The mystery of membrane organization: composition, regulation and roles of lipid rafts

Transcription

1 The mystey of membane oganization: composition, egulation and oles of lipid afts Edinc Sezgin 1, Ilya Levental 2, Satyajit Mayo 3 and Chistian Eggeling 1 Abstact Cellula plasma membanes ae lateally heteogeneous, featuing a vaiety of distinct subcompatments that diffe in thei biophysical popeties and composition. A lage numbe of studies have focused on undestanding the basis fo this heteogeneity and its physiological elevance. The membane aft hypothesis fomalized a physicochemical pinciple fo a subtype of such lateal membane heteogeneity, in which the pefeential associations between cholesteol and satuated lipids dive the fomation of elatively packed (o odeed) membane domains that selectively ecuit cetain lipids and s. Recent studies have yielded new insights into this mechanism and its elevance in vivo, owing pimaily to the development of impoved biochemical and biophysical technologies. Liquid liquid phase sepaation The coexistence of two phases with distinct compositions and biophysical popeties. The components of both phases can diffuse and eaange apidly. 1 MRC Human Immunology Unit, Weatheall Institute of Molecula Medicine, Univesity of Oxfod, Headley Way, Oxfod OX3 9DS, UK. 2 Depatment of Integative Biology and Phamacology, Univesity of Texas Health Science Cente, 6431 Fannin Steet, Houston, Texas 77030, USA. 3 National Cente fo Biological Sciences, Tata Institute fo Fundamental Reseach, Bellay Road, Bangaloe , India. Coespondence to S.M. and C.E. mayo@ncbs.es.in; chistian.eggeling@ dm.ox.ac.uk doi: /nm Published online 30 Ma 2017 Only a yea afte the seminal pape of Singe and Nicolson in which the fluid mosaic model fo bio membane oganization was poposed 1, the fist obsevations that cell membanes can be sepaated into detegentlabile and detegent-esistant factions 2 spaked the idea that distinct membane sub compatments ae pesent in biological membanes (fo a bief histoy of biomembane models, see REF. 3). This finding was followed by a numbe of obsevations that suggested that cellula membanes ae lateally heteo geneous at the submico mete scale 4 9. The membane aft (o lipid aft) hypothesis emeged as a way of explaining this lateal membane inhomogeneity: it poposed that the inteactions between specific lipids (fo example, cholesteol, elatively satuated lipids and glycosylated lipids) in the plane of the membane dive the fomation of functionally impotant, elatively odeed membane egions that ecuit othe lipids and s 10. This concept was suppoted by obsevations of biomimetic model membanes, which povide clea evidence that cetain lipids inteact pefeentially with one anothe, engage in collective behaviou and gene ate lagescale lateal domains as a consequence of liquid liquid phase sepaation 11. Howeve, the pesence and elevance of such odeed membane domains in vivo wee unclea, owing in pat to the lack of diect obsevations of these domains and uncetain definitions of the lipid aft concept. To addess this uncetainty, a consensus opeational defin ition of lipid afts was fomulated in 2006, with the available evidence suggesting that afts ae heteogeneous, dynamic (in tems of both lateal mobility and association dissociation), cholesteoland sphingolipid eniched membane nanodomains ( nm) that have the potential to fom micoscopic domains (>300 nm) upon clusteing induced by and lipid inteactions 12 (FIG. 1). These domains ae pesent in both the inne and the oute leaflets of an asymmetic cell membane, ae pesumably coupled acoss leaflets 13,14 and fom functional platfoms fo the egulation of cellula pocesses 15. Recently, seveal emeging biochemical and biophysical techniques have povided suppot fo the pesence of these domains in cells and suggested key oles fo membane heteo geneity in vaious cellula functions. The consevation of lipid afts thoughout the tee of life has also been demonstated (Supplementay infomation S1 (box)), which has povided futhe suppot fo thei biological significance. Howeve, lipid afts continue to elude diect micoscopic detection; thus, the pesence and exact natue of afts in live cells emain the subject of debate, paticulaly as diffeent methodologies can often yield seemingly contadictoy esults 16. Hee, we define afts as tansient, elatively odeed membane domains, the fomation of which is diven by lipid lipid and lipid inteactions, and we discuss the technological advances that have eignited excitement aound this concept and its in vivo elevance. In paticula, we focus on the cuent undestanding of the mechanisms of aft fomation and maintenance, and conclude with a discussion of the challenges that emain in this dynamic field. NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 18 JUNE

2 REVIEWS a Unsatuated lipids Satuated lipids Glycolipid Cholesteol GPI-anchoed Tansmembane Lipidated Actin b Tansmembane Tansmembane Glycolipid Glyco Tansmembane GPI-anchoed Actin Tansmembane Tansmembane Figue 1 Geneal oveview of lateal heteogeneity the plasma membane. Natue in Reviews Molecula Cell Biology a Lipid aft domains ae usually defined as small, highly dynamic and tansient plasma membane entities that ae eniched in satuated phospholipids, sphingolipids, glycolipids, cholesteol, lipidated s and glycosylphosphatidylinositol (GPI)-anchoed s. Enichment of these hydophobic components endows these lipid domains with distinct physical popeties; these include inceased lipid packing and ode, and deceased fluidity. In addition to membane components, cotical actin plays an active pat in domain maintenance and emodelling. Futhemoe, membane lipids ae asymmetically distibuted in the inne and oute leaflets, which may futhe affect membane oganization. b It is likely that membane oganization is not binay (that is, highly distinct aft and non-aft egions), but instead membanes consist of vaious aft-like and non-aft domains with distinct compositions and popeties. Sphingolipid A class of lipids that compise a long-chain sphingosine base coupled to a fatty acid chain and often a lage pola head goup. Glycosylphosphatidylinositol (GPI)-anchoed s Cell suface s that ae post-tanslationally modified to cay a GPI moiety as an ancho to the membane. Studying lipid afts The definition of afts has been influenced, in lage pat, by the development of methodologies available fo thei investigation. The tem lipid afts has been applied geneically to many distinct, although potentially elated, types of membane assemblies (FIG. 2a). The techniques and tools used to visualize and study membane heteo geneity have evolved consideably since the intoduction of the concept (FIG. 2b d), and with the ecent advent of supe-esolution optical micoscopy (Supplementay infomation S2 (box)) we may now have a key tool fo