Cholesterol Depletion Induces Solid-like Regions in the Plasma Membrane
Stefanie Y. Nishimura,* Marija Vrljic,yLawrence O. Klein,zHarden M. McConnell,*zand W. E. Moerner*z
*Department of Chemistry,yMolecular and Cellular Physiology, andzBiophysics Program, Stanford University, Stanford,
proteins, as well as N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Tritc-
DHPE), are used as probes to determine the effect of cholesterol concentration on the organization of the plasma membrane at
temperatures in the range 22?C–42?C. Cholesterol depletion caused a decrease in the diffusion coefficients for the MHC II
proteins and also for a slow fraction of the Tritc-DHPE population. At 37?C, reduction of the total cell cholesterol concentration
results in a smaller suppression of the translational diffusion for I-Ekproteins (twofold) than was observed in earlier work at 22?C
(five sevenfold) Vrljic, M., S. Y. Nishimura, W. E. Moerner, and H. M. McConnell. 2005. Biophys. J. 88:334–347. At 37?C, the
diffusion of both I-Ekproteins is Brownian (0.9 , a-parameter , 1.1). More than 99% of the protein population diffuses
seen at ;35?C in the Arrhenius plots. Cytoskeletal effects appear to be minimal. These results are consistent with a previously
described model of solid-like domain formation in the plasma membrane.
Glycosylphosphatidylinositol-linked and transmembrane major histocompatibility complex (MHC) class II I-Ek
There is much interest in the relation between lipid organi-
zation found in model membranes and proposals for the lipid
organization in cell membranes. The plasma membrane differs
from most model membranes in that it has an asymmetric lipid
composition, a greater diversity of lipids, a high concentration
of proteins, cytoskeletal elements, and membrane trafficking
processes. The roles of these different factors in determining
lipid organization are under investigation in many laboratories.
In model systems, such as lipid monolayers and bilayers,
liquid-liquid immiscibility for certain mixtures of lipids has
been observed using fluorescence microscopy (1–5). Liquid-
liquid immiscibility has been observed for a number of lipid
compositions and always depends on the presence of choles-
terol. For example, vesicles composed of certain lipid ternary
mixtures show liquid-liquid immiscibility. Cholesterol de-
pletion using b-cyclodextrin (b-CD) changes the relative
proportion of the two coexisting phases (6). The absence of
cholesterol, or its depletion, can also be related to a solid
phase in these systems (6–9).
The size and shape of lipid domains observed in model
systems depends on composition, temperature, and pressure.
Lipid domains can be as large as 10 microns in diameter (6–
9). Deuterium NMR line broadening has been interpreted as
being due to domains as small as several nanometers (10,11).
Molecular complexes termed ‘‘condensed complexes’’ be-
separate phase or as a homogeneous mixture with excess
phospholipid or cholesterol (15,16).
Lipid-mediated structures are thought to exist in cell
membranes, but consensus has not been reached on whether
they are related to a thermodynamic phase separation. A true
phase separation has not been detected in plasma membranes
(however, see Hao et al. (28)), although lipids extracted from
these membranes have been shown to exhibit phase sepa-
Detergent resistance in membrane extracts has been
interpreted as evidence of lateral heterogeneity in the plasma
membrane, with saturated phospholipids, sphingomyelin,
cholesterol, and certain proteins such as GPI-linked proteins
associating with the detergent resistant membrane (DRM)
fraction (18–21). The interpretation of DRM results is con-
troversial (22). In the absence of detergents, DRM-associated
proteins were observed to be homogeneously distributed in
the plasma membrane and to cluster with each other or with
other DRM components only after cross-linking (23,24).
Various studies have attempted to measure domain sizes
in plasma membranes with varying results (25–28). Dif-
ferences in reported domain sizes could be due to variation
in technique, probe selection, cell type, or cholesterol level.
These issues have been discussed in several recent reviews
(16,27,29,30). What is clear is that cholesterol plays an im-
cell signaling, resistance to cross-linking, and the occurrence of
DRM fractions (23,31–37).
In this study, we are concerned with the role of cholesterol
in plasma membrane organization, with the specific goal of
understanding the decrease in the diffusion coefficients of
proteins and lipids mediated by cholesterol extraction (26,
of fluorescently labeled MHC class II I-Ekmembrane-
anchored proteins (labeled by binding of a fluorescently
labeled antigenic peptide) as well as fluorescent lipid analogs,
Submitted July 11, 2005, and accepted for publication October 14, 2005.
Address reprint requests to Stefanie Y. Nishimura, E-mail: snishimu@
? 2006 by the Biophysical Society
Biophysical JournalVolume 90February 2006 927–938927
(Tritc-DHPE). Previous analysis found 30–50% of the
glycosylphosphatidylinositol (GPI)-linked I-Ekand 5–25%
of the transmembrane I-Ekto be localized in the DRM
fraction (25). By comparing the observed translational dif-
fusion of these probes to unconstrained Brownian motion,
we can distinguish modes of motion such as free diffusion,
restricted diffusion, or directed motion. As we then vary
the plasma membrane cholesterol concentration and tem-
perature, the resulting changes in the motion of the mem-
brane probes provide clues as to the molecular aspects of
translational diffusion in cell membranes.
MATERIALS AND METHODS
Chinese hamster ovary (CHO) cells transfected with either mouse MHC
class II protein I-Ek(CHO-I-Ek) or with the I-Ekextracytoplasmic domain
fused with a GPI-linker (CHO-GPI-linked I-Ek) were a generous gift from
M. M. Davis (40). CHO cells were grown in Roswell Park Memorial
Institute (RPMI) 1640 phenol red-free media (Gibco BRL, Grand Island,
NY) supplemented with 10% fetal calf serum (FCS; HyClone, Logan, UT),
10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 1
mM sodiumpyruvate,20 mM 2-mercaptoethanol (2-hydroxy-1-ethanethiol),
and 0.1 mM nonessential amino acids, 0.5 mg/ml geneticin (Gibco BRL),
pH 7.4, and 5% carbon dioxide at 37?C. A more detailed description of the
cell culture is provided in a previous report (25).
Labeling with Cy5-MCC and Tritc-DHPE;
For the I-Ekstudies, cells were labeled by incubation with 0.05–0.5 mg/ml
Cy5-MCC 95-103 peptide for 15–30 min at 37?C. For details, see our
previous work (25,26). Cells were labeled with Cy5-MCC before treatment
with any drug.
Tritc-DHPE or 1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocyanine
(DiIC18) (Molecular Probes, Eugene, OR) was stored in chloroform (1 mg/ml,
stock). Immediately before use, 1–5 mL of dye stock solution was dried into
a film and then reconstituted in 20–100 mL of ethanol. CHO-I-Ekor CHO-
GPI-I-Ekcells were incubated with a final concentration of 100 nM-1 mM of
Tritc-DHPE or 0.1–1 nM DiIC18for 5–10 min at 22?C in supplemented
RPMI 1640 media with FCS. The maximum concentration of ethanol during
incubation was 1% v/v. Cholesterol-depleted cells were labeled after
treatment with 2 h b-CD, but for all other conditions cells were labeled
before the treatment.
2,4,6-trinitrobenzenesulfonic acid (TNBS) was obtained from Sigma
(St. Louis, MO). For quenching experiments, cells were first labeled with
dye, and then treated with 5 mM TNBS (1 M stock in water) in Dulbecco’s
phosphate-buffered saline (Gibco) at 22?C. Imaging of the cells was com-
pleted within 45 min after addition of TNBS. Control cells were also imaged
in Dulbecco’s PBS. Diffusion coefficients obtained from cells in Dulbecco’s
PBS were identical to data taken with the cells in supplemented RPMI.
