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The Origin of Lipid Rafts

Steven L. Regen*

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ABSTRACT: The time-averaged lateral organization of the lipids and

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proteins that make up mammalian cell membranes continues to be the

subject of intense interest and debate. Since the introduction of the fluid
mosaic model almost 50 years ago, the “lipid raft hypothesis” has emerged as
a popular concept that has captured the imagination of a large segment of
the biomembrane community. In particular, the notion that lipid rafts play a
pivotal role in cellular processes such as signal transduction and membrane
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protein trafficking is now favored by many investigators. Despite the

attractiveness of lipid rafts, their composition, size, lifetime, biological
function, and even the very existence remain controversial. The central tenet
that underlies this hypothesis is that cholesterol and high-melting lipids have
favorable interactions (i.e., they pull together), which lead to transient
domains. Recent nearest-neighbor recognition (NNR) studies have
expanded the lipid raft hypothesis to include the influence that low-melting
lipids have on the organization of lipid membranes. Specifically, it has been found that mimics of cholesterol and high-melting lipids
are repelled (i.e., pushed away) by low-melting lipids in fluid bilayers. The picture that has emerged from our NNR studies is that
lipid mixing is governed by a balance of these “push and pull” forces, which maximizes the number of hydrocarbon contacts and
attractive van der Waals interactions within the membrane. The power of the NNR methodology is that it allows one to probe these
push/pull interaction energies that are measured in tens of calories per mole.

■ THE LIPID RAFT HYPOTHESIS

Despite a wealth of information that is currently available on the
composition and dynamic properties of mammalian cell
membranes, the time-averaged lateral organization of the lipids
and proteins that make up these natural enclosures remains
controversial.1−4 A concept that has captured the imagination of
many researchers in this area is that high-melting lipids (i.e.,
lipids having a gel to liquid-crystalline phase transition
temperature, Tm, greater than 37 °C) associate with cholesterol
to form transient domains termed “lipid rafts”.5,6 This Figure 1. Stylized illustration of a raft “phase” coexisting with a nonraft
association appears to be the result of attractive van der Waals region. (Reprinted from ref 9.)
interactions between these two molecules.7 It has also been
posited that lipid rafts play a pivotal role in cellular processes
involving signal transduction and membrane protein traffick- studies of analogous bilayers via fluorescence lifetime, differ-
ing.8 A stylized illustration of a hypothetical lipid raft region ential scanning calorimetry,2H NMR, and Forster resonance
coexisting with a nonraft region is shown in Figure 1.9 energy transfer measurements have yielded additional support

■
for cholesterol favoring association with high-melting lip-
CHOLESTEROL’S AFFINITY TOWARD ids.12−15 Related experiments that we have carried out ourselves
HIGH-MELTING LIPIDS
A large body of evidence now exists that shows that cholesterol Received: October 21, 2020
favors association with high-melting lipids over low-melting Revised: November 13, 2020
lipids (i.e., lipids having Tm values less than 37 °C). This Published: November 23, 2020
evidence can be traced back to early monolayer studies that were
carried out at the air/water interface where discrete cholesterol−
phospholipid complexes were hypothesized.10,11 Subsequent