esolving the continuing contovesy. Biochemical tools. The fist evidence fo a lateally heteo geneous cell membane came fom the obse vation of diffeential solubilization of membane lipids and s by detegents in the 1970s2. The basis of the assay is that cellula membanes can be sepaated into distinct factions containing detegent-soluble membanes (DSMs) o detegent-esistant membanes (DRMs) following extaction with non-ionic dete gents unde specific conditions (most notably, cold tempeatues) (FIG. 2b). These factions have clealy distinct compositions, with DRMs eniched in choles teol, sphingolipids17,18 and glycosylphosphatidylinositol (GPI)-anchoed s5. Although extaction of DRMs became the method of choice fo pobing membane aft composition, it quickly became clea that DRMs do not eflect the native composition and oganization of lipid afts in living cells. Fo example, the com position of DRMs vaies widely depending on the choice of detegent used fo isolation19. Similaly, subtle vai ations in tempeatue o detegent concentation yield diffeent esults and consideably modify the oganiza tion of membane s20, which has led to conta dictoy epots about the composition of afts. Thus, although DRM assays may povide infomation about the popensity of some molecules to associate with specialized membane egions21,22, they do not faithfully eflect the native molecula o biophysical composi tion and oganization of afts23; theefoe, the findings fom these assays equie confimation by moe obust and consistent methods such as those discussed below (fo an excellent ecent example, see REF. 22). Biophysical tools. In paallel with studies of DRMs iso lated fom cells, atificial model membanes have been developed and used to study the liquid liquid phase sepa ation that is believed to undelie the physical pinciple behind lipid aft fomation24 (FIG. 2b). Acoss vaious expeimental set-ups, membanes that consist of elatively satuated lipids with a high melting tem peatue, unsatuated phospholipid species with a low melting tempeatue and cholesteol can sepaate into two distinct liquid phases: a elatively packed, odeed phase eniched in satuated lipid species and choles teol25 (temed the liquid-odeed (Lo) phase), and a moe fluid, disodeed phase compising mainly the unsatuated lipids26,27 (temed the liquid-disodeed (Ld) phase). Owing to its tight molecula packing and enichment of steol and satuated lipids, the Lo phase is consideed to be the model fo lipid afts. Biomimetic monolayes28, suppoted lipid bilayes29, nanoscopic bilaye vesicles30 and giant unilamella vesicles (GUVs)26 have all been used to elucidate the molecula details of this phase sepaation31,32; howeve, despite thei impo tant ole in evealing the physical pinciples of Lo domain fomation, a numbe of caveats and limitations pevent diect tanslation of findings fom these model mem banes to biological ones. Fist, most of these expei ments ae pefomed in lipid-only systems, and although thee ae methods fo incopoating integal membane s into atificial systems33,34, they ae complex, inefficient and vey aely esult in high /lipid atios. This is in contast to biological membanes, in which s ae estimated to constitute up to 25% of the coss- sectional aea of the membane35. Second, pehaps because of the scacity (o even a complete lack) 362 JUNE 2017 VOLUME 18 d e v e s e s t h g i l l A. e u t a N e g n i p S f o t a p, d e t i m i L s e h s i l b u P n a l l i m c a M

3 Cholea toxin Poteinaceous toxin seceted by Vibio choleae that binds to glycolipids on the cell suface and is esponsible fo the symptoms of cholea infection. of s, some featues of domains established in synthetic membanes may not be epesentative of in vivo domains. Fo example, odeed domains in synthetic membanes have extemely high molecula ode and tight packing, wheeas the othe exteme is obseved in the disodeed domains 27,36. These caveats can be avoided by studying moe natual systems such as giant plasma membane vesicles (GPMVs) 37,38. GPMVs ae celldeived, intact plasma membane vesicles that maintain the lipid 39 and 40 divesity of cellula membanes, although it is notable that they lack an assembled cotical actin cytoskeleton, phosphoylated lipids 41 and stict lipid asymmety between sepaate leaflets of the membane bilaye (see REF. 42 fo a detailed discussion of the advantages, caveats and applications of GPMVs). In these biological membanes, the dispaity in molecula ode between coexisting odeed and disodeed domains is much smalle than in synthetic GUVs, fo example. These diffeences in molecula ode between the two phases may account fo the fact that the odeed phase of GUVs excludes almost all tansmembane s and most fluoescent lipid pobes (see below), wheeas the same molecules ae sometimes eniched in the odeed phase in GPMVs (as would be expected fo lipid afts in vivo) 36,43 (BOX 1). Analytical tools. In cells, afts ae believed to be nanoscopic domains (<200 nm) 7,8, and theefoe they cannot be esolved by conventional optical micoscopy, which has an appoximately 250 nm esolution limit that is set by diffaction (see Supplementay infomation S2 (box)). Although the colocalization of cetain molecules with putative lipid aft makes (such as the multivalent cholea toxin) detected by confocal micoscopy has been used as evidence of thei association with afts 44, in geneal the esolution of confocal micoscopy is insufficient to diectly assay aft domain stuctue and composition. To ovecome this limitation, seveal optical tools have a Lipid-associated membane domains Pue lipid clustes (glycolipid clustes, ceamide domains, etc.) Unsatuated lipids Lipid-mediated clustes (GPI domains, RAS domains, etc.) b Tools to study membane domains Synthetic and cell-deived model membanes Liquid liquid phase sepaation of lipids Detegent esistance assays DRM DSM RAS Single-molecule imaging and spectoscopy Fee diffusion Sucose gadient GPI-anchoed Satuated lipids Confined diffusion FRET Hop diffusion Enegy tansfe Dono Liquid liquid phase sepaation of lipids Cholesteol Odeed domain Actin Accepto Disodeed domain Potein-mediated lipid cluste GM1 α β Mass spectomety (lipids and s) Intensity No enegy tansfe Clustes in domain Figue 2 Tools to study membane domain oganization, composition and function. a In pinciple, membane domains can be pue lipid clustes, but in most physiologically elevant cases they also contain s, which include clustes of, fo example, glycosylphosphatidylinositol (GPI)-anchoed s o RAS s. Domains can be puely lipid-diven entities, such as domains that ae established by liquid liquid phase sepaation in model membanes. They can also be induced by aceous clusteing agents such as cholea toxin, which binds to monosialotetahexosylganglioside (GM1), o by antibodies that ecognize suface eceptos (not shown). b Tools that ae commonly