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Annu. Rev. Cell Dev. Biol. 2004. 20:839–66 doi: 10.1146/annurev.cellbio.20.010403.095451 c 2004 by Annual Reviews. All rights reserved Copyright First published online as a Review in Advance on July 2, 2004

MEMBRANE DOMAINS Sushmita Mukherjee and Frederick R. Maxfield Annu. Rev. Cell Dev. Biol. 2004.20:839-866. Downloaded from arjournals.annualreviews.org by University of Pittsburgh on 08/17/07. For personal use only.

Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021; email: [email protected]

Key Words lipid raft, liquid ordered domain, cholesterol ■ Abstract Considerable evidence shows that lateral inhomogeneities in lipid composition and physical properties exist in biological membranes. These membrane lipid domains are proposed to play important roles in processes such as signal transduction and membrane traffic. However, there is not at present an adequate description of the nature of these lipid domains in terms of their size, abundance, composition, or dynamics. We discuss the current analyses of the properties and function of membrane domains in cells and compare their properties with chemically simpler model membrane systems that can be understood in greater detail.

CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MODEL MEMBRANE SYSTEMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lipid Organization in the Membrane Bilayer and the Presence of Lipid Phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of Cholesterol in the Formation of Biologically Relevant Membrane Domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Many Features of Lipid Domains Can Be Demonstrated in Three Component Systems that Include Cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Importance of Phase Boundaries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DESCRIPTION AND ANALYSIS OF PLASMA MEMBRANE DOMAINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detergent Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Direct Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Indirect Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . REGULATION OF MEMBRANE MICRODOMAINS . . . . . . . . . . . . . . . . . . . . . . . . Cholesterol Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lipid Unsaturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Charge on Lipid Head Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Presence of Membrane Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytoskeletal and Extracellular Matrix Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . Organizing Domains in the Outer and Inner Leaflets of the Plasma Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functions of Microdomains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1081-0706/04/1115-0839$14.00

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DOMAINS IN INTRACELLULAR ORGANELLES . . . . . . . . . . . . . . . . . . . . . . . . . 857 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 860

Annu. Rev. Cell Dev. Biol. 2004.20:839-866. Downloaded from arjournals.annualreviews.org by University of Pittsburgh on 08/17/07. For personal use only.

INTRODUCTION Membrane microdomains have been a subject of renewed interest over the past several years because increasing evidence indicates that various types of microdomains play important roles in processes such as signal transduction and membrane traffic (Edidin 2003, Maxfield 2002, Simons & Toomre 2000). It is clear that changes in lipid composition can have dramatic effects on several signal transduction pathways. It is also clear that activation of some signal transduction processes alters the association of signaling molecules with certain types of lipids. Similarly, membrane events such as formation of coated vesicles, viral coat assembly, and several types of membrane traffic can be altered by reduction of cholesterol content. These observations are all highly consistent with a role for lipid organization in these processes. The premise in considering the role of microdomains in such processes is that there are regional differences in the composition and physical properties of the membrane bilayer and that these differences have functional consequences. For various reasons, which are discussed in this review, it has been difficult to provide direct, unequivocal evidence about characteristics such as the size, composition, and stability of membrane microdomains in biological systems, which leads to significant uncertainty about the properties of microdomains. We discuss the current evidence regarding the existence, properties, and functions of membrane microdomains, with an emphasis on the membranes of mammalian cells. The properties of a membrane bilayer arise from the collective effects of a large number of weak noncovalent interactions. These are difficult to study in chemically complex systems such as biological membranes. However, in simpler systems, behaviors such as phase transitions and phase separation can be seen unequivocally. Thus much of our understanding of the properties of membranes comes from studies of chemically defined model membrane systems, and we discuss some recent advances in studies of these systems that may help to understand the more complex biological membranes. From model membrane studies it is known that bilayers containing mostly saturated lipids with similar acyl chain lengths will form more highly ordered membranes than membranes containing mostly unsaturated acyl chains (Gennis 1989). An important observation is that in model membranes containing cholesterol, a lipid organization can be seen at some compositions that is tightly packed yet allows high rates of translational diffusion. This type of lipid organization, called a liquid-ordered (lo) phase, can coexist with a liquid-disordered (ld) phase, which is less tightly packed (Almeida et al. 1993, London 2002). Mammalian cell plasma membranes are composed of roughly 109 molecules, consisting of hundreds of chemically distinct lipid constituents as well as a large

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variety of membrane proteins. A fundamental question to be considered here is whether these complex biological membranes exhibit behaviors similar to phase separations, with distinct biophysical properties in small regions (i.e., microdomains) determined, in part, by their lipid constituents. Recent evidence indicates that a substantial fraction of the plasma membrane has characteristics similar to an lo phase, and it is unclear whether these lo membranes are mainly in small units (i.e., rafts) (Edidin 2003, Maxfield 2002, Simons & Toomre 2000). It is possible that the plasma membrane is kept at a composition that is close to transition points so that perturbations such as cross-linking of some components can have a large effect on local lipid organization (Ritchie et al. 2003). In mammalian cells, cholesterol content is one of the key regulators of membrane properties, and its concentration in various membranes is tightly regulated even as the external availability of cholesterol varies widely. Altering cholesterol levels would affect the formation of lo phases, but it would also be expected to affect general membrane properties such as permeability and stiffness, as well as the shape and size of domains, because cholesterol may be a membrane component that is enriched at domain boundaries (Feigenson & Buboltz 2001, Needham et al. 1988). We discuss current interpretations of the effects of changes in lipid composition on microdomains and other membrane properties. It might seem surprising that we do not have a more precise description of the organization of the membrane bilayer in living cells. There are, however, a number of reasons for this. The molecules that form the bilayer are highly dynamic at physiological temperatures, and most pairwise interactions between individual lipid molecules would likely persist for microseconds to milliseconds (Gennis 1989). Thus small membrane microdomains would be highly dynamic entities with individual molecular components rapidly exchanging in and out. Whereas distinct micrometer-scale phases can be seen in model membranes using fluorescent lipid analogs, such separations are usually not observed in cell membranes. This indicates that the distinct domains in living cells either must be small or do not provide enough compositional variation to be observable within the resolution limits of microscopy. Electron microscopy has not yet been useful in these studies because of difficulties in devising nonperturbing labeling methods and in preserving lipid organization during sample preparation (Chatterjee & Mayor 2001). Methods such as atomic force microscopy and near field scanning optical microscopy have been useful in studying model membranes (Edidin 2001a, Tokumasu et al. 2003), but major technical hurdles need to be overcome before these techniques can be meaningfully applied to cell membranes in living cells. Because of these limitations, our understanding of membrane microdomains is largely inferential on the basis of comparisons with model systems and of biophysical methods that are sensitive to changes in the local environment on the nanometer distance scale. Unfortunately, extensive interpretation of these experimental data is often required in order to relate this information to the organization of membranes in a living cell.

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MODEL MEMBRANE SYSTEMS Model membranes with only a few components can provide valuable insights into the properties of more complex biological membranes. Various sophisticated biophysical tools can be employed to analyze model membrane systems both at the microscopic and molecular levels. Model membranes can be created in various forms. Liposomes are closed surface lipid bilayers similar to soap bubbles. They can be formed in various sizes, and giant (>10 µm diameter) unilamellar vesicles have been particularly useful for microscopy studies. Smaller liposomes, sometimes with an onion-skin multilamellar structure, are often used for methods such as nuclear magnetic resonance (NMR) and electron spin resonance (ESR). Bilayers can also be formed over small apertures with solvent on each side. Lipid monolayers can be supported on hydrophobic surfaces with the aqueous solvent above the monolayer.

Lipid Organization in the Membrane Bilayer and the Presence of Lipid Phases A single-component membrane bilayer can exhibit various types of long-range organization, termed phases, under various physical conditions (Gennis 1989, Vaz 1995). The properties of the lipid bilayer can be influenced by solvent properties (e.g., salts, pH, ionic strength), temperature, and pressure. An alteration in any of these parameters can result in a change of phase for the bilayer. At low temperatures, the membrane bilayer is in a highly ordered gel phase, with the acyl chains stretched out (very few gauche conformations) and tightly packed, and with a small cross-sectional area per lipid. In the gel phase, compared with less-ordered phases, the membrane bilayer is thicker, more laterally compressed, and less permeable to water and solutes. As the temperature is raised, a phase transition temperature (Tm) is reached, and the acyl chains cooperatively melt. There is an increase in the number of gauche conformations, and the bilayer is now in the fluid, liquid crystalline, or liquid-disordered (ld) phase. In this phase, the acyl chains are mobile, the head groups are well hydrated, and the bilayer is thinner and less densely packed, allowing easier percolation of solutes into or through the bilayer. Somewhat more complex behavior is observed in a membrane containing two lipid species with different Tm values (Gennis 1989, Vaz 1995). If this bilayer is placed at a temperature between the two Tms, the lipid with the high Tm will have a propensity to be in the gel phase, whereas the other component will prefer to be in the ld phase. If the fraction of one of the lipid species in the membrane is very small, such that all molecules of this lipid can be accommodated within the matrix of the major component without significant perturbation, then the whole bilayer will exist in the phase preferred by the major component. If, however, the fraction of the minor component is now increased, a threshold will be reached at which there will be a cooperative phase separation, and coexisting lateral phases will result, one in the gel phase, the other in the ld phase. In these coexisting phases, some

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of the low-melting lipid will remain dissolved in the more ordered phase and vice versa. Thus phase separations do not imply a complete chemical separation of the component species, and individual molecules can exchange between the coexisting phases. A system with coexisting phases could be composed of juxtaposed bulk phases or a mixture of microscopic or submicroscopic domains of these phases, depending on the thermodynamic properties of the system. Phases are macroscopic entities reflecting bulk properties of a large ensemble of molecules. A phase is a thermodynamic concept, and a true phase can be adequately described only for a system at thermodynamic equilibrium. The cellular membrane is not at equilibrium, so the different domains in the cellular membranes may have properties similar to various thermodynamic phases but are themselves not true phases. At the molecular scale, a given lipid in a complex mixture may have a preference for certain types of immediate neighbors, which would be reflected in a local compositional difference extending for one or a few molecular diameters. These preferences might be strong and specific, which could cause the formation of transient molecular complexes with properties that differ from their constituent components (McConnell & Radhakrishnan 2003). Larger groupings, containing up to a few thousand molecules, are not large enough to be considered phases, but they can exhibit compositions and properties that differ from the surrounding lipids. Evidence for such submicroscopic domains can be found in studies of model membranes (Anderson & Jacobson 2002).