© 2020 American Chemical Society https://dx.doi.org/10.1021/acs.biochem.0c00851

4617 Biochemistry 2020, 59, 4617−4621
Biochemistry pubs.acs.org/biochemistry
support a “hydrophobic contact mechanism” as a basis for this
affinity.7,9 Specifically, our results support a model in which the
flexible, saturated acyl chains of high-melting lipids establish a
large number of hydrocarbon contacts with the flat and rigid
cholesterol molecule, thereby producing strong attractive van
der Waals interactions.7 In contrast, low-melting phospholipids,
which bear one or more “permanent kinks” (i.e., cis-double
bonds), have a weaker association with cholesterol because they
are unable to create as many hydrocarbon contacts.7
■ EARLY STUDIES INDICATING THE EXISTENCE OF
REPULSIVE FORCES BETWEEN CHOLESTEROL AND
LOW-MELTING LIPIDS
Using a fluorescence-quenching strategy, it has been shown that
low-melting lipids enhance the stability of liquid-ordered
bilayers derived from cholesterol and high-melting lipids such
as 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC).16
Although such experiments do not provide quantitative insight
into lipid−lipid interactions, they imply that low-melting lipids
repel cholesterol and/or DPPC.
In a separate investigation that used FRET measurements and
Monte Carlo simulations to study the interactions among
porcine brain sphingomyelin (SM), cholesterol (Chol), and 1-
palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), it
was concluded that SM/Chol interactions are attractive while
both SM/POPC and Chol/POPC interactions are weakly
repulsive.17
■ NEAREST-NEIGHBOR RECOGNITION
MEASUREMENTS
In an effort to obtain a quantitative assessment of lipid−lipid
interactions in fluid bilayers, we devised a chemical method that
is based on the use of exchangeable, disulfide-based lipid
dimers.9 The essence of this method is illustrated in Figure 2.
Figure 2. Illustration of the nearest-neighbor recognition (NNR)
method. Here, the solid rectangles represent an exchangeable
cholesterol mimic, and the large filled circles with two wavy lines
represent an exchangeable phospholipid.
Thus, a heterodimer, AB, is incorporated in a host membrane
that is derived from (i) low-melting phospholipids and is
maintained in the “liquid-disordered” state or (ii) high-melting
phospholipids plus cholesterol that is maintained in the “liquid-
ordered” state; i.e., a state that is thought to mimic lipid rafts.
Partial reduction with dithiothreitol (DTT) then releases
monomers that undergo exchange with residual dimers via the
thiolate-disulfide interchange. The dimer distribution is then
monitored by high-performance liquid chromatography until
chemical equilibrium has been reached. This equilibrium is
defined by AA + BB ⇌ 2 AB. When an equilibrium constant (K)
for the exchange is found to equal 4, this indicates that A and B
are mixing ideally; i.e., the monomers are randomly distributed,
and the heterodimer, AB, is statistically favored over each of the
4618
Perspective
homodimers, AA and BB, by a factor 2. When values of K deviate
from 4, this is a measure of what we have termed nearest-
neighbor recognition (NNR). Thus, when K is greater than 4,
unlike-nearest-neighbors (i.e., heterodimers) are favored. When
K is less than 4, like-nearest-neighbors (i.e., homodimers) are
favored. The net free energy of the interaction between A and B,
ωAB, is then given by ωAB = −1/2RTln(K/4).9 Those values of
ωAB that are greater than 0 cal per mol of phospholipid are
considered to be repulsive, which means that unlike lipids prefer
to separate from each other. Values of ωAB that are less than 0 cal
per mol of phospholipid are considered to reflect attractive
interactions between unlike nearest-neighbors.
■ EXCHANGEABLE LIPID MIMICS