used to investigate membane domains. These include vaious model membanes such as synthetic giant unilamella vesicles (GUVs) and cell-deived giant plasma membane vesicles (GPMVs); detegent esistance assays, in which aft-like membane egions patition m/z atio Cholea toxin Intensity c Pobes to study aft domains Domain-selective pobes Odeed Disodeed Meged Domain-sensitive pobes (laudan, di-4-aneppdhq, etc.) Wavelength (nm) d Functional studies using aft-tageting dugs Zaagozic acid Methyl-β-cyclodextin Cholesteol oxidase Statins Fumonisins Myiocin Sphingomyelinase Odeed Disodeed Cholesteol Sphingolipids Sphingomyelin into detegent-esistant membane (DRM) factions, wheeas non-aft components ae fully solubilized and ae found in detegent-soluble membane (DSM) factions; single-molecule micoscopy to evaluate the diffusion of membane molecules (the tack of an individual molecule is depicted); and fluoescence spectoscopy methods such as Föste esonance enegy tansfe (FRET) and mass spectomety. c Vaious pobes can be used to study aft domains. Domain-selective pobes patition into one of the domains, wheeas domain-sensitive pobes patition to both domains and change thei photophysical behaviou (fo example, absobance and emission specta) depending on the natue of the suounding lipid envionment. d Teatments that intefee with cholesteol o sphingolipid levels have been used to disupt afts in cells and can shed light on the cellula functions of these domains. NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 18 JUNE

4 Single-paticle tacking (SPT). A single-molecule technique in which the motion of individual molecules is tacked with high tempoal esolution ove elatively long timescales (seconds); these tacks can be used to detemine the diffusion popeties of a molecule. Confined diffusion A mode of diffusion in which the motion of the molecule is tansiently aested by molecula obstacles such as immobile clustes. It is also known as tapped diffusion. Hop diffusion A mode of diffusion in which molecules diffuse feely in the membane except when they encounte a baie (such as a stuctue (o stuctues) associated with actin filaments), the cossing of which hindes diffusion. Intefeometic scatteing micoscopy (iscat). A micoscopy technique to enhance contast by using the intefeence fom coheent light scatteing in the focal plane and of the micoscope cove glass. Fluoescence coelation spectoscopy (FCS). A single-molecule-based technique in which fluoescence intensity fluctuations fom a micoscopic obsevation spot ae used to obtain infomation about molecula diffusion. been developed ecently 45,46 and have been applied to investigate nanoscale stuctues and dynamics in cells. Fo example, supe-esolution optical micoscopy appoaches such as photoactivated localization micoscopy (PALM), stimulated emission depletion (STED) micoscopy (Supplementay infomation S2 (box)) and neafield scanning optical micoscopy (NSOM) have been used to visualize lipid-mediated clusteing Fo moe dynamic measuements, single moleculebased techniques such as single-paticle tacking (SPT) have been used to evaluate the diffusion of membane molecules and elate it to models of heteo geneous oganization of the membane 51. Such studies can eveal oligomeization 52, tansient aest, domain incopoation, and/o confined diffusion and hop diffusion (also known as compatmentalized diffusion) 53 of tacked molecules (FIG. 2b). Recently, intefeometic scatteing micoscopy (iscat) has futhe inceased the sensitivity of SPT 54 and has shown geat potential fo assessing membane heteogeneity. Fo example, iscat was used to show that lipids can tansiently stall and become incopoated into sub 20 nm domains within model membanes 55,56. A technique complementay to SPT, fluoes cence coelation spectoscopy (FCS), has been applied in combination with spot vaiation (svfcs 57 ) o a STED micoscope (STED-FCS 58 ) to pobe the Box 1 Model membanes fo the study of the fomation and oganization of lateal domains Combining a elatively satuated lipid, an unsatuated lipid and cholesteol in a model membane often esults in liquid liquid phase sepaation and the establishment of two distinct phases (which ae still liquid in natue) 11. One of these phases (the liquid-odeed (L o ) phase) is moe viscous than the othe (the liquid-disodeed (L d ) phase) owing to the tighte packing and highe molecula ode of its constituent lipids 91. This L o phase is believed to epesent a potential physical model of lipid afts in cellula membanes. Suppoted lipid bilayes (SLBs; see the figue, pat a) ae plana bilayes fomed on glass o mica sufaces 29. As these membanes ae plana, they ae highly amenable fo micoscopic imaging, eithe by light micoscopy o by atomic foce micoscopy, which allows obsevations of the topology of nanodomains that ae not esolvable by diffaction-limited optical micoscopy (Supplementay infomation S2 (box)). The atefacts caused by the solid suppot in SLBs ae avoided by the use of fee-standing membanes such as giant unilamella vesicles (GUVs) (see the figue, pat b), which have been used fequently to investigate domain dynamics and mophologies 189. The limitation of synthetic model systems is thei simple composition, which does not fully ecapitulate the composition of the cell membane. Giant plasma membane vesicles (GPMVs) ae obtained fom cell membanes 37. Similaly to GUVs, these fom micomete-scale lateal liquid domains (which confims the capacity fo liquid liquid phase sepaation in cellula membanes), but do so while maintaining the boad compositional featues of the native plasma membane. The most notable diffeences between GUVs and GPMVs ae lipid complexity and the pesence (in GPMVs) of abundant tansmembane s 37,38, which ae technically challenging to incopoate into SLBs and GUVs. The biophysical popeties of GPMVs ae somewhat distinct fom those of atificial membanes 37,92. Fo example, the diffeence in packing density between L o and L d domains in GPMVs is much smalle than in GUVs (see the figue, pat c; genealized polaization is a elative index of lipid packing, in which +1 epesents maximally odeed membanes and 1 epesents maximally disodeed membanes), which may explain why tansmembane s can associate with the L o phase in GPMVs 43 but not in GUVs 190. Despite these diffeences, most of the coe featues of the coexisting L o and L d domains in these model systems ae fundamentally simila 24. a SLBs Side view b GUVs Domain-selective pobe Disodeed Odeed Meged L d L o L d Glass o mica suface Aqueous laye Top view Disodeed Odeed Meged L d L o c GUVs GPMVs +1 Unsatuated lipids Satuated lipids Genealized polaization map 1 Cholesteol Disodeed phase make Odeed phase make Natue Reviews Molecula Cell Biology 364 JUNE 2017 VOLUME 18

