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Biophys J, August 1999, p. 925-933, Vol. 77, No. 2
*Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, New York 14853, and #Nutrimed Biotech, Cornell University Research Park, 270-276 Langmuir Laboratory, Ithaca, New York 14850 USA
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
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The dynamic structure of detergent-resistant membranes
(DRMs) isolated from RBL-2H3 cells was characterized using two
different acyl chain spin-labeled phospholipids (5PC and 16PC), a
headgroup labeled sphingomyelin (SM) analog (SD-Tempo) and a
spin-labeled cholestane (CSL). It was shown, by comparison to
dispersions of SM, dipalmitoylphosphatidylcholine (DPPC), and
DPPC/cholesterol of molar ratio 1, that DRM contains a substantial
amount of liquid ordered phase: 1) The rotational diffusion rates
(R
) of 16PC in DRM between 
5°C and
45°C are nearly the same as those in molar ratio DPPC/Chol = 1 dispersions, and they are substantially greater than
R in pure DPPC dispersions in the gel
phase studied above 20°C; 2) The order parameters (S)
of 16PC in DRM at temperatures above 4°C are comparable to those in
DPPC/Chol = 1 dispersions, but are greater than those in DPPC
dispersions in both the gel and liquid crystalline phases. 3)
Similarly, R for 5PC and CSL in DRM is
greater than in pure SM dispersions in the gel phase, and
S for these labels in DRM is greater than in the SM
dispersions in both the gel and liquid crystalline phases. 4)
R of SD-Tempo in DRM is greater than in
dispersions of SM in both gel and liquid phases, consistent with the
liquid-like mobility in the acyl chain region in DRM. However,
S of SD-Tempo in DRM is substantially less than that of
this spin label in SM in gel and liquid crystalline phases (in absolute
values), indicating that the headgroup region in DRMs is less ordered
than in pure SM. These results support the hypothesis that plasma
membranes contain DRM domains with a liquid ordered phase that may
coexist with a liquid crystalline phase. There also appears to be a
coexisting region in DRMs in which the chain labels 16PC and 5PC are
found to cluster. We suggest that other biological membranes containing high concentrations of cholesterol also contain a liquid ordered phase.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
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IgE receptors (Fc
RI) initiate signaling in
RBL-2H3 mast cells after aggregation by antigen. Recently, Field et al.
(1997)
showed that this process involves the association of these
transmembrane receptors and the Src family protein-tyrosine kinase Lyn,
which phosphorylates the 
and 
subunits of FcRI, with
detergent-resistant membranes (DRMs) of a special composition. In
addition, microscopy data show that the same interactions observed
between aggregated IgE-FcRI and DRM components after lysis at low
concentrations of Triton X-100 (TX-100) also occur at the surface of
intact cells (Pierini et al. 1996; Holowka et al., submitted for
publication). These findings provide a structural basis for the initial
coupling between Lyn and FcRI in the cellular signaling process that
utilizes stimulus-sensitive protein-lipid interactions. Recent evidence indicates that DRM-like structures, including morphologically identifiable caveolae, are involved in the signaling of other cell
surface receptors (Anderson, 1998; Brown and London, 1998a) and in
membrane trafficking (Simons and Ikonen, 1997).
Elucidation of the molecular details of the mechanism by which these
cellular signaling processes are initiated and promoted by DRM is a
major challenge to both biochemists and biophysicists. Some progress
has been made toward this goal by investigating the physical properties
of these structures in model membranes. It is known that DRMs from
cells are enriched in SM, gangliosides, and cholesterol (Chol) (Brown
and Rose, 1992). Lund-Katz et al. (1988) showed that Chol packs more
densely with sphingomyelin (SM) monolayers than with
phosphatidylcholine (PC) monolayers. Recently, Schroeder et al. (1994)
found that detergent-resistant liposomes with a composition similar to
that of DRMs are as fluid as
1,2-dipalmitoylphosphatidylcholine/cholesterol (DPPC/Chol) membranes,
which are known to be in a liquid ordered phase. The liquid ordered
phase was defined by Ipsen et al. (1987) as the phase structure of
model membranes containing a high concentration of Chol. Thus it was
also referred to as a high-Chol liquid phase (Ipsen et al., 1987).
Ahmed et al. (1997) further demonstrated that the formation of a liquid
ordered phase is promoted by SM and Chol in model membranes, and they
inferred that the detergent-insoluble membranes isolated from cells are
likely to exist in the liquid ordered phase. Hanada et al. (1995)
showed that both SM and Chol are involved in causing the insolubility
of human placental alkaline phosphatase (a glycosylphosphatidylinositol
(GPI)-linked protein) in TX-100, and they suggested that together, SM
and Chol play a role in the formation of DRM. The role played by
ganglioside GM1 in DRMs was explored by Ferroretto et al.
