Lipid Rafts As a MembraneOrganizing Principle Daniel Lingwood and Kai Simons Cell membranes display a tremendous complexity of lipids and proteins designed to perform the functions cells require To coordinate these functions the membrane is able to laterally segregate its constituents This capability is based on dynamic liquid liquid immiscibility and underlies the raft concept of membrane subcompartmentalization Lipid rafts are fluctuating nanoscale assemblies of sphingolipid cholesterol and proteins that can be stabilized to coalesce forming platforms that function in membrane signaling and trafficking Here we review the evidence for how this principle combines the potential for sphingolipid cholesterol self assembly with protein specificity to selectively focus membrane bioactivity he lipid raft hypothesis proposes that the lipid bilayer is not a structurally passive solvent but that the preferential association between sphingolipids sterols and specific proteins bestows cell membranes with lateral segregation potential The concept has long suffered assessment by indirect means leading to questions of fact or artifact 1 The resistance of sphingolipid cholesterol and a subclass of membrane proteins to cold detergent extraction 2 or mechanical disruption 3 has been widely used as an index for raft association with little or no regard for the artifacts induced by these methods Though the acquisition of resistance to disruption may point to physiologically relevant biases in lateral composition 4 this disruptive measure tells us little about native membrane organization Support from light microscopy was also missing because with the exception of organization into specialized membrane domains such as caveolae or microvilli putative raft components specifically glycosylphosphatidylinositol GPI anchored proteins fluorescent lipid analogs raft transmembrane TM domains and acylated proteins often show a homogeneous distribution at the cell surface 5 Moreover early investigations into submicron membrane organization often yielded conflicting evidence regarding the distribution or motion of these constituents in the living cell 1 Today however the advancement of technology has produced compelling data that self organization of lipids and proteins can induce subcompartmentalization to organize bioactivity of cell membranes T Origins of the Lipid Raft Concept Biochemically it is clear that lipids are sorted within the cell 6 This is particularly notable in polarized epithelia where glycosphingolipids GSLs are enriched at the apical surface 7 Lipid rafts were originally proposed as an exMax Planck Institute for Molecular Cell Biology and Genetics Pfotenhauerstrasse 108 01307 Dresden Germany To whom correspondence should be addressed E mail simons mpi cbg de 46 planation Self associative properties unique to sphingolipid and cholesterol in vitro could facilitate selective lateral segregation in the membrane plane and serve as a basis for lipid sorting in vivo 7 This proposal for compartmentalization by lipid rafts suggested a nonrandom membrane architecture specifically geared to organize functionality within the bilayer This function was initially thought to be membrane trafficking however rafts could influence organization of any membrane bioactivity Fig 1 Here we highlight advances in technology that point to the existence of raft based membrane heterogeneity in living cells and discuss the levels of preferential association underlying dynamic domain structure and biological function s Lipid Interactions in Model Membranes Assembly into two dimensional liquid crystalline biomembranes is a fascinating property characteristic of lipids Long thought to be incapable of coherent lateral structure 8 it is now apparent that principles of lipid self association can also confer organization beyond nonspecific measures of fluidity An important advance in modelmembrane systems was the discovery of phase separation in wholly liquid bilayers 9 10 It is a cholesterol dependent lateral segregation wherein the planarity molecular flatness of the rigid sterol ring favors interaction with straighter stiffer hydrocarbon chains of saturated lipids and disfavors interaction with the more bulky unsaturated lipid species 11 Interaction with cholesterol also forces neighboring hydrocarbon chains into more extended conformations increasing membrane thickness and promoting segregation further through hydrophobic mismatch 12 In purified lipid systems the combined effect is a physical segregation in the membrane plane A thicker liquid ordered Lo phase coexists with a thinner liquid disordered Ld phase 13 Sphingolipids tend to display longer and more saturated hydrocarbon chains thus potentiating interdigitation between leaflets 14 and favoring interaction with cholesterol Moreover unlike glycerophospholi 1 JANUARY 2010 VOL 327 SCIENCE pids the region of chemical linkage between the head group and sphingosine base contains both acceptors and donors of hydrogen bonds thus increasing associative potential both with cholesterol and other sphingolipids 11 Other explanations for cholesterol selectivity include the proposed umbrella effect in which cholesterol hydrophobicity is preferentially shielded by the strongly hydrated head groups of sphingolipid 15 or stoichiometric but reversible complex formation between cholesterol and sphingolipid or saturated glycerophospholipid 16 Immiscible liquid phase coexistence in vitro has been suggested as the physical principle underlying rafts in vivo 17 Of central importance is the demonstration of selectivity in association between certain lipids However phase separation in simple systems at thermodynamic equilibrium in vitro cannot be translated into proof for membrane domain formation in living cells 1 Instead model membrane work emphasizes the fact that certain lipids exhibit preferential association and provides a framework for understanding how heterogeneity in cell membranes may arise 18 In this respect the terms Lo and Ld should not be applied to the living cell as they refer only to the liquid ordered and liquid disordered phases of model membrane systems where parameters relating to translational order lateral diffusion and conformational order trans gauche ratio in the acyl chains can be accurately measured 11 Glimpses of Nano Assemblies in Living Cells Currently lipid rafts are viewed as dynamic nanoscale assemblies enriched in sphingolipid cholesterol and GPI anchored