5 Föste esonance enegy tansfe (FRET). A fluoescence spectoscopy and imaging technique that is based on the distance-dependent tansfe of the excited state enegy of a fluoescent dono molecule to a fluoescent accepto molecule; efficient and widely used to measue intemolecula distances in the ange of 1 10 nm. Amphiphilic popeties Displaying both hydophilic and hydophobic chaacte, such as fo lipids with hydophobic acyl chains and hydophilic head goups. Raman spectoscopy A spectoscopy technique wheeby vibational enegy of the molecules is used as thei fingepint. Ganglioside lipid A class of glycosphingolipids with sialic acid moieties on the head goup. lateal diffusion of membane components ove vaious length scales. Paticulaly in STED-FCS, the size of the obsevation spot can be educed to appoximately nm, which eveals undelying nanoscopic featues of the plasma membane 58,59. Finally, Föste esonance enegy tansfe (FRET; FIG. 2b) is a key tool fo investigating membane aft stuctue and composition 60,61. The spatial egime pobed by this technique makes it ideal fo studying nanoscopic domains, and it has been applied to both model membanes 62 and live cells 63, not only to pobe the existence of domains but also to define thei size 62,64 by using fluoescent pobes with diffeent FRET efficiencies. Fo a detailed eview of these techniques and thei caveats, see REF. 45. Most of the afoementioned methodologies ely on fluoescent labels. This is a paticula issue in the investigation of membanes because the behaviou of lipids is inheently dependent on thei amphiphilic popeties and molecula packing, both of which ae potentially affected by tags such as fluoophoes, which ae often almost the size of the lipid molecules. Thus, the native behaviou of lipids is often alteed consideably by the epote 36. To addess this concen, a numbe of label-fee techniques have been developed. Mass spectoscopy (FIG. 2b), fo example, is one of the most accuate tools fo pobing the lipid and composition of membanes without the necessity of extenal labelling 65, and it has been used fo label-fee detemination of membane domain composition in model membanes and cell deived membanes iscat has also facilitated label-fee obsevation of the dynamics of odeed domains in model membanes 72. Raman spectoscopy is anothe label-fee technique that has been applied successfully to monito membane domain composition 73. Likewise, small-angle neuton scatteing has also been used to detect aft-like domains 74 and detemine thei size 75. Finally, electon micoscopy has the necessay esolution to obtain a snapshot of molecula aangements at the cell suface, and a numbe of studies of oute and inne leaflet lipid-tetheed s (including GPI-anchoed s, glycolipids and RAS s) have evealed the nanoscopic oganization of s in afts 76. One potential caveat of these methods is that they usually equie cell fixation and staining, which ae notoiously poblematic fo visualizing lipid molecules. Theefoe, fluoescence micoscopy emains the pefeed technique fo diect live imaging of putative lipid aft components, and this necessitates the continued optimization of fluoescent labels fo membane components. Pobes selective fo membane domains. Nonpetubing, specific labelling of aft o non-aft domains in cells has been, and emains, one of the foemost challenges in the field. Seveal fluoescent makes including cyanine dyes (fo example, DiO, DiI and DiD) 77, polycyclic aomatic hydocabons (fo example, naphthopyene) 78 and fluoescently labelled lipids 36,79 have been used to distinguish between diffeent membane compatments (FIG. 2c). As mentioned above, the eliability of these fluoescent lipid analogues depends stongly on the choice of both the native lipid and the fluoescent moiety 36. The fluoescent lipids that ae fee fom atefacts linked to fluoescent labelling ae intinsically fluoescent cholesteol analogues such as dehydoegosteol 80 and cholestatienol 81 ; howeve, thei poo photophysical chaacteistics compaed with atificially tagged lipids have pevented thei widespead application. In the case of phospholipids, it is often challenging to peseve the natual physicochemical behaviou of the lipid afte attaching a fluoophoe 82,83. In geneal, the stategy that causes least disuption to lipid behaviou is to label the head goup instead of the acyl chain and to add a hydophilic linke to ensue that the fluoophoes do not affect the head goups of the suounding lipids 84. In addition to lipid analogues that can eveal the geneal oganization of the membane into subdomains of vaiable composition, epotes that selectively bind to coe aft components can potentially be used to visualize domains. These include cholesteol-binding agents such as filipin 85 and pefingolysin O 86, sphingolipid epotes such as osteolysin A 87, lysenin 88 and pleuotoly sin 89, as well as ganglioside lipid ligands such as cholea toxin 90. The majo caveats fo these pobes ae: fist, thei potential petubation of native membane oganiza tion by, fo example, inducing the clusteing of thei binding patnes, as is the case fo cholea toxin; and, second, thei educed specificity in the cellula context, in which they can potentially exhibit off-taget binding that theeby lowes thei specificity fo aft domains. Pobes sensitive to membane envionments. Coexisting lipid domains inheently have diffeent physicochemical popeties. A defining popety of lipid afts is thei tight lipid packing, which is due to the condensing inte actions between elatively satuated lipids and cholesteol 91. Of note, thee is no specific, unique type of molecula packing that is common to the plasma membane and its domains in diffeent cells and contexts 92. The divesity of membane compositions and physical popeties acoss cell types, and within cell types duing physiological events such as secetoy ganule elease 92 o cell cycle pogession 93, implies that a wide ange of diffeent packing states exists in living cells. This lipid packing can be quantified using pobes, such as laudan, that sense the level of hydation in the bilaye 94 in combination with two-photon 32 o conventional confocal 95 micoscopy. The emission specta of these pobes shift depending on the polaity (that is, the aqueous content o hydation) of the envionment 96 (FIG. 2c). This shift povides a atio metic, concentation-independent quantification of the local envionment, which, fo membanes, is detemined lagely by lipid packing 97 (that is, moe tightly packed membanes exclude wate moe efficiently). Imaging of membane packing using these pobes has been applied to investigate membane heteogeneity in live cells 47,98. Moe ecently, in addition to spectal shift, the lifetime 99 and enegy tansfe 100 popeties of simila pobes have been used to futhe investigate lipid packing in living membanes, which has expanded the scope and sensitivity of thei potential applications. Enabling the efficient use of these pobes in supe-esolution micoscopy will be an impotant futue development. NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 18 JUNE

