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Journal of Investigative Medicine:
doi: 10.2310/6650.2005.53608
EB 2005 Symposium

Fleeting Glimpses of Lipid Rafts: How Biophysics Is Being Used to Track Them

Kenworthy, Anne K.

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From the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN.

Presented in part at the American Federation for Medical Research‐sponsored symposium during Experimental Biology 2005, San Diego, CA, April 2‐6, 2005.

Portions of this review appeared in an abstract for the Biophysical Society discussions meeting on “Probing Membrane Microdomains,” October 2004, Asilomar, CA.

Address correspondence to: Dr. Anne K. Kenworthy, Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232; e‐mail:

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ABSTRACT: Cell membranes are fluid but can exhibit local order that gives rise to lateral inhomogeneities, often referred to as membrane microdomains. Among the best studied yet least well understood of these microdomains are lipid rafts. Lipid rafts are hypothesized to participate in a variety of physiologic and pathologic pathways important to human health by causing the spatial segregation of proteins and lipids within the plane of the membrane. Despite the widespread implications of the raft model, major questions remain about raft size, composition, and life span. This article discusses how recent biophysical measurements of the dynamic properties of rafts and putative raft‐associated proteins and lipids are being used to test the hypothesis that confinement of proteins in rafts slows and/or impairs their ability to sample their microenvironment by lateral diffusion.

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Cell membranes are heterogeneous, containing localized regions of specialized lipid and protein composition. The best known of these lateral inhomogeneities, so‐called lipid rafts, are thought to function as platforms to concentrate and segregate proteins within the plane of the bilayer.1‐4 Enriched in glycosphingolipids and cholesterol, lipid rafts also are rich in a variety of proteins. The lipid raft model has been implicated as a general mechanism for the regulation of a variety of cellular processes important to human health, including cell signaling, membrane trafficking events, and the entry and exit of pathogens into and out of cells.5‐8 Pathways are typically defined as raft associated if their components cofractionate with detergent‐resistant membrane fractions or if the pathway can be inhibited by cholesterol depletion.9,10 However, these techniques intrinsically lack the spatial and temporal information required to provide insight into raft function, and the raft model has remained controversial despite its potentially wide‐ranging implications.11 Fueling this controversy is the underlying lack of knowledge about the fundamental properties of these domains, including their size, composition, and dynamic properties. These questions can best be addressed through biophysical studies in living cells.3,12,13 In this overview, I discuss recent studies examining how lipid rafts impact the ability of membrane proteins to sample their environment by lateral diffusion and their implications for the dynamic properties of lipid rafts themselves.

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Rafts are enriched in both cholesterol and lipids with predominantly saturated acyl chains, which contributes to the formation of a so‐called liquid‐ordered phase.14 Studies in simple lipid mixtures indicate that diffusion is more rapid within a liquid‐ordered phase than in a gel phase but less so than in a liquid‐disordered phase.14,15 This decreased lipid mobility in a liquid‐ordered phase versus a liquid‐disordered phase can be observed directly in lipid mixtures mimicking rafts and should extend to the behavior of proteins embedded in these lipid phases.16,17 In cells, several additional mechanisms have been proposed to underlie the slowing of protein diffusion by rafts:

1. Rafts as sites of transient protein trapping. In cells, the selective confinement of proteins in lipid rafts has been hypothesized to lead to their immobilization (Figure 1A) and/or slowed diffusion (Figure 1, B and C).18‐22 This model predicts increased protein mobility on cholesterol depletion (as the result of raft disruption) and/or higher mobility of nonraft versus raft proteins.

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2. Diffusion of raft complexes. Local measurements of the viscous drag on raft proteins suggest that proteins associate with raft domains of ˜50 nm, which themselves are able to diffuse and remain stably associated over minutes (see Figure 1B).23 Association of proteins with raft complexes could also affect their ability to cross cytoskeletal barriers (see Figure 1A).24

3. Induced stabilization of rafts. Under steady‐state conditions, raft proteins may associate with small unstable rafts, allowing them to diffuse essentially as monomers (Figure 1D). However, the dynamics of these molecules can be altered as the result of a stimulus, leading to the formation of stabilized clustered rafts.24 The lipid shell model also allows for the diffusion of monomeric raft proteins (see Figure 1D),25 although the proposed lifetime and size of lipid shells and small unstable rafts differ.24

