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 2006927–938927
(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
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