Chart 1 shows the high-melting DPPC molecule (Tm = 41.5 °C)
and cholesterol, along with their exchangeable mimics Phos (Tm
Chart 1. Structures of DPPC, Cholesterol, and Exchangeable
Mimics
= 41.9 °C) and Chol, respectively.18,19 Chart 2 shows three low-
melting exchangeable lipids, c1-Phos, c2-Phos, and c3-Phos,
which we have investigated in bilayers derived from 1-palmitoyl-
2-dihydrosterculoyl-sn-glycero-3-phosphocholine (PDSPC).20
Experimental evidence that supports the claim that these
exchangeable lipids are excellent mimics of cholesterol and the
corresponding phosphocholines has been obtained from
differential scanning calorimetry, surface-pressure area isotherm,
fluorescence, and nearest-neighbor recognition measure-
ments.9,21
As discussed elsewhere, cis-double bonds undergo cis/trans
isomerization under NNR reaction conditions.22 This isomer-
ization appears to be due to the generation of small amounts of
thiyl radicals and their reversible addition to the double bonds of
phospholipids.23−25 To circumvent this complexity, we have
used cis-cyclopropyl moieties as a means of introducing
permanent “kinks” in the acyl chains to create low-melting
exchangeable lipids. For example, PDSPC has a low Tm value of
−10 °C, which is similar to the Tm value of POPC of −3 °C.26
https://dx.doi.org/10.1021/acs.biochem.0c00851
Biochemistry 2020, 59, 4617−4621
Biochemistry pubs.acs.org/biochemistry
Chart 2. Structures of Low-Melting Exchangeable
Phospholipids
■
QUANTIFYING ATTRACTIVE AND REPULSIVE
FORCES
To assess the interactions between Phos and Chol in
membranes that mimic lipid rafts, we used host membranes
made from DPPC and cholesterol (60:40, mol/mol). At 45 °C,
an attractive force was found that corresponded to ωAB = −260
cal/mol of phospholipid.19 In sharp contrast, related NNR
measurements that were carried out in bilayers made from
PDSPC revealed net repulsive interactions between Chol and
the low-melting lipids, c1-Phos, c2-Phos, and c3-Phos (Figure
3).20 In addition, the magnitude of these repulsive interactions
Figure 3. Net free energies of the interaction between Chol and the
low-melting lipids, c1-Phos, c2-Phos, and c3-Phos, in host membranes
made from PDSPC at 45 °C. Also shown is the net free energy of the
interaction between Chol and Phos in host membranes made from
PDSPC at 45 °C.20
was found to increase as the number of permanent kinks
increased. It is noteworthy that the magnitude of the repulsive
forces between Chol and these low-melting lipids was virtually
identical with those of Phos interacting with these same low-
melting lipids (Figure 3). Since the net free energy of interaction
between Chol and Phos in bilayers of PDSPC is essentially 0 cal/
mol, these findings indicate that Chol and Phos have similar
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Perspective
attractive and repulsive forces in fluid bilayers made from
PDSPC (Figure 3).
■ THE ORIGIN OF LIPID RAFT FORMATION
The picture that has emerged from our NNR studies is that lipid
mixing in fluid bilayers is governed by “push and pull” forces.
More specifically, high-melting lipids favor binding to flat, rigid
cholesterol molecules by using their flexible, saturated acyl
chains to make a high number of hydrocarbon contacts with
them.7 For low-melting lipids, the number of hydrocarbon
contacts that can be made with this sterol is significantly reduced
due to permanent kinks in their acyl chains. Despite this
reduction in hydrocarbon contact, it is sufficient for cholesterol
to exert its long-studied condensing effect, as evidenced by
monolayer measurements.27 Thus, in bilayers containing
cholesterol, high-melting lipids, and low-melting lipids, the
number of hydrocarbon contacts and attractive van der Waals
interactions are maximized by having the low-melting lipids