Cholesterol depletion and repletion
For cholesterol depletion, cells were incubated in 10 mM b-CD (Sigma) or
2 3 10?6moles b-CD/10,000 cells, in supplemented RPMI with FCS at
37?C for the times indicated in the figures. The total cell cholesterol con-
centration after treatment of the CHO cells for various times with b-CD
was determined using the Amplex Red Cholesterol Assay (Molecular
Probes), and the results were reported in previous work (26). After the times
indicated in the figures, the b-CD solution was rinsed and cells were imaged
in media without b-CD. For details, see Vrljic et al. (25,26).
Long-term viability of b-CD-treated cells was not affected. At 24 h after
b-CD treatment, the cell morphology resembled that of untreated cells and
cells were dividing (data not shown). As reported previously, a small frac-
and these cells were excluded from the analysis (26). For additional detail, see
Cholesterol was added to the cells using cholesterol-loaded methyl-b-CD
(chol-mb-CD) (Sigma) at 1 mM cholesterol (0.2 3 10?6moles of choles-
terol/10,000 cells). Chol-mb-CD was dissolved in supplemented RPMI
1640 with 10% FCS, and the cells were incubated at 37?C for the times
indicatedin the figures.The cholesterol solutionwas refreshed every 30 min.
For the cholesterol repletion studies, cells were first incubated with 10 mM
b-CD for 60 or 90 min and then incubated with chol-mb-CD. Chol-mb-CD
was not present in the media during imaging. Cholesterol loading did not
cause an increase in the number of apoptotic cells and did not restore the
morphology of the cells to normal on the timescale of the experiments (3 h)
(data not shown). The treated cells remained less elongated and more
spherical than the cells at normal cell cholesterol concentration. This sug-
gests that the reversal of the cell signaling process responsible for initial loss
of cell adhesion points takes longer than 3 h even in the presence of
Stock solutions of nocodazole (Sigma; 20 mM stock) and cytochalasin D
(Sigma; 1 mg/ml stock solution) were prepared in DMSO. Control cells
were treated with an equivalent amount of dimethyl sulfoxide (DMSO)
alone. For tubulin depolymerization, cells were treated for 30 min at 37?C
with 100 mM nocodazole before imaging. To disrupt the actin cytoskeleton,
cells were treated with 4, 13, or 40 mM cytochalasin D (25,35,41) and
imaged immediately at 37?C. The cells were then imaged at 37?C until they
became rounded, indicating extensive cytoskeletal rearrangement (;15–30
min). Cell edges remained smooth and cytoplasm displayed a similar
number of vacuoles, indicating that cells are not apoptotic. Both drugs were
present in the media during imaging. The effect of these drugs has been
described elsewhere (42–44).
Cells were culturedon a chamberedcoverglass (Nalgene NuncInternational,
Naperville, IL) and imaged in supplemented RPMI 1640 phenol red-free
medium. FCS was excluded from the media during imaging to lower the
background fluorescence signal. Data taken with and without FCS in the
imaging media were identical (26). An enzymatic oxygen scavenger system
was used when imaging Cy5: 1% v/v glucose (Sigma; 500 mg/ml stock), 1%
v/v glucose oxidase (Sigma; 5000 U/ml stock), 1% v/v catalase (Sigma;
Diffusion coefficients taken with and without the enzymatic oxygen
scavenger system were identical except that the lifetime of the fluorophore
before photobleaching was extended in the presence of oxygen scavengers
(Supplementary Material). No oxygen scavengers were present in the media
when Tritc-DHPE was imaged. For further details, see Vrljic et al. (25,26).
The fluorescence imaging of the cells was performed with wide-field epi-
illumination in an area of 8-by-8 mm, using an inverted microscope (Eclipse
TE300, Nikon, Burlingame, CA). Laser illumination at 633 (106-1, Spectra-
Physics, Mountain View, CA) or 532 nm (GS32-20, Intelite Laser, Genoa,
NV) provided an intensity of ;2 kW/cm2at the sample plane. The
epifluorescence was collected with a 1003 magnification, 1.4 numerical
928Nishimura et al.
Biophysical Journal 90(3) 927–938
aperture oil-immersion objective (PlanApo, Nikon) and infinity-corrected
objective adaptor (Nikon). A 645 nm dichroic mirror and 640 nm ALPHA-
Epsilon long-pass filter (Omega Optical, Brattleboro, VT) or a 545 nm
dichroic mirror and 540 nm long-pass filter (Omega Optical) were used to
filter the emission from Cy5 and Tritc-DHPE/DiIC18, respectively. The
emission was collected using an on-chip intensified frame-transfer CCD
camera (Cascade 512B, Roper Scientific, Trenton, NJ) at a frame rate of
65 Hz. For further details, see Vrljic et al. (25,26).
The temperature of the sample was varied using a homemade heating stage
in combination with an objective heating collar (Bioptechs, Butler, PA). The
microscopestagewas heated aboveroom temperature usingresistive heating
tape connected through a variable power supply (Variac, Superior Electric,
Bristol, CT), and a Petri dish was placed over the 8-well chambered cover-
glass on the stage to reduce cooling through air currents. The air temperature
was determined using a PT-100 thermocouple (Omega) placed under the
plastic dish, and for the 37?C measurements, the air temperature was
maintained at 37.1?C 6 0.50?C. The temperature of the solution within each
concentration was determined each time the sample was imaged, and this
value was used as a secondary calibration for the temperature of the sample
(data not shown). The temperature of the stage was varied above room
temperature from 27?C–42?C.
For a detailed description of the data analysis methods, see Vrljic et al. (25).
Single-molecule positional trajectories were mapped by determining the
centerofthe fluorescentspotineachframeby eyewithanaccuracyof;39.5
nm (diameter of one pixel). This spatial resolution was sufficient in this
experiment sincethe average displacement of the I-Ekproteins from frame to
frame was ;100–300 nm (Ær2æ ¼ 4 Dt, where Ær2æ ¼ mean squared radial
displacement for a time lag, D ¼ 0.2–1.0 mm2/s, t ¼ 15.4 ms). The average
trajectory lengths in this study were ;0.5 s long before photobleaching.
The diffusion coefficients were determined in two ways. In Figs. 2, 3, and
6, the radial displacements from all trajectories for a particular time lag were
combinedand fitted to the cumulativeradial probability distribution function
(CDF) for a Brownian diffuser to extract an estimate of the mean diffusion
coefficient (see Supplementary Material for the exact number of trajectories
and cells used for each case). In this analysis, the individuality of the tra-
jectories was disregarded and the combined distribution of all independent
displacements from all trajectories at a specific time lag was analyzed. In
determining the diffusion coefficient, the largest time lag was taken to be the
last time at which at least 50 displacements contributed to the fit. In Figs. 1
and 7, the diffusion coefficients were averaged over all time lags to obtain a
mean diffusion coefficient.
In the diffusion coefficient versus time lag plots, deviation from
Brownian motion was characterized by determining the anomalous diffusion
parameter, a. For a two-dimensional random walk a ¼ 1 and for anomalous
diffusion 0 , a , 1 (D ¼ D0ta?1;Ær2æ ¼ 4D0ta; where D is the observed
diffusion coefficient and D0is the true diffusion coefficient) (45–47).
In Figs. 4 and 5, the mean-squared displacements from an individual
single-molecule trajectory were used according to D ¼ Ær2æ=4t; with t ¼
30.8 ms. In this method, each single-molecule trajectory was used to
calculate an apparent diffusion coefficient, and the distribution of apparent
diffusion coefficients for the individual trajectories was constructed to test
for heterogeneity from molecule to molecule. The number of displacements
were made uniform for all trajectories by clipping the trajectories such that
the first N points from each trajectory were included in the analysis (N ¼ 20
for I-Ekand DiIC18 and 10 for Tritc-DHPE data). Histograms were
normalized by dividing by the total number of trajectories. The expected
distribution of apparent diffusion coefficients for a homogeneous popula-
tion of diffusers was computed using the arithmetic mean of the diffusion
coefficients from the individual trajectories (25,48).
