Article

Baumgart, T. et al. Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles. Proc. Natl Acad. Sci. USA 104, 3165-3170

Department of Chemistry and Chemical Biology, Cornell University, Итак, New York, United States
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/2007; 104(9):3165-70. DOI: 10.1073/pnas.0611357104
Source: PubMed
ABSTRACT
The membrane raft hypothesis postulates the existence of lipid bilayer membrane heterogeneities, or domains, supposed to be important for cellular function, including lateral sorting, signaling, and trafficking. Characterization of membrane lipid heterogeneities in live cells has been challenging in part because inhomogeneity has not usually been definable by optical microscopy. Model membrane systems, including giant unilamellar vesicles, allow optical fluorescence discrimination of coexisting lipid phase types, but thus far have focused on coexisting optically resolvable fluid phases in simple lipid mixtures. Here we demonstrate that giant plasma membrane vesicles (GPMVs) or blebs formed from the plasma membranes of cultured mammalian cells can also segregate into micrometer-scale fluid phase domains. Phase segregation temperatures are widely spread, with the vast majority of GPMVs found to form optically resolvable domains only at temperatures below approximately 25 degrees C. At 37 degrees C, these GPMV membranes are almost exclusively optically homogenous. At room temperature, we find diagnostic lipid phase fluorophore partitioning preferences in GPMVs analogous to the partitioning behavior now established in model membrane systems with liquid-ordered and liquid-disordered fluid phase coexistence. We image these GPMVs for direct visual characterization of protein partitioning between coexisting liquid-ordered-like and liquid-disordered-like membrane phases in the absence of detergent perturbation. For example, we find that the transmembrane IgE receptor FcepsilonRI preferentially segregates into liquid-disordered-like phases, and we report the partitioning of additional well known membrane associated proteins. Thus, GPMVs now provide an effective approach to characterize biological membrane heterogeneities.

Full-text

Available from: David Holowka
Large-scale fluid/fluid phase separation of proteins
and lipids in giant plasma membrane vesicles
Tobias Baumgart*, Adam T. Hammond
, Prabuddha Sengupta
, Samuel T. Hess
, David A. Holowka
, Barbara A. Baird
,
and Watt W. Webb*
§
*School of Applied and Engineering Physics and
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853; and
Department of Physics and Astronomy, University of Maine, Orono, ME 04469
Contributed by Watt W. Webb, December 26, 2006 (sent for review September 21, 2006)
The membrane raft hypothesis postulates the existence of lipid
bilayer membrane heterogeneities, or domains, supposed to be
important for cellular function, including lateral sorting, signaling,
and trafficking. Characterization of membrane lipid heterogene-
ities in live cells has been challenging in part because inhomoge-
neity has not usually been definable by optical microscopy. Model
membrane systems, including giant unilamellar vesicles, allow
optical fluorescence discrimination of coexisting lipid phase types,
but thus far have focused on coexisting optically resolvable fluid
phases in simple lipid mixtures. Here we demonstrate that giant
plasma membrane vesicles (GPMVs) or blebs formed from the
plasma membranes of cultured mammalian cells can also segregate
into micrometer-scale fluid phase domains. Phase segregation
temperatures are widely spread, with the vast majority of GPMVs
found to form optically resolvable domains only at temperatures
below 25°C. At 37°C, these GPMV membranes are almost exclu-
sively optically homogenous. At room temperature, we find diag-
nostic lipid phase fluorophore partitioning preferences in GPMVs
analogous to the partitioning behavior now established in model
membrane systems with liquid-ordered and liquid-disordered fluid
phase coexistence. We image these GPMVs for direct visual char-
acterization of protein partitioning between coexisting liquid-
ordered-like and liquid-disordered-like membrane phases in the
absence of detergent perturbation. For example, we find that the
transmembrane IgE receptor Fc
RI preferentially segregates into
liquid-disordered-like phases, and we report the partitioning of
additional well known membrane associated proteins. Thus,
GPMVs now provide an effective approach to characterize biolog-
ical membrane heterogeneities.
liquid-disordered liquid-ordered membrane domains
membrane heterogeneity rafts
T
he membrane raft hypothesis proposes that membrane lipid
domains enriched in cholesterol and sphingolipids form
dynamic sorting and signaling platforms for membrane proteins
(1). Consensus data on the size, lifetime, and even existence of
lipid rafts in intact plasma membranes, however, have been
elusive (2, 3). Controversy arises due to current experiment al
ambiguit y and to operational defin itions of membrane lipid
heterogeneit y.