Role of Cholesterol in the Formation of Biologically Relevant Membrane Domains The two component systems described above illustrate the basic principles of domain formation in model membranes, but it is clear that biological membranes do not exist in the gel phase, except possibly in some specialized conditions. It is thus necessary to invoke fluid-fluid immiscibility, rather than gel-fluid immiscibility, to understand domain formation in biological membranes. Such fluid-fluid immiscibility is familiar, for example, as the immiscibility of oil and water. In the biological membrane, one type of fluid domain would probably be similar to the ld phase described above, and other domains could resemble the lo phase (London 2002). Coexisting lo and ld phases can be seen in model membranes composed of ternary lipid mixtures containing cholesterol and two lipids, one with a high Tm and one with a low Tm. Before we describe such ternary phase diagrams, it is worthwhile to consider how cholesterol interacts with membrane lipids at the molecular level. In contrast to amphipathic lipid molecules, cholesterol is almost entirely nonpolar, with only a single –OH group attached to the stiff, fused ring system. When incorporated into the bilayer, this very small head group is not enough to shield the nonpolar part of cholesterol from interfacial water, so cholesterol must use the head groups of the adjacent lipids as “umbrellas” to shield itself from interfacial water (Huang & Feigenson 1999). The main consequence of this is that it

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enhances cholesterol-lipid lateral interactions and precludes cholesterol-cholesterol interactions within the bilayer, which would increase cholesterol exposure to water. At low-cholesterol concentrations, cholesterol distribution can be essentially random (as long as two cholesterols are not next to each other). As the cholesterol concentration increases, fewer of the possible lateral distributions are consistent with keeping all cholesterol shielded from water. Above the solubility limit, the adjacent phospholipid head groups can no longer cover the cholesterol, and excess cholesterol precipitates out as cholesterol monohydrate crystals. Experiments show that if a phosphatidylethanolamine (PE) membrane contains >1:1 mol/mol cholesterol:PE, or a phosphatidylcholine (PC) membrane contains >2:1 mol/mol cholesterol:PC, then the excess cholesterol precipitates out (Huang et al. 1999). Note that PE has a smaller head group cross-sectional area than PC and is able to accommodate only one cholesterol molecule per PE, whereas PC can accommodate two molecules of cholesterol per PC. It is interesting that the outer leaflet of the plasma membranes of most mammalian cells contains a significant fraction of glycosphingolipids, where the head group cross-sectional area could be larger than a phospholipid, thus potentially providing an umbrella not only over a single cholesterol molecule but also over cholesterol multimers. In addition to the purely steric considerations described above, specific interactions of cholesterol and phospholipids, especially sphingomyelin, possibly through forming hydrogen-bonded condensed complexes, have been proposed to be another structural feature of cholesterol-containing membranes (McConnell & Radhakrishnan 2003). Whereas these complexes need not necessarily constitute a separate phase (or domain), they may, under certain conditions, coalesce and serve as a nucleating point for a high-cholesterol content domain. Similar complex formation between transmembrane proteins and a lipid shell has also been proposed to play an important role in initiating or stabilizing domains in the biological membrane (Anderson & Jacobson 2002).

Many Features of Lipid Domains Can Be Demonstrated in Three Component Systems that Include Cholesterol Most mammalian cell plasma membranes contain three classes of lipids: (a) glycerophospholipids (most often with one or both acyl chains unsaturated, containing one or more cis double bonds), (b) sphingolipids (containing a sphingosine backbone and typically a long, saturated acyl chain), and (c) cholesterol (London 2002). There is great diversity in the head groups and the acyl chains in both the glycerophospholipids and sphingolipid classes. Ternary lipid phase diagrams have been constructed by several groups (de Almeida et al. 2003, Feigenson & Buboltz 2001, Silvius et al. 1996). The components in most cases are a high-melting lipid, a low-melting lipid, and cholesterol. In a recent study (de Almeida et al. 2003), a mixture representing the most biologically abundant lipids from each class was used (palmitoylsphingomyelin (PSM)/palmitoyloleoylphosphatidylcholine (POPC)/cholesterol). Although there

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are differences in the phase diagrams obtained by various groups using different lipid mixtures, several common characteristics are of interest in understanding domain behavior in mammalian cell membranes. As an example, the phase diagram for the ternary mixture, saturated long chain dipalmitoylphosphatidylcholine (DPPC; C16:0)/saturated short chain dilauroylphosphatidylcholine (DLPC; C12:0)/cholesterol, is shown in Figure 1 (Feigenson & Buboltz 2001). This phase diagram was obtained at room temperature using several complementary techniques, including confocal fluorescence microscopy on giant unilamellar vesicles (GUVs), fluorescence resonance energy transfer (FRET) measurements, and Dipyrene-PC monomer/excimer ratios. The ternary phase diagram shows several interesting properties. As expected, the phase diagram contains several regions of varying phase compositions. At high DLPC and low DPPC/low cholesterol regimes (region A), a single DLPC ld phase exists that goes through a coexisting ld and DPPC-rich gel phase (region B) to a pure DPPCrich gel phase (region C), as the DPPC concentration increases while the cholesterol concentration remains low. An interesting type of phase emerges at relatively high DPPC concentrations, where the cholesterol concentration in the membrane is at an intermediate level (mole fraction between 0.16 and 0.25 in this system) (region D). This region consists of a single phase, which changes continuously from a gel phase at the region C/D boundary to a ld phase at the region D/E boundary. Within this region, ordered lipids are somewhat separated from disordered lipids, as seen by energy transfer experiments, with fluorescent lipid probes preferential for these types of lipid organization. A further increase in DLPC concentration leads to a pure lo phase (region E). Finally, at any DLPC/DPPC ratio, provided enough cholesterol is present (mole fraction of cholesterol between 0.25 and 0.66), the membrane exists in a lo phase (region F) but with properties different from the fluid-ordered phase observed at lower cholesterol and DPPC concentrations (region E). Analysis of this phase diagram shows that even with only three components in the membrane, several different types of lo domains can be produced. In biological membranes, different lipids and proteins will exhibit various partitioning preferences into different types of lo domains (Pike 2003). Furthermore, a membrane can undergo a continuous phase transition, such that the membrane may slowly evolve from one phase property to another as composition is altered, without the two phases separately coexisting at any time.

Importance of Phase Boundaries When a membrane contains coexisting domains, the boundary of each domain will have an energy that is proportional to its length. In the absence of additional variables, the domain will tend to acquire a circular shape to minimize its edge energy. The edge energy can also be described in terms of the line tension, which is the edge energy per unit length (Lipowsky 1993). Line tension can be significantly reduced by edge-active molecules that preferentially associate with domain edges

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(Lipowsky 1993, Sackmann & Feder 1995). (This is similar to the emulsification of an oil/water mixture by adding detergents/surfactants.) Cholesterol has been shown to localize in domain edges, at least in systems with coexisting solid and fluid domains (Gaub et al. 1986, McConnell et al. 1986). Thus it is possible that, in addition to forming cholesterol-rich domains, one of the functions of cholesterol in membranes could be reducing the line tension between ld and lo domains, thereby allowing the domains to be more highly intermixed. Evidence for this role for cholesterol is that in model membranes (Feigenson & Buboltz 2001) and in cells (Hao et al. 2001) reduction in cholesterol levels can lead to formation of micrometer-scale phase separations.