6 Ceamides A class of lipids composed of sphingosine and a fatty acid. Coase-gained simulations Simulations that ely on simplified epesentations of the simulated components. Hydogen bonding Non-covalent chemical bonds between a hydogen covalently bound to an electonegative atom (as in the -NH goup of sphingolipids) and anothe electonegative atom (such as the oxygen in the -OH goup of cholesteol). Raft-tageting dugs. A common paadigm in the study of the physiological oles of lipid afts has been the use of dugs o enzymes to impai the stuctue and function of these domains (FIG. 2d). As cholesteol is thought to be eniched in afts, the most common aft-disupting agent in use is methyl-β-cyclodextin (MβCD), which selectively and efficiently extacts cholesteol fom membanes 101. Howeve, it is impotant to conside that MβCD-mediated cholesteol emoval has boad pleiotopic effects that extend beyond aft disuption. Fo example, it inceases membane pemeability to ions and theeby disupts membane potential 102, and it is potentially cytotoxic 103. Moeove, this eagent appeas to pefeentially deplete cholesteol fom L d (non-aft) domains in model membanes 104, which can poduce unexpected and inconsistent 21,67 effects on lipid packing in moe complex membanes. Dugs that taget cholesteol synthesis (statins 105 and zaagozic acid 106 ), o cholesteol-modifying enzymes (fo example, cholesteol oxidase 107 ), have the potential to eplace the use of MβCD to disupt afts, but thei specificity and effectiveness emain to be demonstated conclusively. Sphingolipids ae anothe coe component of afts in cells, and a numbe of eagents can intefee with thei synthesis (fo example, fumonisin B1 (REF. 108) and myiocin 109 ) o stability (fo example, sphingomyelinases 110 ). Howeve, these eagents suffe fom potential off-taget effects on pocesses such as geneal sphingolipid metabolism and the geneation of ceamides, which can then alte membane popeties in othe ways. Molecula dynamics simulations. One of the biggest challenges emaining in ou undestanding of bio membanes is how the myiad of inteactions between membane molecules detemines membane oganization. Ovecoming this challenge equies a combin ation of complementay expeimental appoaches as well as in silico techniques that integate expeimental obsevations (fo example, data about the stuctue and enegetics of the system) into a simu lation famewok that ideally can econstitute the natual behaviou in silico solely on the basis of physical inteactions 111. An inheent advantage of such in silico appoaches is that they simultaneously model a multitude of molecules at a high spatial (atomic level) and tempoal (nanosecond micosecond) esolution without elying on exogenous pobes o labels. Thus, in silico molecula dynamics simulations can be egaded as a computational micoscope (REF. 112) that is capable of visualizing molecula behaviou with unpecedented pecision. Cuently, such computational micoscopes have the opposite limitations to optical micoscopes, in that they eveal only shot pocesses (ove micoseconds) at a nanoscopic scale (thousands of molecules), as opposed to pocesses that occu ove longe timescales and at lowe esolution that ae accessible by optical micoscopy 112,113. To close the gap between computational and expeimental appoaches, methods such as coase-gained simulations have been developed to extend the spatiotempoal scale of molecula dynamics simulations without sacificing the molecula details 114. Such simulations have been used successfully to study lipid lipid and lipid inteactions 115,116 and lipid domains in complex membanes 14,117,118. It is impotant to note that such in silico obsevations ae inheently model-diven and must ultimately be veified by expeiments. Unfotunately, in the case of membane domains, the spatiotempoal gap between the simulated and expeimental obsevables is still too lage to allow diect compaisons 111. Howeve, effots to bidge this divide will ensue pogess towads a molecula undestanding of how complex membane components self-oganize into functional substuctues. Natue and composition of lipid afts Dissecting the physical popeties the lifetime, size, and coveage aea of lipid afts in the cellula envionment emains one of most vexing issues in the field. Computational models have confimed the intuitive assumption that both the tempoal and spatial compatmentalization of membane molecules into domains is cucial fo membane function 119. Unfotunately, both the small size and shot lifetime of putative aft domains in vivo complicate diect measuement of thei popeties in living cells. Futhemoe, the complexity of plasma membanes suggests that a ange of aft-like domains with vaying sizes and lifetimes can be established in vivo 92,98,120, futhe complicating intepetations of expeimental measuements. The oiginal model of lipid afts suggested the existence of a L d (non-aft) membane punctuated by moe-odeed (aft) domains with minimal coveage 121. Howeve, ecent data indicate a much geate extent of odeed aft-like egions in membanes (which suggests that odeed membane domains might in fact pedominate and possibly cove the majoity of the plasma membane) with intespesed less-odeed (non-aft) domains 47,59 (FIG. 3a). The elative aea as well as the size and lifetime of membane domains may be futhe tuned by cellula pocesses such as signalling and membane tafficking (FIG. 3b), which makes it even moe challenging to daw conclusions egading these membane domains. In the oiginal fomulation of the lipid aft model, aft fomation was based on pefeential inteactions between sphingolipids and cholesteol 10. Consistent with this notion, sphingomyelin has been identified as a coe component of DRMs 2 and odeed lipid phases 122, owing in pat to stong hydogen bonding of lipids with cholesteol 123,124 (FIG. 4a). Howeve, the patitioning of cholesteol between moe- and less-odeed domains is less clea: expeimental 30 and computational 125 studies suggest that it is abundant in both odeed (aftlike) and disodeed (non-aft) phases, although it is eniched in moe-odeed domains. Ganglioside lipids wee also found to inteact with cholesteol, which esults in the fomation of cholesteol-ich domains in model membanes 70, and these lipids have been detected consistently in the odeed domains of model membanes 90. In addition, othe lipids such as elatively satuated phospholipids have often been associated with aft-like envionments, especially in model membanes. 366 JUNE 2017 VOLUME 18