One common definition has relied on biochemical assessment
of membrane heterogeneity involving membrane lysis using
cert ain nonion ic detergents, such as Triton X-100 at 4°C (4). A
sec ond, operational definition for lipid raf ts has been cellular
processes that are perturbed by either cholesterol or sphingo-
myelin depletion. Problems of these approaches have been
extensively discussed (2, 3).
The reports that membranes composed of lipids that are
resistant to detergent solubilization are apparently in a liquid-
ordered (L
o
) state (5–7) have been the basis for proposing that
lipid rafts in plasma membranes represent the L
o
phase of
c oexisting L
o
and liquid-disordered (L
d
) fluid/fluid phases (2, 8).
Biological membrane heterogeneity, however, cannot be re-
duced to partitioning between c oexisting lipid domains, because
reciprocal protein–lipid, as well as protein–protein interactions
necessarily modulate the thermodynamics of lipid heterogeneity
(2, 9, 10), emphasizing the need for examining membranes with
sign ificant protein content, as suggested by protein segregation
measurements on living cells (11).
Several studies have suggested the existence of membrane
heterogeneities with size scales below optical resolution (300
nanometers), but estimates of lipid raft sizes in the literature
range from several micrometers, as in cholesterol-depleted cells
(12), to just a few molecules (13). Fluid/fluid phase separation
in biological cell membranes has thus far not been unequivocally
demonstrated in live cells, perhaps because coupling of the
plasma membrane to the underlying c ytoskeleton may prevent
fluid phase segregation into distinctive domains, as can be
observed in lipid-only model membranes with appropriate com-
positions. Differences from model membranes could arise from
the pinning of plasma membrane domains by c ytoskeleton-
att ached, membrane-associated proteins or from complex mem-
brane rugosities induced by protein–protein interactions. Fur-
ther more, the enor mous c omplex it y of cellular plasma
membrane protein and lipid compositions might preclude the
simple fluid/fluid phase separations observed in ternary lipid
mixtures (14).
To investigate fluid/fluid phase coexistence in systems with
realistic biological membrane compositions, we here use giant
plasma membrane vesicles (GPMVs) that are derived from
biological cells, RBL mast cells, and fibroblasts by chemically
induced plasma membrane vesiculation or ‘‘blebbing’’ (15–17).
These GPMVs show simple, low-curvature geometries of giant
un ilamellar vesicles and appear free of cytoskeletal constraints.
By using fluorophores providing recently characterized mem-
brane fluid phase partitioning behavior (T.B., G. Hunt, E. R.
Fark as, W.W.W., and G. W. Feigenson, unpublished data) for
lipid phase characterization here, we find that these GPMVs can
segregate into multi-micrometer-scale coexisting fluid phases,
thereby enabling extension of early cell blebbing studies in our
laboratories in the 1980s (15, 18–20). These results are noted and
used throughout this paper.
Results
In initial experiments to study phase separation in plasma
membrane-derived, micrometer-scale vesicles, mammalian cells
Author contributions: T.B., S.T.H., and W.W.W. initiated research; T.B. P.S., S.T.H., and
A.T.H. performed research; T.B., A.T.H., P.S., S.T.H., D.A.H., and B.A.B. analyzed data;
and T.B., D.A.H., B.A.B., and W.W.W. wrote the paper.
The authors declare no conflict of interest.
Abbreviations: CM, laser scanning confocal microscopy; CTB, cholera toxin subunit B;
GPMV, giant plasma membrane vesicle; GUV, giant unilamellar vesicle; L
d
, liquid-disor-
dered; L
o
, liquid-ordered; Nap, naphthopyrene; R-DOPE, rhodamine B sulfonyl dioleoyl
phosphatidyl ethanolamine.
§
To whom correspondence should be addressed at: Cornell University, 212 Clark Hall,
Ithaca, NY 14853. E-mail: www2@cornell.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0611357104/DC1.