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In a planar bilayer with two liquid phases, the boundaries between domains will take a circular shape in order to minimize edge energy. However, the length of the edges can be reduced further if a domain buds out of the plane (Lipowsky 1993, Sackmann & Feder 1995). If the domain edge forms the neck of the bud, the domain edge will become shorter during the budding process. The propensity toward curvature will be determined by the balance between the resistance of the membrane to curvature and the line tension at the edge of the domain (Lipowsky 1993, Sackmann & Feder 1995). Such curvature would be facilitated if the two coexisting domains have opposite spontaneous curvature preferences, or if one of the domains has a curvature preference whereas the other prefers flat membranes. Many glycosphingolipids, which constitute a significant fraction of the outer leaflet lipids of mammalian cell plasma membranes, as well as cholesterol, have strong preference for curved membranes because of their shapes (see below). Thus if these lipids are enriched in a domain, they may facilitate biologically relevant membrane curvature. An elegant study (Baumgart et al. 2003) has provided experimental estimates of boundary tension between coexisting ld/lo domains in GUVs with a variety of lipid compositions and has experimentally validated the link between elasticity of domains and their boundary properties to the shape adopted by the membranes and the formation of particular domain patterns. In their studies, the authors find smaller amplitudes of thermally excited out-of-plane undulations for the lo domains, and, ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 1 Lowering of cholesterol has similar effects on the domain properties of model membranes [giant unilamellar vesicles (GUV)] and mammalian cells [Chinese hamster ovary (CHO) cells]. (a) Ternary phase diagram for DPPC/DLPC/cholesterol at 24◦ C. Each vertex represents a pure component. The numbers labeling the DLPCDPPC axis correspond to mole fraction of DPPC, whereas the numbers labeling the PC-cholesterol axes correspond to cholesterol mole fraction. (Reproduced by permission from Feigenson & Buboltz 2001.) (b) Composition dependence of phase behavior in GUVs of DPPC/DLPC/cholesterol at 24◦ C, visualized using confocal fluorescence microscopy. Fluid-preferring Bodipy-PC (green) and the more order-preferring DiIC20 (red) are incorporated into the vesicles at mole fractions ∼0.001. At cholesterol mole fractions above 0.16, no apparent phase separation exists (left). Lowering the cholesterol mole fraction to 0.08, at the same DPPC mole fraction, gives rise to microscopically distinguishable domains (right). Bars are 5 µm. (Reproduced by permission from Feigenson & Buboltz 2001.) (c) Effect of cholesterol modulation on the distribution of lipid analogs in the plasma membranes of living CHO cells. The images presented are single confocal sections near the bottom adherent surfaces of living cells. Cells labeled with C6-NBD-SM (green, fluid domain–preferring lipid analog) and DiIC16 (red, ordered domain–preferring analog) label the cells uniformly under normal growth conditions (c, a–c). Cells where the cholesterol content is lowered to 40% of the control levels (either by acute treatment with methyl-β-cyclodextrin, or by 3-day metabolic depletion), exhibit microscopically visible domains (c, d–f ). Bar 10 µm. (Reproduced by permission from Hao et al. 2001.)

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in the case of bud formation, the ld domains bend toward the lo domains in the neck region. In the presence of cone-shaped molecules such as cholesterol, steep local curvature gradients can cause lateral lipid redistribution and therefore locally modify the membrane’s mechanical properties. Also, in liposomes containing large curvatures, the ld phase is observed preferentially in the saddle shapes and the tips of tubular structures, whereas the lo phase segregates to the low curvature tubular parts. This is consistent with greater bending flexibility in the ld domains compared with that of the lo domains.

DESCRIPTION AND ANALYSIS OF PLASMA MEMBRANE DOMAINS Detergent Resistance The most common way in which components of functionally important domains or rafts are operationally identified in cellular systems is by the partitioning of these constituents into low-density fractions when the cells, or their isolated membranes, are extracted with cold nonionic detergents (typically, 1% Triton X-100 at 4◦ C) and then floated on sucrose density gradients (Brown & London 2000, Brown & Rose 1992). These domains have been variously referred to as rafts, detergentresistant membranes (DRMs), detergent-insoluble glyocolipid fractions (DIGs), or glycolipid-enriched membranes (GEMs). Lipid bilayers in tightly packed configurations (e.g., in the lo state) are less susceptible to solubilization by low amounts of cold nonionic detergents, presumably because the tight packing reduces access to the hydrophobic core by the detergent molecules (Brown & London 2000, London 2002). Sphingolipids and glycosyl phosphatidylinositol (GPI)-anchored proteins (whose acyl chains are long and saturated), as well as cholesterol, are enriched in the cold detergent–insoluble fractions (Brown & London 2000, London 2002). Depletion of cholesterol or sphingomyelin from biological membranes causes a reduction in detergent insolubility, which is consistent with the idea that lo-like lipid organizations are responsible for the DRMs in cell membranes (Brown & London 2000, London 2002). The inclusion of a lipid in a DRM depends both on the properties of the lipid itself and on the surrounding membrane. In model membranes, when low levels of raft lipids such as DPPC were included in ld membranes, they were solubilized along with the rest of the bilayer (London 2002). One problem with using this method to analyze lipid organization in cells is that lipid reorganization occurs during solubilization, so the detergent-resistant membrane structures differ from the membranes prior to detergent extraction (Edidin 2003). This can be seen directly by treating cells with cold Triton X-100 on a microscope stage (Hao et al. 2001, Mayor & Maxfield 1995). If fluorescent lipids with differing preferences for ordered and disordered domains are used to label the cells, they are almost completely intermixed prior to detergent treatment. After exposure to the cold Triton X-100, the lipid analogs with a preference for disordered BASIS (ORDER AND CHEMICAL COMPOSITION)

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domains are completely extracted; the residual membrane retains the analogs with a preference for ordered domains and covers about 70–80% of the original area of the cell, and holes are seen in the membrane that are often a few micrometers in diameter. This indicates that the more detergent-resistant membrane components coalesced to maintain a bilayer as other components were extracted. A recent study using NMR and three different types of calorimetric measurements showed that cold Triton X-100 could induce domain formation in otherwise miscible lipid bilayers (Heerklotz 2002), which suggests that, in some cases, the extraction procedure could cause domain formation rather than simply providing an assay for pre-existing domains. The use of other nonionic detergents, such as Lubrol, can result in different sets of nonextracted proteins and lipids (Drobnik et al. 2002, Slimane et al. 2003), which may indicate that different sets of proteins exist in various types of membrane organizations in the untreated membranes. Given the uncertainties about the interpretation of resistance to cold Triton X-100, it seems prudent to be cautious about equating this detergent resistance with specialized membrane domains and to look for confirmation of this hypothesis using other complementary techniques (Pike 2003). In its initial descriptions, the raft hypothesis considered that rafts were a minor component of the plasma membrane, which was mostly ld-like. This was based, in part, on the observation that the large majority of membrane proteins were solubilized by cold Triton X-100. However, in terms of the lipids, the majority of lipids in the outer leaflet of the plasma membrane have a preference for the lo phase, so from a lipid viewpoint, most of the plasma membrane might be in an lo-like organization. There is increasing evidence that a high fraction, and perhaps the majority, of the plasma membrane is in an lo-like lipid organization (Gidwani et al. 2001, Hao et al. 2001).

EXTENT: HOW MUCH OF THE PLASMA MEMBRANE IS DETERGENT-RESISTANT?

Direct Observation Both widefield and confocal laser scanning microscopy show that under normal growth conditions, mammalian cells show no distinguishable domains when their plasma membranes are labeled with lipid analogs or proteins that would preferentially partition into an ld- or an lo-type domain (Hao et al. 2001, Harder et al. 1998). As noted above, extraction with cold Triton X100 leaves about 70–80% of the surface of the cell covered by detergent-resistant membrane components, indicating that the majority of the plasma membrane is raft-like by this criterion. It should be noted, however, that this extraction is at 4◦ C, and may not reflect the situation at 37◦ C. Although discrete lipid domains are not observable by optical microscopy under normal conditions, they can be induced under various conditions. As discussed above, microscopically observable domain separation is often lost in the three component membrane systems as cholesterol levels increase. The cholesterol content of the plasma membrane is above this threshold for observing discrete domains. When cholesterol is reduced in living cells, separation of domains labeled

OPTICAL MICROSCOPY

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with DiI-C16 or DiI-C18 (ordered domain markers) from domains containing C6-NDB-SM (disordered domain marker) can be seen (Hao et al. 2001). The DiI-C16-labeled domains cover the majority of the cell surface, which is again consistent with a high fraction of the plasma membrane being in an ordered phase. This domain separation was observed at 33–34◦ C. Cross-linking of raft-associated components can also lead to the formation of observable domains in the plasma membrane (Harder et al. 1998, Mayor et al. 1994). For example, when the cell surface distribution of a GPI-anchored protein (the human folate receptor type α) is probed with fluorescently labeled folate or monovalent antibody, the protein is seen to label the plasma membrane uniformly. However, when the same receptors are cross-linked with secondary antibodies, they segregate into microscopically resolvable domains. If another GPI-anchored protein is cross-linked at the same time, the two GPI-anchored proteins will cocluster, indicating that there is a preference for occupying the same domains (Mayor et al. 1994). When the IgE receptor, FcεRI, is cross-linked on RBL cells at 4◦ C, visible patches of DiI-C16 can be seen to codistribute with the FcεRI patches (Pierini et al. 1996, Thomas et al. 1994). Similar results have been obtained in copatching experiments in BHK cells transfected with a variety of proteins (Harder et al. 1998). Several GPI-anchored proteins and transmembrane proteins with known raft partitioning preference (e.g., influenza HA protein) copatch on the cell surface, but patched nonraft proteins such as the transferrin receptor are excluded from these clusters (Harder et al. 1998). These observations show that cross-linking raft components can create larger domains capable of including or excluding other proteins and lipids, on the basis, at least in part, of membrane organization preferences. One optical technique that has contributed significantly to both the detection of domains and the estimation of their size and stability is single-particle tracking (SPT) (Ritchie et al. 2003). A membrane protein or lipid is tagged with a small particle (40–200 nm), and the trajectories of these particles are followed by optical microscopy, with 10–20 nm precision. Molecules on the plasma membrane, instead of exhibiting a simple Brownian random walk, appear to undergo random walk in a transient confinement zone, with infrequent transitions (hops) to neighboring compartments. Using very fast cameras, it was found that lipids can be confined for an average of 11 ms in 230-nm compartments (Fujiwara et al. 2002). At longer times, confinements in larger structures with confinement times of approximately seconds have also been seen (Dietrich et al. 2002, Fujiwara et al. 2002). Confinement of lipids in these compartments is poorly correlated with cholesterol content of membranes or with the order preferences of the lipids being tracked, indicating that this confinement is not solely from inclusion into or exclusion from lipid rafts.