Diffusion is suppressed after cholesterol
depletion when imaged at 37?C
To understand the origin of the suppression of diffusion
observed for certain membrane molecules upon cholesterol
depletion, the diffusion of I-Ekproteins was monitored as the
total cell cholesterol concentration was incrementally re-
duced by incubating the CHO cells with b-CD. The cell
cholesterol was thereby gradually reduced to ;50% of nor-
mal (26). The cells were then imaged at physiological tem-
perature (37?C) and results compared with the previous data
obtained at room temperature (26) to test whether the sup-
pression of protein motion upon cholesterol depletion also
occurs at physiological temperature.
As expected, the mean diffusion coefficients of the GPI-
linked and transmembrane I-Ekproteins were higher when
the cells were imaged at 37?C than when the cells were
imaged at 22?C for all cholesterol concentrations (Fig. 1, C
and D and Table 1).
Cholesterol-depletion caused a decrease in the mean
diffusion coefficients for both the GPI-linked and trans-
membrane I-Ekproteins when imaged at 37?C. Also, the
mean diffusion coefficients for both proteins decrease as the
incubation time with b-CD is increased (Fig. 1, A and B),
correlating with the decrease in the total cell cholesterol
concentration (Fig. 1, C and D). However, the percentage
decrease in the mean diffusion coefficients after cholesterol
depletion was only twofold when the cells were imaged at
37?C, a smaller change than the five- or sevenfold decrease
observed when cells were imaged at 22?C (Fig. 1, C and D).
Diffusion is restored after cholesterol
reintroduction when imaged at 37?C
To confirm that the suppression of the diffusion was
reversible and due to changes in cholesterol concentration,
TABLE 1Diffusion coefficients
(mm2/s) at 37?CGPI-linked I-Ek
1.1 6 0.06
0.93 6 0.08*
0.49 6 0.03z
0.59 6 0.04
0.63 6 0.05y
0.34 6 0.03z
0.39 6 0.02§
0.19 6 0.02§
Normal cholesterol0.19 6 5.9 3 10?3
0.15 6 3.3 3 10?3
*1 h b-CD followed by 2 h chol-mb-CD.
y90 min b-CD followed by 2 h chol-mb-CD.
z2 h b-CD.
§2 h b-CD followed by nocodazole treatment.
Chol Depletion Induces Solid Formation929
Biophysical Journal 90(3) 927–938
cholesterol was reintroduced to the plasma membrane using
chol-mb-CD. The cells were first treated with b-CD for 60
min (GPI-linked I-Ek) or 90 min (transmembrane I-Ek), and
then the cholesterol-depleted cells were incubated with chol-
mb?CD for 2h. The cell cholesterol concentration increased
from ;50% of normal to above 100% of normal using this
method (26). After cholesterol reintroduction, the diffusion
coefficients for both proteins increased to the value observed
under normal cholesterol concentrations (Fig. 2, A and B,and
On the timescale of our experiments, the cholesterol-
depleted cells were unable to metabolically restore the cell
cholesterol concentration (26) or diffusion coefficients to
the original values, even after incubation for 2 or 4 h in
cholesterol-containing FCS supplemented RPMI media at
37?C (Fig. 2, A and B). After incubation for 2 h in sup-
plemented RPMI media, the total cell cholesterol concen-
tration was ;40% of normal (26). The average diffusion
coefficient for the GPI-linked I-Ekpopulation after 2 h b-CD
followed by 4 h in supplemented RPMI media was found
to be 0.70 6 0.06 mm2/s. The average diffusion coefficient
for the transmembrane I-Ekpopulation after 90 min b-CD
followed by 2 h in supplemented RPMI media was found to
be 0.40 6 0.02 mm2/s.
Protein diffusion is predominately Brownian when
imaged at 37?C
The diffusion coefficients of GPI-linked and transmembrane
I-Ekwere examined as a function of time lag from 0.0154 to
;0.5 s to determine if the motion of these proteins was
constrained at reduced cholesterol concentrations when the
cells were imaged at 37?C (Fig. 3, A and B). The a-values for
these proteins at room temperature were reported to be be-
tween 0.9 and 1.1 (26).
In a few experiments (,1% of the trajectories), it was
apparent that a fluorescent spot was not localized to the
plasma membrane, as it either appeared or disappeared as
a defocused spot in an area where there were other fluo-
rescent spots in focus. These fluorescent spots then followed
a nearly linear trajectory and either moved into or out of
focus. These fluorescent spots were clearly located in a focal
plane within the cell. Such fluorescent spots included both
single molecules of Cy5 that exhibited digital photobleach-
ing and spots that were brighter than expected for a single
molecule (data not shown). These fluorescent spots, which
exhibited directed motion outside of the plane of the plasma
membrane, were not included in the analysis as it was as-
sumed that these molecules were undergoing active transport
on cytoskeletal elements. Directed motion was observed at
all cholesterol concentrations, but after treatment with a
tubulin-disrupting drug (nocodazole), directed motion out-
side of the plane of the plasma membrane was not observed
(data not shown). All molecules were included in the
analysis except those above or below the plasma membrane,
even if the molecule appeared to be undergoing directed
D, versus time lag calculated fromfits tothe cumulative distribution function
for GPI-linked I-Ekafter cholesterol is reintroduced (B) D versus time lag
for transmembrane I-Ekafter cholesterol is reintroduced. The diffusion
coefficients at normal cholesterol and reduced cholesterol are shown for
comparison. The control case (cholesterol depletion followed by incubation
in RPMI media) is also shown. The number of trajectories contributing to
each data point may be found in the Supplementary Material.
Cholesterol reconstitution at 37?C. (A) Diffusion coefficients,
coefficients, D, calculated from fits to the cumulative distribution function
averaged over all time lags plotted againstthe correspondingincubationtime
with b-CD for (A) GPI-linked I-Ekand (B) transmembrane I-Ek. See Fig. 3
for D versus time lags for these data. In all panels, black circles represent
data taken at 37?C and white squares represent data taken at 22?C (26).
Cholesterol reduction at 22?C results in a fivefold decrease in the D for GPI-
linkedI-Ek(C) anda sevenfolddecrease inthe Dfortransmembrane I-Ek(D)
versusan ;2-folddecrease at37?C for bothproteins.The percentagenormal
cell cholesterol concentration as a function of b-CD incubation time has
been reported (26). For the number of trajectories contributing to each data
point, see Table S1 in the Supplementary Material.
Diffusion coefficients after cholesterol depletion. Diffusion
930 Nishimura et al.
Biophysical Journal 90(3) 927–938
motion. Directed motion outside of the plane of the plasma
membrane was not observed in cells imaged at 22?C.
The diffusion coefficients for the GPI-linked and trans-
membrane I-Ekwere found to be predominately Brownian at
all cholesterol concentrations when imaged at 37?C. Agree-
ment with Brownian motion was determined by calculating
the a-parameter from the diffusion coefficient as a function
of time lag plots. The a-values for both the GPI-linked and
transmembrane I-Ek, at all observed cholesterol concen-
trations, were between 0.9 and 1.1, indicating that the motion
of the proteins is primarily Brownian for up to ;0.5 s (Fig. 3,
C and D) and yielding no evidence for confinement.