7 a Vaying aft coveage Isolated domains Pecolating (continuous) domains b Raft clusteing and declusteing Cellula pocesses such as signalling and tafficking Addition of clusteing agents Nanodomains Raft platfoms Rafts Non-aft egions Figue 3 Aea coveage of membane domains and domain size. a Models of membanes with vaying aft coveage. Total aft coveage in a given membane may vay boadly, anging fom small isolated domains to pecolating (continuous) aft phases of inceasing size. The specific oganizational state depends on a vaiety of factos, which include cell type, specific cellula conditions (fo example, cell cycle phase) and/o the identity of the membane (fo example, plasma membane vesus intacellula membanes). b Anothe mode of modulation of membane oganization can occu without changing oveall aft abundance. Fo example, the size and/o lifetime of individual domains may be influenced by cellula pocesses such as endocytosis and exocytosis, lipid metabolism, and so on. In addition, the binding of clusteing agents (antibodies and toxins) to thei eceptos can pomote the fomation of lage membane domains. Wheeas the biophysical basis fo the lipid composition of afts can be explained by these simple pinciples, the basis fo the selective incopoation of s into moe-odeed (aft-like) domains lagely emains a mystey. In geneal, s that inteact with the membane via lipid anchos follow the ules set by the lipids: satuated lipid anchos such as GPI o palmitoyl moieties geneally favou odeed membane envionments, wheeas banched o unsatuated anchos such as penyl goups pefe disodeed (non-aft) egions 126. In fact, GPIanchoed s wee some of the fist s to be identified in DRMs 5 and late in the odeed domains of model membanes 33,127. Lateal GPI-anchoed domains have been extensively chaacteized by singlemolecule appoaches 128,129. Although thei elationship to membane afts emains unesolved 130, the inteactions between these lipid-anchoed s and lipids almost cetainly egulate membane stuctue and function 14,22,69. Howeve, lipidated s ae cetainly not the only species that associate with aft-like domains. In fact, in a ecent expeiment, 35% of all plasma membane s wee found in odeed domains in GPMVs 43. These aftophilic s included GPIanchoed s and palmitoylated s, as expected (each constituting appoximately one-thid of the identified s) 43. Howeve, the emaining one-thid of aftophilic s contained neithe a GPI no a palmitoyl ancho, and the mechanism of association of many of these s to aft-like domains is cuently unclea. Some s ae known to become moe aftophilic upon oligomeization, which may modulate thei activity 131. Recently, a database of putative aftophilic s identified in mass spectomety studies of isolated DRMs has been established (RaftPot) 132, although it is impotant to emphasize that these studies may be subject to the DRM-associated atefacts descibed above. As the actual content of membane domains is uncetain, few genealizable insights into the stuctual deteminants of aftophilic behaviou of tansmembane s ae available 133. Inteestingly, a ecent study demonstated that the length of the tansmembane domain (TMD) appeas to be a key featue detemining the aftophilic popeties of a longe TMDs pefeentially taget the to the thicke, odeed domains 134. Mechanisms of domain egulation Although the aft concept and its in vivo elevance have been contovesial, the pinciple of lateal membane compatmentalization by lipids is intuitive: thee ae clea diffeences in the inteaction affinities between vaious lipids, and these diffeences may be sufficient to poduce a heteogeneous lipid distibution. Fo systems in themo dynamic equilibium (including synthetic and biological model membanes 42 ), the manifestation of these phenomena is macoscopic phase sepaation, which can be egulated by tempeatue 135, lipid composition 21,26,67 o specific inteactions that enhance the inheent connectivity of paticula components and thus lead to enhanced clusteing 136. Howeve, cell membanes in vivo ae not closed systems in chemical and themodynamic equili bium, and many potential modes of egulation contibute to the ultimate output of the inheent self-oganizing capacity of biological lipids and thei sepaation into distinct domains (FIG. 4). Lipid lipid and lipid inteactions. In the tadi tional aft model, the fomation of aft domains is diven mainly by the pefeential binding of cholesteol to sphingo lipids 124 and possibly othe lipids such as gangliosides 69 (FIG. 4a). Howeve, an inheent limitation of studying the factos that egulate aft domain popeties is the difficulty of measuing these popeties in situ. NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 18 JUNE

8 Epithelial mesenchymal tansition A developmental tansciptional pogamme that impats mesenchymal chaacteistics (fo example, motility) to epithelial cells. To addess this limitation, seveal ecent studies 93,137 have focused on factos that egulate the tempeatue at which macoscopic aft-like domains fom in GPMVs, with the undelying infeence that highe phase sepaation tempeatues suggest moe stable domains. This paadigm is based on obsevations of a specific type of phase sepaation in GPMVs that occus nea a compositional citical point and involves lage-scale fluctuations at tempeatues close to the phase tansition 135. Such citical fluctuations ae pesent in all systems that exhibit citical behaviou, which suggests that thee ae scaling laws that allow extapolation of domain size and stability to living cells 135,139. It is impotant to note that this hypothesis has yet to be fomally evaluated; howeve, if validated, it will povide an impotant methodological tool fo assaying aft popeties. Fo example, it was ecently demonstated that the stability of moe-odeed domains in GPMVs is affected by dietay fatty acids. In paticula, exogenously supplied polyunsatuated fatty acids such as the fish oil component docosahexaenoic acid ae obustly incopoated into cellula membanes, in which they induce extensive changes in lipid composition and biophysical popeties, including inceasing the stability of aft-like domains 67. A study elating these effects to cell behaviou showed that incopoation of docosa hexaenoic acid into membanes, and the concomitant incease in the stability of aft-like domains, can epess the stem cell popeties and motility of beast cance cells by intefeing with the plasma membane emodelling that is necessay fo the epithelial mesenchymal tansition 140. Although vaiations in lipid composition ae cetainly key dives of lipid membane heteogeneity, lipid inteactions also have impotant oles in aft egulation. Fo example, some s, including the HIV glyco gp41 (REF. 141), have cholesteolbinding motifs that egulate thei membane distibution (FIG. 4b). Othe s specifically bind glycosphingolipids 138 o sphingomyelin 142, which potentially mediates thei ecuitment to aft-like membane domains. Futhemoe, a vaiation on the ole of palmitoylation in a Lipid lipid inteactions Sphingomyelin