© 2007 by The National Academy of Sciences of the USA
www.pnas.orgcgidoi10.1073pnas.0611357104 PNAS
February 27, 2007
vol. 104
no. 9
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BIOPHYSICS
Page 1
were treated with combinations of polar organ ic solvents to
induce GPMV formation and selected membrane dyes to label
lipid phases. Fig. 1 illustrates t ypical fluorescence-labeled cell-
att ached GPMVs imaged by stacks of laser scanning confocal
microsc opy (CM) or two-photon microscopy sections. The
GPMV in Fig. 1A was induced by addition of 4% (vol/vol)
ethanol to suspended RBL cells, and GPMV in Fig. 1B was
induced by addition of DMSO (1% vol/vol) to adherent fibro-
blasts. Under these c onditions, substantial membrane dye is
rapidly internalized, delivering fluorescence to the cell body, as
illustrated in Fig. 1 A, C, and D. These GPMVs, having dimen-
sions comparable to cell sizes, occur in low fractions (5%) of
treated cells and are possibly induced by transient, local high
solvent concentration during solvent injections into the sur-
rounding buffer. The cell-attached GPMV on an RBL cell in Fig.
1 A was prelabeled by the plasma membrane inner-leaflet-
t argeted protein, geranylgeranyl-EGFP (21), and it shows a
homogenous membrane bilayer at optical resolution when im-
aged at room temperature. Fig. 1B shows a lipid phase-separated
GPMV from NIH 3T3 fibroblasts labeled with lissamine rhoda-
mine B sulfonyl dioleoyl phosphatidyl ethanolamine (R-DOPE)
to demonstrate that plasma membrane fluid/fluid phase sepa-
ration at room temperature is not a unique property of GPMVs
obt ained from RBL mast cells.
At temperatures of 4–10°C, cell-attached GPMV membranes
of ten separate into two distinct phases, as shown by the con-
trasting fluorescence patterns of naphthopyrene (Nap) in Fig. 1C
and R-DOPE in Fig. 1D. That these coex isting domains are in
fluid phase states at 4°C is indicated by frequent coalescence
activity and circular morphology. The fluid phase separations
shown in Fig. 1 C and D, as well as all other phase-separated
GPMVs examined here, show only two dif ferent fluorescence
intensities, suggesting that the lipid domains are c oupled across
the bilayer, as previously observed for inner- and outer-leaflet-
labeled domain registration in phase-separated model mem-
brane giant unilamellar vesicles (GUVs) (22).
Nap preferentially labels the L
o
phase in model membranes
containing ternary lipid mixtures of brain sphingomyelin, dioleoyl
phosphatidylcholine, and cholesterol, whereas R-DOPE strongly
prefers L
d
membranes coexisting with L
o
domains over wide range s
of differing lipid compositions (T.B., G. Hunt, E. R. Farkas,
W.W.W., and G. W. Feigenson, unpublished data). Thus, GPMVs
from RBL cells appear to phase-separate into coexisting fluid
membrane phase s with L
o
-like and L
d
-like phase state s. Because
membrane phase behavior is likely to be modified in the presence
of organic solvents, we also developed alternative protocols for
GPMV formation and membrane staining.
Membrane blebbing induced by chemical methods that modify
amino and sulfhydryl groups (15, 17, 23) allows isolating large
numbers of GPMVs f rom adhering cells for characterization (15,
24). GPMVs isolated after treatment of RBL mast cells with 25
mM for maldehyde, together with 2 mM DTT, or by treatment
with2mMN-ethylmaleimide for1hat37°C phase separate in
a temperature-dependent manner. Represent ative low-
magn ification views of cell-free GPMVs isolated after formal-
dehyde/DTT treatment and labeled w ith the L
d
phase-preferring
fluorescent phospholipid R-DOPE were imaged at three differ-
ent temperatures [supporting information (SI) Fig. 4]. At 5°C,
essentially all vesicles phase separate (SI Fig. 4A), but at room
temperature only 10–25% of all vesicles show large-scale
fluid/fluid phase coex istence (SI Fig. 4B). At 37°C, fluid phase
c oexistence is rare: 1% of all imaged GPMVs show optically
resolvable domains. One of these exceptional cases is depicted in
SI Fig. 4C Inset. The large variation in phase separation observed
in different vesicles, especially at intermediate temperatures,
may indicate compositional variations among GPMVs in these
preparations.
Fig. 2 compares the fluid-phase partitioning behavior of
several lipid analogues (Nap, R-DOPE, and DiI C16:0) and
lipid-binding proteins [cholera toxin subunit B (CTB) and
annexin V] in cell-free GPMVs imaged at room temperature
(Fig. 2 AC) and at 12°C (Fig. 2D). Nap and R-DOPE show
c ontrasting partitioning in coex isting fluid phases, both at room
temperature as shown in Fig. 2 A and at 5°C (Fig. 1 C and D). Fig.