Protein diffusion is homogeneous
If the I-Ekproteins partitioned into different membrane
environments, then heterogeneity in the distribution of dif-
fusion coefficients should be observed. To probe for possible
cholesterol concentrations, histograms were constructed using
the apparent diffusion coefficient for each single-molecule
positional trajectory at a particular cholesterol concentration
(Fig. 4). Individual trajectories were clipped to be 20 steps
long (308 ms), and the mean square displacement at time lag
30.8mswas usedto estimatethe diffusion coefficient, De. The
solid line plotted in each histogram is not a fit, but is the
expected distribution of measurements for a homogeneous
population with a diffusion coefficient given by the mean De
for each case (48).
At 37?C, the observed distribution of diffusion coeffi-
cients for GPI-linked and transmembrane I-Ekfor all cho-
lesterol concentrations is described well by a homogeneous
distribution. Reduction in cholesterol concentration caused
the entire population of diffusers to shift to a lower diffusion
coefficient for both GPI-linked I-Ek(Fig. 4, A–E) and trans-
the entire population shifts to a higher average diffusion
coefficient (Fig. 4, F and L).
Cholesterol depletion has a varied effect on the
diffusion of fluorescent lipid analogs
Histograms of the single-molecule diffusion coefficients for
Tritc-DHPE and DiIC18 incorporated into the CHO cell
plasma membrane at 22?C are shown in Fig. 5. The full
distribution of Tritc-DHPE diffusion coefficients is not
described well by a homogeneous population of diffusers
(Fig. 5, A and B, solid lines). For the 22?C data (before and
after cholesterol depletion), fits to a two population CDF (49)
show that 20% of the Tritc-DHPE had a diffusion coefficient
of ;2 mm2/s and 80% of the population had D ; 0.2 mm2/s
(Fig. 5, A and B, dashed lines) (25,49). The diffusion coef-
ficient of the faster population did not significantly change
after cholesterol depletion, whereas the slower diffusion coef-
ficient decreased by a factor of ;6 to a diffusion coefficient of
0.03 mm2/s (Fig. 5, A and B). When the Tritc-DHPE diffusion
coefficients were measured in the presence of a fluorescence
quencher (TNBS) with low membrane permeability at 22?C,
the relative size of the slower population decreased by 25%
(Fig.5C).Duringthe timeofthe experiment,TNBS primarily
quenches the fluorescence of Tritc-DHPE and DiI localized in
the outermembrane leaflet(Supplementary Material, (50,51)).
Cholesterol depletion had no effect on the observed dis-
Diffusion coefficients at different cholesterol concen-
trations appear Brownian when measured at 37?C for
GPI-linked I-Ek(A) and transmembrane I-Ek(B). The
corresponding a-values calculated for the diffusion
coefficients at each b-CD incubation time for GPI-
linked I-Ek(C) and transmembrane I-Ek(D).
Brownian diffusion of proteins at 37?C.
Chol Depletion Induces Solid Formation 931
Biophysical Journal 90(3) 927–938
was present (Fig. 5 D), indicating that diffusion of Tritc-
DHPE on the inner leaflet is unaffected by cholesterol de-
pletion. Data for Tritc-DHPE at 37?C are shown in the
The distributions of diffusion coefficients for DiC18before
and after cholesterol depletion are shown in Fig. 5, E and F.
The DiIC18distributions were predominately homogeneous.
The distributions of diffusion coefficients for DiIC18in the
presence of TNBS are shown in Fig. 5, G and H. The mean
diffusion coefficient of DiIC18 at the normal cholesterol
concentration did not change when TNBS was present
during imaging: D (no TNBS) ¼ 1.08 6 0.09 mm2/s and D
(with TNBS) ¼ 1.12 6 0.06 mm2/s (one population fit).
After cholesterol depletion, the mean of the full DiIC18
distribution shifted to 0.69 6 0.06 mm2/s (Fig. 5 F). When
TNBS was present, the mean diffusion coefficient for the
cholesterol-depleted case was found to be 0.90 6 0.11 mm2/s
(Fig. 5 H).
Reduction in mobility is not due to
Treatment of the cholesterol-depleted cells with cyto-
chalasin D, a drug that prevents actin polymerization,
did not restore the mobility of the GPI-linked or
transmembrane I-Ek(Fig. 6, A and B). Cholesterol-depleted
cells (pretreated with 2 h b-CD) were exposed to 4, 13, or
40 mM cytochalasin D according to several protocols
recently reported (26,35,41). The cells were imaged with
cytochalasin D present in the media, at 37?C, only until
the cells began to look more spherical—a criterion used
for cytochalsin D experiments in the Kusumi lab (52). Cell
sphericity indicates more extensive actin cytoskeletal
rearrangements and loss of actin stress fibers. The mean
diffusion coefficients obtained after treatment with each
of these three protocols were identical to the 2 h b-CD
efficients for individual MHC proteins at
37?C. Individual trajectories were clipped to
be 20 steps long and a time lag of 30.8 ms
was chosen such that 10 displacements from
each track were used to calculate each
diffusion coefficient. The solid line repre-
sents the expected distribution of diffusion
coefficients for a homogeneous population
of Brownian diffusers (25). The effect of
cholesterol depletionon the distributionof Ds
for GPI-linked I-Ekis depicted in panels A–E
and for transmembrane I-Ekin panels G–K.
The distributions of Ds are shown for GPI-
linked I-Ek(F) and for transmembrane I-Ek
(L) after cholesterol is reintroduced. See
Supplementary Material for the number of
tracks used for each histogram.
Distributions of diffusion co-
932 Nishimura et al.
Biophysical Journal 90(3) 927–938
After a 2 h b-CD treatment, the tubulin network was
disrupted with nocodazole and the cells were imaged with
the drug present in the media at 37?C. Treatment of the
cholesterol-depleted cells with nocodazole also did not re-
store the mobility of the GPI-linked I-Ek(Fig. 6 C) or
transmembrane I-Ek(Fig. 6 D), but instead caused a slight
decrease in the diffusion coefficient (Table 1).
Temperature dependence of the diffusion
coefficients at various cholesterol concentrations
The temperature dependence of the mean diffusion coef-
ficients for GPI-linked and transmembrane I-Ekwas inves-
tigated at normal and reduced cholesterol concentrations
(Fig. 7, A and B). Arrhenius plots of these data were used to
determine the activation energy for diffusion, Ea, using the
relationship: lnðDÞ ¼ lnðD0Þ ? ðEa=RTÞ; where R ¼ 8.314
Jmol?1K?1(53–55). For normal cholesterol concentrations,
the Eafor diffusion has a constant value for GPI-linked and
transmembrane I-Ekover the temperature range of 22?C–
42?C (Table 2). When the total cell cholesterol is reduced,
the Eachanges at ;35?C for both proteins, thus suggesting
a change in the phase behavior of the membrane at this tem-
perature (Table 2). The change in the Eavalue is observed
when the cholesterol is depleted using either a 10 min or a 2h
b-CD treatment. The break in the Arrhenius plots was
estimated to occur at ;34?C for the GPI-linked I-Ekafter 2 h
b-CD, ;36?C for the transmembrane I-Ekafter 10 min
No evidence of confinement was observed for any of the
proteins or lipid analogs employed in our diffusion studies
given a 15 ms integration time and ;0.5 s Cy5 on-time
before photobleaching of the fluorophore. However, the
diffusion of probes in the outer and inner leaflets of the
plasma membrane responded differently to cholesterol de-
Several membrane molecules exhibited a decrease in their
diffusion coefficients upon cholesterol extraction. These
included the GPI-linked and transmembrane I-Ekproteins
with large extracellular domains as well as the slow Tritc-
DHPE population. Diffusion of probes located in the inner
leaflet, the DiIC18and fast Tritc-DHPE population, were
unaffected by cholesterol depletion. From Arrhenius plots of
diffusion coefficient, we conclude that solid-like regions
form in the outer leaflet of the plasma membrane and sup-
press diffusion of membrane molecules that are at least
partially located in the outer leaflet.
