and(400 mN/m) and (600 mN/m) for C24:1Cer and C16Cer respectively[21]. For the sake of comparison, we recall that fluid phases exhibitmuch smaller moduli; for instance, compressibility values of ca.80 mN/m for POPC, 100 mN/m for E. coli polar lipid extract, 200 mN/m for C16SM; in contrast with more ordered phases containingcholesterol (900 mN/m for C16SM:Chol 1:1 molar ratio) [63,64]. Asimilar behavior is found for breakthrough forces (see Section 2.1.4).The presence of ceramide in DOPC:ESM:Chol bilayers increased thebreakthrough forces from 1.4 to 4.1 nN in the Ld phase and from 3.2 to5.0 nN in the Lo. A lower limit of 8–12 nN for the ceramide-rich phasewas estimated [39].

3.2. Shear viscoelasticity

Unpublished results on the shear rheology of SM and ceramideidentify interesting differences on their mechanical behavior whichcould have important biological implications [65]. Whilst pure SM(in the gel phase) and SM:Chol (in Lo phase) behave as viscoelasticfluids with vanishing shear rigidity, ceramide possesses a hard solidbehavior characterized by a shear modulus of 90 mN/m and anextremely high shear viscosity [65]. The group of Maggio has recentlyshown how ceramide-rich domains can diffuse within a SM:Cer (9:1molar ratio) monolayer characterized by a very low microviscosity(η∼10−7 N·s/m at 30 mN/m) [66]. Here we highlight the verydifferent viscosity within the ceramide-rich domains which is 102

bigger than in a SM monolayer [65]. Consequently, protein diffusionwithin a ceramide-rich domain would be extremely slowed-down.We can anticipate that ceramide-rich domains could thereforeconstitute platforms to immobilise proteins due to their particularintrinsic rigidity.

3.3. Curvature

Curvature is another physicochemical property which is verydifferent in both SM and ceramide molecules. Ceramides, due to theirsmall headgroup, have a negative curvature, whereas SM is classifiedamong the planar bilayer promoting lipids [67]. The newly formedceramide molecules generated by enzymatic activity should promoteinitially a highly negative curvature in the membrane. In fact, therapid equilibration of ceramide within both monolayers by flip-flopwould lead to a zero curvature, as long as the rate of ceramideformation is slower than the spontaneous flip-flop. In addition,ceramides with very asymmetrical hydrocarbon chains, as C24:1Cer,can cause hydrocarbon chain interdigitation which may compensatecurvature stress [68,69].

3.4. Lamellar to hexagonal phase transition and lipid scramblingpromotion

In contrast to SM, ceramide has been shown to promote lamellar tohexagonal phase transition [56,70]. Veiga et al. found that in lipidvesicles made of PC:PE:Chol (2:2:1 molar ratio) and SM:PE:Chol(2:2:1 molar ratio), the PE lamellar to hexagonal transition temper-ature was shifted from 80–90 °C to 60–70 °C respectively by thepresence of 10 mol% of ECer [56]. In pure DPPE or DOPE-Me [64]vesicles, the PE transition temperature was decreased from 65–63 °Cto 51–53 °C respectively by the presence of 15–20 mol% of ECer.Finally, a striking effect on membrane perturbation was found eitherwhen liposomes were asymmetrically treated by SMase or when alarge amount of ceramides (up to 10 mol%)was asymmetrically addedto preformed large unilamellar vesicles: i.e., the promotion oftransbilayer movement of other lipids, the so called lipid scrambling[71,72]. A similar explanation (lipid scrambling) was given to theobservation that externally added SMase to SM-containing liposomesconverted into ceramide more than the expected 50 mol% of the totalSM available in the outer leaflet [50].

3.5. Surface tension

Negative curvature and hexagonal phase promotion have beeninvoked in several works to explain the membrane destabilisation (inparticular, lipid scrambling) when lipid bilayers were incubated withSMase. This interpretation is however unlikely since a) the temper-ature range in most of experiments is much lower than the range oftemperatures where non-lamellar formation takes place and b) thenegative spontaneous curvature promoted by the new formedceramide is compensated by the fact that ceramides equilibrate intoboth monolayers by rapid flip-flop or chain interdigitation[37,38,68,69]. In addition, as pointed out by Goñi et al., “no lipid flip-flop is observed in case ceramides are incorporated in the initialliposome lipid mixture” [72]. Alternatively, a driving force of verydifferent nature can be invoked to explain the observed lipidtranslocation: the difference in surface tension (lateral pressure)between the two leaflets produced by the asymmetrical ceramide toSM conversion [44,50,72]. The surface tension increase promoted bySM to ceramide conversion is directly measured by Langmuir balancein experiments carried out on lipid monolayers. In order to maintainthe surface pressure constant during SMase activity [40,41], theavailable surface area was reduced according to the observedmonolayer surface pressure decrease. It is therefore important tokeep inmind the fact that the enzymatic activity of SMase significantlymodifies the mechanical properties of model lipid membranes. Anadditional and interesting point is that these localized perturbationsproduced enzymatically in the membranes can propagate instanta-neously over distances larger than the molecular dimension. The localmechanical stress induced by the conversion of SM to ceramide isdirectly related to the difference in themeanmolecular area of the SM