2B shows that CTB bound to the ganglioside GM
1
partitions into
membrane regions complementary to those labeled with L
d
-
preferring R-DOPE. CTB is frequently used as a marker for
cellular lipid rafts and caveolae based on sucrose gradient
f ractionation experiments (25), and GUV model membrane
studies have shown that fluorescent CTB bound to GM
1
pref-
erentially labels L
o
phases that are coexisting with L
d
phases (26,
27). Partitioning of CTB bound to GM
1
, in contrast to DiI C16:0,
indicates that DiI preferentially partitions into the L
d
-like phase
(Fig. 2C). Although DiI C16:0 is deemed a marker for lipid rafts
on cells (12, 28, 29), it has been observed to partition preferen-
tially into L
d
phases in selected ternary mixtures of brain
sphingomyelin/dioleoyl phosphatidylcholine/cholesterol and in
mixtures of distearoyl phosphatidylcholine/dioleoyl phosphati-
dylcholine/cholesterol (data not shown).
We compared the partitioning of CTB bound to GM
1
with
fluorescent annexin V, which binds phosphatidylserine (PS) in a
calcium-dependent manner (30). A nnexin V is an indicator of
transbilayer flipping of the negatively charged PS that is a
hallmark of cellular apoptosis (31). In phase-separated GPMVs,
annexin V labeling is in contrast to L
o
-like domains that are
preferentially labeled with fluorescent CTB (Fig. 2D). This
indicates that PS, and possibly other negatively charged phos-
pholipids, partition preferentially into more disordered regions
of these GPMVs. Noting that phase separation in vesicles labeled
with fluorescent CTB and annexin V derivatives is similar to
lipid-bound fluorophores (R-DOPE, DiIC16:0, and Nap) ex-
cludes the possibility that the fluid phase segregation we detect
is due to membrane insertion of fluorescent lipids or to photo-
dec omposition products of membrane-embedded fluorophores.
Fig. 1. Cell-attached GPMVs can laterally segregate into coexisting fluid
phases. (A, C, and D) RBL cells treated with 4% (vol/vol) ethanol. Cells con-
taining fluorescent membrane probes show formation of large GMPVs at-
tached to the cell bodies by superposition of CM or two-photon microscopy
image z-stacks. (A) Cell expressing geranylgeranyl-EGFP (GG-GFP) with at-
tached GPMV measured by CM-imaged stack at 23°C. Note the absence of
internal membranes within the GPMV and significant partitioning of GG-GFP
into the GPMV compared with the cell body structures. (B) CM images of an
NIH 3T3 GPMV incubated at room temperature with 1% (vol/vol) DMSO and
R-DOPE, showing large-scale fluid/fluid phase coexistence similar to the RBL
cells. (C and D) Two-photon microscopy images of a cell-attached GPMV
colabeled with the membrane markers Nap (C) and R-DOPE (D), imaged at 5°C.
Note their contrasting wavelength-selected labeling of the separated phases.
Cell bodies show large fluorescence (attenuated here) due to internalized
membrane probes. (Scale bars, 5
m.)
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Photoinduced lipid oxidation, which can interfere with mem-
brane phase behavior (32), was min imized by the antioxidant
DTT.
As seen by CM, soluble fluorescently labeled proteins, such as
CTB and annexin V, are mostly excluded f rom the interior of
GPMVs, suggesting that their membranes are sufficiently sealed
to prevent internalization of protein-sized solutes. However,
some GPMVs are slightly leaky to these proteins, as judged by
the occasional observation of slow internal labeling of GPMVs
(dat a not shown). Nevertheless, determin ing whether fluores-
cent annexin V on GPMVs is labeling phosphatidylserine flipped
to the outer leaflet or on the inner leaflet of these GPMVs is
uncert ain. Earlier experiments on formaldehyde induced plasma
membrane vesicles of neuroblastoma cells indicated partial
retention of lipid asymmetry for as long as 3 days after blebbing
at 37°C for 1.5–2 h and subsequent storage at 4°C (33), suggesting
that GPMVs may retain significant leaflet asymmetry during our
imaging, but it remains for future studies to deter mine the extent
of lipid asymmetry preservation in GPMVs.