SINGLE-PARTICLE TRACKING

Indirect Methods If lipid microdomains are 100 nm or smaller in size, techniques with spatial resolution on this scale are required.

FLUORESCENCE RESONANCE ENERGY TRANSFER

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FRET, which is sensitive to the distance between fluorophores in the 3–10-nm range, is one applicable method. In a study using a FRET technique, which measured fluorescence depolarization resulting from fluorescein:fluorescein energy transfer, a constant fraction of the GPI-anchored human folate receptors was found to be within energy transfer range of each other (i.e., within 10 nm) on the plasma membranes of CHO fibroblasts, independent of the plasma membrane concentration of the folate receptors over a greater than 10-fold range (Varma & Mayor 1998). However, when FRET between Cy3- and Cy5-labeled antibodies was measured in Madin-Darby canine kidney cells expressing GPI-anchored 5 nucleotidase, no evidence for clustering was found (Kenworthy & Edidin 1998). This latter study may not have been sensitive to less than 20% clustering, which is approximately the amount later estimated to be present in the CHO cells (Edidin 2001b). The motion of lipid molecules in bilayers occurs on a time scale that can be observed by fluorescence anisotropy and ESR. Various types of motions such as rotations and wagging motions can be detected because these motions affect the ESR spectrum of nitroxide-labeled lipids. Using a variety of labeled lipids, it was found that vesicles isolated from the plasma membrane show two types of lipid behavior. The major component corresponds most closely to the behavior of tightly packed lipids similar to lo domains, whereas the minor component is more like that of ld domains (Ge et al. 2003). Similar results have been obtained from fluorescence anisotropy measurements of 2-[3-(diphenylhexatrienyl)propanoyl]-1hexadecanoyl-sn-glycero-3-phosphocholine (DPH-PC) incorporated into the plasma membrane. By comparison with measurements in model membranes, it was estimated that about 40% of the lipid probe was incorporated into ordered domains at 37◦ C (Gidwani et al. 2001). Estimates of the fraction of lipids in an ordered domain in the plasma membrane varies depending on the method of measurement. This may reflect variables such as the differential partitioning of various probes into different types of domains or the influence of other factors such as interactions with the cytoskeleton. Nevertheless, all the methods indicate that the fraction of lipids in the ordered domain is at least 30% and perhaps more than 50%, even at physiological temperatures (Gidwani et al. 2001, Hao et al. 2001, Mayor 1999).

ELECTRON SPIN RESONANCE AND FLUORESCENCE ANISOTROPY

REGULATION OF MEMBRANE MICRODOMAINS Cholesterol Levels Cholesterol is a major lipid component of the plasma membrane, with estimates of its abundance in the range of 30–40% of the phospholipids on a molar basis (Yeagle 1985). In model membranes, cholesterol is essential for the formation of lo phases in association with other lipids. The effects of cholesterol on membrane organization arise from its unique chemistry, with its stiff fused ring system and its very small

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head group, which affects its packing with other lipids and proteins. Cholesterol could participate in the formation of lo domains through specific interactions with the saturated acyl chains of lipids or with sphingolipid head groups (Brown 1998, McConnell & Radhakrishnan 2003). Alternatively, on the basis of nonspecific effects such as exclusion from the ld phase owing to packing mismatch, cholesterol could play a role in domain formation (Ritchie et al. 2003). The transbilayer distribution of cholesterol and several aspects of its intracellular transport remain active areas of investigation (Maxfield & Wüstner 2002). Recent studies indicate that cholesterol can exchange between membrane leaflets within seconds (Steck et al. 2002) and that cholesterol can exchange among membrane organelles by vesicular and nonvesicular mechanisms within a few minutes (Hao et al. 2002). It is important to note that cholesterol, by itself, does not partition very strongly into the lo domains. A recent study (Veatch & Keller 2003) showed that in model membranes composed of 1:1 DOPC/DPPC + 30% cholesterol, ∼80% of the DPPC was found in ordered phase. In contrast, only ∼66% of the cholesterol resided in the ordered phase.

Lipid Unsaturation In addition to cholesterol, the acyl chain composition of the lipids in the membrane is the major determinant of domain segregation. As discussed earlier, cholesterol orders a fluid bilayer by restricting the number of gauche conformations the acyl chains can assume. This ordering is essential, according to the umbrella model (discussed above), for the cholesterol molecules to be covered by the adjacent lipid head groups. It is clear that it is easier to reduce the degrees of freedom for gauche conformations of saturated chains than to pack a stiff cholesterol ring next to a cis double bond of an unsaturated lipid. NMR, ESR, and diffraction techniques have revealed that in fluid bilayers the stiff fused ring of cholesterol extends beyond carbon 9 position in the adjacent lipid (Gennis 1989). Because C9:C10 is the most common double-bond position in mono- and poly-unsaturated acyl chains of lipids in mammalian cells, cholesterol would not pack favorably in a domain that is enriched in unsaturated acyl chains.

Charge on Lipid Head Groups Charge on lipid head groups plays an especially important role in recruiting peripheral membrane proteins to raft domains on the inner leaflet of the plasma membrane, which is relatively enriched in negatively charged lipids such as phosphatidylserine (McLaughlin & Aderem 1995, McLaughlin et al. 2002). Both hydrophobic interactions of the acyl chains with the membrane interior, as well as electrostatic interaction of basic amino acids on the lipid-binding sites of membrane proteins with acidic phospholipid head groups, are essential for the partitioning of Src-family kinases to raft domains (Deschenes et al. 1990, McLaughlin & Aderem 1995, McLaughlin et al. 2002). Also, signaling events produce highly charged lipids at the inner leaflet, possibly in locally high concentrations (e.g.,

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polyphosphorylated phosphatidylinositols), which might recruit other proteins at these sites and/or generate local curvature leading to budding or fusion.


Presence of Membrane Proteins Membrane proteins add significant complexity to domain organization and can contribute in several ways to domain segregation. Most transmembrane proteins are excluded from lipid rafts (Simons & Ikonen 1997), possibly because their incorporation into such domains would perturb local order of the lo domains. In contrast, some membrane proteins preferentially partition into the raft domains, either because they are anchored into the membranes via a lipid anchor that prefers to partition into ordered domains (Deschenes et al. 1990, Simons & Ikonen 1997) or because they have a transmembrane region that specifically binds lipids that are enriched in the rafts (e.g., Na+K+-ATPase and the influenza HA protein) (Anderson & Jacobson 2002). Boundary lipids associated with transmembrane proteins may promote association with certain types of membrane domains. Even though individual boundary lipid molecules exchange rapidly, a preference for lipids of a certain length or shape may facilitate association with different types of lipid organization. Membrane proteins may create or stabilize domain boundaries (e.g., by being anchored to the underlying membrane skeleton) (Ritchie et al. 2003). In many cases, cross-linking of some proteins to create oligomers significantly enhances their association with raft domains (Holowka & Baird 2001). In addition, such cross-linking may cause small microdomains to coalesce into larger structures, which could more effectively separate proteins into distinct domains.

Cytoskeletal and Extracellular Matrix Interactions Many membrane proteins are linked to the cytoskeleton, and this can influence the properties of the bilayer, such as the generation of the transient confinement zones or picket fences seen in SPT studies (Ritchie et al. 2003). Whereas most SPT studies have focused on monomeric lipid or protein molecules, such barriers would be expected to more severely restrict the motion of cross-linked proteins or lipid microdomains and result in their exclusion from or inclusion into large coalesced domains. The extracellular matrix may play a role analogous to the cytoskeletal network, especially for motile or weakly adherent cells, as well as for adherent cells during cell spreading (Gomez-Mouton et al. 2001, Miceli et al. 2001).

Organizing Domains in the Outer and Inner Leaflets of the Plasma Membrane There is experimental evidence for some codistribution of membrane domains in the external and cytoplasmic leaflets (Pyenta et al. 2001), but it is not clear how the two membrane leaflets interact to generate synchronous domains. The two leaflets have different lipid compositions and a different profile of additional influences (membrane-bound cytoskeleton and peripheral membrane proteins, versus the

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extracellular matrix, and signal dependent conformation change or oligomerization of receptors). Although model lipid systems with a composition similar to the outer leaflet of the mammalian plasma membrane exhibit domain segregation (Silvius 2003), such segregation does not happen for a lipid mixture mimicking the inner leaflet, even in the presence of significant amounts of cholesterol (Wang & Silvius 2001). Several hypotheses have been put forth regarding possible ways in which the two leaflets could be interconnected (Edidin 2003). The most obvious would be via transmembrane proteins, where a conformation change or oligomerization of the transmembrane protein through ligand binding on the outer leaflet would be transmitted to the inner leaflet. This would not explain the effects of cross-linking GPI-anchored proteins unless every GPI-anchored protein had a transmembrane protein associated with it. In model membranes with coexisting ordered and disordered domains, the regions of order codistribute in both leaflets, so there must be effective trans-bilayer coupling in the absence of any proteins (Korlach et al. 1999) that could be the result of partial interdigitation of the two leaflets.