1354 I. López-Montero et al. / Biochimica et Biophysica Acta 1798 (2010) 1348–1356

precursor molecule and the final produced ceramide. The meanmolecular area occupied by this lipid differs from that of SM byapproximately 15% (measured in monolayers at 30 mN/m) [21,63].The condensing effect of new ceramides on the molecular packing ofone leaflet (relative to the initial situation) generates a lateral pressureasymmetry which can be relaxed through one of several mechanisms:by membrane bending or by creating membrane defects throughwhich lipid scrambling occurs [73,74]. In case of high tensionasymmetry, instabilities cannot be smoothly relaxed and the lipidbilayer collapses.

All the arguments presented above illustrate the fact thatconsidering only the peculiar and unique physicochemical propertiesof ceramides might not be enough to understand their biologicalfunction.When localized SMase activity produces ceramide directly inthe membrane, additional mechanical perturbations come into playthat could explain the important and diverse physiological roleattributed to ceramides in cells.

4. Effects of ceramide generation on cells: a biophysical viewpoint

In this section we will try to correlate the effects of local SMaseactivity and ceramide generation on membrane mechanical proper-ties described in model membranes with three main membrane-associated events observed in plasma biomembranes after SMasestimulation: a) domain clustering and protein aggregation, b)vesiculation (or blebbing) and c) lipid scrambling. When extrapolat-ing experimental observations on model membranes to real situa-tions, one must keep in mind all oversimplifications that modelsystems represent [75]. First, eukaryotic cell membranes are consti-tuted by thousands of different lipids (with varying chain length andsaturation). In addition, cells are not in equilibrium and the eukaryoticplasma membrane is also composed of membrane proteins andassociated to the cytoskeleton. Moreover, lipids are asymmetricallydistributed within the plasma membrane. Briefly, PS and PE aremainly located in the inner leaflet, whereas most of SM and PC arefound in the outer monolayer [76]. Cholesterol, although presenting afast transbilayer motion [77], could be enriched in the non-cytosolicleaflet of plasma membranes due to its high affinity for sphingolipids.Conversely, lipid mixtures in model membranes are constructedsymmetrically and composed commonly of 1 to 4 well chemicallydefined lipid species. Consequently, the phase behavior and themechanical properties of real membranes can be very different fromthose found in ideal systems. We will however dare to analyze someexperimentally observed biological processes within the frameworkof the evidence reported in model systems reviewed above: theunique mechanical properties of ceramides and the mechanical stressinduced by SM to ceramide conversion.

4.1. Clustering and capping

In vivo, it is believed that ceramide concentrates signal receptorproteins within ceramide-enriched domains. Experimental evidenceshows that fluorescent antibodies against acid SMase (FITC-coupledanti-Smase) and ceramide (Cy3-labelled anti-ceramide) colocalizein cell plasma membranes, visualised as large fluorescent regions,after cell activation by numerous stimulation mechanisms of signalreceptors (CD95, CD40, infection or γ-irradiation among others)[78–80]. Moreover, it is worth noting that the formation offluorescent ceramide regions is completely restored by externaladdition of natural C16-Cer in deficient SMase cells [80]. The newmodel for transmembrane signalling (readers are referred to fourrecent extensive reviews and references therein [18,81–83])suggests that SMase is activated after stimulation of single signallingreceptors. The newly synthesised ceramide would form soliddomains which coalescence into large platforms (clustering),which should be symmetric due to the rapid flip-flop of ceramides

in both fluid and lo phases [37,38]. The ceramide-rich platforms mayserve to aggregate the stimulated receptors (capping), thereforeacting as signal amplifiers. In this sense, ceramides do not directlyparticipate in transmembrane signalling but in amplifying theprimary signal by aggregating the signalling receptors. Such aninteresting scenario is only possible if the physical properties ofceramides are adequate for, first, enabling the coalescence of smallceramide domains and second, for having the capacity of seques-tering signal receptor proteins.

