, 46 (2009);
et al.Daniel Lingwood,
Lipid Rafts As a Membrane-Organizing Principle
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Lipid Rafts As a Membrane-
Daniel Lingwood and Kai Simons*
Cell membranes display a tremendous complexity of lipids and proteins designed to perform the
functions cells require. To coordinate these functions, the membrane is able to laterally
segregate its constituents. This capability is based on dynamic liquid-liquid immiscibility and
underlies the raft concept of membrane subcompartmentalization. Lipid rafts are fluctuating
nanoscale assemblies of sphingolipid, cholesterol, and proteins that can be stabilized to coalesce,
forming platforms that function in membrane signaling and trafficking. Here we review the
evidence for how this principle combines the potential for sphingolipid-cholesterol self-assembly
with protein specificity to selectively focus membrane bioactivity.
tion between sphingolipids, sterols, and specific
proteins bestows cell membranes with lateral seg-
regation potential. The concept has long suffered
assessmentbyindirect means,leading toquestions
of fact or artifact (1). The resistance of sphingo-
lipid, cholesterol, and a subclass of membrane
proteins to cold detergent extraction (2) or me-
chanical disruption (3) has been widely used as
an index for raft association with little or no re-
gard for the artifacts induced by these methods.
may point to physiologically relevant biases in
lateral composition (4), this disruptive measure
tells us little about native membrane organization.
Support from light microscopy was also missing
because, with the exception of organization into
specialized membrane domains such as caveolae or
glycosylphosphatidylinositol (GPI)–anchored pro-
teins, fluorescent lipid analogs, raft transmembrane
a homogeneous distribution at the cell surface (5).
Moreover, early investigations into submicron
membrane organization often yielded conflicting
evidence regarding the distribution or motion of
these constituentsin the living cell(1). Today, how-
ever, the advancement of technology has produced
proteins can induce subcompartmentalization to
organize bioactivity of cell membranes.
Origins of the Lipid Raft Concept
Biochemically, it is clear that lipids are sorted
within the cell (6). This is particularly notable
in polarized epithelia where glycosphingolipids
(GSLs) are enriched at the apical surface (7).
Lipid rafts were originally proposed as an ex-
he lipid raft hypothesis proposes that the
lipid bilayer is not a structurally passive
solvent, but that the preferential associa-
planation: Self-associative properties unique to
sphingolipid and cholesterol in vitro could facili-
tate selective lateral segregation in the membrane
plane andserve asa basisfor lipid sortinginvivo
(7). This proposal for compartmentalization by
lipid rafts suggested a nonrandom membrane ar-
chitecture specifically geared to organize func-
tionality within the bilayer. This function was
initially thought to be membrane trafficking; how-
ever, rafts could influence organization of any
membrane bioactivity (Fig. 1). Here, we highlight
advances in technology that point to the existence
of raft-based membrane heterogeneity in living
cells and discuss the levels of preferential asso-
ciation underlying dynamic domain structure and
Lipid Interactions in Model Membranes
Assembly into two-dimensional liquid crystalline
biomembranes is a fascinating property charac-
teristic of lipids. Long thought to be incapable of
coherent lateral structure (8), it is now apparent
that principles of lipid self-association can also
confer organization beyond nonspecific measures
of fluidity. An important advance in model-
membrane systems was the discovery of phase
separation in wholly liquid bilayers (9, 10). It is a
cholesterol-dependent lateral segregation, where-
in the planarity (molecular flatness) of the rigid
hydrocarbon chains of saturated lipids and dis-
favors interaction with the more bulky unsatu-
rated lipid species(11).Interactionwith cholesterol
also forces neighboring hydrocarbon chains into
more extended conformations, increasing mem-
through hydrophobic mismatch (12). In purified
lipid systems, the combined effect is a physical
segregation in the membrane plane: A thicker,
liquid-ordered, Lo phase coexists with a thinner,
liquid-disordered, Ld phase (13). Sphingolipids
tend to display longer and more saturated hydro-
carbon chains, thus potentiating interdigitation
between leaflets (14) and favoring interaction with
cholesterol. Moreover, unlike glycerophospholi-
pids, the region of chemical linkage between the
head group and sphingosine base contains both
acceptors and donors of hydrogen bonds, thus
increasing associative potential, both with cho-
lesterol and other sphingolipids (11). Other
explanations for cholesterol selectivity include
the proposed umbrella effect,inwhichcholesterol
hydrophobicity is preferentially shielded by the
strongly hydrated head groups of sphingolipid
(15) or stoichiometric, but reversible, complex
formation between cholesterol and sphingolipid
or saturated glycerophospholipid (16).