Functions of Microdomains Although we are only now starting to understand membrane microdomains, several types of structurally and/or functionally specialized microdomains have long been known to exist in mammalian cells. Caveolae are typical examples of structurally defined domains. Several types of signaling domains have also been described, although their structural characteristics often remain uncertain. Caveolae are one of the best-characterized types of microdomains (Kurzchalia & Parton 1999, Minshall et al. 2003). These structures are associated with the plasma membrane and with internal membranes of many cell types, and they are abundant in some cell types such as lung endothelial cells. A single caveola forms a flask-shaped membrane invagination that is about 55 nm in diameter. In many cases, caveolae are joined together in clusters that resemble a bunch of grapes and can penetrate several micrometers into a cell while maintaining open contact with the surface. Under most circumstances, plasma membrane caveolae appear to be relatively stable structures that do not pinch off, but they can be induced to pinch off by stimuli such as interaction with SV40 virus particles (Pelkmans et al. 2002). Caveolae are associated on their cytoplasmic surface with caveolins-1 and -2, which partially penetrate into the membrane, but caveolins are not transmembrane proteins. Caveolae have been isolated and analyzed biochemically (Kurzchalia & Parton 1999, Minshall et al. 2003). They are rich in sphingomyelin, glycosphingolipids, and cholesterol; they are resistant to solubilization at low temperatures by nonionic detergents such as TritonX-100 and float on sucrose density gradients with the low-density fractions. GPI-anchored proteins can be recruited into caveolae by incubation with two layers of antibody, indicating that these proteins have a weak affinity for caveolae that is enhanced upon cross-linking (Mayor et al. 1994).

CAVEOLAE

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Because caveolae cover but a few percent of the surface of most cell types, they can be only a small fraction of the DRMs or raft-like membranes. A large number of functions have been attributed to caveolae. In many early studies the distinction between caveolae and other DRMs was not made, so it is likely that most of those functions are not specifically associated with caveolae. The availability of knockout mice that lack expression of caveolin-1 and caveolin-2 provide a good system for further exploration of the actual roles of caveolae (Drab et al. 2001, Razani et al. 2002). The fact that the knockout mice survive to adulthood suggests that caveolae are not essential for a large number of cellular processes, but alterations have been observed in nitric oxide signaling, lipid metabolism, and glucose regulation in these knockout mice (Sotgia et al. 2002). One of the most important proposed functions for lipid microdomains is the regulation of signal transduction processes (Baird et al. 1999, Resh 1999, Ritchie et al. 2003). There is abundant evidence suggesting that lipid organization can play a critical role in signal transduction, but in most cases the function of lipid domains remains to be characterized in detail. Most models describing the role of lipid domains in signaling have been based on the idea that recruitment to a specific type of domain will increase the local concentration. The type of domain considered in these studies is usually a raft-like or lo domain (Ritchie et al. 2003). An example would be recruitment of Src-family kinases that are myristoylated and palmitoylated so that they have a preference for association with ordered lipid domains. Recruitment of these molecules into signal transduction complexes could result in enhanced cross-phosphorylation and activation, which would lead to significant signal amplification. However, if a large fraction of the plasma membrane is made up of raft-like domains, the degree of enrichment by recruitment to these domains would be relatively small. Once again, the inability to directly observe lipid domains and recruitment of molecules into them has required interpretation of indirect evidence about the role of lipid domains. There are essentially two types of evidence that support a role for recruitment to raft-like domains as part of an activation process: increased amounts of signaling proteins in DRMs and the effects of lipid modifications on signaling (Baird et al. 1999, Resh 1999, Ritchie et al. 2003). Both observations provide strong evidence for a role for lipid organization in signaling, but they do not provide a precise description of how lipids affect signaling. The easiest lipid component to modulate is the cholesterol content of membranes. This can be done by metabolic depletion (i.e., incubating cells in low cholesterol medium in the presence of an inhibitor of HMG-CoA reductase), which blocks cholesterol synthesis. Cholesterol can also be removed effectively and rapidly from membranes by incubation with certain cyclodextrins, such as methyl-β-cyclodextrin, which have some selectivity for removing cholesterol from membranes. Because cyclodextrins can also remove other lipid components from membranes, it is useful to check that any effects can be reversed by using cholesterol-loaded cyclodextrin carrier to deliver cholesterol back to cells. Cholesterol depletion does affect many signaling pathways, which is consistent with lipid

SIGNALING DOMAINS

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organization being important for these processes (Baird et al. 1999, Resh 1999, Ritchie et al. 2003). For some of these processes it is possible to associate the effects of cholesterol depletion with the association of signaling molecules with raft-like lo domains. For example, Src-family kinases that are myristoylated and palmitoylated do not associate with DRMs after cholesterol depletion, and many signaling pathways involving these kinases are blocked by cholesterol depletion (Baird et al. 1999, Resh 1999, Ritchie et al. 2003). However, precisely how these observations are linked is not certain. Clustering of IgE-FcεRI complexes on rat basophilic leukemia (RBL) cell plasma membranes upon the addition of multivalent antigens has been used in many studies of the role of raft-like membranes in signaling (Holowka & Baird 2001). Cross-linking causes FcεRI to become associated with DRMs, and cholesterol depletion interferes with recruitment to DRMs and activation of signaling through cross-linked IgE-FcεRI complexes. The cross-linking of raft-associated FcεRI could induce coalescence of small, transient raft-like domains into larger domains (Holowka & Baird 2001). This could bring FcεRI into contact with the Src-family kinase, Lyn, which is raft-associated because of its acylation. However, as noted above, the enrichment through recruitment to rafts results in only a small increase in the concentration of FcεRI in proximity to Lyn. Furthermore, there is not a measurable overall increase in activation of Lyn caused by cross-linking the receptors (Young et al. 2003). The Lyn that is associated with DRMs has higher kinase activity (and higher Tyr phosphorylation in the active site loop of the kinase domain) compared with the Lyn that is not in the DRM fractions, but this again is true in both the stimulated and the unstimulated cells. One plausible explanation for this is that Lyn and FcεRI in the larger raft-like domains caused by cross-linking receptors are protected from phosphatases related to CD45, which have a preference for nonraft membranes (Young et al. 2003). This model remains somewhat speculative at present, but it is an interesting alternative to the more prevalent models in which activation is solely dependent on recruitment of positive signal regulators into rafts. Here, it is the exclusion of some proteins from raft-like membranes that is actually the key to increased signal transduction. Increasing evidence indicates that lipid domains are associated with elements of the underlying cytoskeleton (Pierini & Maxfield 2001). Cross-linked IgE-FcεRI complexes initially colocalize with GM1 patches labeled with multivalent fluorescent cholera toxin subunit B, but within ∼10 min, the two types of patches segregate from each other (Stauffer & Meyer 1997). Cytochalasin D, an actin polymerization inhibitor, prolongs the overlap of the two types of cross-linked rafts (Holowka et al. 2000). This sustained association also sustains Lyn-mediated FcεRI tyrosine phosphorylation (Holowka et al. 2000). When these cells are extracted with cold Triton X-100, in the absence of cytochalasin D, the FcεRI-carrying fraction moves to a higher density than the GM1-cholera toxin-carrying fraction, although both are still low enough in their density to qualify as rafts. It thus appears that raft-associated receptors can segregate from other raft components and that the actin cytoskeleton plays an important role in this process (Holowka et al. 2000,

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Raucher et al. 2000). In polarized neutrophils (Pierini et al. 2003, Seveau et al. 2001) and lymphocytes (Gomez-Mouton et al. 2001), the distribution of lipid domains is closely associated with the organization of the actin cytoskeleton. In neutrophils, DRMs are preferentially found over the cortical actin cytoskeleton at the rear of the cell, whereas membranes associated with the protruding leading edge are solubilized by cold Triton X-100 (Pierini et al. 2003, Seveau et al. 2001). In polarized T-lymphocytes, GM3 is enriched at the leading edge, and GM1 is enriched toward the rear (Gomez-Mouton et al. 2001). Another family of signaling proteins, the small GTPases, are membrane anchored by isoprenyl modifications (sometimes also with an acyl chain anchor). These isoprenyl units are highly unsaturated and would not be expected to fit well in ordered raft-like domains. Several studies indicate that molecules with these anchors are not associated with DRMs (Melkonian et al. 1999, Pyenta et al. 2001, Wang et al. 2000), but other studies report that these molecules are included in DRMs (Hekman et al. 2002). Of course, some of these proteins might associate with DRMs as a consequence of protein:protein interactions that override the effects of the ispoprenyl group. In model membranes, proteins with isoprenyl groups do not associate well with lo lipids (Wang et al. 2000). Although the Rho-family GTPases are generally not associated with lo-like domains, their signaling can be abrogated by cholesterol depletion (Garred et al. 2001, Lacalle et al. 2002, Pierini et al. 2003). This is another illustration that cholesterol depletion has complex effects on membrane organization. It is worth noting that a large fraction of cholesterol is in fact in the ld-like membrane domains, which coexist with cholesterol-enriched raft-like membranes in the plasma membrane. Ras-membrane interactions provide another interesting example of the role of microdomain distribution in signaling. H-Ras and K-Ras have different membrane anchoring moieties that can direct them to different nonraft membrane compartments (Niv et al. 2002). A recent study using immunogold electron microscopy of plasma membrane sheets, coupled with spatial point pattern analysis (Prior et al. 2003), shows that inactive H-Ras is distributed between lipid rafts and a cholesterol-independent microdomain. Conversely, activated H-Ras and K-Ras reside predominantly in nonoverlapping, cholesterol-independent microdomains.