OH O NH O P O O O OH Cholesteol Hydogen bond CH 3 CH3 + N CH 3 b Lipid inteactions Tansmembane with a cholesteol-binding motif Unsatuated lipids Satuated lipids Cholesteol c Lipidated s as domain oganizes d Hydophobic match o mismatch x > y e Tansbilaye coupling GPI-anchoed Long-chain inne leaflet lipid (e.g. PS) x y Palmitoylated Long TMD Shot TMD Adapto s Figue 4 Regulation of membane domains. a Lipid lipid inteactions in paticula, inteactions between cholesteol and sphingolipids (but also between othe elatively satuated lipids) ae the defining featue of lipid-diven odeed domain fomation. The pefeential inteaction between sphingolipids and steols is due to the satuation of sphingolipid hydophobic tails, but also to hydogen bonding between these lipid species. The amide of the sphingolipid backbone can both donate and accept a hydogen bond, and these hydogen bonds ae within the intefacial egion of the membane, in which the elative paucity of wate inceases the stability of these bonds. b Some s contain lipid-binding domains though which they inteact with cholesteol o sphingolipids, and these lipid inteactions may detemine the affinity of s fo odeed lipid domains. c Lipidated s, which ae modified by the attachment of a satuated acyl chain (such as a palmitoyl moiety), ae ecuited to aft-like domains, but they may also nucleate and ecuit membane domains if they ae integated into a elatively static scaffold. d Hydophobic inteactions can contibute to membane domain oganization and composition. In paticula, s that diffe in the length of thei Long TMD Actin tansmembane domains (TMDs) segegate into diffeent lipid envionments, which ensues that thei hydophobic Natue TMDs Reviews ae potected Molecula fom Cell exposue Biology to the aqueous suoundings. Fo example, s with long TMDs associate with domains eniched in long-chain satuated lipids (top panel). When thee is a mismatch between the length of the TMD and the local lipid envionment in which the esides, inteaction might be favoued instead, which leads to a local incease in concentation (bottom panel). e Inne leaflet lipids containing long satuated acyl chains ae immobilized by actin clustes, which ae fomed owing to the inteactions between actin and membane lipids (fo example, phosphatidylseine (PS)) o adapto s (such as those that possess PS-binding domains and actin-binding domains). This immobilization esults in the engagement of long acyl chain-containing lipid-anchoed s (such as glycosylphosphatidylinositol (GPI)-anchoed s) located in the oute leaflet, in the pesence of cholesteol. This esults in the fomation of locally odeed tansbilaye domains, which ae dynamic owing to the dynamics of actin clustes and may fom even unde conditions that do not favou liquid liquid phase sepaation of lipids o othe suppoting inteactions. 368 JUNE 2017 VOLUME 18

9 egulation of aft-like domains was ecently poposed fo post synaptic density 95 (PSD95; also known as DLG4). Compehensive lipidomic analysis of neuonal synapses suggested that aft-like domains ae specifically ecuited to synaptic sites, which was poposed to be mediated by the integation of palmitoylated PSD95 into the post synaptic density scaffold 143. In this scenaio, instead of aft-like domains ecuiting palmitoyl ated s, it is the immobilized palmitoylated s that ecuit satuated lipids and thus nucleate odeed domains at specific cellula sites 143 (FIG. 4c). This hypothetical mechanism and its applicability to othe cellula contexts emain to be confimed. Howeve, the evidence that anothe palmitoylated, membane palmitoylated 1 (MPP1; also known as p55), nucleates aft-like envionments in eythoid cells 144,145 suggests the existence of a moe geneal mechanism wheeby s dictate, o at least consideably influence, the localization and stability of oganized domains. Hydophobic match o mismatch. Mammalian membane lipids can contain hydocabon acyl chains that ae cabons in length; thus, thee is the potential to yield dastically diffeent hydophobic tail lengths fo individual lipids. To minimize the unfavouable exposue of hydophobic tails to the aqueous envionment, lipids segegate accoding to thei acyl chain length, which can potentially intoduce lateal heteogeneity. In phasesepaated model membanes, this thickness mismatch between longe satuated (aft) and shote unsatuated (non-aft) lipids appeas to egulate the size of the coexisting domains, such that lage mismatches give ise to lage domains, and vice vesa 146. Similaly, the TMDs of nealy all eukayotic integal membane s consist of α-helices with hydophobic amino acid side chains, which ae buied inside the hydophobic coe of the membane. Hydophobic matching between these TMDs and the suounding membane lipids minimizes the enegetically unfavouable exposue of hydophobic esidues to aqueous envionments 147 (FIG. 4d). In the case of a significant length mismatch between TMDs and the suounding lipids, lateal -ich aggegates can potentially be induced 148. Howeve, the ole of hydophobic mismatch in membane domain dynamics in the plasma membane of living cells needs to be demonstated unambiguously. Cotical actin cytoskeleton. The cotical actin cytoskeleton is undoubtedly one of the most impotant factos that influence membane oganization 149 and mechanics 150. The actin scaffold has been shown to detemine molecula diffusion dynamics (fo example, tapped and hop diffusion) and supamolecula aangements in the membane 129, In in vito cholesteolcontaining membane systems that ae capable of lage-scale phase sepaation, actin can diectly stabilize o abogate this sepa ation depending on the natue of the lipid species that ae coupled to actin 153, If actin filaments ae coupled to, fo example, satuated acyl chain containing lipid species, they tend to stabilize L o domains, but pevent lage-scale phase sepaation 153. In a living cell, it is likely that the dynamics of actin filaments will influence the oganization of the membane components that ae associated with actin. In fact, a theoetical famewok fo undestanding the inteplay between the oganization of the cotical actin cyto skeleton and living asymmetic membanes has emeged fom studies of the actomyosin-dependent clusteing behaviou of GPI-anchoed s in the oute leaflet of the plasma membane. It was poposed that such clusteing is the esult of dynamic self-oganization of actomyosin into nanoscopic contactile assemblies temed astes 158,159. These assemblies bind to and tansiently immobilize the chaged lipid phosphatidylseine in the inne membane leaflet, possibly via specific inteactions between actin and membane adapto s. This lipid species contains long satuated acyl chains that engage in cholesteol mediated tansbilaye inteactions with long acyl chain containing GPI-anchoed s located in the oute leaflet, which esults