(1997), who found that GM1/SM, SM/Chol, and
GM1/GM1 interactions all contribute to the
formation of DRMs. Other recent studies show that the liquid ordered
phase and not specific molecular interactions is responsible for the
detergent insolubility of GPI-anchored proteins (Schroeder et al.,
1998).
Despite these efforts, so far, no experimental results on the physical
properties of DRMs directly isolated from cell membranes have been
reported. Thus, for example, the question remains whether DRMs from
cell membranes have a liquid ordered structure that could facilitate
their phase segregation from the surrounding membranes. The mechanism
of how DRMs are involved in the various biological processes is only
partially understood (Sheets et al., 1999). We have conducted an
electron spin resonance (ESR) spin-labeling study that characterizes
the ordering and dynamics of DRM vesicles, isolated from RBL-2H3 cells
after lysis by TX-100. Our results provide a detailed analysis of ESR
spectra from a headgroup-labeled SM analog (SD-Tempo), two
chain-labeled PCs (5PC and 16PC), and a spin-labeled Chol analog (CSL)
incorporated into the DRM vesicles and vesicle dispersions of SM, DPPC,
and DPPC/Chol = 1. By comparing the structural and dynamic
properties of the DRM vesicles with these model membranes, and by
comparing our ESR results with those from previous studies of the
effects of Chol on the physical properties of model membranes, we find
that the ordering and dynamics in DRM vesicles are very similar to
those in dispersions of DPPC/Chol = 1 between 4°C and 45°C. We
chose this concentration of Chol in the model membrane samples because
the DPPC/Chol = 1 membrane is known to be in a liquid ordered
phase (Brown and London, 1998b). Furthermore, the concentration of Chol
in plasma membranes is known to be in the range of 30-50 mol% Chol
(Gennis, 1989). In addition, this concentration of Chol is less than
the maximum solubility of Chol in DPPC bilayers (Huang et al., 1999).
Thus we confirm for the first time that the DRMs, derived from RBL-2H3 cell detergent lysates, contain the liquid ordered phase. We also find
evidence for structural inhomogeneity in DRM, and this might play a
role in IgE receptor signaling.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
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Materials
Chain-labeled PCs (5PC and 16PC) and non-labeled lipids were
purchased from Avanti (Alabaster, AL). The headgroup-labeled SM analog,
SD-Tempo, was synthesized at Nutrimed Biotech (Ithaca, NY). The spin
label 3-doxyl-5
-cholestane (CSL), a Chol analogue, was purchased
from Sigma Chemical Co. (St. Louis, MO). The chemical structures of
these spin labels are shown in Fig. 1.
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Sample preparations
Isolation of DRM from RBL-2H3 mast cells
RBL-2H3 cells were maintained in cell culture, harvested, and lysed in TX-100 as previously described (Field et al., 1995, 1997). For
most experiments, 0.05% TX-100 (v/v) was used for lysis to produce
conditions that preserve interactions with cross-linked IgE receptors
(Field et al., 1997). For these, the lysates (4 × 106
cells/ml) were adjusted to 40% (wt/v) sucrose by diluting with an
equal volume of 80% (wt/vol) sucrose for a final volume of 20 ml. The
lysates were then layered with 5 ml each of 35% (w/v) and 5% (w/v)
sucrose containing 25 mM Tris (pH 7.5), 125 mM NaCl, and 2 mM EDTA.
Gradients were centrifuged in a SW27 rotor (Beckman Instruments, Palo
Alto, CA) at 100,000 × g for 16 h at 4°C. The opaque material containing DRM vesicles at the interface of the 35%
and 5% sucrose layers was harvested in ~3.0 ml. The DRM fractions from two or three identically prepared gradients were typically pooled
before labeling. For experiments with CSL, 0.25% TX-100 (v/v) was used
for cell lysis, and sucrose gradients of cell lysates were carried out
as described by Field et al. (1995).