DOMAINS IN INTRACELLULAR ORGANELLES Considerable evidence points to lipid microdomains residing within intracellular organelles. Some of the earliest evidence supporting microdomains in biological membranes came from studies of the sorting of lipids and lipid-anchored proteins (Simons & Fuller 1985). It is difficult to envision, for example, the efficient sorting of GPI-anchored proteins to the apical domain of many polarized epithelial cells without efficient sorting of lipids as they exit the Golgi apparatus or endocytic recycling compartments. Every step of membrane traffic involves the budding of a vesicle or tubule from the donor organelle and then fusion of the vesicle or tubule with the target

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organelle. Certain types of lipid microdomains could be effectively included or excluded from forming vesicles or tubules, and this would result in lipid sorting. Various general mechanisms for sorting of lipids during vesicular membrane traffic processes can be envisioned. One mechanism is based on the shapes of the lipids. The sites of vesicle or tubule budding are often regions of high curvature, and the shape of lipids can affect their ability to be accommodated in regions of high curvature (see Figure 2) (Lipowsky 1993, Mukherjee et al. 1999, Sackmann & Feder 1995). For example, cone-shaped lipids (with a head group area smaller than the acyl chain area) might be accommodated in the cytoplasmic leaflet of the neck region of a vesicle bud, whereas inverted cone lipids (with a head group area larger than the acyl chain area) might be better accommodated in the lumenal leaflet of the neck region. This shape dependence is independent of whether a given lipid has a preference for lo or ld domains, but it could provide an efficient mechanism for lipid sorting. The lo and ld domains themselves have some curvature preferences, with lo domains favoring flat parts of the membrane and ld domains better able to accommodate curved regions (Baumgart et al. 2003). Membrane proteins can also influence the sorting of lipids in vesicle traffic. This could be accomplished by relatively specific interactions of transmembrane

Figure 2 Schematic representation of how lipid shapes affect their curvature preferences.

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domains with certain classes of lipids, but this may affect only a small fraction of the lipids because tightly bound lipids are generally found only in a single boundary layer around a transmembrane domain (Marsh & Horváth 1998). Longer-range effects could be accomplished by hydrophobic thickness matching between transmembrane proteins and their surrounding lipids (Sperotto & Mouritsen 1993). For example, it has been proposed that proteins of the trans Golgi could have an average transbilayer length that is shorter than that found for plasma membrane proteins (Bretscher & Munro 1993). Optimal matching with the lipids would similarly require smaller bilayer thickness for the lipids, either by using shorter acyl chains or by having greater unsaturation, which diminishes bilayer thickness. This type of hydrophobic matching could contribute to mutual sorting of some proteins and lipids. The energetic feasibility of such a bilayer-dependent sorting mechanism has been evaluated using a model of elastic bilayer deformation (Lundbæk et al. 2003). It was proposed that raft-preferring lipids are differentially sorted at several steps of membrane traffic (Gruenberg 2001, Mayor & Riezman 2004, Mukherjee & Maxfield 2000, Simons & Ikonen 1997). An early example of this idea was that cholesterol, sphingomyelin, and glycosphingolipids would be preferentially sorted to apically targeted vesicles upon exit from the Golgi apparatus in polarized epithelial cells (Simons & Fuller 1985). As such, this mechanism could account for the relative enrichment of these lipid components in the apical domains. Although the proposal is attractive, several problems have developed in regard to the simplest forms of this model. Cholesterol is mostly delivered to the plasma membrane, including apical membranes in polarized epithelia, by nonvesicular pathways that do not require passage through the Golgi (Wustner et al. 2002). Thus this essential component of lo domains is transported mainly to the apical membrane independently of any delivery of rafts. Although the sphingolipid concentration is higher in the apical membrane of polarized epithelia (van Meer & Lisman 2002), the surface area of the basolateral membrane is often larger, so a large fraction of sphingolipids exiting the Golgi must be directed to basolateral membranes. Nevertheless, the sorting efficiency of many GPI-anchored proteins for delivery to apical membranes is quite high and relatively independent of the protein portion (Rodriguez-Boulan & Powell 1992). This certainly indicates that the glycolipid portion of the GPI-anchor contains important sorting information. Reduction of cellular cholesterol affects GPI-anchored protein sorting upon exit from both the Golgi apparatus (Simons 1993) and the endocytic recycling compartment (ERC) (Mayor et al. 1998), and this is consistent with the idea that lipid organization plays an important role in this sorting. The mechanisms underlying this sorting remain to be determined. Raft-preferring lipids have also been proposed to use specialized pathways for endocytic removal from the plasma membrane, in addition to the well-characterized clathrin-coated pit pathway. For example, in lymphocytes the IL-2 receptor, which partitions to DRMs, enters cells by a clathrin- and caveolin-independent pathway but not by a RhoA-dependent pathway (Lamaze et al. 2001, Mayor & Riezman

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2004). It has also been proposed that a fraction of GPI-anchored proteins enter cells by a dynamin-independent Cdc42-regulated pathway (Sabharanjak et al. 2002). Many of these independent entry mechanisms deliver some of their contents to endosomes formed by the clathrin-dependent pathway. There is additional evidence for specialized lipid domains within intracellular organelles. One of the clearest examples is the internal membranes of late endosome multivesicular bodies (MVBs) (Kobayashi et al. 1999). Although originally described as internal vesicles, some of these internal membranes are in continuity with the limiting membrane of the organelle. These membranes are enriched in an unusual lipid, lyso-bisphosphatidic acid. In several lipid storage disorders, this membrane compartment expands enormously, and these internal membranes are the main site of accumulation of cholesterol and sphingolipids (Kobayashi et al. 1999, Pagano et al. 2000). It is interesting to note that different enzyme deficiencies, with differing primary effects on lipid metabolism, result in strikingly similar types of lipid accumulations in the MVBs (Pagano et al. 2000). This suggests that expansion of the pool of one lipid type leads to compensating accumulation of the other lipids that are needed to form the internal membranes of the MVBs. Further evidence for membrane domain separation in the endocytic trafficking pathways comes from studies of the fates of fluorescent lipid analogs that differ only in their hydrocarbon chains (Mukherjee et al. 1999). DiI-C16 (with two 16 carbon chains) is sorted efficiently to late endosomes, whereas DiI-C12 (with 12 carbons per chain) is transferred to the ERC. Either increasing the lipid unsaturation in a cell or lowering cholesterol levels causes the DiI-C16 to be redirected to the ERC, consistent with the idea that the efficient sorting is related to lipid organization in the endosomes. In nonpolarized cells such as fibroblasts, the ERC is highly enriched in cholesterol (Hao et al. 2002, Mukherjee et al. 1998). This organelle seems to be a much larger internal store of the cholesterol than the trans Gogli network or other organelles. Consistent with this fact, the ERC membrane is relatively resistant to extraction by cold TX-100, indicating that a large fraction of the lipids in the ERC are in lo-type lipid organizations (Hao et al. 2004). In contrast, the late endosomes are not particularly enriched in cholesterol, and their membranes are largely solubilized by cold TX-100 (Hao et al. 2004). The role of lipid organization in exit from the ER is uncertain. In yeast cells, ERderived vesicles that contain GPI-anchored proteins are distinct from the vesicles that carry most other proteins to the Golgi, but it in unclear whether this is related to the formation of rafts or the inclusion of GPI-anchored proteins in DRMs within the ER (Bagnat et al. 2000, Mayor & Riezman 2004).

CONCLUSIONS There is considerable evidence that lipid domains or lateral inhomogeneities exist in biological membranes and that they play important roles in processes such as signal transduction and membrane traffic. Nevertheless, the organization of the

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plasma membrane lipids is very complex and only partially understood. Progress will depend upon better analytical techniques for examining membrane composition and properties at high-spatial resolution, further studies using increasingly complex model membranes, and development of computational methods for simulating compositionally diverse membranes with large numbers of molecules over long time periods to allow approach to equilibrium. These are likely to be significant challenges for many years. In the absence of a satisfyingly complete description, metaphors are useful to help understand various characteristics of membrane organization. The original fluid-mosaic model (Singer & Nicolson 1972) largely envisioned the lipids as a two-dimensional fluid permeability barrier and support for proteins. The raft model (Simons & Ikonen 1997) has made a valuable contribution in focusing attention on lateral inhomogeneities in lipid composition and organization, which can affect and be affected by the proteins in the membrane. The raft metaphor, however, breaks down upon detailed examination because a large fraction of the plasma membrane is in the raft-like state, the components of microdomains exchange in and out rapidly, and there appear to be many different types of microdomains with varying characteristics. Unfortunately, it is not easy to replace the raft metaphor with a simple alternative because the properties that we wish to describe are based upon large numbers of relatively weak interactions. Such systems have proven to be challenging in other areas of investigation as well. In fact, adequate models of lipid organization in biological membranes may be a more complex problem than protein folding in a computer—a problem widely acknowledged as a challenge for the largest current computer systems. An interesting comparison can also be made between lipid domains and the mechanisms of cloud formation, an area that is beginning to yield to large computer simulations, but until recently has been in the realm of poets. ACKNOWLEDGMENTS We are grateful to Olaf Andersen for critical reading of the manuscript and to Gerry Feigenson for permission to reproduce Figure 1. Supported by grants from the W.M. Keck Foundation and the National Institutes of Health. The Annual Review of Cell and Developmental Biology is online at http://cellbio.annualreviews.org

LITERATURE CITED Almeida PF, Vaz WL, Thompson TE. 1993. Percolation and diffusion in threecomponent lipid bilayers: effect of cholesterol on an equimolar mixture of two

phosphatidylcholines. Biophys. J. 64:399– 412 Anderson RG, Jacobson K. 2002. A role for lipid shells in targeting proteins to caveolae,