Immiscible liquid phase coexistence in vitro
has been suggested as the physical principle under-
lying rafts in vivo (17). Of central importance is
the demonstration of selectivity in association be-
tween certain lipids. However, phase separation in
simple systems at thermodynamic equilibrium in
vitro cannot be translated into proof for mem-
model-membrane work emphasizes the fact that
certain lipids exhibit preferential association and
provides a framework for understanding how het-
erogeneity in cell membranes may arise (18). In
this respect, the terms Lo and Ld should not be
applied to the living cell, as they refer only to the
liquid-ordered and liquid-disordered phases of
model-membrane systems where parameters
relating to translational order (lateral diffusion)
and conformational order (trans/gauche ratio in
the acyl chains) can be accurately measured (11).
Glimpses of Nano-Assemblies in Living Cells
Currently, lipid rafts are viewed as dynamic nano-
scale assemblies enriched in sphingolipid, choles-
terol, and GPI-anchored proteins (19) (Fig. 2A).
To reach this viewpoint, membrane research has
had to contend with the observer’s effect, akin to
and/or induce heterogeneity in membranes simply
by trying to observe it. Initially, this required mov-
ing away from detergent extraction as a means to
infer native organization. In a first step, detergent-
free, chemical cross-linking of GPI-anchored pro-
teins at the plasma membrane suggested that the
intrinsic heterogeneity by rafts was present in
nanoscale complexes below the optical resolution
limit set by the diffraction of light (19). This
nanometer-size scale was later supported by vis-
cous drag measures of antibody-bound raft pro-
teins (21) and electron microscopic observation
of immunogold-labeled raft antigens (20). Indeed,
recent near-field scanning optical microscopy has
confirmed a nanoscale bias in the distribution of
raft-associating proteins in fixed cells (22). Less
perturbing measures of spatial and temporal dy-
namics in living cells have also provided corre-
lating data. For example, single-particle tracking
reveals “stimulation-induced temporary arrest of
50-nm areas as a bioactive feature of receptor
function (23). Parallel advances in microscopy and
spectroscopy have revealed similar heterogeneity
Max Planck Institute for Molecular Cell Biology and Genetics,
Pfotenhauerstrasse 108, 01307 Dresden, Germany.
*To whom correspondence should be addressed. E-mail:
1 JANUARY 2010VOL 327
on January 4, 2010
for raftmolecules in uncross-linked,“resting”con-
ditions. For GPI-anchored proteins, variable waist
fluorescence correlation spectroscopy points to
<120-nm assemblages that fluctuate on a sub-
second time scale (24). High spatial and temporal
resolution fluorescence resonance energy transfer
(25) has generated a more conservative size es-
timate with GPI-anchored receptors residing in
bly formation is always cholesterol-dependent,
and, in some cases, an actin requirement has also
been seen (23, 25). However, other techniques
have indicated that nanoheterogeneity is actin-
independent (26). The situation for TM proteins
is not yet clear. However, fluorescence photo-
activation localization microscopy has revealed a
dynamically clustered nanoscale distribution of
hemagglutinin (27), a TM protein previously de-
scribed as raft-associating (21). The role of the
association between cholesterol and sphingolip-
ids in assembly formation has been analyzed
recently by stimulated emission depletion mi-
croscopy. This study revealed
that, unlike glycerophospholipids,
display transient cholesterol-
dependent confinement in areas
of <20 nm (28). In this case,
differences in diffusion were at-
tributed to differential hydrogen-
bonding capacities of glycerol-
versus sphingosine-based lipids.
at the cell surface have also re-
vealed heterogeneity in membrane
time scale (29).
Different techniques are yield-
inga range of values for different
molecular constituents in diverse
cell types. However, these meth-
ods all point to the existence of
small, dynamic and selective
in the plasma membranes of
living cells. Recent data point to
critical behavior as a potential
fluctuating nanoscale assemblies
in plasma membranes (30).