Diffusion of I-Ekproteins is unconstrained
Diffusion coefficients from both the GPI-linked and trans-
membrane I-Ekproteins show homogeneous diffusion at all
cholesterol concentrations when imaged at 37?C, indicating
that the entire population of each protein diffuses in a similar
environment regardless of the cholesterol concentration.
individual fluorescent lipid analogs. Individual trajectories
were clipped to be 10 steps long for Tritc-DHPE and
20 steps for DiIC18. A time lag of 30.8 ms was chosen such
that 5 or 10 displacements were used to calculate a dif-
fusion coefficient for each track. The solid lines represent
the expected homogeneous distribution of Ds given the
average diffusion coefficient for the full population. The
dashed lines represent the expected two population dis-
tribution (see Supplementary Material for fit parameters).
Histogram of diffusion coefficients for Tritc-DHPE at
normal total cell cholesterol concentration (A) and after 2 h
b-CD (B). Histogram of diffusion coefficients for Tritc-
DHPE in the presence of TNBS at normal total cell
cholesterol concentration (C) and after 2 h b-CD (D).
DiIC18at normal total cell cholesterol concentration (E)
and after 2 h b-CD (F). DiIC18with TNBS at normal total
cell cholesterol concentration (G) and after 2 h b-CD (H).
Distributions of diffusion coefficients for
Chol Depletion Induces Solid Formation933
Biophysical Journal 90(3) 927–938
Also, positional trajectories showed no evidence of confine-
ment for GPI-linked or transmembrane I-Ekat any choles-
terol concentration studied, for cells imaged at either room
temperature (25,26) or 37?C. The diffusion for both proteins
is predominately Brownian for the length of the trajectories
at all studied cholesterol concentrations, as judged by our
measured a-parameter, although instances of non-Brownian
motion were observed for portions of single-molecule tra-
jectories in very rare cases (;1%) (data not shown). The
average on-time for a Cy5 molecule (0.5 s) and the inte-
gration time of one frame (15.4 ms) can be used to place
bounds on the region of free diffusion. Given the diffusion
coefficient at normal and ;50% normal total cell cholesterol,
we observe unconstrained diffusion of the GPI-linked I-Ek
protein in areas up to 2.2 mm2at normal or 1.0 mm2at ;50%
normal total cell cholesterol. We observe unconstrained
diffusion in areas of up to 1.2 mm2at normal or 0.9 mm2at
;50% normal total cell cholesterol for transmembrane I-Ek
protein (,r2. ¼ 4Dt). Using a more conservative estimate
of the time required for a molecule to sense the confinement
boundary for a stationary, impermeable domain (Ær2ðNÞæ ¼
Dt=4) (56), the upper limit is 1.4 mm2at normal or 0.06 mm2
at ;50% normal total cell cholesterol for GPI-linked I-Ek
and 0.08 mm2or 0.05 mm2for transmembrane I-Ek. Our
results do not preclude the existence of larger domains,
mobile domains, domains with permeable boundaries, or
At 37?C, the diffusion coefficients for the GPI-linked and
transmembrane I-Ekproteins are cholesterol dependent,
decreasing by a factor of 2.2 and 1.7, respectively, when
the total cell cholesterol is reduced to ;50% of normal. The
twofold decrease is similar to the magnitude of the decrease
lesterol depletion at 37?C. Cells were pretreated with 2 h
b-CD, then either the actin cytoskeleton was disrupted
using 4, 13, or 40 mM cytochalasin D treatment (A and B)
or microtubules were disrupted using nocodazole (C and
D). (A) Diffusion coefficients as a function of time lag for
GPI-linked I-Ekafter actin cytoskeleton disruption. The D
values at normal cholesterol (D ¼ 1.1 6 0.06 mm2/s) and
after 2 h b-CD (D ¼ 0.49 6 0.03 mm2/s) are shown for
comparison (lines with error bars). (B) D values as
a function of time lag for transmembrane I-Ekafter actin
cytoskeleton disruption. The D values at normal choles-
terol (D ¼ 0.59 6 0.04 mm2/s) and after 2 h b-CD (D ¼
0.34 6 0.03 mm2/s) are shown for comparison (lines with
error bars). (C) D values for GPI-linked I-Ekat 37?C after
the tubulin network was disrupted. The D values at normal
cholesterol and after 2 h b-CD are again shown for
comparison (lines with error bars). (D) D values for
transmembrane I-Ekat 37?C after the tubulin network was
disrupted. The D values at normal cholesterol and after 2 h
b-CD are again shown for comparison (lines with error
Effect of cytoskeletal disruption after cho-
the MHC proteins. Data were taken at temperatures between
22?C and 42?C. Normal refers to normal cholesterol
concentration. (A) GPI-linked I-Ek. (B) Transmembrane
I-Ek. Activation energies are reported in Table 2. Diffusion
coefficients were calculated from fits to the cumulative
distribution function averaged over all time lags. The number
of tracks used in each case is reported in the Supplementary
Arrhenius plot of the diffusion coefficients for
934 Nishimura et al.
Biophysical Journal 90(3) 927–938
in diffusion coefficients reported by Kenworthy et al. (38) for
GPI-linked and transmembrane proteins imaged in cells with
;30% of normal cholesterol levels at 37?C.
Heterogeneity in the diffusion of fluorescent
The distributions of diffusion coefficients for Tritc-DHPE at
normal and reduced cholesterol concentrations were hetero-
geneous. At 22?C, there are two populations of diffusers with
average values centered at ;2 and 0.2 mm2/s. Histograms of
the diffusion coefficients for Tritc-DHPE show 80% of the
population contributing to the slower diffusion coefficient. In
the presence of TNBS, which quenches fluorophores in the
outer leaflet, the relative size of the slower population is
reduced by 25%, indicating that Tritc-DHPE in the slower
population were present in the outer leaflet. The diffusion of
Tritc-DHPE in the outer leaflet decreased ;6-fold after
cholesterol depletion, whereas diffusion in the inner leaflet
did not significantly change. Distributions of diffusion
coefficients for DiIC18were homogeneous, and not signif-
icantly altered by the presence of TNBS. It is unlikely that all
of the DiIC18is present in the inner leaflet. We speculate that
the diffusion of DiIC18is similar in the inner and outer
leaflets, and thus introduction of TNBS does not change the
distribution of diffusion coefficients.
These results suggest that the regions in the inner leaflet
probed by the fast Tritc-DHPE population and DiIC18
respond differently to cholesterol depletion than the regions
in the outer leaflet probed by the GPI-linked and transmem-
brane I-Ekproteins and the slower Tritc-DHPE population.
The composition of biological membranes is thought to be
asymmetric; phosphatidylcholine and sphingomyelin lipids
are enriched in the outer leaflet and lipids with phosphatidyl-
ethanolamine and phosphatidylserineheadgroups are enriched
in the inner leaflet (57). In erythrocyte plasma membranes, the
inner leaflet is reported to contain more unsaturated lipids than
the outer leaflet (58,59). The diffusion of fluorescent lipid
analogs in the inner leaflet is reportedly faster than in the outer
leaflet of the plasma membrane of human erythrocytes (60),
Chinese hamster lung fibroblasts (61), and bovine aortic
(63) at 37?C to minutes at 4?C (64,65), so it is likely that cho-
lesterol may redistribute between the two leaflets during the 2 h
of b-CD treatment utilized here.
