ReviewInsolubility of lipids in Triton X-100: physical origin and relationship tosphingolipid/cholesterol membrane domains (rafts)Erwin Londona;b;*, Deborah A. BrownaaDepartment of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, NY 11794-5215, USAbDepartment of Chemistry, State University of New York at Stony Brook, Stony Brook, NY 11794-5215, USAReceived 11 April 2000; received in revised form 29 June 2000; accepted 29 June 2000AbstractThe insolubility of lipids in detergents is a useful method for probing the structure of biological membranes. Insolubility indetergents like Triton X-100 is observed in lipid bilayers that exist in physical states in which lipid packing is tight. The TritonX-100-insoluble lipid fraction obtained after detergent extraction of eukaryotic cells is composed of detergent-insolublemembranes rich in sphingolipids and cholesterol. These insoluble membranes appear to arise from sphingolipid- andcholesterol-rich membrane domains (rafts) in the tightly packed liquid ordered state. Because the degree of lipid insolubilitydepends on the stability of lipid-lipid interactions relative to lipid-detergent interactions, the quantitative relationshipbetween rafts and detergent-insoluble membranes is complex, and can depend on lipid composition, detergent andtemperature. Nevertheless, when used conservatively detergent insolubility is an invaluable tool for studying cellular raftsand characterizing their composition. ß 2000 Elsevier Science B.V. All rights reserved.Keywords: Sphingomyelin ; Dipalmitoylphosphatidylcholine; Liquid ordered state; Detergent resistant membrane1. IntroductionThe interaction of detergents with membranes wasactively explored 25^30 years ago, when the useful-ness of detergent solubilization for isolating andcharacterizing integral membrane proteins was ¢rstappreciated [1,2]. In recent years it has become ap-parent that detergent can solubilize membrane do-mains in di¡erent physical states di¡erentially. Thisproperty has provided signi¢cant support for theproposal that separate domains with di¡erent lipidand protein compositions can exist in cellular mem-branes [3,4]. These domains are important becauserecent work has shown that the a¤nity of certainproteins and lipids for speci¢c membrane domainshas important physiological consequences in process-es as diverse as cell surface signaling, cell adhesionand motility, and intracellular sorting [5,6]. In thisreview, we will summarize the e¡ects of lipid physicalstate upon detergent (speci¢cally Triton X-100)-membrane interactions, and explore the implicationsof these e¡ects for the domain organization of modeland cellular membranes.2. Physical states of lipid bilayers2.1. The liquid crystalline and gel statesLipid bilayers can exist in several physical states0005-2736 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved.PII: S0304-4157(00)00007-1* Corresponding author, at address a. Fax: (631) 6328575;E-mail: [email protected] 77966 10-11-00Biochimica et Biophysica Acta 1508 (2000) 182^195www.elsevier.com/locate/bba[7,8]. The `solid' gel (LL) and £uid, liquid crystallinestates (LK, Ld, Lc) are the two most familiar states.In the solid-like gel state lipids are tightly packed,with the acyl chains extended. There is little lateraldi¡usion. In contrast, in the LK state the acyl chainsare more kinked, packing is loose, and lateral motionis relatively rapid.2.2. Melting temperatureTm, the temperature of the gel to LK melting tran-sition for a pure lipid, is an important parameter inunderstanding lipid physical state [7,8]. It can bethought of as a measure of the stability of the tightlypacked gel state relative to the £uid state. The higherthe Tmvalue, the more stable the tightly packed gelphase. Tmdepends strongly on acyl chain structure.Saturated acyl chains impart a high Tmto lipids. Tmalso increases with chain length [9]. In contrast, un-saturated acyl chains, which have cis double bonds,interfere with tight packing, and they impart a rela-tively low Tmvalue to lipids. Sphingolipids (which,as we will see, are likely to be important in domainformation in biological membranes) tend to have rel-atively saturated chains and thus high Tmvalues[10,11]. In contrast, natural glycerophospholipidstend to be enriched in mono- or polyunsaturatedchains attached to the 2-position of the glycerol,and thus have relatively low Tm. (Some sphingolipidsdo contain a trans double bond in the sphingoidbase, but it is in a position close to the bilayer sur-face. Double bonds at this position do not greatlyinterfere with tight packing, and lipids containingsuch double bonds maintain a high Tm[11,12]. Beingin the trans state further reduces the e¡ect of thisdouble bond. Other sphingolipids have one doublebond located far down a very long chain fatty acid,which again would allow facile tight packing [13,14].)Another important factor a¡ecting Tmis polar headgroup structure. For example, sphingomyelin, whichhas a phosphocholine attached to the hydrophobicceramide core, tends to have a lower Tmthan glyco-sphingolipids, which have carbohydrates attached toceramide [11].2.3. The liquid ordered stateMore recent work suggests that lipids can exist inanother physical state that may be of biological sig-ni¢cance. This is the liquid ordered state (Lo state,also called Q state in early studies). The Lo state hasbeen identi¢ed in some mixtures of lipids and choles-terol in model membranes [15^20]. In the Lo state,acyl chains of lipids are extended and tightly packed.In this sense, the Lo state is similar to the gel state,and lipids that favor gel state formation, and thushave a high Tmin the absence of cholesterol, tend toform the Lo state in the presence of cholesterol [17].On the other hand, lateral di¡usion in the Lo stateappears to be almost as rapid as in the £uid LK state[21,22]. Thus, in a sense the Lo state has propertiesintermediate between gel and LK states.3. Interaction of the detergent Triton X-100 with lipid3.1. IntroductionThe polyoxyethylene detergent Triton X-100 is oneof the most widely used non-ionic detergents. WhenTriton X-100 micelles and lipid vesicles are mixed,they generally form mixed Triton-lipid aggregates.In excess Triton X-100, these aggregates often takethe form of mixed detergent/lipid micelles [23,24].Mixed micelles are much smaller than vesicles, andthus lipid incorporated into mixed micelles is