Incorporation of 16PC and 5PC into DRM vesicles
Pooled DRM fractions (6-9 ml) from sucrose gradients of cells (8-10 × 107) lysed in 0.05% TX-100 (v/v) were diluted to 20 ml with 40 mM HEPES (pH 7.4) and centrifuged at 300,000 × g for 30 min in a Ti60 rotor (Beckman Instruments, Palo Alto, CA). Pelleted DRMs were resuspended in 1-3 ml of HEPES buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM EDTA). For each sample, 10-20 µl of 0.07 mM 16PC or 5PC in methanol was added dropwise to 1-2 ml of washed DRM, and methanol was added to a final concentration of 2% (v/v). Following incubation for 30 min to 2 h at 4°C, these samples were diluted to 4 ml with 25 mM HEPES buffer and centrifuged at 250,000 × g for 30 min in an SW60 rotor (Beckman Instruments). The DRM pellets were resuspended in 4 ml HEPES buffer and recentrifuged, and the pellets were transferred to a capillary of 1.5 mm internal diameter for ESR measurements. This amount of spin label was found to lead to optimal ESR spectra. Higher concentrations cause severe exchange broadening (cf. discussion below), whereas lower concentrations greatly reduce the signal-to-noise ratio. Using Fourier transform mass spectrometry, it was determined that DRMs washed by centrifugation and resuspension as in the labeling procedure above contain no detectable TX-100 (i.e., <0.0001 (v/v)).Incorporation of SD-Tempo into DRM vesicles
Eighty microliters of 0.03 mM SD-Tempo in methanol was added dropwise to DRM vesicles, which were pooled from sucrose gradients of cells (6-10 ml of 8-12 × 107 cell equivalents) lysed in 0.05% TX-100 as described above. Methanol was added to a final concentration of 2% (v/v), and the samples were incubated for 1 h at 4°C, then diluted to 4 ml in HEPES buffer. The labeled DRM vesicles were pelleted, washed, and analyzed as for DRM vesicles labeled with 16PC or 5PC described above. The amount of spin label used was found to optimize the ESR spectra (cf. above).Incorporation of CSL into DRM vesicles
DRM vesicles prepared from ~7 × 107 cells lysed in 0.25% TX-100 were recovered in 0.8 ml, and 30 µl of saturated CSL (~0.04 mM) in methanol was added to the DRM sample to optimize the ESR spectra as described above for SD-Tempo and the PC derivatives. DRM vesicles were then pelleted as described above. For some experiments, CSL-labeled DRM vesicles were washed by ultracentrifugation (250,000 × g for 10 min) and resuspended in 25 mM Tris (pH 7.5), 125 mM sodium chloride, and 2 mM EDTA, before a second ultracentrifugation and analysis of the pellet by ESR. Identical ESR spectra were obtained for pellets with and without the wash step.Preparation of model membranes
Measured stock solutions of lipid (SM in chloroform:methanol 1:1 (v/v), and DPPC and chol in chloroform) and the spin label (SD-Tempo in chloroform:methanol 1:1 (v/v); other spin labels in chloroform) were mixed thoroughly in a glass tube. The total weight of dry lipids was 2 mg, and the concentration of spin labels was 0.5 mol% of the lipids for all samples except for the case of SD-Tempo, where it was 0.1 mol%. Additional experiments to check the effects of spin concentration were also performed. As the solvent was evaporated by N2 flow, the dried lipids formed a thin film on the wall of the tube, then the sample was evacuated with a mechanical pump for at least 2 h to remove trace amounts of the solvent. After the addition of 2 ml of 50 mM Tris (pH 7.0), 160 mM sodium chloride, and 0.1 mM EDTA, the lipids were scraped off the wall, and the solution was stirred for 1 min and kept in the dark at room temperature for at least 2 h.ESR spectroscopy and nonlinear least-squares analysis of ESR spectra
Spectra were obtained at a frequency of 9.55 GHz on a Bruker
Instruments ER-200 ESR spectrometer equipped with a Varian temperature control unit. The field sweeps were calibrated with a Bruker ER 035M
NMR gaussmeter. All spectra were digitized to 512 points and had
~120G sweep widths. Nonlinear least-squares (NLLS) analyses of the
spectra based on the stochastic Liouville equation (Meirovitch et al.,
1982; Schneider and Freed, 1989) were performed using the latest
fitting program (Budil et al., 1996), which yields the following
parameters. R is the rotational diffusion rate of the nitroxide radical around an axis perpendicular to the mean
symmetry axis for the rotation. This symmetry axis is also the
direction of preferential orientation of the spin-labeled molecule
(Schneider and Freed, 1989). For 16PC and 5PC,
R represents the rotational wagging motion of
the long axis of the acyl chains, whereas for CSL it is the rotational
wagging motion of the long molecular axis (Ge et al., 1994). For the
headgroup label SD-Tempo, it represents the rotational motion of the
nitroxide headgroup (Ge and Freed, 1998). These are illustrated in Fig. 1. For the simulation of ESR spectra of spin labels incorporated into
multilamellar vesicles, a MOMD model (which stands for microscopic order and macroscopic disorder; Meirovitch et al., 1984; Budil et al.,
1996) was used. This model is based on the characteristics of the
dynamic structure of lipid dispersions, i.e., locally in a lipid
bilayer segment, lipid molecules are preferentially oriented by the
structure of the bilayer, but globally the lipid bilayer segments are
distributed randomly (Meirovitch et al., 1984). The order parameter,
S, is a measure of the angular extent of the rotational
diffusion of the nitroxide moiety; the larger S is, the more
restricted is the motion. Therefore, S reflects the local ordering of lipid molecules in the disordered membrane dispersions. The
magnetic A tensor and g tensor components needed
for the simulations were obtained from fits to rigid limit spectra of
the samples, which were taken at temperatures below 150°C.