In model systems, domain clustering and protein aggregation havealso been described. First, the results reported by the group ofJohnston revealed that the action of SMase on supported bilayersmade of DOPC:SM:Chol induced the generation of heterogeneousregions composed by larger ceramide-rich domains [53]. Although,the mechanism by which small ceramide domains fuse to form largeplatforms remains unclear, it seems that it could be related to themechanical stress generated by the SM to ceramide conversion. Infact, if the same proportion of ceramide is previously mixed to thelipid composition, ceramide domains do not fuse to form largerpatches. Second, several membrane proteins have been seen topartition into ceramide domains [35]. Although, none of the studiedproteins is involved in signalling processes, these results are of greatinterest because they represent the first demonstration of proteinpartition and immobilisation into ceramide-rich domains.

The described aggregation of membrane proteins into ceramidedomains could also be a consequence (or associated) to the peculiarceramide domains shear viscoelasticity. As we have already men-tioned, ceramide bilayers present much higher shear elasticitycompared to the one found in membranes of different compositions.Its shear modulus has been estimated to be on the order of 105 largerthan other biologically relevant lipid mixtures (even the typical raft-forming combination). Therefore, ceramide domains could be partic-ularly efficient in concentrating the signal receptors due to theirexceptional shear viscoelastic properties [65]. Fluidity is one of themain biological membranes attributes relevant for maintaining theproper distribution and diffusion of membrane-embedded proteins.In fact, in fluid membranes (Ld as well as Lo phase is characterized byvanishing shear modulus) thermal energy would suffice to destabilisethe protein–lipid interaction in protein–lipid domain association.However, ceramides present such a high resistance to shear (shearmodulus ∼90 mN/m), that proteins embedded in ceramide-richplatforms will be entrapped (frozen) within the domains and proteincapping could take place.

4.2. Blebbing and lipid scrambling

Ceramide levels on plasmamembrane are increasedwhen cells aresubmitted to different stresses. The SM to ceramide conversion isassociated therefore with different physiological processes as signal-ling and apoptosis. Apoptosis includes a large membrane reorganisa-tion like receptor aggregation (Section 4.1), blebbing, lipid scramblingand cholesterol release. The physiological role attributed to ceramidesin these processes has been revisited in the last years to propose amembrane restructuring role for the newly formed ceramide from SM.For this structural component of ceramides, topological considera-tions must be kept in mind [84,85]. As mentioned above, lipids in theplasmamembrane are asymmetrically distributed on both leaflets andmost of SM is located in the non-cytosolic monolayer. Only a 10% ofSM is found in the cytosolic side of themembrane. Thus, themain poolof SM remains inaccessible for enzymatic conversion by intracellularSMases and a topological problem emerges. A very interesting model,proposed by Tepper et al. [84] suggested the activation of a lipidscramblase to enrich inner leaflet SM content. Later SM degradationon the cell interior would alter the SM-Chol domains (rafts) andpromote subsequent cholesterol release and later morphologicalalterations within the plasma membrane.

1355I. López-Montero et al. / Biochimica et Biophysica Acta 1798 (2010) 1348–1356

However, a complementary explanation can be suggested accordingto the new biophysical results obtained in model systems that tightlycorrelate the SM to ceramide conversion with lipid scrambling[50,71,72]. An asymmetrically SM to ceramide conversion of only5 mol% suffices to produce an important increase in surface tensionwithin the monolayer where SM is degraded. The surface tensionmismatch generated by the differences in lipid packing induced by theSM to ceramide conversion, triggers a mechanical stress which can leadto membrane rupture in model systems [31,43–45,53]. In vivo, thepresence of the cytoskeleton, may contribute to release the asymmet-rical surface tension generated by cellular SMases preventing mem-brane rupture and allowing only the formation of transient defectsinterconnecting both monolayers which facilitate mass equilibrationbetween them [74]. This SMase-mediated lipid scramblingwouldmakethe SM located in the outer monolayer accessible to intracellularSMases. Subsequent SM conversion would lead to first, cholesterolrelease, either by a SM concentration decrease or by the cholesteroldisplacement by ceramidedescribed above [32]; and second, to producemembrane blebbing, vesiculation and apoptotic body efflux [42,44,57].A major consequence of this interpretation is that the response to themechanical perturbation needs not be spatially restricted to where theenzyme activity takes place. The local ceramide generation does notentail a colocalisation of membrane defects. The mechanical perturba-tion is simultaneously felt over all molecules integrating themembrane,and a local perturbation can be propagated over large distances. Certaindegree of spatial location could however still be achieved by thedifferent sensitivity to stress provided by different lipid compositions:the less rigid zones in themembrane, as liquid-disordered regions withlower compressibility modulus or breakthrough force, would collapsemore easily.