Each of the above models makes the implicit assumption that the fraction of continuous nonraft membrane is larger than that of raft domains. However, rafts could potentially act as barriers to the diffusion of nonraft proteins and lipids as well.26 This concept can be understood in terms of a percolating raft model in which the liquid‐ordered phase becomes continuous and the liquid‐disordered phase forms isolated domains.27

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Biophysical techniques sensitive to lateral diffusion, such as fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and single particle tracking (SPT), can be used to distinguish between these models. Each of these techniques is sensitive to different aspects of lateral mobility; they complement one another to provide information over a wide range of temporal and spatial scales.

In FRAP, a population of fluorescently labeled molecules is irreversibly photobleached in a defined region by a rapid, high‐intensity laser pulse (Figure 2A). Recovery of fluorescence, resulting from diffusive exchange with molecules in the surrounding region, is then monitored over time.28 The bleached area can be defined either by a tightly focused laser or a larger region of interest using a scanning laser confocal microscope. In either case, the resulting fluorescence recovery curves are typically characterized by a diffusion coefficient (D) and mobile fraction, the fraction of molecules that are able to recover over the time course of the experiment (Figure 2B). In contrast, FCS monitors fluctuations in fluorescence resulting from the movement of a small number of molecules in and out of a minute volume element defined using confocal optics.29,30 Through autocorrelation analysis, FCS data can provide a measure of the typical residence time of molecules within the volume, which is related to their diffusion coefficient D. Like FCS, SPT is also a technique with single‐molecule sensitivity. Here a protein or lipid of interest is directly labeled with a fluorophore or with a gold bead to which antibodies against the protein of interest have been adsorbed at low densities, allowing for the visualization of individual molecules. Such measurements allow for highly temporally and spatially resolved measurements of the movements of individual molecules. Detailed analysis of the trajectories of individual molecules allows for classification of their modes of motion, including free diffusion, confined diffusion and/or transient confinement, and immobilization.31

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We are still in the early stages of understanding how lipid rafts regulate the lateral mobility of their protein components (and the converse, what diffusion measurements can tell us about raft dynamics). Below I summarize recent studies addressing these questions in cells.

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A subclass of lipid rafts, caveolae are 50 to 100 nm flask‐shaped invaginations of the cell surface. Unlike lipid rafts, the structure and function of caveolae can be directly studied by virtue of their requirement for the protein caveolin.32,33 Studies using green fluorescent protein (GFP)‐tagged caveolin as a marker for caveolae are in general agreement that caveolae are immobile structures.34‐37 The static nature of caveolae may impact the diffusional mobility of other molecules as well. For example, we found that cholera toxin B subunit diffuses quite slowly, perhaps reflecting its association with caveolae prior to its eventual internalization.38

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Glycosylphosphatidylinositol‐Anchored Proteins

Glycosylphosphatidylinositol (GPI)‐anchored proteins are strongly associated with detergent‐resistant membrane fractions, suggesting that they are predominantly localized to lipid raft domains. Does their diffusional mobility reflect this? Early FRAP studies showed that GPI‐anchored proteins can diffuse almost as rapidly as lipids and are not subject to the same diffusional constraints experienced by transmembrane proteins.39‐41 Consistent with these results, we found that GFP‐tagged GPI‐anchored proteins diffuse faster than most transmembrane proteins, whether or not they are raft associated.38 However, Henis and colleagues found that the mobility of a GPI‐linked form of influenza hemagglutinin (HA) is slowed compared with a transmembrane nonraft form.19 They also observed differences in the interaction of wild‐type and GPI‐anchored HA with raft patches. Clearly, the mode of anchorage of a raft protein (transmembrane or GPI anchored) impacts its mobility, but whether this is due to proteins associating with rafts to different extents, different kinds of rafts, or the fact that GPI‐anchored proteins generally experience fewer constraints to their diffusion is not yet known.