Antibody cross-linking at the
cell surface causes raft proteins
and lipids to co-patch and exclude
non-raft proteins (31). This selec-
tivity in patching is cholesterol-
dependent and can be transmitted
lets (32). The nonrandom coales-
cence behavior observed in these
artifactual cross-linking studies
suggests how raft organizing po-
tential may be functionalized to
larger, more physiologically rele-
vant temporal and spatial scales
(Fig. 2B). A contention of the
lipid raft hypothesis is that dy-
namic nanoscale heterogeneity can
be stabilized to coalesce into larger
protein-lipid, and protein-protein
interactions (20). In this sense, cell
membranes would possess an un-
multimerization promotes the sorting of GPI-
anchored proteins into sphingolipid/cholesterol-
enriched carriers during clathrin-independent
endocytosis (33). Along similar lines, cluster-
ing of cell surface Gb3or GM1 (both GSLs) by
their respective ligands Shiga toxin and cholera
toxin induces energy-independent tubular in-
vaginations of sphingolipid-biased membrane
composition (34, 35). Similar behavior has
and budded viral
selective sorting of
in cell membranes
display selective sterol-
dependent coalescence at
the cell surface
separate in model
membranes and cell
Lipidomics reveals that
sphingolipids and sterol are
sorted in the TGN during
transport to the plasma
Advances in microscopy and
spectroscopy (e.g. SPT, FCS,
FRET, STED, FPALM) reveal
dynamic nanoassemblies of sterol,
sphingolipid, and protein in living
785 785 790 795 800 805 810 815 820 875
Rel. Int. (%)
Fig. 1. Evolutionoftheraftconceptforsubcompartmentalizationincellmembranes.AboldHindicateshydrogenbonding.
VSV, vesicular stomatitus virus; DRMs, detergent-resistant membranes; GUV, giant unilamellar vesicle; m/z, mass/charge
ratio; SPT, single-particle tracking; FCS, fluorescence correlation spectroscopy; FRET, fluorescence resonance energy
transfer; STED, stimulated emission depletion; FPALM, fluorescence photoactivation localization microscopy.
VOL 3271 JANUARY 2010
on January 4, 2010
also been reported during the multi-
valent binding of SV40 virus to its
GM1 receptor (35). Invagination
from the plasma membrane was
mon to sphingolipid, and suggests
that the effect is mediated by line
tension arising between membrane
domains of different compositional
order (35). Coalescence of dynamic
naling, for example, during the for-
mation of B cell receptor (BCR) or
T cell receptor (TCR) foci. Antigen
binding induces the dynamic asso-
ciation of BCR to its signaling ef-
fector Lyn kinase and leads to the
formation of an immune synpase.
The interaction is dependent on the
nature of Lyn lipid anchorage, with
drocarbon chains preventing associa-
tion with the BCR (36). During TCR
activation, raft components of this
proteins) become selectively immo-
bilized in nanoscale clusters (37),
seeding the accumulation of choles-
terol, sphingomyelin, and saturated
into the synapse, effectively sorting
proteins according to their affinity
for raft domains (38). Rafts in this
“activated” or coalesced condition
constitute a more ordered assem-
in which proteins can be modulated
specifically (39), yet that exists sepa-
rich in unsaturated glycerophospho-
lipid. Raft activation is often stabi-
lized or nucleated by scaffolding
elements such as cortical actin (40)
and may become dominating when
the mole fraction of sphingolipids
and cholesterol increases, as is the
case in the apical membrane of
epithelial cells (41).