5. Conclusions and perspectives

The conversion of SM to ceramide by the action of SMase triggers anumber of metabolic and cellular events [11,78,82]. Although themechanism behind each process is probably complex and unique, wehave emphasized in this review that twophysical aspects of ceramide'sformation in biological membranes, its aggregation in solid viscousdomains and the mechanical stress produced on the membrane as SMis enzymatically converted into ceramide, are important to understandceramides physiological role in living cells. The signal amplificationobtained through the aggregation of protein receptors associated toceramide domains [18,81–83] could be strongly facilitated by thehighly viscous character of these solid domains described in modelsystems. Other biologically relevant membrane events as invagina-tions or lipid scrambling triggered by SMase activity [86,87] can also bepartially explained merely considering the mismatched surfacetension induced on the asymmetric cell membranes exposed toSMase enzymatic activity. More work should be done in simple modelsystems of increasing complexity to mimic bilayer asymmetry oraggregation of membrane receptors associated to ceramide domainsin vivo. Such research could allow discriminating between cellresponses mediated by specific ceramide interactions from those inwhich the role played by ceramide is mediated through physicalmembrane perturbations.

Acknowledgements

This work was supported by grant nos. FIS2006-01305, FIS2009-14650-C02-01 (F.Mand I.L-M.) and BIO2008-04478-C03-00 (M.Vélez)from MICINN. We thank the support of CONSOLIDER CSD2007-0010(F.M and M.Vélez) and the Comunidad Autónoma de Madrid undergrant S-0505/MAT-0283 (F.M, M.Vélez and I.L-M.). I.L-M. wassupported by Juan de la Cierva program 2007 from MICINN. Theauthors especially thank Sarah Schnack for editorial facilities.

References

[1] I. Pascher, Molecular arrangements in sphingolipids. Conformation and hydrogenbonding of ceramide and their implication on membrane stability and perme-ability, Biochim. Biophys. Acta (BBA) — Biomembr. 455 (1976) 433–451.

[2] H. Löfgren, I. Pascher, Molecular arrangements of sphingolipids. The monolayerbehaviour of ceramides, Chem. Phys. Lipids 20 (1977) 273–284.

[3] J. Shah, J.M. Atienza, A.V. Rawlings, G.G. Shipley, Physical properties of ceramides:effect of fatty acid hydroxylation, J. Lipid Res. 36 (1995) 1945–1955.

[4] S.J. Singer, G.L. Nicolson, The fluid mosaic model of the structure of cellmembranes, Science 175 (1972) 720–731.

[5] K. Simons, E. Ikonen, Functional rafts in cellmembranes, Nature 387 (1997) 569–572.[6] D.M. Engelman, Membranes are more mosaic than fluid, Nature 438 (2005)

578–580.[7] L.J. Pike, Rafts defined: a report on the Keystone symposium on lipid rafts and cell

function, J. Lipid Res. 47 (2006) 1597–1598.[8] S. Munro, Lipid rafts: elusive or illusive? Cell 115 (2003) 377–388.[9] D.T. Parrott, J.E. Turner, Mesophase formation by ceramides and cholesterol: a

model for stratum corneum lipid packing? Biochim. Biophys. Acta (BBA) —

Biomembr. 1147 (1993) 273–276.[10] R. Kolesnick, Y.A. Hannun, Ceramide and apoptosis, Trends Biochem. Sci. 24

(1999) 224–225.[11] Y.A. Hannun, C. Luberto, Ceramide in the eukaryotic stress response, Trends Cell

Biol. 10 (2000) 73–80.[12] A.H. Futerman, Y.A. Hannun, The complex life of simple sphingolipids, EMBO Rep.

5 (2004) 777–782.[13] H.-W. Huang, E.M. Goldberg, R. Zidovetzki, Ceramide induces structural defects

into phosphatidylcholine bilayers and activates phospholipase A2, Biochem.Biophys. Res. Commun. 220 (1996) 834–838.