A n advantage of GPMVs is that membrane protein partition-
ing can be reliably studied by optical microscopy in vesicles with
clearly identified fluid/fluid phase coexistence. Fig. 3 demon-
strates selective partitioning of lipid-anchored proteins from
outer and inner leaflets of cellular plasma membranes, as well as
partition ing of a transmembrane protein complex. Glycosylphos-
phatidylinositol-anchored proteins, including Thy-1, are com-
mon markers for outer leaflets of lipid rafts, based on detergent
f ractionation, (34, 35), intact cell membrane (36, 37), and model
membrane studies (38). Fig. 3A shows Thy-1 labeled with
Cy3-c onjugated Ox7 mAb partitioning preferentially into the
L
o
-like phase in GPMVs with fluid/fluid phase coexistence. In
c ontrast, several lipid-anchored proteins that localize to the
inner leaflet of the plasma membrane were found to partition
preferentially into the L
d
-like phase.
Fig. 3B shows that Lyn-GFP, which rec onstitutes FcRI-
mediated signaling (39, 40), partitions preferentially into the L
d
phase of GPMVs. However, several sucrose gradient analysis
studies had suggested that Lyn itself preferentially associates
with ‘‘lipid raf ts’’ under a variety of cell lysis conditions (39, 41,
42). Similarly, the GFP chimera PM-GFP, which is anchored to
the plasma membrane inner leaflet by myristate and palmitate
ac yl chains attached to the first 20 amino acid residues of Lyn
(21), preferentially partitions into the L
d
-like phase in GPMVs
(Fig. 3C), and a geranylgeranyl-anchored chimera of GFP (21)
similarly partitions into L
d
-like phases (Fig. 3D). Sucrose gra-
dient fractionation of detergent-lysed cells suggests that the GFP
module enhances L
d
partition ing (39). However, the strong L
d
partition ing observed with these inner-leaflet lipid-anchored
proteins in GPMVs represents a sign ificant quantitative differ-
ence from detergent fractionation predictions.
We find that A488-labeled IgE bound to FcRI strongly
partitions into the L
d
-like phase of GPMVs, as shown in Fig. 3E.
These results are c onsistent with those obtained from sucrose
gradient analysis of TX-100-lysed RBL cells over a large range
of detergent concentrations (41, 43, 44). Cross-linking of this
IgE-receptor complex has been shown to substantially increase
its partitioning into detergent-resistant lipid rafts (41), and it will
be interesting to investigate the effects of FcRI cross-linking on
its phase partitioning in GPMVs.
Fig. 3F shows that the FITC-conjugated lectin, Con A, which
binds to a large number of
-methyl mannoside-containing
glyc oproteins and glyc olipids, preferentially partitions into the
L
d
-like phase as well, as evidenced by colabeling w ith R-DOPE.
Comparison of the fluorescence intensity of FITC-Con A label-
ing of t wo GPMVs attached to a cell in Fig. 3G shows that the
glyc oproteins and glycolipids that segregate into the emerging
GPMVs represent a substantial fraction of the Con A binding
sites on the cell surface. However, quantitative estimation of bleb
protein content versus cellular membrane protein content is
uncert ain because of the highly folded nature of the cellular
membrane, compared with the smooth bleb membranes.
GUV model membranes enable examination of mechanical
aspects of membranes with fluid/fluid phase coexistence, in-
cluding curvature coupling to membrane phase patterns and line
tension at phase boundaries (14, 45–47), as well as experimental
tests of mechanical membrane theories (14, 48). GUVs with
L
o
/L
d
phase coexistence and relatively monodisperse domain
sizes show hexagonal arrays of phase domains in superstructures
that are reminiscent of modulated phase separation patterns
observed in many other physical systems (49). SI Fig. 5A
illustrates the hexagonally modulated phase patterns that also
are oc casionally observed in GPMVs. Modulated phase patterns
can be stabilized by competing short-range attractive and long-
range repulsive forces, and model membrane systems have
indicated compositional variations arising from short-range at-
tractions that couple to membrane curvature modulations, pos-
sibly leading to long-range repulsion (14, 49). These modulated
patterns are likely to be k inetically trapped nonequilibrium
str uctures subject to domain coalescence and possibly other
c oarsening mechanisms (50) due to line tension at phase bound-
aries typically leading to complete phase separation after long
observation times.
Fig. 2. Plasma membrane lipids and lipid fluorophores in GPMVs demon-
strating characteristic phase preferences resembling the L
o
/L
d
partitioning
behavior in model membranes. GPMVs, all prepared by formaldehyde/DTT
treatment of RBL cells, were colabeled by Nap, R-DOPE, or DiIC16 and by
A488-CTB bound to GM
1
or A568-annexin V bound to phosphatidylserine.