Temperature dependence of diffusion at reduced
cholesterol concentrations suggests formation of
Arrhenius plots were used to analyze the temperature
dependence of the mean diffusion coefficients for GPI-
linked and transmembrane I-Ek
cholesterol concentrations in the range of 22?C to 42?C. At
normal cell cholesterol concentration, the Arrhenius plots of
the diffusion coefficients for both proteins are linear as
the temperature of the cells is raised from room temperature
to above physiological temperature (22?C–42?C). The
activation energies (Ea) for diffusion for the I-Ekproteins
are higher than the values of 12.6–54.4 kJ/mol reported for
fluorescent lipid analogs with chain lengths from C6 to C18
diffusing in liquid multibilayers composed of dimyristoyl-
phosphatidylcholine (DMPC), dioleoylphosphatidylcholine
(DOPC), egg phosphatidylcholine, or bovine brain phos-
phatidylserine over a similar temperature range (53,54,66).
When the cell cholesterol was incrementally reduced, Ea
gradually increased over the temperature range of from 22?C
to ;35?C. After a 2 h b-CD treatment, the Eavalues for the
I-Ekproteins were similar to the Eaof 151 kJ/mol reported
for NBD-PE diffusion in the gel phase of a DMPC multi-
bilayer (55). Above ;35?C after cholesterol depletion, the
Eavalues for the diffusion of both the I-Ekproteins were
similar to those reported for fluorescent lipid analogs
(53,54,66). The change in Eaat ;35?C, for the proteins after
cholesterol depletion, is consistent with a pseudo ‘‘phase
transition’’ for the plasma membrane lipids.
In a previous work, we proposed that the observed
decrease in the diffusion coefficients after cholesterol
depletion at room temperature could be understood in terms
of the model of condensed complexes in which cholesterol
associates with high melting phospholipids to form a liquid
(26). Removal of cholesterol then releases the high melting
lipids from complexes to form a solid-like phase. These
solid-like regions act as obstacles to diffusion. The constant
Eafor diffusion at normal cell cholesterol and the subsequent
increases in the observed Ea after cholesterol depletion
support the model of solid-like domain formation in the
plasma membrane caused by freezing of high melting
phospholipids when cholesterol is removed. It is interesting
to note that the biochemical and physical properties of fatty
acid auxotrophs of Escherichia coli also display breaks in
Arrhenius plots near the growth temperatures (67,68).
Cholesterol extraction affected various membrane probes
to different extents. The diffusion coefficient of ;80–90% of
the Tritc-DHPE molecules (the slow fraction) decreased by
a factor of ;6 at 22?C and ;3 for the full population at 37?C
when the cell cholesterol was reduced to ;50% of normal.
However, the diffusion coefficient for ;10–20% of the
Tritc-DHPE molecules (the fast fraction) was unaffected by
cholesterol depletion over this range at both temperatures.
In contrast, the DiIC18probe showed only a small drop in
proteins at different
TABLE 2Activation energies
Ea(kJ/mol) GPI-linked I-Ek
83.7 6 4.4
68.4 6 0.82Normal
2 h b-CD
10 min b-CD
143.6 6 10.2 47.6 6 1.1
(37?C–42?C) (22?C–32?C) (37?C–42?C)
150.5 6 3.6
48.7 6 13.4
52.0 6 8.5
Chol Depletion Induces Solid Formation935
Biophysical Journal 90(3) 927–938
diffusion upon cholesterol depletion. Taken together, the
differences in cholesterol dependence for the GPI-linked and
transmembrane I-Ekproteins, Tritc-DHPE, and DiIC12(26)
or DiIC18indicate the presence of regional heterogeneity in
the CHO cell plasma membrane and the differences in the
region of the membrane sensed by the different probes. The
effective viscosity of the membrane does not appear to have
increased homogeneously after cholesterol depletion.
The decrease in the diffusion coefficients for the GPI-
linked and transmembrane I-Ekcould be ascribed to an
increaseineitherthe sizeofthe diffusingobjectorthe concen-
tration or size of immobile obstacles present in the plasma
membrane (45,69,70). The size of the diffusing object would
need to increase to several microns in diameter to account for
the observed decrease in the diffusion coefficient for the I-Ek
proteins at room temperature (26). Alternately, the shape of
the putative solid-like regions could be filamentous, thus
suppressing the mobility of certain membrane molecules.
However, no evidence of clustering or spatial heterogeneity
has been observed for the GPI-linked I-Ek, transmembrane
fluorescence microscope images (data not shown). Certainly
the heterogeneity in the Tritc-DHPE motion suggests that
some of the lipid probes are in a more restricted environment
than others. Nevertheless, we cannot make a definitive
statement as to the shape of these solid-like regions, which
appear to act as obstacles to the diffusion ofcertain molecules.
The solid-like regions might be a solid solution, or glass. In
the latter case the breaks in Arrhenius plots could be regarded
as a glass transition.
Cytoskeletal effects are negligible
The decrease in the diffusion coefficients of the MHC pro-
teins does not appear to be mediated by cytoskeletal interac-
tions since neither prevention of actin polymerization nor
depolymerization of the tubulin network led to an increase in
the diffusion of the proteins after cholesterol depletion when
imaged at 15.4 ms per frame. This result is consistent with
our previous work at room temperature (25).
In the literature, actin disruption using varying cytocha-
lasin D treatments has been reported to have an effect on the
diffusion of certain membrane molecules. Kwik et al. (35)
reported that a 10 mM (5 mg/ml) cytochalasin D treatment for
30 min after cholesterol depletion to 50–60% of normal cell
cholesterol led to an increase in the mobile fraction of the
human leukocyte antigen (HLA) proteins. In the same report,
a 4 mM (2 mg/ml) cytochalasin D treatment for 3 min at room
temperature and 20 min at 31?C after cholesterol depletion
was reported to decrease the number of HLA molecules con-
fined by the cholesterol-dependent, PIP2-mediated, reorga-
nization of cytoskeleton (35). Murase et al. (41) reported
that a 13 mM cytochalasin D treatment for 5 min at 37?C
resulted in a fourfold increase in the area of confinement
for Cy3-labeled 1,2-dioleoyl-sn-glycero-3-phosphoethanol-
amine (DOPE). In this study, we found no change in the
diffusion coefficient for the GPI-linked or transmembrane
I-Ek proteins after treatment with various concentrations of
cytochalasin D after cholesterol depletion.
In this work, a temperature increase from 22?C to 42?C
showed faster diffusion of transmembrane and GPI-linked
MHC II proteins and lipid analogs, and for the case with
lowered cholesterol, a change in activation energy for the
motion, suggesting a change in the phase behavior of the
plasma membrane. Whereas analysis of the diffusion for
MHC II proteins showed homogeneous behavior, Tritc-
DHPE motion showed a clear presence of two populations,
the slower of which became even slower in its diffusion upon
cholesterol extraction. The slow population is likely to arise
from Tritc-DHPE localized in the outer leaflet. The
variations in behavior are likely due to the differing inner-
outer leaflet localization of the lipid analogs and the MHC II
proteins in the plasma membrane. Taken together, these re-
sults provide evidence for the presence of solid-like regions
in the plasma membrane at reduced cholesterol levels.
An online supplement to this article can be found by visiting
BJ Online at http://www.biophysj.org.
The authors thank Michael Edidin for suggesting the use of a membrane
impermeable fluorescence quencher.
This work was supported in part by National Science Foundation grant No.
and National Institutes of Health grant No. 1 P20 HG003638-01 (W.E.M.).
H.M.McC. acknowledges support from the Dept. of Chemistry at Stanford
1. Subramanian, S., and H. M. McConnell. 1987. Critical mixing in
monolayer mixtures of phospholipid and cholesterol. J. Phys. Chem.