[14] J.M. Holopainen, J.Y.A. Lehtonen, P.K.J. Kinnunen, Lipidmicrodomains in dimyristoyl-phosphatidylcholine-ceramide liposomes, Chem. Phys. Lipids 88 (1997) 1–13.

[15] K. Venkataraman, A.H. Futerman, Ceramide as a second messenger: stickysolutions to sticky problems, Trends Cell Biol. 10 (2000) 408–412.

[16] W.J. van Blitterswijk, A.H. van der Luit, R.J. Veldman, M. Verheij, J. Borst, Ceramide:second messenger or modulator of membrane structure and dynamics? Biochem.J. 369 (2003) 199–211.

[17] F.M. Goñi, A. Alonso, Biophysics of sphingolipids I. Membrane properties ofsphingosine, ceramides and other simple sphingolipids, Biochim. Biophys. Acta(BBA) — Biomembr. 1758 (2006) 1902–1921.

[18] H. Grassmé, J. Riethmüller, E. Gulbins, Biological aspects of ceramide-enrichedmembrane domains, Prog. Lipid Res. 46 (2007) 161–170.

[19] Y. Zhang, X. Li, K.A. Becker, E. Gulbins, Ceramide-enriched membrane domains—structure and function, Biochim. Biophys. Acta (BBA) — Biomembr. 1788 (2009)178–183.

[20] F.M. Goñi, A. Alonso, Effects of ceramide and other simple sphingolipids onmembrane lateral structure, Biochim. Biophys. Acta (BBA) — Biomembr. 1788(2009) 169–177.

[21] J.M. Holopainen, H.L. Brockman, R.E. Brown, P.K.J. Kinnunen, Interfacial interac-tions of ceramide with dimyristoylphosphatidylcholine: impact of the N-acylchain, Biophys. J. 80 (2001) 765–775.

[22] L.C. Silva, R.F.M. de Almeida, A. Fedorov, A.P. Matos, M. Prieto, Ceramide-platformformation and -induced biophysical changes in a fluid phospholipid membrane,Mol. Membr. Biol. 23 (2006) 137–148.

[23] M. Karttunen, M.P. Haataja, M. Saily, I. Vattulainen, J.M. Holopainen, Lipid domainmorphologies in phosphatidylcholine–ceramide monolayers, Langmuir 25 (2009)4595–4600.

[24] M. Fidorra, L. Duelund, C. Leidy, A.C. Simonsen, L.A. Bagatolli, Absence of fluid-ordered/fluid-disordered phase coexistence in ceramide/POPC mixtures contain-ing cholesterol, Biophys. J. 90 (2006) 4437–4451.

[25] G. Staneva, A. Momchilova, C. Wolf, P.J. Quinn, K. Koumanov, Membranemicrodomains: role of ceramides in the maintenance of their structure andfunctions, Biochim. Biophys. Acta (BBA) — Biomembr. 1788 (2009) 666–675.

[26] E. ten Grotenhuis, R.A. Demel, M. Ponec, D.R. Boer, J.C. van Miltenburg, J.A.Bouwstra, Phase behavior of stratum corneum lipids in mixed Langmuir–Blodgettmonolayers, Biophys. J. 71 (1996) 1389–1399.

[27] K. Ekelund, L. Eriksson, E. Sparr, Rectangular solid domains in ceramide-cholesterol monolayers — 2D crystals, Biochim. Biophys. Acta (BBA)— Biomembr.1464 (2000) 1–6.

[28] E. Sparr, L. Eriksson, J.A.Bouwstra, K. Ekelund, AFMstudyof lipidmonolayers: III. Phasebehavior of ceramides, cholesterol and fatty acids, Langmuir 17 (2000) 164–172.

[29] J. Sot, L.A. Bagatolli, F.M. Goñi, A. Alonso, Detergent-resistant, ceramide-enricheddomains in sphingomyelin/ceramide bilayers, Biophys. J. 90 (2006) 903–914.

[30] B.M. Castro, L.C. Silva, A. Fedorov, R.F.M. de Almeida, M. Prieto, Cholesterol-richfluid membranes solubilize ceramide domains: implications for the structure anddynamics of mammalian intracellular and plasma membranes, J. Biol. Chem. 284(2009) 22978–22987.

[31] S. Chiantia,N. Kahya, J. Ries, P. Schwille, Effects of ceramide on liquid-ordereddomainsinvestigated by simultaneous AFM and FCS, Biophys. J. 90 (2006) 4500–4508.

[32] E. London Megha, Ceramide selectively displaces cholesterol from ordered lipiddomains (rafts): implications for lipid raft structure and function, J. Biol. Chem.279 (2004) 9997–10004.