A second characteristic diffusional behavior of GPI‐anchored proteins is their association with transient confinement zones (TCZs). TCZs are regions of membrane in which the particle is “trapped” for seconds as assessed by single‐molecule tracking techniques. An initial study showing that GPI‐anchored proteins undergo transient confinement raised the possibility that these domains correspond to the in vivo equivalent of detergent‐insoluble membrane fractions.22 In a study evaluating the nature of TCZs, Jacobson and colleagues show that GPI‐anchored proteins spent more time in these zones than a lipid analog.20 Interestingly, they also found that diffusion was slowed more or less twofold within these regions, and the TCZs could be revisited. However, Subczynski and Kusumi reported that the GPI‐anchored protein CD59 normally diffuses as rapidly as a nonraft phospholipid and that transient confinement is observed primarily in response to crosslinking.42 Moreover, single‐molecule tracking studies of major histocompatibility class II using a fluorescently labeled peptide showed little evidence of confinement of either a conventional transmembrane or GPI‐anchored form.43 It will be interesting to further determine the nature of TCZs and define the events that lead to their formation, especially given the possibility that they correspond to sites of cellular signaling.24

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The small guanosine triphosphatase Ras is a key player in signal transduction pathways regulating cell proliferation, death, and differentiation. The diffusional mobility of GFP‐tagged forms of Ras, a resident of the cytoplasmic leaflet of the plasma membrane, has been the subject of several recent studies.18,21,44‐46 Even more mobile than GPI‐anchored proteins, the steady‐state mobility of Ras proteins at the cell surface is extremely high, with D's ranging from 0.33 to over 1 μm2 /s and mobile fractions of 75 to 95% depending on the temperature, cell type, and observation method. For example, we found that HRas (a putative raft marker) and KRas (a nonraft marker) exhibited diffusion coefficients of ˜1 μm2 /s, with mobile fractions of > 95% when expressed in COS‐7 cells.38 This exceptionally high mobility seems inconsistent with the notion that the protein is confined to microdomains. Yet a combination of biochemical, functional, and morphologic data suggests that HRas and KRas reside in separate microdomains and can change their microlocalization in a regulated manner.47‐49

A more detailed analysis of the diffusional behavior of Ras supports the concept that the Ras proteins associate with several new classes of microdomains in addition to lipid rafts. For example, saturable D values were observed as a function of increasing expression of constitutively active HRas or KRas but not wild‐type HRas, and only HRas diffusion was sensitive to cholesterol depletion.18 Single‐particle tracking measurements of yellow fluorescent protein (YFP) appended with HRas membrane targeting sequence indicate that 30 to 40% of the molecules are constrained in domains of 200 nm that are insensitive to cholesterol depletion or actin depolymerization.45 Using an elegant combination of fluorescence resonance energy transfer and SPT, Kusumi and colleagues recently showed that diffusion of HRas is substantially slowed on activation and that this effect was not blocked by cholesterol depletion.46 FRAP studies further indicate that multiple domains within HRas contribute to its overall microdomain localization.21 The implication that Ras isoforms can undergo regulated interactions with several distinct types of domains is intriguing and begs the question of whether rafts are the only types of microdomains for which we should be looking.

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Dependence of Diffusional Mobility on Cholesterol Availability

Cholesterol depletion is commonly used to disrupt lipid rafts. The effects of cholesterol depletion on protein diffusion in cell membranes, however, are unexpectedly complex. Several studies report that cholesterol depletion leads to increased diffusion of raft proteins, as if the proteins were released from confining domains.18‐21,23 However, this effect is not uniformly observed. For example, we found that methyl‐β‐cyclodextrin (MBCD) treatment caused a twofold drop in D for both raft and nonraft proteins (Figure 2C).38 Others have also reported immobilization and/or slowing of proteins or lipid probes in MBCD‐treated cells.45,50,51 Of particular interest is a study by Edidin and colleagues showing that both acute and chronic cholesterol depletion led to immobilization of human leukocyte antigen molecules.50 This effect was reversed by cytochalasin D treatment and mimicked by the sequestration of phosphoinositol 4,5‐bisphosphate, linking cholesterol depletion to reorganization of the actin cytoskeleton. These observations suggest that cholesterol depletion may not function solely by dissipating raft domains and highlight the utility of diffusional mobility measurements in providing new and unexpected insights into rafts.