2. Dietrich, C., L. A. Bagatolli, Z. N. Volovyk, N. L. Thompson, M. Levi,
K. Jacobson, and E. Gratton. 2001. Lipid rafts reconstituted in model
membranes. Biophys. J. 80:1417–1428.
3. Dietrich, C., Z. N. Volovyk, M. Levi, N. L. Thompson, and K.
Jacobson. 2001. Partitioning of Thy-1, GM1, and cross-linked phos-
pholipid analogs into lipid rafts reconstituted in supported model
membrane monolayers. Proc. Natl. Acad. Sci. USA. 98:10642–
4. Samsonov, A. V., I. Mihalyov, and F. S. Cohen. 2001. Characterization
of cholesterol-sphingomyelin domains and their dynamics in bilayer
membranes. Biophys. J. 81:1486–1500.
5. Veatch, S. L., and S. L. Keller. 2002. Organization in lipid membranes
containing cholesterol. Phys. Rev. Lett. 89:2681011–2681014.
6. Veatch, S. L., and S. L. Keller. 2003. Separation of liquid phases in
giant vesicles of ternary mixtures of phospholipids and cholesterol.
Biophys. J. 85:3074–3083.
936Nishimura et al.
Biophysical Journal 90(3) 927–938
7. Bagatolli,L.A.,andE.Gratton.2000.Twophotonfluorescence micros-
copy of coexisting lipid domains in giant unilamellar vesicles of binary
phospholipid mixtures. Biophys. J. 78:290–305.
8. Korlach, J., P. Schwille, W. W. Webb, and G. W. Feigenson. 1999.
Characterization of lipid bilayer phases by confocal microscopy and
fluorescence correlation spectroscopy. Proc. Natl. Acad. Sci. USA.
9. Feigenson, G. W., and J. T. Buboltz. 2001. Ternary phase diagram of
dipalmitoyl-PC/dilauroyl-PC/cholesterol: nanoscopic domain forma-
tion driven by cholesterol. Biophys. J. 80:2775–2788.
10. Vist, M. R., and J. H. Davis. 1990. Phase equilibria of cholesterol/
dipalmitoylphosphatidylcholine mixtures: 2H nuclear magnetic reso-
nance and differential scanning calorimetry. Biochemistry. 29:451–
11. Veatch, S. L., I. V. Polozov, K. Gawrisch, and S. L. Keller. 2004.
Liquid domains in vesicles investigated by NMR and fluorescence
microscopy. Biophys. J. 86:2910–2922.
12. Radhakrishnan, A., and H. M. McConnell. 1999. Cholesterol-phospho-
lipid complexes in membranes. J. Am. Chem. Soc. 121:486–487.
13. Radhakrishnan, A., and H. M. McConnell. 1999. Condensed complexes
of cholesterol and phospholipids. Biophys. J. 77:1507–1517.
14. McConnell, H. 2005. Complexes in ternary cholesterol-phospholipid
mixtures. Biophys. J. 88:L23–L25.
15. McConnell, H. M., and A. Radhakrishnan. 2003. Condensed com-
plexes of cholesterol and phospholipids. Biochim. Biophys. Acta. 1610:
16. McConnell, H. M., and M. Vrljic. 2003. Liquid-liquid immiscibility in
membranes. Annu. Rev. Biophys. Biomol. Struct. 32:469–492.
17. Keller, S. L., W. H. Pitcher III, W. H. Huestis, and H. M. McConnell.
1998. Red blood cell lipids form immiscible liquids. Phys. Rev. Lett.
18. Simons, K., and E. Ikonen. 1997. Functional rafts in cell membranes.
19. Brown, D. A., and J. K. Rose. 1992. Sorting of GPI-anchored proteins
to glycolipid-enriched membrane subdomains during transport to the
apical cell surface. Cell. 68:533–544.
20. Schroeder, R., E. London, and D. Brown. 1994. Interactions between
saturated acyl chains confer detergent resistance on lipids and
glycosylphosphatidylinositol (GPI)-anchored proteins: GPI-anchored
proteins in liposomes and cells show similar behavior. Proc. Natl.
Acad. Sci. USA. 91:12130–12134.
21. Brown, D. A., and E. London. 2000. Structure and function of
sphingolipid- and cholesterol-rich membrane rafts. J. Biol. Chem. 275:
22. Heerklotz, H., H. Szadkowska, T. Anderson, and J. Seelig. 2003. The
sensitivity of lipid domains to small perturbations demonstrated by the
effect of triton. J. Mol. Biol. 329:793–799.
23. Harder, T., P. Scheiffele, P. Verkade, and K. Simons. 1998. Lipid
domain structure of the plasma membrane revealed by patching of
membrane components. J. Cell Biol. 141:929–942.
24. Mayor, S., K. G. Rothberg, and F. R. Maxfield. 1994. Sequestration of
GPI-anchored proteins in caveolae triggered by cross-linking. Science.
25. Vrljic, M., S. Y. Nishimura, S. Brasselet, W. E. Moerner, and H. M.
McConnell. 2002. Translational diffusion of individual class II MHC
membrane proteins in cells. Biophys. J. 83:2681–2692.
26. Vrljic, M., S. Y. Nishimura, W. E. Moerner, and H. M. McConnell.
2005. Cholesterol depletion suppresses the translational diffusion of
class II major histocompatibility complex proteins in the plasma
membrane. Biophys. J. 88:334–347.
27. Anderson, R. G. W., and K. Jacobson. 2002. A role for lipid shells in
targeting proteins to caveolae, rafts and other lipid domains. Science.
28. Hao, M., S. Mukherjee, and F. R. Maxfield. 2001. Cholesterol
depletion induces large scale domain segregation in living cell mem-
branes. Proc. Natl. Acad. Sci. USA. 98:13072–13077.
29. Lommerse, P. H. M., H. P. Spaink, and T. Schmidt. 2004. In vivo
plasma membrane organization: results of biophysical approaches.
Biochim. Biophys. Acta. 1664:119–131.
30. Edidin, M. 2003. The state of lipid rafts: from model membranes to
cells. Annu. Rev. Biophys. Biomol. Struct. 32:257–283.
31. Xavier, R., T. Brennan, Q. Li, C. McCormack, and B. Seed. 1998.
Membrane compartmentation is required for efficient T cell activation.
32. Field, K., D. Holowka, and B. Baird. 1997. Compartmentalized
activation of the high affinity immunoglobulin E receptor within mem-
brane domains. J. Biol. Chem. 272:4276–4280.
33. Sheets, E. D., D. Holowka, and B. Baird. 1999. Critical role for
cholesterol in Lyn-mediated tyrosine phosphorylation of FceRI and
their association with detergent-resistant membranes. J. Cell Biol.
34. Vidalain, P. O., O. Azocar, C. Servet-Delprat, C. Rabourdin-Combe,
D. Gerlier, and S. Manie. 2000. CD40 signaling in human dendritic
cells is initiated within membrane rafts. EMBO J. 19:3304–3313.
35. Kwik, J., S. Boyle, D. Fooksman, L. Margolis, M. P. Sheetz, and M.
Edidin. 2003. Membrane cholesterol, lateral mobility, and the phos-
phatidylinositol 4,5-bisphosphate-dependent organization of cell actin.
Proc. Natl. Acad. Sci. USA. 100:13964–13969.
36. Shvartsman, D. E., M. Kotler, R. D. Tall, M. G. Roth, and Y. I. Henis.
2003. Differently anchored influenza hemagglutinin mutants display
distinct interaction dynamics with mutual rafts. J. Cell Biol. 163:879–888.