[33] S. Chiantia, N. Kahya, P. Schwille, Raft domain reorganization driven by short- and long-chain ceramide: a combined AFM and FCS study, Langmuir 23 (2007) 7659–7665.

[34] J. Popov, D. Vobornik, O. Coban, E. Keating, D. Miller, J. Francis, N.O. Petersen, L.J.Johnston, Chemical mapping of ceramide distribution in sphingomyelin-richdomains in monolayers, Langmuir 24 (2008) 13502–13508.

1356 I. López-Montero et al. / Biochimica et Biophysica Acta 1798 (2010) 1348–1356

[35] S. Chiantia, J. Ries, G. Chwastek, D. Carrer, Z. Li, R. Bittman, P. Schwille, Role ofceramide in membrane protein organization investigated by combined AFM andFCS, Biochim. Biophys. Acta (BBA) — Biomembr. 1778 (2008) 1356–1364.

[36] K. Bacia, P. Schwille, T. Kurzchalia, Sterol structure determines the separation ofphases and the curvature of the liquid-ordered phase in model membranes, Proc.Natl Acad. Sci. USA 102 (2005) 3272–3277.

[37] I. Lopez-Montero, N. Rodriguez, S. Cribier, A. Pohl, M. Velez, P.F. Devaux, Rapidtransbilayer movement of ceramides in phospholipid vesicles and in humanerythrocytes, J. Biol. Chem. 280 (2005) 25811–25819.

[38] A. Pohl, I.n. López-Montero, F. Rouvière, F. Giusti, P.F. Devaux, Rapid transmem-brane diffusion of ceramide and dihydroceramide spin-labelled analogues in theliquid ordered phase, Mol. Membr. Biol. 26 (2009) 194–204.

[39] R.M.A. Sullan, J.K. Li, S. Zou, Direct correlation of structures and nanomechanicalproperties of multicomponent lipid bilayers, Langmuir 25 (2009) 7471–7477.

[40] M.L. Fanani, S. Härtel, R.G. Oliveira, B. Maggio, Bidirectional control ofsphingomyelinase activity and surface topography in lipid monolayers, Biophys.J. 83 (2002) 3416–3424.

[41] S. Härtel, M. Laura Fanani, B. Maggio, Shape transitions and lattice structuring ofceramide-enriched domains generated by sphingomyelinase in lipid monolayers,Biophys. J. 88 (2005) 287–304.

[42] J.M. Holopainen, M.I. Angelova, P.K.J. Kinnunen, Vectorial budding of vesicles byasymmetrical enzymatic formation of ceramide in giant liposomes, Biophys. J. 78(2000) 830–838.

[43] Y. Taniguchi, T. Ohba, H. Miyata, K. Ohki, Rapid phase change of lipidmicrodomains in giant vesicles induced by conversion of sphingomyelin toceramide, Biochim. Biophys. Acta (BBA) — Biomembr. 1758 (2006) 145–153.

[44] I. López-Montero, M. Vélez, P.F. Devaux, Surface tension induced by sphingomye-lin to ceramide conversion in lipid membranes, Biochim. Biophys. Acta (BBA) —Biomembr. 1768 (2007) 553–561.

[45] L.J. Ira, Johnston, ceramide promotes restructuring of model raft membranes,Langmuir 22 (2006) 11284–11289.

[46] L. De Tullio, B. Maggio, M.L. Fanani, Sphingomyelinase acts by an area-activatedmechanism on the liquid-expanded phase of sphingomyelin monolayers, J. LipidRes. 49 (2008) 2347–2355.

[47] M.L. Fanani, L. De Tullio, S. Hartel, J. Jara, B. Maggio, Sphingomyelinase-induceddomain shape relaxation driven by out-of-equilibrium changes of composition,Biophys. J. 96 (2009) 67–76.

[48] M. Jungner, H. Ohvo, J.P. Slotte, Interfacial regulation of bacterial sphingomyelinaseactivity, Biochim. Biophys. Acta (BBA) — Lipids Lipid Metab. 1344 (1997) 230–240.

[49] F.X. Contreras, J. Sot, M.B. Ruiz-Argüello, A. Alonso, F.M. Goñi, Cholesterolmodulation of sphingomyelinase activity at physiological temperatures, Chem.Phys. Lipids 130 (2004) 127–134.

[50] L.C. Silva, A.H. Futerman,M. Prieto, Lipid raft compositionmodulates sphingomyelinaseactivity and ceramide-induced membrane physical alterations, Biophys. J. 96 (2009)3210–3222.