Phase Separation in
Despite their selective co-patching
with raft markers at the cell surface, raft TM
proteins are depleted from the tightly packed Lo
Thus, the Lo phase as it exists in simple model
heterogeneity in plasma membranes that selec-
tively includes TM proteins (44, 45). Giant
plasma-membrane vesicles isolated by a chem-
ical membrane blebbing procedure can be
cooled to phase separate into Lo- and Ld-like
phases (46), and here also, raft TM proteins are
typically excluded from the ordered membrane
phase (47). Remarkably, this phase coexistence
indicates that after chemical modification of
protein (e.g., formaldehyde cross-linking, thiol
treatment), the capacity for physical or lipid-
based liquid-liquid phase separation can be
manifested by the plasma membrane, despite its
compositional complexity. Now the question is,
how might phase length–scale separation take
place in plasma membranes at physiological
Some insight has come from a cell-swelling
procedure to separate plasma-membrane spheres
from the influence of cytoskeletal, endocytic, or
exocytic processes in a cell line enriched in the
raft ganglioside GM1. Pentavalent clustering by
cholera toxin resulted in sterol-dependent coales-
cence of a micron-scale raft “phase” at 37°C,
selectively reorganizing the lateral distribution of
proteins and lipids according to their predicted
affinity for raft domains (44). In this case, selec-
tive incorporation of TM proteins was achieved
at a lipid-ordering level far below that observed
in model-membrane Lo phases (45). Thus, where-
as preferential lipid-lipid associations do under-
Raft dissociatingRaft associating
Raft platformRaft platform
GSL / SM
Fig. 2. Hierarchy of raft-based heterogeneity in cell membranes. (A) Fluctuating nanoscale assemblies of sterol- and
sphingolipid-related biases in lateral composition. This sphingolipid/sterol assemblage potential can be accessed
and/or modulated by GPI-anchored proteins, certain TM proteins, acylated cytosolic effectors, and cortical actin.
Gray proteins do not possess the chemical or physical specificity to associate with this membrane connectivity and
are considered non-raft. GPL, glycerophospholipid; SM, sphingomyelin. (B) Nanoscale heterogeneity is functionalized
to larger levels by lipid- and/or protein-mediated activation events (e.g., multivalent ligand binding, synapse for-
mation, protein oligomerization) that trigger the coalescence of membrane order–forming lipids with their accom-
panying selective chemical and physical specificities for protein. This level of lateral sorting can also be buttressed
by cortical actin. (C) The membrane basis for heterogeneity as revealed by the activation of raft phase coalescence
at equilibrium in plasma-membrane spheres. Separated from the influence of cortical actin and in the absence of
membrane traffic, multivalent clustering of raft lipids can amplify the functional level to a microscopic membrane
phase. Membrane constituents are laterally sorted according to preferences for membrane order and chemical
1 JANUARY 2010VOL 327
on January 4, 2010
lie raft clustering, at physiological temperature
they do not form a Lo phase when raft proteins
become included,andspecific lipid-protein inter-
actions must come into play to modify organi-
zation (Fig. 2C). Along these lines, a comparison
of lipid-packing in vesicles formed from lipids of
the plasma membrane versus the plasma mem-
brane itself reveals that lipid domain–forming
order is tightly regulated by the presence of pro-
Rafts as Entities of Physical and
Selective coordination of TM protein organiza-
tion suggests that cells functionalize lipid order–
based sorting by including another specificity,
most likely interactions involving proteins (45).
Cell membranes are crowded with membrane
proteins and their associated biases in regional
composition (49). Proteins can specifically orga-
nize the distribution of lipids, a property that
combines with sphingolipid-cholesterol assem-
blage potential to produce raft-based membrane
During vertical distortion of the bilayer, cer-
tain lipids of varying chain length are perturbed
by the protein surface to different extents via the
hydrophobic matching condition. Generally, it is
part of the protein is stiff with no appreciable
internal flexibility (50). However, by distorting
lipids in the vertical direction, it would be pos-
sible to counter mismatch. The lipid species best
adapted to the matching condition will have an
increasedprobabilityofbeing close totheprotein-
lipid interface (50). Defined as “wetting” (51), the
membrane protein surface is proposed to stabilize
a sterically favored lipid environment. Electron
spin resonance has identified a highly dynamic
selection of boundary lipids for a number of pro-
teins (52). However, some membrane proteins
retain tightly bound lipids, even in the detergent
solution present after purification (53). In such
cases, lipids may have defined binding sites,
where specific intercalation into protein structure
is achieved (54).
Because lipids must vertically complement
the rigid hydrophobic surface of the membrane
domain of integral membrane proteins, variation
in the protein boundary also has direct conse-
quences for the thickness and conformational
order of the bilayer (50). In model membranes,
long amphiphilic peptides order and thicken the
bilayer in the absence of cholesterol, whereas
shorter peptides offset the membrane order and
thickness induced by the presence of cholesterol
based increases in membrane thickness influence
the subcellular distribution of membrane proteins
(56). Conversely, changes in protein TM length
itself have been argued to be the thickness-
determining factor (57).