Images are equatorial CM sections obtained at 23°C. (A) GPMVs colabeled
with Nap and R-DOPE show contrasting partitioning. (B) GPMVs colabeled
with CTB and R-DOPE show contrasting partitioning. (C) GPMVs
colabeled with CTB and DiI C16:0 show contrasting partitioning. (D) CTB/
annexin V-colabeled GPMV shows contrasting partitioning, indicating that
annexin V labels an L
d
-like phase.
Baumgart et al. PNAS
February 27, 2007
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BIOPHYSICS
Page 3
A lthough domains of our phase-separated GPMVs show small
curvature gradients, i.e., the membrane geometries approach
circular domains on a smooth sphere, a notable exception is
GPMVs with small domains that are labeled with CTB, as seen
in Fig. 2B and SI Fig. 5B. Upon cholera toxin binding, preexisting
L
o
-like domains develop sign ificant inward curvature. Interest-
ingly, internalization of CTB bound to GM
1
oc curs in cells in a
cholesterol-dependent fashion (51), and it is possible that CTB
promotes internalization by inducing a spontaneous inward
curvature that facilitates budding and fission. Similar inward
curvature is occasionally observed for CTB-labeled L
o
domains
in GUV model membranes (A.T.H., unpublished data).
Oc casionally, phase-separated GPMVs show domain shapes
fluctuating via in-plane domain boundary undulations. SI Fig.
5C shows sequential images of a vesicle at 23°C with domain
undulations. These two-dimensional undulations also are ob-
served in GUV model membranes with fluid/fluid phase coex-
istence near mixing/demixing transition temperatures, where
line tension can become relatively small (14, 45). These undu-
lations further highlight the presence of fluid/fluid phase coex-
istence in GPMVs. Fluid phase c oexistence also is supported by
fast recovery of spot photobleaching of all labeled lipids and
membrane proteins examined here (data not shown).
Discussion
Plasma membrane vesicles, commonly called blebs, are naturally
for med in a variety of physiological processes that include
blebbing in locomoting cells, in cells undergoing mitosis, and in
apoptotic cells. Bleb formation can be artificially induced by a
variet y of methods (52), including our addition of solvents or
sulfhydryl group-blocking reagents. Depending on the method,
dif ferent types of blebs can be obtained (52, 53). Ours were
for med through cell swelling and blebbing, as in oncosis (53).
These blebs have been shown to be free of cellular organelles,
with lipid compositions representative of the plasma membrane,
with phospholipid/cholesterol ratios of 2:1 (6, 16, 24). Our
GPMVs from RBL cells cont ain constitutively active tyrosine
k inase Lyn (N. Smith, D.A.H., and B.A.B., unpublished data).
A possible question is the cellular source of GPMV mem-
branes, considering the apparent increased plasma membrane
area during blebbing (see Figs. 1 and 3G). Membrane capaci-
t ance measurements via patch-clamping of mast cells have
revealed that osmotic inflation of cells by approximately 4 times
the resting volume did not change membrane conductance and
only caused small reversible changes of total cell membrane
capacit ance (54). These results combined with the finding of
exoc ytosis inhibition by inflation (54) suggest that GPMV mem-
branes are likely to accr ue from excess membrane area stored in
membrane microv illi and various protrusions including coated
pits, lamellipodia, and ruffles, but more direct comparisons are
needed.
The overall protein content of GPMVs must influence
membrane phase behav ior. GPMVs from RBL cells have been
reported to c ontain 20–25% of the cellular IgE-receptor
c omplexes (15), roughly c omparable with the apparent f rac-
tion of cellular plasma membrane extruded as GPMV. This
c onclusion is supported by abundant Con A labeling v ia their
glyc osylated protein binding sites ret ained in cell-attached
GPMVs (Fig. 3G ) and by recent ESR measurements that
prov ided evidence for similar phase c oexistence behavior in
GPMV and in liv ing cell plasma membranes (55). Earlier
IgE-receptor dif fusion measurements have indicated sign ifi-
cant mobility increase due to observed reductions of molecular
crowding relative to cell membranes on hyperton ically swollen
cells and chemically induced membrane blebs (56, 57). Exten-
sive ex periments will be required to establish the import ant
relations between membrane phases and protein c ontents. We
find that the F-actin probe A lexa Fluor 488 phalloidin does
Fig. 3. GPMVs reveal membrane protein phase preferences in detergent-
free GPMVs. Fluorescence images of equatorial confocal sections through
GPMVs comparing the fluid phase partitioning of membrane-associated pro-
teins (left column) to the lipid probes R-DOPE and CTB bound to GM
1
(right
column). All GPMVs were prepared by formaldehyde/DTT treatment of RBL
cells and imaged at 23°C. (A) GPI-anchored protein Thy-1 labeled with
A488-anti-Thy1 mAb is preferentially in L
o
phase, showing fluorescence in
regions contrasting to R-DOPE partitioning in the L
d
-like phase. (B) Lyn-GFP
partitions preferentially into the L
d
-like phase, in contrast with the CTB-
labeled L
o
-like phase. (C) PM-GFP partitions strongly into the L
d
-like phase, in
contrast to the CTB-enriched phase, in a cell-attached GPMV. (D) GG-GFP
partitions preferentially into the L
d
-like phase, complementary to CTB. (E)
FcRI labeled with A488-IgE partitions into the L
d
-like phase colabeled with
R-DOPE. (F) FITC-Con A labels glycoproteins and glycolipids that preferentially
cosegregate with the L
d
phase marker R-DOPE. (G) A significant fraction of
Con A receptors populate GPMVs, compared with those in the attached cell.