[51] S.L. Veatch, S.L. Keller, Miscibility phase diagrams of giant vesicles containingsphingomyelin, Phys. Rev. Lett. 94 (2005) 148101.

[52] R.F.M. de Almeida, A. Fedorov, M. Prieto, Sphingomyelin/phosphatidylcholine/cholesterol phase diagram: boundaries and composition of lipid rafts, Biophys. J.85 (2003) 2406–2416.

[53] L.J.Johnston Ira, Sphingomyelinase generation of ceramide promotes clustering ofnanoscale domains in supported bilayer membranes, Biochim. Biophys. Acta(BBA) — Biomembr. 1778 (2008) 185–197.

[54] L.C. Silva, R.F.M. de Almeida, B.M. Castro, A. Fedorov, M. Prieto, Ceramide-domainformation and collapse in lipid rafts: membrane reorganization by an apoptoticlipid, Biophys. J. 92 (2007) 502–516.

[55] G. Basáñez, M.B. Ruiz-Argüello, A. Alonso, F.M. Goñi, G. Karlsson, K. Edwards,Morphological changes induced by phospholipase C and by sphingomyelinase onlarge unilamellar vesicles: a cryo-transmission electron microscopy study ofliposome fusion, Biophys. J. 72 (1997) 2630–2637.

[56] M.B. Ruiz-Arguello, G. Basanez, F. Goni, A. Alonso, Different effects of enzyme-generated ceramides and diacylglycerols in phospholipid membrane fusion andleakage, J. Biol. Chem. 271 (1996) 26616–26621.

[57] L.R. Montes, M.B. Ruiz-Arguello, F.M. Goni, A. Alonso, Membrane restructuring viaceramide results in enhanced solute efflux, J. Biol. Chem. 277 (2002) 11788–11794.

[58] J. Sot, F.M. Goñi, A. Alonso, Molecular associations and surface-active properties ofshort- and long-N-acyl chain ceramides, Biochim. Biophys. Acta (BBA) —Biomembr. 1711 (2005) 12–19.

[59] C.G. Simon, P.W. Holloway, A.R.L. Gear, Exchange of C16-ceramide betweenphospholipid vesicles, Biochemistry 38 (1999) 14676–14682.

[60] D.C. Carrer, B. Maggio, Phase behavior and molecular interactions in mixtures ofceramide with dipalmitoylphosphatidylcholine, J. Lipid Res. 40 (1999)1978–1989.

[61] D.C. Carrer, B. Maggio, Transduction to self-assembly of molecular geometry andlocal interactions in mixtures of ceramides and ganglioside GM1, Biochim.Biophys. Acta (BBA) — Biomembr. 1514 (2001) 87–99.

[62] B. Maggio, Favorable and unfavorable lateral interactions of ceramide, neutralglycosphingolipids and gangliosides in mixed monolayers, Chem. Phys. Lipids 132(2004) 209–224.

[63] X.-M. Li, M.M. Momsen, J.M. Smaby, H.L. Brockman, R.E. Brown, Cholesteroldecreases the interfacial elasticity and detergent solubility of sphingomyelins,Biochemistry 40 (2001) 5954–5963.

[64] I. Lopez-Montero, L.R. Arriaga, F. Monroy, G. Rivas, P. Tarazona, M. Velez, High fluidityand soft elasticity of the inner membrane of Escherichia coli revealed by the surfacerheology of model Langmuir monolayers, Langmuir 24 (2008) 4065–4076.

[65] Unpublished results. PhD thesis of G. Espinosa, Paris 2009.[66] N. Wilke, B. Maggio, The influence of domain crowding on the lateral diffusion of

ceramide-enriched domains in a sphingomyelin monolayer, J. Phys. Chem. B 113(2009) 12844–12851.

[67] J.N. Israelachvili (Ed.), Intermolecular and Surface Forces, 2 ed., Academic Press,London, 1985.

[68] D.C. Carrer, S. Härtel, H.L. Mónaco, B. Maggio, Ceramide modulates the lipidmembrane organization at molecular and supramolecular levels, Chem. Phys.Lipids 122 (2003) 147–152.

[69] D.C. Carrer, S. Schreier, M. Patrito, B. Maggio, Effects of a short-chain ceramide onbilayer domain formation, thickness, and chain mobililty: DMPC and asymmetricceramide mixtures, Biophys. J. 90 (2006) 2394–2403.