Heterogeneity at the protein boundary is in-
tensified by the fact that most membrane proteins
are oligomeric, acting in specific macromolecular
complexes to organize function (49). Superficially,
these complexes are a source of steric restrictions
and molecular crowding (49), but they can also
incorporate specific lipids as integral features of
their quaternary structure, thus functionally unit-
ing protein-protein and lipid-protein interactions
(54). Lipid incorporation is a function of specific
polar–head group interactions and hydrocarbon-
chain space-filling functions within the oligo-
meric complex (54). Many of these oligomeric
resistant to detergents, with the binding of cho-
lesterol to oligomers of caveolin-1 being a prom-
inent example (58).
In the raft field, we should be asking what it
means for proteins to be wetted or, as we define
the term in this context, “lubricated” (59), by
specific lipids or lipid environments, particularly
when it involves constituents that are important
components of heterogeneity by rafts. A number
of sphingolipid binding motifs have been de-
scribed for membrane proteins (60). We propose
that specific protein interaction with membrane-
ordering “raft lipids” provides a functionalizing
connection to the sphingolipid-cholesterol basis
for raft assembly (Fig. 3). Interestingly, cholera
toxin–cross-linking of GM1 was found to in-
crease the partitioning of the raft enzyme beta-
secretase to the Lo phase in giant unilamellar
does not reproduce the modestly ordered, TM
protein–inclusive raft structure of cell membranes.
Thus, the fact that an undefined specificity for
GSLs overcomes this stringent lateral sorting
condition suggests that the specific lubrication of
based heterogeneity. Under this scheme, the func-
tionalization of this heterogeneity depends on both
lipid physical parameters and specific interactions
that may include or even require proteins. For
example, the TM protein LATis an obligate com-
ponent of raft-based accumulation of membrane-
ordering lipids during the formation of the
tioned, membrane proteins work in functional
complexes, so it is not surprising that evolution
has crafted additional specificity to a lipid-based
minimal energy input. A cholesterol-binding
pocket, as well as six palmitate residues, has
recently been identified in the crystal structure of
the b2-adrenergic receptor dimer interface (61).
Palmitoylation of some membrane proteins has
been shown to enhance association with raft
nano-assemblies (62). Perhaps in the context of
forming functional protein oligomers, the propen-
sity of palmitate for raft association is augmented
the b2-adrenergic receptor harnesses this lipid to
connect with raft-based heterogeneity is not yet
Given the contribution of both physical and
chemical specificities to lateral selectivity in the
bilayer, lipid rafts are probably functionalized by
both lipid-lipid and specific protein-lipid inter-
actions. These lateral associations are governed
by both physical and chemical specificity. Lipid-
protein interactions alone cannot describe lipid
rafts (63), because these do not account for the
preferentially connecting lipid-lipid interactions
that have so convincingly been demonstrated in
model lipid membranes. Rather, we assert that
sphingolipid-cholesterol assemblage potential
forms a core raft connectivity that can be pre-
cisely modulated by protein specificity. In this
view, raft-based membrane heterogeneity cou-
ples specific chemistries of association to the
physical order preferences of lipids and proteins.
Moreover, the assembly of proteins into rafts may
be accompanied by conformational changes that
modify protein activity.
Rafts Inside the Cell
The propensity to form heterogeneity by rafts is
positively correlated with sterol content, which is
maximized in the plasma membrane (6, 64),
where the actin cortex also plays a central role in
influencing or organizing sphingolipid-cholesterol
assemblage potential (23, 25). However, for intra-
cellular membranes the situation is less clear. But
the emerging field of lipidomics is proving an
important tool in evaluating both surface and
intracellular membrane heterogeneity (38, 65).
Fig. 3. The lubrication of a raft TM protein by lipid. Membrane proteins bind and/or enrich certain
lipids through chemical and physical specificities. These lipids may themselves exhibit sphingolipid/
sterol assemblage potential. In this scheme, a TM raft protein (light blue) specifically interacts with
sterol and GSL, an interaction that lubricates its inclusion to and the assembly of functionalized
(coalesced) raft membrane.