push away cholesterol and also the high-melting lipids, thereby
allowing them to pull together to form less fluid domains, i.e.,
lipid rafts (Figure 4). At the same time, this increased
Figure 4. Illustration summarizing the “push and pull” forces among
cholesterol, low-melting lipids, and high-melting lipids that drive the
formation of lipid rafts.
hydrocarbon contact minimizes unfavorable exposure of the
hydrophobic portion of the lipids toward water. In other words,
both van der Waals interactions and the hydrophobic effect
contribute to the formation of lipid rafts.
■ THE HIDDEN ROLE THAT POLYUNSATURATED
LIPIDS ARE LIKELY TO PLAY IN FORMING LIPID
RAFTS
Although substantial attention has focused on favorable
interactions between high-melting lipids and cholesterol in
forming lipid rafts over the past 20 years, relatively little
attention has been paid to the role that low-melting lipids may
play. Given the abundance of polyunsaturated lipids in
mammalian cell membranes and the fact that repulsive
interactions increase with increasing numbers of permanent
kinks, it appears likely that the repulsive interactions that
cholesterol and high-melting lipids experience with polyunsatu-
rated lipids are a major driving force for lipid raft formation.28−33
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Biochemistry 2020, 59, 4617−4621
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■ ■
pubs.acs.org/biochemistry
PUSH AND PULL FORCES AS A MEASURE OF LIPID
MISCIBILITY
In this Perspective, I have referred to values of ωAB that are
greater than 0 cal per mol of phospholipid as being repulsive
(representing a “push”) and values of ωAB that are less than 0 cal
per mol of phospholipid as being attractive (representing a
“pull”). An alternative way of thinking about these push and pull
forces is to consider them as a measure of miscibility where like-
attracts-like. Thus, a value of ωAB that is strongly positive reflects
two lipids that are dissimilar and do not want to become nearest-
neighbors. In other words, they are immiscible at the molecular
and at the supramolecular level. In contrast, a value of ωAB that is
strongly negative reflects two different lipids that want to
become nearest-neighbors and are miscible. Figure 5 shows how
Figure 5. Snapshots of Monte Carlo simulations of mixtures of Chol
with (A) c1-Phos and (B) c3-Phos. The mixtures contain 40% Chol
(red) and 60% of c1-Phos (black) or c3-Phos (black). The total number
of lipids in each snapshot is 10,000. (Reprinted from ref 34).
two different repulsive forces can influence lipid mixing on a
macroscopic scale. Here, snapshots of Monte Carlo simulations
are shown for 40:60 (mol/mol) mixtures of Chol/c1-Phos and
Chol/c3-Phos. These simulations were based, directly, on our
nearest-neighbor recognition measurements. Each snapshot
encompasses a total of 10,000 lipids. In the case of Chol and c1-
Phos, where ωAB = +160 cal/mol, the two lipids are found to mix
very well. However, a comparison of this snapshot with a similar
Monte Carlo snapshot that was based on a value of ωAB = 0 cal/
mol indicates that this mixing is not ideal (not shown).34 In the
case of Chol and c3-Phos, where ωAB = +430 cal/mol, the mixing
of the two lipids is clearly very poor.34 It is noteworthy, in this
regard, that small-angle neutron scattering experiments have
revealed that lipid domain size can increase with the extent of
acyl chain unsaturation.35
■ CONCLUSIONS
Although the composition, size, lifetime, and biological function
of lipid rafts remain to be defined, the nearest-neighbor
recognition measurements that I have described in this
Perspective provide a basis for their likely existence. Specifically,
these measurements have revealed the presence of push and pull