If cholesterol is limiting for raft formation, one might expect extra cholesterol to increase the number of rafts and therefore cause decreased diffusional mobility of raft markers. Alternatively, the formation of an increased area fraction of rafts by excess cholesterol could potentially limit the diffusion of nonraft proteins. Our initial studies in cholesterol‐loaded cells did not support either of these possibilities because we observed little effect on the mobility of either raft proteins or a nonraft protein.38 In current studies, we are testing whether these findings extend to proteins localized to the inner leaflet of the plasma membrane.

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The concept that lipid rafts function to compartmentalize molecules within the plane of the membrane strongly suggests that raft association should lead to a measurable decrease in diffusional mobility. Recent biophysical measurements support this model, at least to a point. To date, only caveolae appear to have the diffusional characteristics of a stable membrane domain. Even when studied with the very high spatial and temporal resolution of techniques such as SPT, protein components of rafts do not necessarily behave as if they are solely confined to rafts. Instead, a picture is emerging of transient associations of proteins with unstable domains, at least under steady‐state conditions. Understanding if cells regulate these fleeting interactions by virtue of the preferential interactions of cholesterol and glycosphingolipids with one another or if they contribute to the generation of these domains remains a challenge for the future.

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37 Thomsen P, Roepstorff K, Stahlhut M, van Deurs B. Caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic trafficking. Mol Biol Cell 2002;13:238-50.

38 Kenworthy AK, Nichols BJ, Remmert CL, et al. Dynamics of putative raft-associated proteins at the cell surface. J Cell Biol 2004;165:735-46.

39 Edidin M, Stroynowski I. Differences between the lateral organization of conventional and inositol phospholipid-anchored membrane proteins. A further definition of micrometer scale membrane domains. J Cell Biol 1991;112:1143-50.

40 Zhang F, Crise B, Su B, et al. Lateral diffusion of membrane-spanning and glycosylphosphatidylinositol-linked proteins: toward establishing rules governing the lateral mobility of membrane proteins. J Cell Biol 1991;115:75-84.

41 Ishihara A, Hou Y, Jacobson K. The Thy-1 antigen exhibits rapid lateral diffusion in the plasma membrane of rodent lymphoid cells and fibroblasts. Proc Natl Acad Sci U S A 1987;84:1290-3.

42 Subczynski WK, Kusumi A. Dynamics of raft molecules in the cell and artificial membranes: approaches by pulse EPR spin labeling and single molecule optical microscopy. Biochim Biophys Acta 2003;1610:231-43.

43 Vrljic M, Nishimura SY, Brasselet S, et al. Translational diffusion of individual class II MHC membrane proteins in cells. Biophys J 2002;83:2681-92.

44 Niv H, Gutman O, Henis YI, Kloog Y. Membrane interactions of a constitutively active GFP-Ki-Kas 4B and their role in signaling. Evidence from lateral mobility studies. J Biol Chem 1999;274:1606-13.

45 Lommerse PH, Blab GA, Cognet L, et al. Single-molecule imaging of the H-ras membrane-anchor reveals domains in the cytoplasmic leaflet of the cell membrane. Biophys J 2004;86:609-16.

46 Murakoshi H, Iino R, Kobayashi T, et al. Single-molecule imaging analysis of Ras activation in living cells. Proc Natl Acad Sci U S A 2004;101:7317-22.

47 Hancock JF. Ras proteins: different signals from different locations. Nat Rev Mol Cell Biol 2003;04:373-84.

48 Prior IA, Hancock JF. Compartmentalization of Ras proteins. J Cell Sci 2001;114:1603-8.

49 Parton RG, Hancock JF. Lipid rafts and plasma membrane microorganization: insights from Ras. Trends Cell Biol 2004;14:141-7.

50 Kwik J, Boyle S, Fooksman D, et al. Membrane cholesterol, lateral mobility, and the phosphatidylinositol 4,5-bisphosphate-dependent organization of cell actin. Proc Natl Acad Sci U S A 2003;100:13964-9.

51 Hao M, Mukherjee S, Maxfield FR. Cholesterol depletion induces large scale domain segregation in living cell membranes. Proc Natl Acad Sci U S A 2001;98:13072-7.

Key Words:: lipid rafts; membrane microdomains; lateral diffusion; fluorescence recovery after photobleaching; green fluorescent protein; caveolae; Ras

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© 2005 American Federation for Medical Research


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