[70] M.P. Veiga, J.L.R. Arrondo, F.M. Goñi, A. Alonso, Ceramides in phospholipidmembranes: effects on bilayer stability and transition to nonlamellar phases,Biophys. J. 76 (1999) 342–350.

[71] F.X. Contreras, A.-V. Villar, A. Alonso, R.N. Kolesnick, F.M. Goni, Sphingomyelinaseactivity causes transbilayer lipid translocation in model and cell membranes,J. Biol. Chem. 278 (2003) 37169–37174.

[72] F.X. Contreras, G. Basañez, A. Alonso, A. Herrmann, F.M. Goñi, Asymmetric additionof ceramides but not dihydroceramides promotes transbilayer (flip-flop) lipidmotion in membranes, Biophys. J. 88 (2005) 348–359.

[73] M. Traïkia, D.E. Warschawski, O. Lambert, J.-L. Rigaud, P.F. Devaux, AsymmetricalMembranes and Surface Tension, vol. 83, 2002, pp. 1443–1454.

[74] R.M. Raphael, R.E. Waugh, S.a. Svetina, B.t. Zeks, Fractional occurrence of defects inmembranes and mechanically driven interleaflet phospholipid transport, PhysicalReview E 64 (2001) 051913.

[75] G. van Meer, D.R. Voelker, G.W. Feigenson, Membrane lipids: where they are andhow they behave, Nat. Rev. Mol. Cell Biol. 9 (2008) 112–124.

[76] M. Seigneuret, P.F. devaux, ATP-dependent asymmetric distribution of spin-labeled phospholipids in the erythrocyte membrane: relation to shape changes.Proc. Natl. Acad. Sci. USA 81 (1984) 3751–3755.

[77] T.L. Steck, J. Ye, Y. Lange, Probing red cell membrane cholesterol movement withcyclodextrin, Biophys. J. 83 (2002) 2118–2125.

[78] H. Grassme, A. Jekle, A. Riehle, H. Schwarz, J. Berger, K. Sandhoff, R. Kolesnick, E.Gulbins, CD95 signaling via ceramide-rich membrane rafts, J. Biol. Chem. 276(2001) 20589–20596.

[79] H. Grassme, V. Jendrossek, J. Bock, A. Riehle, E. Gulbins, Ceramide-rich membranerafts mediate CD40 clustering, J. Immunol. 168 (2002) 298–307.

[80] H. Grassme, V. Jendrossek, A. Riehle, G. von Kurthy, J. Berger, H. Schwarz, M.Weller, R. Kolesnick, E. Gulbins, Host defense against Pseudomonas aeruginosarequires ceramide-rich membrane rafts, Nat. Med. 9 (2003) 322–330.

[81] A.E. Cremesti, F.M. Goni, R. Kolesnick, Role of sphingomyelinase and ceramide inmodulating rafts: do biophysical properties determine biologic outcome? FEBSLett. 531 (2002) 47–53.

[82] E. Gulbins, S. Dreschers, B. Wilker, H. Grassmé, Ceramide, membrane rafts andinfections, J. Mol. Med. 82 (2004) 357–363.

[83] C.R. Bollinger, V. Teichgräber, E. Gulbins, Ceramide-enriched membrane domains,Biochim. Biophys. Acta (BBA) — Mol. Cell Res. 1746 (2005) 284–294.

[84] A.D. Tepper, P. Ruurs, T. Wiedmer, P.J. Sims, J. Borst, W.J. van Blitterswijk,Sphingomyelin hydrolysis to ceramide during the execution phase of apoptosisresults from phospholipid scrambling and alters cell-surface morphology, J. CellBiol. 150 (2000) 155–164.

[85] D.R.Green,Apoptosis and sphingomyelinhydrolysis: theflip side, J. Cell Biol. 150 (2000)5F–8F.

[86] J. Herz, J. Pardo, H. Kashkar, M. Schramm, E. Kuzmenkina, E. Bos, K. Wiegmann, R.Wallich, P.J. Peters, S. Herzig, E. Schmelzer, M. Krönke, M.M. Simon, O. U., Acidsphingomyelinase is a key regulator of cytotoxic granule secretion by primary Tlymphocytes, Nat. Immunol. 10 (2009) 761–768.

[87] K. Trajkovic, C. Hsu, S. Chiantia, L. Rajendran, D. Wenzel, F. Wieland, P. Schwille, B.Brugger, M. Simons, Ceramide triggers budding of exosome vesicles intomultivesicular endosomes, Science 319 (2008) 1244–1247.