10 Sep 2004 13:10


862

AR

AR226-CB20-30.tex

MUKHERJEE

AR226-CB20-30.sgm

LaTeX2e(2002/01/18)

P1: GCE

MAXFIELD

rafts, and other lipid domains. Science 296: 1821–25 Bagnat M, Keranen S, Shevchenko A, Simons K. 2000. Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast. Proc. Natl. Acad. Sci. USA 97:3254– 59 Baird B, Sheets ED, Holowka D. 1999. How does the plasma membrane participate in cellular signaling by receptors for immunoglobulin E? Biophys. Chem. 82:109– 19 Baumgart T, Hess ST, Webb WW. 2003. Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension. Nature 425:821–24 Bretscher MS, Munro S. 1993. Cholesterol and the Golgi apparatus. Science 261:1280–81 Brown DA, London E. 2000. Structure and function of sphingolipid- and cholesterolrich membrane rafts. J. Biol. Chem. 275: 17221–24 Brown DA, Rose JK. 1992. Sorting of GPIanchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68:533–44 Brown RE. 1998. Sphingolipid organization in biomembranes: what physical studies of model membranes reveal. J. Cell Sci. 111:1– 9 Chatterjee S, Mayor S. 2001. The GPI-anchor and protein sorting. Cell Mol. Life Sci. 58: 1969–87 de Almeida RF, Fedorov A, Prieto M. 2003. Sphingomyelin/phosphatidylcholine/ cholesterol phase diagram: boundaries and composition of lipid rafts. Biophys. J. 85: 2406–16 Deschenes RJ, Resh MD, Broach JR. 1990. Acylation and prenylation of proteins. Curr. Opin. Cell Biol. 2:1108–13 Dietrich C, Yang B, Fujiwara T, Kusumi A, Jacobson K. 2002. Relationship of lipid rafts to transient confinement zones detected by single particle tracking. Biophys. J. 82:274– 84 Drab M, Verkade P, Elger M, Kasper M, Lohn M, et al. 2001. Loss of caveolae, vascular dys-

function, and pulmonary defects in caveolin1 gene-disrupted mice. Science 293:2449–52 Drobnik W, Borsukova H, Bottcher A, Pfeiffer A, Liebisch G, et al. 2002. Apo AI/ABCA1dependent and HDL3-mediated lipid efflux from compositionally distinct cholesterolbased microdomains. Traffic 3:268–78 Edidin M. 2001a. Near-field scanning optical microscopy, a siren call to biology. Traffic 2:797–803 Edidin M. 2001b. Shrinking patches and slippery rafts: scales of domains in the plasma membrane. Trends Cell Biol. 11:492–96 Edidin M. 2003. The state of lipid rafts: from model membranes to cells. Annu. Rev. Biophys. Biomol. Struct. 32:257–83 Feigenson GW, Buboltz JT. 2001. Ternary phase diagram of dipalmitoyl-PC/dilauroylPC/cholesterol: nanoscopic domain formation driven by cholesterol. Biophys. J. 80: 2775–88 Fujiwara T, Ritchie K, Murakoshi H, Jacobson K, Kusumi A. 2002. Phospholipids undergo hop diffusion in compartmentalized cell membrane. J. Cell Biol. 157:1071– 81 Garred O, Rodal SK, van Deurs B, Sandvig K. 2001. Reconstitution of clathrin-independent endocytosis at the apical domain of permeabilized MDCK II cells: requirement for a Rho-family GTPase. Traffic 2:26–36 Gaub HE, Moy VT, McConnell HM. 1986. Reversible formation of plastic twodimensional lipid crystals. J. Phys. Chem. 90: 1721–25 Ge M, Gidwani A, Brown HA, Holowka D, Baird B, Freed JH. 2003. Ordered and disordered phases coexist in plasma membrane vesicles of RBL-2H3 mast cells. An ESR study. Biophys. J. 85:1278–88 Gennis RB. 1989. Biomembranes: Molecular Structure and Function. New York: SpringerVerlag Gidwani A, Holowka D, Baird B. 2001. Fluorescence anisotropy measurements of lipid order in plasma membranes and lipid rafts from RBL-2H3 mast cells. Biochemistry 40:12422–29

10 Sep 2004 13:10

AR

AR226-CB20-30.tex

AR226-CB20-30.sgm

LaTeX2e(2002/01/18)


MEMBRANE DOMAINS Gomez-Mouton C, Abad JL, Mira E, Lacalle RA, Gallardo E, et al. 2001. Segregation of leading-edge and uropod components into specific lipid rafts during T cell polarization. Proc. Natl. Acad. Sci. USA 98:9642–47 Gruenberg J. 2001. The endocytic pathway: a mosaic of domains. Nat. Rev. Mol. Cell Biol. 2:721–30 Hao M, Lin SX, Karylowski OJ, Wustner D, McGraw TE, Maxfield FR. 2002. Vesicular and non-vesicular sterol transport in living cells. The endocytic recycling compartment is a major sterol storage organelle. J. Biol. Chem. 277:609–17 Hao M, Mukherjee S, Maxfield FR. 2001. Cholesterol depletion induces large scale domain segregation in living cell membranes. Proc. Natl. Acad. Sci. USA 98:13072– 77 Hao M, Mukherjee S, Sun Y, Maxfield FR. 2004. Effects of cholesterol depletion and increased lipid unsaturation on the properties of endocytic membranes. J. Biol. Chem. 279:14171–78 Harder T, Scheiffele P, Verkade P, Simons K. 1998. Lipid domain structure of the plasma membrane revealed by patching of membrane components. J. Cell Biol. 141:929–42 Heerklotz H. 2002. Triton promotes domain formation in lipid raft mixtures. Biophys. J. 83:2693–701 Hekman M, Hamm H, Villar AV, Bader B, Kuhlmann J, et al. 2002. Associations of Band C-Raf with cholesterol, phosphatidylserine, and lipid second messengers: preferential binding of Raf to artificial lipid rafts. J. Biol. Chem. 277:24090–102 Holowka D, Baird B. 2001. FcεRI as a paradigm for a lipid raft-dependent receptor in hematopoietic cells. Semin. Immunol. 13: 99–105 Holowka D, Sheets ED, Baird B. 2000. Interactions between FcεRI and lipid raft components are regulated by the actin cytoskeleton. J. Cell Sci. 113:1009–19 Huang J, Buboltz JT, Feigenson GW. 1999. Maximum solubility of cholesterol in phosphatidylcholine and phosphatidylethanola-

P1: GCE

863

mine bilayers. Biochim. Biophys. Acta 1417: 89–100 Huang J, Feigenson GW. 1999. A microscopic interaction model of maximum solubility of cholesterol in lipid bilayers. Biophys. J. 76:2142–57 Kenworthy AK, Edidin M. 1998. Distribution of a glycosylphosphatidylinositol-anchored protein at the apical surface of MDCK cells examined at a resolution of 100 A˚ using imaging fluorescence resonance energy transfer. J. Cell Biol. 142:69–84 Kobayashi T, Beuchat M, Lindsay M, Frias S, Palmiter R, et al. 1999. Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport. Nat. Cell Biol. 1: 113–18 Korlach J, Schwille P, Webb WW, Feigenson GW. 1999. Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy. Proc. Natl. Acad. Sci. USA 96:8461–6 Kurzchalia TV, Parton RG. 1999. Membrane microdomains and caveolae. Curr. Opin. Cell Biol. 11:424–31 Lacalle RA, Mira E, Gomez-Mouton C, Jimenez-Baranda S, Martinez AC, Manes S. 2002. Specific SHP-2 partitioning in raft domains triggers integrin-mediated signaling via Rho activation. J. Cell Biol. 157:277–89 Lamaze C, Dujeancourt A, Baba T, Lo CG, Benmerah A, Dautry-Varsat A. 2001. Interleukin 2 receptors and detergent-resistant membrane domains define a clathrin-independent endocytic pathway. Mol. Cell 7:661– 71 Lipowsky R. 1993. Domain-induced budding of fluid membranes. Biophys. J. 64:1133–38 London E. 2002. Insights into lipid raft structure and formation from experiments in model membranes. Curr. Opin. Struct. Biol. 12:480–86 Lundbäk JA, Andersen OS, Werge T, Nielsen C. 2003. Cholesterol-induced protein sorting: an analysis of energetic feasibility. Biophys. J. 84:2080–89 Marsh D, Horváth LI. 1998. Structure, dynamics and composition of the lipid-protein

10 Sep 2004 13:10


864

AR

AR226-CB20-30.tex

MUKHERJEE

AR226-CB20-30.sgm

LaTeX2e(2002/01/18)