forces that can maximize attractive van der Waals forces within a
membrane. Based on the extreme sensitivity of the nearest-
neighbor recognition method, which allows for interaction
energies to be measured in tens of calories per mole, I believe
that, with careful molecular design, it could also provide unique
insight into lipid-membrane protein interactions.36
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Perspective
AUTHOR INFORMATION
Corresponding Author
Steven L. Regen − Department of Chemistry, Lehigh University,
Bethlehem, Pennsylvania 18015, United States; orcid.org/
0000-0001-6192-7916; Email: slr0@lehigh.edu
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.biochem.0c00851
Notes
The author declares no competing financial interest.
■ ACKNOWLEDGMENTS
I am most grateful to my co-workers who are listed in our
publications that are cited in this Perspective. I am also grateful
to the National Institutes of Health (PHS GM56149) and the
National Science Foundation (CHE-1145500) for their
financial support, which has made our research in this area
and this Perspective possible.
■ REFERENCES
(1) Singer, S., and Nicolson, G. (1972) The fluid mosaic model of the
structure of cell membranes. Science 175, 720−731.
(2) Engelman, D. M. (2005) Membranes are more mosaic than fluid.
Nature 438, 578−580.
(3) Levental, I., Levental, K. R., and Heberle, F. A. (2020) Lipid rafts:
controversies resolved, mysteries remain. Trends Cell Biol. 30 (5), 341−
353.
(4) Levental, I. (2020) Lipid rafts come of age. Nat. Rev. Mol. Cell Biol.
21, 420.
(5) Simons, K., and Ikonen, E. (1997) Functional rafts in cell
membranes. Nature 387 (6633), 569−572.
(6) Ahmed, S. N., Brown, D. A., and London, E. (1997) On the origin
of sphingolipid/cholesterol-rich detergent-insoluble cell membranes:
physiological concentrations of cholesterol and sphingolipid induce
formation of a detergent-insoluble, liquid-ordered phase in model
membranes. Biochemistry 36, 10944−10953.
(7) Daly, T. A., Wang, M., and Regen, S. L. (2011) The origin of
cholesterol’s condensing effect. Langmuir 27, 2159−2161.
(8) Lingwood, D., and Simons, K. (2010) Lipid rafts as a membrane-
organizing principle. Science 327, 46−50.
(9) Krause, M. R., and Regen, S. L. (2014) The structural role of
cholesterol in cell membranes: from condensed bilayers to lipid rafts.
Acc. Chem. Res. 47, 3512−3521.
(10) Radhakrishnan, A., and McConnell, H. M. (1999) Cholesterol-
phospholipid complexes in membranes. J. Am. Chem. Soc. 121, 486−
487.
(11) Radhakrishnan, A., and McConnell, H. M. (1999) Condensed
complexes of cholesterol and phospholipids. Biophys. J. 77, 1507−1517.
(12) Radhakrishnan, A., and McConnell, H. M. (2005) Condensed
complexes in vesicles containing cholesterol and phospholipids. Proc.
Natl. Acad. Sci. U. S. A. 102, 12662−12666.
(13) Engberg, O., Hautala, V., Yasuda, T., Dehio, H., Murata, M.,
Slotte, J. P., and Nyholm, T. K.M. (2016) The affinity of cholesterol for
different phospholipids affects lateral segregation in bilayers. Biophys. J.
111, 546−556.
(14) Nyholm, T. K. M., Engberg, O., Hautala, V., Tsuchikawa, H., Lin,
K.-L., Murata, M., and Slotte, J. P. (2019) Impact of acyl chain
mismatch on the formation and properties of sphingomyelin-
cholesterol domains. Biophys. J. 117, 1577−1588.
(15) Engberg, O., Lin, K.-L., Hautala, V., Slotte, J. P., and Nyholm, T.
K. M. (2020) Sphingomyelin acyl chains influence the formation of
sphingomyelin- and cholesterol-encriched domains. Biophys. J. 119,
913−923.
(16) Bakht, O., Pathak, P., and London, E. (2007) Effect of the
structure of lipids favoring disordered domain formation on the stability
https://dx.doi.org/10.1021/acs.biochem.0c00851
Biochemistry 2020, 59, 4617−4621
Biochemistry pubs.acs.org/biochemistry Perspective