in the fomation of local aft-like domains 14 (FIG. 4e). Thus, an actin-diven clusteing mechanism may be esponsible fo the fomation of odeed domains in membanes of living cells, even unde conditions (fo example, tempeatue and/o lipid composition) that ae not nomally conducive fo phase sepaation. A poof of pinciple fo this mechanism has been demonstated ecently in vito 160 by showing that dynamically emodelling actomyosin netwoks can oganize and segegate associated lipids in a synthetic suppoted membane bilaye system. In addition, ecent live-cell wok has shown that self-oganizing cotical actin pattens such as astes geneate moe-odeed membane envionments in the immediate plasma membane aeas 159. As an addition to the chemical pinciples of lipid lipid inteactions, this actin-diven mechanism of membane odeing povides a consistent explanation fo the dynamic popeties and non-equilibium distibution of nanoclustes that ae fomed by seveal lipid o species. These include GPI-anchoed s, glycolipids in the oute leaflet and RAS s in the inne leaflet of live-cell membanes 161. The molecula machiney that geneates these actinbased nanoclustes has not been identified, and futhe wok is necessay to undestand how these small actinbased nanoclustes may give ise to lage-scale odeed membane domains with functional significance 161. Physiological functions of afts The most appaent function of aft-like domains (o heteogeneity in membane lipid ode in geneal) is to segegate specific elements in ode to egulate thei inteactions with othe membane components and hence thei activity. In addition, inteactions with aftophilic lipids (cholesteol o glycosphingolipids), o with the distinct biophysical envionment of afts, may change the confomation of a aft-esident and thus its activity 162,163 (FIG. 5a). These geneal modes of egulation may be boadly employed in cellula physiology, and a few examples ae descibed hee. Howeve, it should be emphasized that the diect mechanistic effects of lipid afts on cell function and dysfunction ae unclea owing to the inheent difficulties in defining aft composition and popeties and in achieving specificity when petubing thei function. NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 18 JUNE

10 a Inceasing the concentation of signalling molecules Induction of a confomational change Unsatuated lipids Satuated lipids Cholesteol Raft Non-aft Raft Non-aft b Immune signalling Immune ecepto complex Host pathogen inteactions Binding of bacteial toxin o vius Downsteam signalling P Phosphoylation SRC family kinases Phosphatases GSLs Raft Vial eceptos (e.g. CD4) Non-aft Gag Vius budding Raft Raft Non-aft Figue 5 Cellula functions of lipid afts. a Mechanisms by which membane domains can potentially egulate the activity of thei associated components. Raft-like domains can facilitate an incease in the concentation of cetain molecules, which esults in the establishment of functional catalytic platfoms. Fo example, enzymes and substates can be bought togethe to incease thei encounte pobability and theeby tigge eactions (fo example, signal tansduction). A elated possibility is that distinct physicochemical envionments povided by lipid afts diectly affect confomation, and theeby egulate activity. b Examples of physiological functions of membane domains. Kinases of the SRC family ae eniched in aft-like domains owing to thei palmitoylation, wheeas tansmembane phosphatases ae geneally excluded fom them. This segegation has been found to be impotant fo immune signalling, in which aft-associated SRC kinases ae involved in egulating the phosphoylation state, and hence the signal tansduction activity, of vaious immune eceptos that include the T cell ecepto and the high-affinity immunoglobulin E ecepto (FcεRI). Many pathogens and thei poducts (such as bacteial toxins) selectively bind to membane afts owing to the pesence of thei specific eceptos, such as glycosphingolipids (GSLs; fo cholea toxin) o CD4 (fo HIV), in these domains, and theeby gain access to host cells. Vius budding is also thought to occu pefeentially at aft-like domains. Although the mechanism behind this selective budding is not yet clea, vial s such as the Gag of HIV ae believed to be sensitive to membane fluidity and to associate with cholesteol-eniched domains. Vial envelope The lipid membane that coves the vial capsid and is deived fom the plasma membane of the host cell. Immune signalling. Compatmentalization of cellula signalling in membane domains may be used to concentate positive egulatoy components (such as kinases 164 ), togethe with excluding negative egulatoy elements (such as phosphatases 165 ) (FIG. 5b). Immunoglobulin E (IgE)-mediated signalling was the fist signalling pathway that was shown to be associated with lipid afts 166. Since then, seveal studies have implicated these domains in vaious innate and adaptive immune esponses 167. In these contexts, the key immune eceptos, including the high-affinity IgE ecepto (FcεRI), the T cell ecepto 168 and the B cell ecepto 44, wee found in DSMs in esting o immatue cells, but these shifted to DRMs following ecepto activation, which suggests that the tanslocation to membane afts is associated with active signalling though these eceptos This notion is suppoted by the co enichment in DRMs of the poximal signal tansduction machiney that lies downsteam of the immune eceptos, which includes lymphocyte cell specific tyosine kinase (LCK) and a potoonco, the tyosine kinase FYN 164, as well as the signalling adapto linke fo activation of T cells (LAT) 43. Futhemoe, seveal othe immune-associated s ae GPI-anchoed (suggesting that they ae pefeentially tageted to afts) and have been found in DRMs 172 ; these include CD14, the ecepto fo bacteial lipopolysaccha ides, and THY1 (also known as CD90), which is cucial fo T cell activation 173. Host pathogen inteactions. Inteest in lipid afts as modulatos of host pathogen inteactions has been boosted by the ecent discovey of a high level of satuated lipids (in paticula, sphingolipids) and cholesteol in the vial envelope (of HIV 174, fo example) and by finding odeed membane domains in pathogenic mico oganisms 175. Thee is now substantial evidence that viuses and bacteial poducts such as toxins bind pefe entially to detegent-esistant highly odeed plasma membane egions to penetate the cell. This could be due to the enichment of thei eceptos in afts, as is the case fo glycolipids 176 (which function as eceptos fo cholea toxin 90, fo example) o vius eceptos 177. Futhemoe, binding of HIV Gag (which is necessay fo vius budding and elease fom host cells) has been shown to occu pefeentially in membane domains with high cholesteol content 178, which suggests that afts might be the pefeed sites fo vius budding 178 (FIG. 5b). 370 JUNE 2017 VOLUME 18