(Scale bar, 5
m.)
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Page 4
penetrate the membranes and un ifor mly st ains the volume of
the GPMV plus the actin c ytoskeleton in the residual cell body
(dat a not shown), confirming the absence of a c ortical actin
assembly on GPMVs, as shown prev iously by electron micros-
c opy (23) and flourescence microscopy (20). This uncoupling
f rom the cortex is likely to be critical for the observed
membrane phase separations. Polyphosphoinositide lipids (24,
58) and c y toskeletally immobilized proteins may be excluded
f rom GPMVs (59, 60), a future question that needs to be
addressed.
Import ant results of this study are the defined mixing/
demixing transition temperatures in the protein-containing
membranes of indiv idual GPMVs. The significant temperature
dependence of plasma membrane phase behav ior also has
been demonstrated recently by photobleaching rec overy mea-
surements (61). We find that individual vesicles show large
variations in transition temperatures, with the vast majorit y of
vesicles undergoing phase separations only at temperatures
bet ween 10°C and 25°C. These low-temperature phase sep-
arations are defined by the c oexistence of two fluid membrane
phases with sharp phase boundaries at optical resolution. Two
distinct fluid phases do form despite the c omplex GPMV
membrane composition, relative to simple ternary lipid mix-
tures of model membranes w ith c omposition-dependent fluid-
phase-c oexistence temperatures (38, 45, 47). Coexisting fluid
membrane phases in GPMVs show fluorescence probe parti-
tion ing behav ior similar to model membranes with L
o
/L
d
phase
c oexistence.
Demixing phase separations, particularly in binary or ternary
systems, can be considered in regular solution theory, where an
enthalpic term due to preferential molecular interactions balances
against the entropic contribution proportional to temperature to
determine the free energy change of mixing. Highly preferential
interactions lead to lower demixing temperature s. Local concen-
tration fluctuations associated with nonideal mixing (62) may
diverge on approaching critical (consolute) points of multicompo-
nent phase diagrams (63). Our finding of L
o
/L
d
-like phase separa-
tion only below physiological temperatures suggests correlated
concentration fluctuations above the demixing temperatures that
may lead to local composition fluctuations amenable to protein
interactions. Correlated concentration fluctuations in native, mac-
roscopically homogenous plasma membranes could yield an in-
creased encounter probability of signaling molecules with similar
membrane phase preference and, likewise, decreased encounter
probabilities of signaling components with differing phase prefer-
ences. Our experiments do not yet include lifetimes or length scales
of such composition fluctuations.
The large-scale development of membrane phase domains is
driven by the interphase energy of the phase boundaries.
Whereas this line tension at fluid phase boundaries can be
subst antial in lipid model membrane systems (14), line tension
magn itudes c ould differ sign ificantly and be much smaller in
more complex membrane compositions, possibly favoring small-
scale cell membrane heterogeneities (64), because line tensions
are generally reduced significantly by solute segregation to the
interphase boundaries.
