Biophysical Journal Volume 84 March 2003 1827–1832 1827Real-Time Analysis of the Effects of Cholesterol on Lipid Raft BehaviorUsing Atomic Force MicroscopyJared C. Lawrence, David E. Saslowsky, J. Michael Edwardson, and Robert M. HendersonDepartment of Pharmacology, University of Cambri dge, Cambridge, United Ki ngdomABSTRACT Cholesterol plays a crucial role in cell membranes, and has been implicated in the assembly and maintenance ofsphingolipid-rich rafts. We have examined the cholesterol-dependence of model rafts (sphingomyelin-rich domains) insupported lipid monolayers and bilayers using atomic force microscopy. Sphingomyelin-rich domains were observed in lipidmonolayers in the absence and presence of cholesterol, except at high cholesterol concentrations, when separate domainswere suppressed. The effect of manipulating cholesterol levels on the behavior of these sphingomyelin-rich domains in bilayerswas observed in real time. Depletion of cholesterol resulted in dissolution of the model lipid rafts, whereas cholesterol additionresulted in an increased size of the sphingomyelin-rich domains and eventually the formation of a single raftlike lipid phase.Cholesterol colocalization with sphingomyelin-rich domains was confirmed using the sterol binding agent filipin.INTRODUCTIONCholesterol is an essential component of eukaryotic cellmembranes, and plays numerous roles in membrane function(Simons and Ikonen, 2000). Recently, it has been suggestedthat cholesterol is involved in the assembly and maintenanceof sphingolipid-rich microdomains or ‘‘rafts,’’ which areproposed to act as platforms for the preferential sorting ofproteins (Simons and Ikonen, 1997). The raft hypothesis isbased on the observation that detergent-resistant membranes,which are enriched in sphingolipids and cholesterol, canbe isolated using cold non-ionic detergents (Brown andLondon, 1998, 2000; Brown and Rose, 1992).Model membrane studies have shown that lipid-lipidinteractions are sufficient to induce the formation of raftlikedomains (Dietrich et al., 2001a; Saslowsky et al., 2002). Itis well established that phase separation can occur in bi-nary lipid mixtures consisting of lipids that have diff erentphase transition temperatures. Typically, a gel phase, whichis characterized by tightly-packed lipids that have limitedlateral mobility, co-exists with a fluid or liquid-disorderedphase in which the lipids are loosely packed and have a highdegree of lateral mobility. Addition of cholesterol has beenreported to modi fy the gel phase component of such systemsresulting in the so-called liquid-ordered phase in which thelipids are still tightly packed but acquire a relatively highdegree of lateral movement (Sankaram and Thompson,1990). Lipid rafts are proposed to exist in a state similar tothe liquid-ordered phase surrounded by a fluid lipid matrix.To investigate lipid raft characteristics in model mem-branes, sphingomyelin (SM) is commonly combined witha fluid-phase lipid, such as dioleoylphosphatidylcholine(DOPC), and cholesterol. SM lipids typically have long,saturated acyl chains that facilitate close packing, animportant feature of lipid raft organization (Ahmed et al.,1997; Brown and London, 2000). For this reason, SM-enriched domains in a bilayer are thicker than areas enrichedin more fluid, unsaturated lipids, which have kinked chainsthat effectively shorten the molecules. SM lipids also havesignificantly higher phase transition temperatures than phos-phocholine lipids (e.g., the transition temperature of brainSM is 37–418C). In addition, there is a favorable interac-tion between SM and cholesterol, and there is strong evi-dence that they are colocalized in cell membranes, wherecholesterol is thought to promote the formation and stabilityof lipid rafts (Simons and Ikonen, 2000; Slotte, 1999). TheSM/cholesterol interaction is most likely strengthened byhydrogen bonding betwee n the 39-OH group of cholesteroland the amide of the SM head group (Bittman et al., 1994).Currently, the requirement for cholesterol in raft formationis unclear. For instance, it has been reported that raft do-mains disappear after cholesterol depletion from the plasmamembrane in experiments using cultured cells (Cerneuset al., 1993; Ilangumaran and Hoessli, 1998). However,cholesterol-independe nt raft domains have also been re-ported in both model membranes (Milhiet et al., 2002;Saslowsky et al., 2002), and in the brush border membrane ofenterocytes (Hansen et al., 2001).In vitro studies of lipid raft behavior have mainly usedfluorescence microscopy to monitor the distribution of fluo-rescent raft markers (Dietrich et al., 2001a,b; Samsonov et al.,2001; Wang et al., 2000). However, recently, the directvisualization of raftlike domains in model membranes hasbeen achieved using atomic force microscopy (AFM; Milhietet al., 2001, 2002; Rinia et al., 2001; Saslowsky et al., 2002).AFM is a particularly suitable technique for studyingsupported lipid layers because of its ability to discriminateA˚ngstrom-scale height differences between lipid domains,and also to visualize surfaces under aqueous conditions(Dufreˆne et al., 1997). Previously, AFM has been usedto investigate model raft domains at a number of fixedSubmitted August 14, 2002, and accepted for publication November 20,2002.Address correspondence to Dr. R. M. Henderson, Dept. of Pharmacology,University of Cambridge, Tennis Court Road, CB2 1PD, UK. Tel.: 44-1223-334033; Fax: 44-1223-334040; E-mail: [email protected].Ó 2003 by the Biophysical Society0006-3495/03/03/1827/06 $2.00cholesterol concentrations. These studies have provideduseful information about the effect of cholesterol on raftthickness and area. In the present study, we have used AFMto analyze in real time the effects of manipulating cholesterollevels in supported model membranes containing DOPC andSM. In addition, we have used the cholestero l binding agentfilipin, to reveal the lateral distribution of cholesterol insupported lipid bilayers containing model rafts.MATERIALS AND METHODSMaterials1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), brain sphingo-myelin (SM), and cholesterol (Avanti Polar Lipids, Birmingham, AL,USA) were used as received. Methyl-b-cyclodextrin (MbCD), water-soluble cholesterol (MbCD loaded with cholesterol) and filipin complex(minimum 75% filipin III) were purchased from Sigma (UK). Water wasobtained from a Millipore water purification system.Formation of supported