Lipid Rafts As a Membrane-Organizing PrincipleDaniel Lingwood and Kai Simons*Cell membranes display a tremendous complexity of lipids and proteins designed to perform thefunctions cells require. To coordinate these functions, the membrane is able to laterallysegregate its constituents. This capability is based on dynamic liquid-liquid immiscibility andunderlies the raft concept of membrane subcompartmentalization. Lipid rafts are fluctuatingnanoscale assemblies of sphingolipid, cholesterol, and proteins that can be stabilized to coalesce,forming platforms that function in membrane signaling and trafficking. Here we review theevidence for how this principle combines the potential for sphingolipid-cholesterol self-assemblywith protein specificity to selectively focus membrane bioactivity.The lipid raft hypothesis proposes that thelipid bilayer is not a structurally passivesolvent, but that the preferential associa-tion between sphingolipids, sterols, and specificproteins besto ws cell membr anes with lateral seg-regation potential. The concept has long sufferedassessment by indirect means, leading to questionsof fact or artifact (1). The resistance of sphingo-lipid, cholesterol, and a subclass of membraneproteins to cold detergent extraction (2)orme-chanical disruption (3) has been widely used asan index for raft association with little or no re-gard for the artifacts induced by these methods.Though the acquisition of resistance to disruptionmay point to physiologically relevant biases inlateral composition (4), this disruptive measuretells us little about native membrane organization.Support from light microscopy was also miss ingbecause, with the exception of organization intospecialized membrane domains such as caveolae ormicrovilli, putative raft components—specificallyglycosylphosphatidy linositol (GPI)–a nchored pro-teins, fluorescent lipid analogs, raft transmembrane(TM) domain s, and acylat ed protein s—often showa homogeneous distribution at the cell surface (5).Moreover, early investigations into submicronmembrane or ganization often yielded conflictingevidence regarding the distribution or motion ofthese constituents in the living cell (1). T oday, how-ever , the advancement of technology has producedcompellin g data that self-org anization of lipids andproteins can induce subcompartmentalization toorganize bioactivity of cell membranes.Origins of the Lipid Raft ConceptBiochemically, it is clear that lipids are sortedwithin the cell (6). This is particularly notablein polarized epithelia where glycosphingolipids(GSLs) are enriched at the apical surface (7).Lipid rafts were originally proposed as an ex-planation: Self-associative properties unique tosphingolipid and cholesterol in vitro could facili-tate selective lateral segregation in the membraneplane and serve as a basis for lipid sorting in vivo(7). This proposal for compartmentalization bylipid rafts suggested a nonrandom membrane ar-chitecture specifically geared to organize func-tionality within the bilayer. This function wasinitially thought to be membrane trafficking; how-ever , rafts could influence organization of anymembrane bioactivity (Fig. 1). Here, we highlightadvances in technology that point to the existenceof raft-based membrane heterogeneity in livingcells and discuss the levels of preferential asso-ciation underlying dynamic domain structure andbiological function(s).Lipid Interactions in Model MembranesAssembly into two-dimensional liquid crystallinebiomembranes is a fascinating property charac-teristic of lipids. Long thought to be incapable ofcoherent lateral structure (8), it is now apparentthat principles of lipid self-association can alsoconfer organization beyond nonspecific measuresof fluidity. An important advance in model-membrane systems was the discovery of phaseseparation in wholly liquid bilayers (9, 10). It is acholesterol-dependent lateral segregation, where-in the planarity (molecular flatness) of the rigidsterol ring favors interaction with straighter , stifferhydrocarbon chains of saturated lipids and dis-favors interaction with the more bulky unsatu-rated lipid spe ci e s (11). Interaction with cholesterolal s o for c e s neighboring hydrocarbon chains in t omore extended conformations, increasing mem-bran e thickness and promoting segregation furtherthrough hyd r o p h o b ic mism a t ch (12). In purifiedlipid systems, the combined effect is a physicalsegregation in the membrane plane: A thicker ,liquid-ordered, Lo phase coexists with a thinner ,liquid-disordered, Ld phase (13). Sphingolipidstend to display longer and more saturated hydro-carbon chains, thus potentiating interdigitationbetween leaflets (14) and favoring interaction withch oles t e r o l . Mor e over, unlike glycerophospholi-pids, the region of chemical linkage between thehead group and sphingosine base conta ins bothac ce p t o r s a n d do nors of hydrogen bonds, thusincreasing associative potential, both with cho-lesterol and other sphingolipids (11). Otherexplanations for cholesterol selectivity includethe proposed umbrella ef f ec t , in wh i c h chol e s t er o lhydrophobicity is preferentially shielded by thestrongly hydrated head groups of sphingolipid(15) or stoichiometric, but reversible, complexformation between cholesterol and sphingolipidor saturated glycerophospholipid (16).Immiscible liquid phase coexistence in vitrohas been suggested as the physical principle under-lying rafts in vivo (17). Of central importance isthe demonstration of selectivity in association be-tween certain lipids. However , phase separation insimple systems at thermodynamic equilibrium invitro cannot be translated into proof for mem-brane domain formation in living cells (1). Instead,model-membrane work emphasizes the fact thatcertain lipids exhibit preferential association andprovides a framework for understanding how het-erogeneity in cell membranes may arise (18). Inthis respect, the terms Lo and Ld should not beapplied to the living cell, as they refer only to theliquid-ordered and liquid-disordered phases ofmodel-membrane systems where parametersrelating to translational order (lateral diffusion)and conformational order (trans/gauche ratio inthe acyl chains) can be accurately measured (11).Glimpses of Nano-Assemblies in Living CellsCurrently, lipid rafts are viewed as dynamic nano-scale assemblies enriched in sphingolipid, choles-terol, and GPI-anchored proteins (19)(Fig.2A).To reach this viewpoint, membrane