In summary, we find that GPMVs f rom RBL mast cells
undergo fluid phase segregation that permits the characteriza-
tion of lipid and protein partitioning in c omplex biological
membranes. These GPMVs, which contain integral and periph-
eral membrane proteins and hundreds of different lipids, seg-
regate into L
o
/L
d
-like fluid membrane phases at low tempera-
tures, indicating nonideal, nonrandom lateral distributions of
membrane components and permitting deter mination of mem-
brane phase preferences of labeled lipids and of membrane
proteins. The high-affin ity IgE receptor partitions into L
d
-like
phases, whereas a glycosylphosphatidylinositol-anchored protein
and CTB bound to GM
1
segregate into L
o
-like phases. Several
ac yl-chain-anchored, GFP-labeled proteins, including palmitoyl/
myristoyl-anchored Lyn, partition preferentially to L
d
-like (i.e.,
‘‘non-raf t’’) phases in GMPVs, in contrast to their reported
association with detergent-resistant membrane particles, that
have been hypothesized to represent ‘‘rafts.’’ These inner-leaflet
ac yl-chain-anchored proteins may be affected by changes in lipid
asy mmetry during the formation of GPMVs.
Our method avoids membrane disruption by detergent treat-
ment or depletion of membranes of cholesterol or sphingomy-
elin. The approach is suit able for examining the distribution of
lipids and proteins between membrane domains and therefore
allows testing predictions from the raf t hypothesis in laterally
int act cellular bilayer membranes. Our results present clear
evidence that these complex biological cell plasma membranes
can phase segregate into coexisting fluid phases but primarily at
temperatures below 25°C. The strong temperature dependence
of this segregation is c onsistent with expectations from model
membrane studies and implies that, at temperatures above the
demixing transition temperature, diffusing membrane hetero-
geneities may persist. Further investigation will be necessary to
clarif y the differences bet ween GPMV and cellular plasma
membranes in temperature-dependent phase behavior. Future
studies can use GPMVs to examine the dependence of mem-
brane protein phase partitioning on biochemical activities, in-
cluding the regulation of membrane-associated kinases and
phosphat ases in cell signaling.
Materials and Methods
For lipid probes, antibodies, GFP constructs, cell culture and trans-
fection, and imaging methods, see SI Materials and Methods.
GPMV Formation. GPMVs were formed by three different meth-
ods aimed to produce either cell-attached or free GPMVs of
RBL cell plasma membranes.
Cell-attached GPMVs.
Cell-att ached GPMVs were prepared by
addition of 1–5% (vol/vol) ethanol, acetone, or DMSO to RBL
cells suspended at 10
6
cells per milliliter in PBS for 15 min at
room temperature (23°C) or by addition of solvents to adherent
NIH 3T3 cells in four-well plates. Solvents used to induce
membrane vesiculation typically c ontained R-DOPE or
DiIC16:0 at 200
g/ml or Nap at 50
g/ml.
Cell-Free GPMVs.
Cell-f ree GPMVs were prepared by either of the
t wo following methods. The primary protocol for GPMV for-
mation used a procedure modified from Scott (16, 17) and
described in ref. 15. Briefly, cells were grown to confluency in a
25-cm
2
tissue culture flask, then cells were washed twice w ith
GPMV buffer (2 mM CaCl
2
/10 mM Hepes/0.15 M NaCl, pH 7.4),
and 1.5 ml of freshly prepared GPMV reagent was added,
c onsisting of 25 mM formaldehyde and 2 mM DTT in GPMV
buf fer. The flasks were then incubated for1hat37°C while
slowly shaking (6080 cycles per minute). Af ter incubation,
GPMVs that had detached from the cells were gently decanted
into a conical tube. Previously published protocols (15) further
purified the vesicles by centrifugation and dialysis, but we found
that these procedures caused f ragmentation of large GPMVs,
mak ing microscopic analysis difficult. For the present experi-
ments, we allowed GPMVs to settle on ice for 10- 45 min and
c ollected them by removing 20% of the total volume f rom the
bottom of the tube. By using this method, a single confluent
25-cm
2
flask yields sufficient GPMVs to create several dozen
microsc opy samples.
In an alternative protocol, formaldehyde and DTT were replaced
by2mMN-ethylmaleimide, a reagent previously shown to cause
GPMV formation (17). All other steps are identical. The yield of
GPMV is lower, and more cells detach under these conditions.
Therefore, this protocol was used primarily to verify that the small
amount of formaldehyde and DTT present in the primary method
did not affect the results obtained.
Baumgart et al. PNAS
February 27, 2007
vol. 104
no. 9
3169
BIOPHYSICS
Page 5
This work was supported by Science and Technology Centers Prog ram
Agreement ECS-9876771 f rom the Nanobiotechnology Center of
the National Science Foundation and by National Institute of A l-
lerg y and Infectious Diseases Grant R01 AI18603 and National
Institute of Biomedical Imaging and Biotechnolog y Grant P41
EB001976.
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www.pnas.orgcgidoi10.1073pnas.0611357104 Baumgart et al.
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