VOL 327 1 JANUARY 2010
on January 4, 2010
The concentration of sphingolipids and sterols Download full-text
increases along the biosynthetic pathway from the
endoplasmic reticulum to the trans-Golgi network
of immunoisolated post-Golgi carriers has revealed
that order-forming sphingolipids, long and satu-
rated glycerophospholipids, and sterols are selec-
the TGN to the plasma membrane (66).
Thus, functional raft clustering probably under-
lies the lateral sorting of cell surface–destined
constituents within the TGN, in keeping with the
hypothesis of raft “phase” segregation principles
as a means to selective carrier formation.
Compositional Evolution of the
The cellularlipodome is theoretically made up of
9600 species ofglycerophospholipids;morethan
100,000 species of sphingolipids; thousands of
fatty-acid and sterol-based structures (67). This
amounts to an abundance of composition that
in the membrane. However, this is not the case.
potential in thermodynamically equilibrated plasma-
membrane spheres leads to demixing of only two
“phases” (P) as presently observed (44). The Gibbs
phase rule states that the number of de-mixed
entities (P) for a system at equilibrium is strictly
correlated with the number of chemically inde-
pendent components (C) by the expression P =
C – F + 2, where F is the number of
independently variable intensive properties (i.e.,
pressure, temperature, and mole fractions of
phase components). Thus, one could venture to
postulate that these physical segregation princi-
ples have guided the coevolution of both
membrane lipid and protein species, such that
instead of having the vast P complexity possible
from the phase rule, very few different P have
survived. This could be explainedbythefactthat
many components of the plasma membrane are
not chemically independent, often forming spe-
cific complexes to reduce the lateral dimension-
ality of function. How then can long-range
collective behavior arise from a chemically
cross-talking plasma membrane? The answer is
physicochemical teamwork. Activating the
sphingolipid-cholesterol assemblage potential
does not involve a purely physical phase transi-
tion with defined melting points and the like, but
rather the coalescence of raft membrane arises
through the functionally relevant cooperation of
physical order (from lipid hydrocarbon chains,
sterols, and the protein boundary) with specific
chemical interactions (between proteins and lip-
ids). In this sense, the cell appears to have de-
signed a membrane composition that manipulates
the physically selective behavior of lipids in a
chemically specific manner, enabling organized
heterogeneity to occur in the living condition.
The introduction of membrane-organizing
cholesterol seems to have coincided with the
evolution of multicellular complexity after the
oxidation of our atmosphere (68). This correla-
tion may imply that, in the pre-sterol era, other
chemical means of reducing lateral dimensionality
could have evolved. Interestingly, Caenorhabditis
elegans does not use sterol as a structural element
in its membranes (69). Principles of organized
heterogeneity in such organisms are unknown but
cal toolkit for membrane subcompartmentalization.
Cell membranes are complicated in composition
but precise in purpose: to selectively compart-
mentalize the constituents of life away from en-
vironmental lifelessness. Thus, it is not surprising
that membranes have innovated a means to later-
ally organize gatekeepers of this task. In living
cells, there is strong evidence for dynamic raft-
based membrane heterogeneity at the nanoscale,
which can be functionally coalesced to more sta-
ble membrane-ordered assemblies. At its core,
sphingolipid-cholesterol assemblage potential sup-
plies membranes with a subcompartmentalization
propensity that can be accessed or organized by
proteinaceous input at little energetic cost. Raft
proteins are envisioned as being equipped with a
dynamic sterol-sphingolipid–dependent bias in
composition at the nanoscale, allowing for the
partitioning to and assembly of more stable raft
platforms in the functionalized state. During raft
activation, protein-lipid interactions are coupled
to lipid-order–based sorting, generating hetero-
geneity serving to functionalize, focus, and coor-
dinate the bioactivity of membrane constituents.
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Simons lab for critical reading of this paper. A special thank
you goes to M. Surma, M. Gerl, and I. Levental for their
construction of and contribution to the figures. This work was
supported by European Union FP6 Lipid PRISM grant no.
LSHB-CT2007-037740, Deutsche Forschungsgemeinschaft
Schwerpunktprogramm 1175, and Bundesministerium für
Bildung und Forschung BioChance Plus grant no. 0313827.
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