of cholesterol-containing ordered domains (lipid rafts): identification (35) Heberle, F. A., Petruzielo, R. S., Pan, J., Drazba, P., Kuceerka, N.,
of multiple raft-stabilization mechanisms. Biophys. J. 93, 4307−4318. Standaert, R. F., Feigenson, G. W., and Katsaras, J. (2013) Bilayer
(17) Frazier, M. L., Wright, J. R., Pokorny, A., and Almeida, P. F. F. thickness mismatch controls domain size in model membranes. J. Am.
(2007) Investigation of domain formation in sphingomyelin/ Chem. Soc. 135, 6853−6859.
cholesterol/popc mixtures by fluorescence resonance energy transfer (36) Huster, D. (2014) Solid-state NMR spectroscopy to study
and monte carlo simulations. Biophys. J. 92, 2422−2433. protein-lipid interactions. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids
(18) Krisovitch, S. M., and Regen, S. L. (1992) Nearest-neighbor 1841, 1146−1160.
recognition in phospholipid membranes: a molecular-level approach to
the study of membrane suprastructure. J. Am. Chem. Soc. 114, 9828−
9835.
(19) Turkyilmaz, S., Almeida, P. F., and Regen, S. L. (2011) Effects of
isoflurane, halothane and chloroform on the interactions and lateral
organization of lipids in the liquid-ordered phase. Langmuir 27, 14380−
14385.
(20) Wang, C., Yu, Y., and Regen, S. L. (2017) Lipid raft formation:
key role of polyunsaturated phospholipids. Angew. Chem., Int. Ed. 56,
1639−1642.
(21) Mukai, M., and Regen, S. L. (2015) Exchangeable mimics of dppc
and dppg exhibiting similar nearest-neighbor interactions in fluid
bilayers. Langmuir 31, 12674−12678.
(22) Dewa, T., Vigmond, S. J., and Regen, S. L. (1996) Lateral
heterogeneity in fluid bilayers composed of saturated and unsaturated
phospholipids. J. Am. Chem. Soc. 118, 3435−3440.
(23) Chatgilialoglu, C., Ferreri, C., Ballestri, M., Mulazzani, Q. G., and
Landi, L. (2000) cis-trans Isomerization of monounsaturated fatty acid
residues in phospholipids by thiyl radicals. J. Am. Chem. Soc. 122, 4593−
4601.
(24) Ferreri, C., Costantino, C., Perrotta, L., Landi, L., Mulazzani, Q.
G., and Chatgilialoglu, C. (2001) cis-trans Isomerization of poly-
unsaturated fatty acid residues in phospholipids catalyzed by thiyl
radicals. J. Am. Chem. Soc. 123, 4459−4468.
(25) Ferreri, C., Samadi, A., Sassatelli, F., Landi, L., and Chatgilialoglu,
C. (2004) Regioselective cis-trans isomerization of arachidonic double
bonds by thiyl radicals: the influence of phospholipid supramolecular
organization. J. Am. Chem. Soc. 126, 1063−1072.
(26) Koynova, R., and Caffrey, M. (1998) Phases and phase
transitions of the phosphatidylcholines. Biochim. Biophys. Acta, Rev.
Biomembr. 1376, 91−145.
(27) Jurak, M. (2013) Thermodynamic aspects of cholesterol effect
on properties of phospholipid monolayers: langmuir and langmuir-
blodgett monolayer study. J. Phys. Chem. B 117, 3496−3501.
(28) Kucerka, N., Marquardt, D., Harroun, T. A., Nieh, M.-P., Wassall,
S. R., de Jong, D. H., Schafer, L. V., Marrink, S. J., and Katsaras, J. (2010)
Cholesterol in bilayers with pufa chains: doping with dmpc or popc
results in sterol reorientation and membrane-domain formation.
Biochemistry 49, 7485−7493.
(29) Shaikh, S. R., Kinnun, J. J., Leng, X., Williams, J. A., and Wassall, S.
R. (2015) How polyunsaturated fatty acids modify molecular
organization in membranes: insight from NMR studies of model
systems. Biochim. Biophys. Acta, Biomembr. 1848, 211−219.
(30) Lin, X., Lorent, J. H., Skinkle, A. D., Levental, K. R., Waxham, M.
N., Gorfe, A. A., and Levental, I. (2016) Domain stability in biomimetic
membranes driven by lipid polyunsaturation. J. Phys. Chem. B 120,
11930−11941.
(31) Levental, K. R., Lorent, J. H., Lin, X., Skinkle, A. D., Surma, M. A.,
Stockenbojer, E. A., Gorfe, A. A., and Levental, I. (2016)
Polyunsaturated lipids regulate membrane domain stability by tuning
membrane order. Biophys. J. 110, 1800−1810.
(32) Bennett, W. F. D., Shea, J.-E., and Tieleman, D. P. (2018)
Phospholipid chain interactions with cholesterol drive domain
formation in lipid membranes. Biophys. J. 114, 2595−2605.
(33) Engberg, O., Hautala, V., Yasuda, T., Dehio, H., Murata, M.,
Slotte, P. J., and Nyholm, T. K. M. (2016) (2016) The affinity of
cholesterol for different phospholipids affects lateral segregation in
bilayers. Biophys. J. 111, 546−556.
(34) Wang, C., Almeida, P. F., and Regen, S. L. (2018) Net
interactions that push cholesterol away from unsaturated phospholipids
are driven by enthalpy. Biochemistry 57, 6637−6643.

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Biochemistry 2020, 59, 4617−4621