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RESULTS AND DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
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Comparison of DRM vesicles with SM, DPPC, and DPPC/Chol = 1 dispersions
ESR spectra from 16PC in DRM vesicles were collected from 5°C
to 45°C; those in dispersions of pure DPPC were collected from 25°C
to 50°C, which includes the temperature for the DPPC gel-to-liquid crystalline phase transition, 41°C (Marsh, 1990). Those in
dispersions of DPPC/Chol = 1 were collected from 5°C to
50°C, to compare with the spectra from DRM vesicles and pure DPPC
dispersions. Spectra (experimental and simulated) of 16PC at selected
temperatures from DPPC, DPPC/Chol = 1 dispersions and DRM vesicles
are shown in Fig. 2. All of the spectra
from DPPC and DPPC/Chol = 1 dispersions can be simulated very well
with just one component. However, the spectra from the DRM vesicles
showed additional features characteristic of a broadened component.
These spectra were simulated, allowing for two components in achieving
the best least-squares fits. We find that one component is a spectrum
with the normal three hyperfine lines, which is very similar to those
obtained from the model membranes. The other spectrum is a single broad
line, which is a clear indication of strong spin-spin interactions,
which would be expected from clustering of the 16PC molecules (Fajer et
al., 1992; Earle et al., 1994; and cf. below). The two components
obtained from the simulation of the spectrum at 24°C are shown in
Fig. 3. The relative population of the
sharper component with three hyperfine lines is ~0.30 at all
temperatures above 5°C, but it drops to 0.18 at 5°C. (Because
the ESR signal height is inversely proportional to the square of the
linewidth, we note that the broad component only makes a small
contribution to the composite lineshape, as shown in Fig. 3.) The best
fit parameters for R and S of 16PC
in the DRM vesicles and in dispersions of DPPC, DPPC/Chol = 1 are
listed in Table 1, and their variations
with temperature are shown in Fig. 4.
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As shown in Fig. 4 A and Table 1, there is a sharp increase
in R of 16PC in DPPC dispersions from 40°C
to 41°C, indicative of the gel-to-liquid crystalline
(L
) phase transition of DPPC bilayers. In contrast, the
values of R of 16PC in DRM vesicles increase
smoothly from 8.71 × 107 s
1 at 5°
to 3.22 × 108 s1 at 45°C. They are
greater than the values of R of 16PC in DPPC
dispersions of the gel phase, which are between 5.05 × 107 s1 and 6.61 × 107
s1, and they are larger than or comparable to those in
DPPC dispersions in the liquid crystalline phase, 1.00-4.00 × 108 s1. For DPPC/Chol = 1 dispersions,
the values of R are close to those for the
DRM vesicles at the corresponding temperatures. Similarly, they show no
sign of a gel-to-L phase transition.
As shown in Fig. 4 B and Table 1, the curves of S
versus temperature for DRM and for DPPC/Chol = 1 are comparable to
each other above 4°C, with values of S that are
significantly larger than those for DPPC dispersions in both its gel
and L phases. It is also seen in Fig. 4 B
that the value of S of 16PC in DPPC dispersions decreases
from 0.16 at 25°C to 0.06 at 40°C, then drops to zero at 41°C, a
further indication of the gel-to-L phase transition,
with S becoming zero in the liquid crystalline phase. No
such drop in S is observed in DRM vesicles, nor for DPPC/Chol = 1 dispersions over the whole temperature range studied.
The differences in the temperature dependence of
R and S for DRM and DPPC/Chol = 1 versus pure DPPC are consistent with the differences in their ESR
lineshapes, as they must be. As shown in Fig. 2, when the temperature
goes from 40°C to 41°C, the spectrum of 16PC in DPPC dispersions
suddenly narrows. In contrast, over the wide temperature ranges
studied, the spectra of 16PC in DRM vesicles and in DPPC/Chol = 1 dispersions change only gradually with temperature.