37. Sooksawate, T., and M. A. Simmonds. 2001. Effects of membrane cho-
lesterol on the sensitivity of the GABA(A) receptor to GABA in acutely
dissociated rat hippocampal neurones. Neuropharmacology. 40:
38. Kenworthy, A. K., B. J. Nichols, C. L. Remmert, G. M. Hendrix, M.
Kumar, J. Zimmerberg, and J. Lippincott-Schwartz. 2004. Dynamics
of putative raft-associated proteins at the cell surface. J. Cell Biol.
39. Bacia, K., D. Scherfeld, N. Kahya, and P. Schwille. 2004. Fluorescence
correlation spectroscopy relates rafts in model and native membranes.
Biophys. J. 87:1034–1043.
40. Wettstein, D. A., J. J. Boniface, P. A. Reay, H. Schild, and M. M.
Davis. 1991. Expression of a class II major histocompatibility complex
(MHC) heterodimer in a lipid-linked form with enhanced peptide/
soluble MHC complex formation at low pH. J. Exp. Med. 174:219–
41. Murase, K., T. Fujiwara, Y. Umemura, K. Suzuki, R. Iino, H.
Yamashita, M. Saito, H. Murakoshi, K. Ritchie, and A. Kusumi. 2004.
Ultrafine membrane compartments for molecular diffusion as revealed
by single molecule techniques. Biophys. J. 86:4075–4093.
42. Rotsch, C., and M. Radmacher. 2000. Drug-induced changes of
cytoskeletal structure and mechanics in fibroblasts: an atomic micros-
copy study. Biophys. J. 78:520–535.
43. Huby, R. D. J., A. Weiss, and S. C. Ley. 1998. Nocodazole inhibits
signal transduction by the T cell antigen receptor. J. Biol. Chem. 273:
44. Wakatsuki, T., B. Schwab, N. C. Thompson, and E. L. Elson. 2000.
Effects of cytochalasin D and latranculin B on mechanical properties of
cells. J. Cell Sci. 114:1025–1036.
45. Saxton, M. J. 1994. Anomalous diffusion due to obstacles: a Monte
Carlo study. Biophys. J. 66:394–401.
46. Smith, P. R., I. E. G. Morrison, K. M. Wilson, N. Fernandez, and
R. J. Cherry. 1999. Anomalous diffusion of major histocompatibility
complex class I molecules on HeLa cells determined by single particle
tracking. Biophys. J. 76:3331–3344.
47. Feder, T. J., I. Brust-Mascher, J. P. Slattery, B. Baird, and W. W.
Webb. 1996. Constrained diffusion of immobile fraction on cell sur-
faces: a new interpretation. Biophys. J. 70:2767–2773.
48. Qian, H., M. P. Sheetz, and E. L. Elson. 1991. Single particle tracking.
Analysis of diffusion and flow in two-dimensional systems. Biophys. J.
Chol Depletion Induces Solid Formation937
Biophysical Journal 90(3) 927–938
49. Schu ¨tz, G. J., H. Schindler, and T. Schmidt. 1997. Single-molecule mi-
croscopy on model membranes reveals anomalous diffusion. Biophys.
50. Wolf, D. E. 1985. Determination of the sidedness of carbocyanine dye
labeling of membranes. Biochemistry. 24:582–586.
51. Schwiegelshohn, B., J. F. Presley, M. Gorecki, T. Vogel, Y. A.
Carpentier, F. R. Maxfield, and R. J. Deckelbaum. 1995. Effects of
apoprotein E on intracellular metabolism of model triglyceride-rich
particles are distinct from effects on cell particle uptake. J. Biol. Chem.
52. Murakoshi, H., R. Iino, T. Kobayashi, T. Fujiwara, C. Ohshima, A.
Yoshimura, and A. Kusumi. 2004. Single-molecule imaging analysis of
Ras activation in living cells. Proc. Natl. Acad. Sci. USA. 101:7317–
53. Tamm, L. K., and H. M. McConnell. 1985. Supported phospholipid
bilayers. Biophys. J. 47:105–113.
54. Vaz, W. L., Z. I. Derzko, and K. A. Jacobson. 1982. Photobleaching mea-
surements of the lateral diffusion of lipids and proteins in artificial phos-
pholipid bilayer membranes. In Membrane Reconstitution. G. Poste and
G. L. Nicolson, editors. Elsevier Biomedical Press, Amsterdam. 83–136.
55. Smith, B. A., and H. M. McConnell. 1978. Determination of molecular
motion in membranes using periodic pattern photobleaching. Proc.
Natl. Acad. Sci. USA. 75:2759–2763.
56. Saxton, M. J. 1993. Lateral diffusion in an archipelago. Single-particle
diffusion. Biophys. J. 64:1766–1780.
57. Devaux, P. F. 1992. Protein involvement in transmembrane lipid asym-
metry. Annu. Rev. Biophys. Biomol. Struct. 21:417–439.
58. Myher, J. J., A. Kuksis, and S. Pind. 1989. Molecular species of
glycerophospholipids and sphingomyelins of human erythrocytes im-
proved methods of analysis. Lipids. 24:396–407.
59. Hullin, F., M.-J. Bossant, and N. Salem Jr. 1991. Aminophospholipid
molecular species asymmetry in the human erythrocyte plasma mem-
brane. Biochim. Biophys. Acta. 1061:15–25.
60. Morrot, G., S. Cribier, P. F. Devaux, D. Geldwerth, J. Davoust, J. F.
Bureau, P. Fellmann, P. Herve, and B. Frilley. 1986. Asymmetric
lateral mobility of phospholipids in the human erythrocyte membrane.
Proc. Natl. Acad. Sci. USA. 83:6863–6867.
61. Chahine, J., S. Cribier, and P. Devaux. 1993. Phospholipid trans-
membrane domains and lateral diffusion in fibroblasts. Proc. Natl.
Acad. Sci. USA. 90:447–451.
62. Julien, M., J.-F. Tournier, and J.-F. Tocanne. 1993. Differences in the
transbilayer and lateral motions of fluorescent analogs of phosphati-
dylcholine and phosphatidylethanolamine in the apical plasma
membrane of bovine aortic endothelial cells. Exp. Cell Res. 208:387–
63. Steck, T. L., J. Ye, and Y. Lange. 2002. Probing red cell mem-
brane cholesterol movement with cyclodextrin. Biophys. J. 83:
64. Schroeder, F., G. Nemecz, W. Gibson Wood, C. Joiner, G. Morrot, M.
Ayraut-Jarrier, and P. F. Devaux. 1991. Transmembrane distribution
of sterol in the human erythrocyte. Biochim. Biophys. Acta. 1066:
65. Muller, P., and A. Herrmann. 2002. Rapid transbilayer movement of
spin-labeled steroids in human erythrocytes and in liposomes. Biophys.
66. Derzko, Z. I., and K. A. Jacobson. 1980. Comparative lateral diffusion
of fluorescent lipid analogues in phospholipid multibilayers. Bio-
67. Tsukagoshi, N., and C. F. Fox. 1973. Transport system assembly and
the mobility of membrane lipids in Escherichia coli. Biochemistry.
68. Linden, C. D., K. L. Wright, H. M. McConnell, and C. F. Fox. 1973.
Lateral phase separations in membrane lipids and the mechanism of
sugar transport in Escherichia coli. Proc. Natl. Acad. Sci. USA.
69. Saffman, P. G., and M. Delbruck. 1975. Brownian motion in biological
membranes. Proc. Natl. Acad. Sci. USA. 72:3111–3113.
70. Hughes, B. D., B. A. Pailthorpe, and L. R. White. 1981. The trans-
lational and rotational drag on a cylinder moving in a membrane. J. Fluid
938Nishimura et al.
Biophysical Journal 90(3) 927–938