P1: GCE

MAXFIELD

interface. Perspectives from spin-labeling. Biochim. Biophys. Acta 1376:267–96 Maxfield FR. 2002. Plasma membrane microdomains. Curr. Opin. Cell Biol. 14:483– 87 Maxfield FR, Wüstner D. 2002. Intracellular cholesterol transport. J. Clin. Invest. 110: 891–98 Mayor S. 1999. Analysis of the cell-surface distribution of GPI-anchored proteins. Methods Mol. Biol. 116:23–36 Mayor S, Maxfield FR. 1995. Insolubility and redistribution of GPI-anchored proteins at the cell surface after detergent treatment. Mol. Biol. Cell 6:929–44 Mayor S, Riezman H. 2004. Sorting GPIanchored proteins. Nat. Rev. Mol. Cell Biol. 5:110–20 Mayor S, Rothberg KG, Maxfield FR. 1994. Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking. Science 264:1948–51 Mayor S, Sabharanjak S, Maxfield FR. 1998. Cholesterol-dependent retention of GPIanchored proteins in endosomes. EMBO J. 17:4626–38 McConnell HM, Keller D, Gaub H. 1986. Thermodynamic models for the shapes of monolayer phospholipid crystals. J. Phys. Chem. 90:1717–21 McConnell HM, Radhakrishnan A. 2003. Condensed complexes of cholesterol and phospholipids. Biochim. Biophys. Acta 1610: 159–73 McLaughlin S, Aderem A. 1995. The myristoyl-electrostatic switch: a modulator of reversible protein-membrane interactions. Trends Biochem. Sci. 20:272–76 McLaughlin S, Wang J, Gambhir A, Murray D. 2002. PIP2 and proteins: interactions, organization, and information flow. Annu. Rev. Biophys. Biomol. Struct. 31:151–75 Melkonian KA, Ostermeyer AG, Chen JZ, Roth MG, Brown DA. 1999. Role of lipid modifications in targeting proteins to detergentresistant membrane rafts. Many raft proteins are acylated, while few are prenylated. J. Biol. Chem. 274:3910–17

Miceli MC, Moran M, Chung CD, Patel VP, Low T, Zinnanti W. 2001. Co-stimulation and counter-stimulation: lipid raft clustering controls TCR signaling and functional outcomes. Semin. Immunol. 13:115–28 Minshall RD, Sessa WC, Stan RV, Anderson RG, Malik AB. 2003. Caveolin regulation of endothelial function. Am. J. Physiol. Lung Cell Mol. Physiol. 285:L1179–83 Mukherjee S, Maxfield FR. 2000. Role of membrane organization and membrane domains in endocytic lipid trafficking. Traffic 1:203– 11 Mukherjee S, Soe TT, Maxfield FR. 1999. Endocytic sorting of lipid analogues differing solely in the chemistry of their hydrophobic tails. J. Cell Biol. 144:1271–84 Mukherjee S, Zha X, Tabas I, Maxfield FR. 1998. Cholesterol distribution in living cells: fluorescence imaging using dehydroergosterol as a fluorescent cholesterol analog. Biophys. J. 75:1915–25 Needham D, McIntosh TJ, Evans E. 1988. Thermomechanical and transition properties of dimyristoylphosphatidylcholine/cholesterol bilayers. Biochemistry 27:4668–73 Niv H, Gutman O, Kloog Y, Henis YI. 2002. Activated K-Ras and H-Ras display different interactions with saturable nonraft sites at the surface of live cells. J. Cell Biol. 157:865–72 Pagano RE, Puri V, Dominguez M, Marks DL. 2000. Membrane traffic in sphingolipid storage diseases. Traffic 1:807–15 Pelkmans L, Puntener D, Helenius A. 2002. Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolae. Science 296:535–39 Pierini L, Holowka D, Baird B. 1996. FcεRImediated association of 6-µm beads with RBL-2H3 mast cells results in exclusion of signaling proteins from the forming phagosome and abrogation of normal downstream signaling. J. Cell Biol. 134:1427–39 Pierini LM, Eddy RJ, Fuortes M, Seveau S, Casulo C, Maxfield FR. 2003. Membrane lipid organization is critical for human neutrophil polarization. J. Biol. Chem. 278:10831–41

10 Sep 2004 13:10

AR

AR226-CB20-30.tex

AR226-CB20-30.sgm

LaTeX2e(2002/01/18)


MEMBRANE DOMAINS Pierini LM, Maxfield FR. 2001. Flotillas of lipid rafts fore and aft. Proc. Natl. Acad. Sci. USA 98:9471–73 Pike LJ. 2004. Lipid rafts: Heterogeneity on the high seas. Biochem. J. 378:281–92 Prior IA, Muncke C, Parton RG, Hancock JF. 2003. Direct visualization of Ras proteins in spatially distinct cell surface microdomains. J. Cell Biol. 160:165–70 Pyenta PS, Holowka D, Baird B. 2001. Crosscorrelation analysis of inner-leaflet-anchored green fluorescent protein co-redistributed with IgE receptors and outer leaflet lipid raft components. Biophys. J. 80:2120–32 Raucher D, Stauffer T, Chen W, Shen K, Guo S, et al. 2000. Phosphatidylinositol 4,5bisphosphate functions as a second messenger that regulates cytoskeleton-plasma membrane adhesion. Cell 100:221–28 Razani B, Wang XB, Engelman JA, Battista M, Lagaud G, et al. 2002. Caveolin-2-deficient mice show evidence of severe pulmonary dysfunction without disruption of caveolae. Mol. Cell. Biol. 22:2329–44 Resh MD. 1999. Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim. Biophys. Acta 1451:1–16 Ritchie K, Iino R, Fujiwara T, Murase K, Kusumi A. 2003. The fence and picket structure of the plasma membrane of live cells as revealed by single molecule techniques. Mol. Membr. Biol. 20:13–18 Rodriguez-Boulan E, Powell SK. 1992. Polarity of epithelial and neuronal cells. Annu. Rev. Cell Biol. 8:395–427 Sabharanjak S, Sharma P, Parton RG, Mayor S. 2002. GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway. Dev. Cell 2:411–23 Sackmann E, Feder T. 1995. Budding, fission and domain formation in mixed lipid vesicles induced by lateral phase separation and macromolecular condensation. Mol. Membr. Biol. 12:21–28 Seveau S, Eddy RJ, Maxfield FR, Pierini LM. 2001. Cytoskeleton-dependent mem-

P1: GCE

865

brane domain segregation during neutrophil polarization. Mol. Biol. Cell 12:3550–62 Silvius JR. 2003. Fluorescence energy transfer reveals microdomain formation at physiological temperatures in lipid mixtures modeling the outer leaflet of the plasma membrane. Biophys. J. 85:1034–45 Silvius JR, del Giudice D, Lafleur M. 1996. Cholesterol at different bilayer concentrations can promote or antagonize lateral segregation of phospholipids of differing acyl chain length. Biochemistry 35:15198– 208 Simons K. 1993. Biogenesis of epithelial cell surface polarity. Harvey Lect. 89:125–46 Simons K, Fuller SD. 1985. Cell surface polarity in epithelia. Annu. Rev. Cell Biol. 1:243– 88 Simons K, Ikonen E. 1997. Functional rafts in cell membranes. Nature 387:569–72 Simons K, Toomre D. 2000. Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol. 1:31–39 Singer SJ, Nicolson GL. 1972. The fluid mosaic model of the structure of cell membranes. Science 175:720–31 Slimane TA, Trugnan G, Van ISC, Hoekstra D. 2003. Raft-mediated trafficking of apical resident proteins occurs in both direct and transcytotic pathways in polarized hepatic cells: role of distinct lipid microdomains. Mol. Biol. Cell 14:611–24 Sotgia F, Razani B, Bonuccelli G, Schubert W, Battista M, et al. 2002. Intracellular retention of glycosylphosphatidyl inositol-linked proteins in caveolin-deficient cells. Mol. Cell. Biol. 22:3905–26 Sperotto MM, Mouritsen OG. 1993. Lipid enrichment and selectivity of integral membrane proteins in two-component lipid bilayers. Eur. Biophys. J. 22:323–28 Stauffer TP, Meyer T. 1997. Compartmentalized IgE receptor-mediated signal transduction in living cells. J. Cell Biol. 139:1447– 54 Steck TL, Ye J, Lange Y. 2002. Probing red cell membrane cholesterol movement with cyclodextrin. Biophys. J. 83:2118–25

10 Sep 2004 13:10


866

AR

AR226-CB20-30.tex

MUKHERJEE

AR226-CB20-30.sgm

LaTeX2e(2002/01/18)

P1: GCE

MAXFIELD

Thomas JL, Holowka D, Baird B, Webb WW. 1994. Large-scale co-aggregation of fluorescent lipid probes with cell surface proteins. J. Cell Biol. 125:795–802 Tokumasu F, Jin AJ, Feigenson GW, Dvorak JA. 2003. Nanoscopic lipid domain dynamics revealed by atomic force microscopy. Biophys. J. 84:2609–18 van Meer G, Lisman Q. 2002. Sphingolipid transport: rafts and translocators. J. Biol. Chem. 277:25855–58 Varma R, Mayor S. 1998. GPI-anchored proteins are organized in submicron domains at the cell surface. Nature 394:798–801 Vaz WL. 1995. Percolation properties of twocomponent, two-phase phospholipid bilayers. Mol. Membr. Biol. 12:39–43 Veatch SL, Keller SL. 2003. A closer look at the canonical ‘Raft Mixture’ in model membrane studies. Biophys. J. 84:725–26 Wang TY, Leventis R, Silvius JR. 2000. Fluorescence-based evaluation of the parti-

tioning of lipids and lipidated peptides into liquid-ordered lipid microdomains: a model for molecular partitioning into “lipid rafts.” Biophys. J. 79:919–33 Wang TY, Silvius JR. 2001. Cholesterol does not induce segregation of liquid-ordered domains in bilayers modeling the inner leaflet of the plasma membrane. Biophys. J. 81:2762– 73 Wustner D, Herrmann A, Hao M, Maxfield FR. 2002. Rapid nonvesicular transport of sterol between the plasma membrane domains of polarized hepatic cells. J. Biol. Chem. 277:30325–36 Yeagle PL. 1985. Cholesterol and the cell membrane. Biochim. Biophys. Acta 822:267– 87 Young RM, Holowka D, Baird B. 2003. A lipid raft environment enhances Lyn kinase activity by protecting the active site tyrosine from dephosphorylation. J. Biol. Chem. 278:20746–52