The above results with 16PC indicate that the physical properties of
DRM vesicles are similar to those of DPPC/Chol = 1 dispersions and
are quite different from those of DPPC dispersions. Specifically: 1)
The ordering of the acyl chains near the center of the bilayers in DRM
vesicles and in DPPC/Chol = 1 dispersions is higher than that in
DPPC dispersions in the gel phase. 2) The rotational mobility of acyl
chains near the center of the bilayers in DRM vesicles and in
DPPC/Chol = 1 dispersions is comparable over the whole temperature
range, but is significantly greater than that in pure DPPC dispersions
in the gel phase (20-40°C); only in the L phase
(>41°C) do the latter become comparable. 3) The characteristic gel-to-L phase transition at 41°C in DPPC dispersions
is not observed in DPPC/Chol = 1 dispersions over the whole range of temperatures studied. Similarly, there is no sign of any phase transition for DRM vesicles in the corresponding temperature range.
We used additional spin labels to probe the dynamic structure over the
acyl chain region and the headgroup region of the DRM: SD-Tempo, 5PC,
and CSL. SD-Tempo is a good reporter of the headgroup region, 5PC is a
good spin label for exploring the dynamic structure of acyl chains near
the headgroup region, and CSL with its rigid molecular structure
provides information about the overall dynamic and ordering properties
of the acyl chain region (Kar et al., 1985; Tanaka and Freed, 1984).
ESR spectra from these three spin labels in DRM vesicles at 20°C,
22°C, and 37°C are shown in Fig. 5
(experimental, solid lines; simulated, dashed
lines). For purposes of comparison, ESR spectra from the same
three spin labels in pure SM dispersions in the gel phase (20°C or
37°C) and in the L phase (50°C) are also shown in
Fig. 5. The spectra from SD-Tempo and CSL in DRM were simulated using
only one component. The spectrum from 5PC in DRM exhibited a broadening
similar to that in the spectra from 16PC in DRM and was therefore
simulated allowing for two components; again we found that one
component has a normal three hyperfine pattern, and the other is just a single broad line. Relative populations of the two components are 0.28 and 0.72, respectively, which are close to those of the two components
found from the simulations of the spectra from 16PC in the DRM
vesicles.
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The best fit parameters of R and S
from the NLLS analyses for the spectra in Fig. 5 are listed in Table
2. Note that SM and DPPC belong to
sphingolipid and glycerophospholipid families, respectively. They are
different in backbone structure, but the gel-to-L phase
transition temperatures for SM with different N-acyl chain lengths
(16-24) are 41-48°C (Marsh, 1990), and these are comparable to that
for DPPC. Since SM is also an important component of DRMs (Fridriksson
et al., 1999), we used it as an alternative model membrane system in
our further comparisons with DRMs. From Table 2 it is seen that in DRM
vesicles the value of S for 5PC at 22°C is 0.52, and for
CSL at 37°C it is 0.85. These are even greater than those obtained in
the gel phase of SM dispersions from 5PC at 20°C (0.46) and from CSL
at 37°C (0.53), respectively. In addition, in DRM vesicles the values
of R of 5PC at 22°C, 4.07 × 107 s1, and of CSL at 37°C, 3.84 × 107 s1, are 2.5-4.6 times larger than those
in the gel phase of SM dispersions at the corresponding temperatures.
It is only in the L phase (50°C) that the
R of the two spin labels in the SM
dispersions become comparable to those in the DRM at the lower
temperatures; at the elevated temperatures, we would expect even larger
values in the DRM, because R increases
significantly with temperature in all cases studied (cf. Fig. 4 and
Shin and Freed, 1989a; Freed, 1994). These results from spin labels 5PC
and CSL show that in the bilayers of DRM vesicles the acyl chains,
either in the region near the headgroup or overall, are as ordered as
in the gel (solid-like) phase of SM, but are as fluid as in the liquid
crystalline (liquid-like) phase of SM. These results are consistent
with those from 16PC presented above.
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Molecular packing and interactions in the headgroup region have been
explored less than in the acyl chain region. Recently we found that
there is a strong orienting potential in the DPPC bilayer surface that
is dependent on the phase structure of the bilayers (Ge and Freed,
1998). In our present study we have observed very similar behavior in
SM bilayers, viz., we find that the change in order parameter of
SD-Tempo in SM dispersions ranges from 0.32 at 20°C (gel phase) to
0.23 at 50°C (L phase) (see Table 2). The change in
sign of S of SD-Tempo from positive to negative indicates
that the nitroxide moeity changes its preferential orientation from
perpendicular to parallel to the bilayer surface as SM dispersions go
from the gel to the L phase. This type of behavior has previously been observed for another headgroup-labeled lipid in DPPC
dispersions (Ge and Freed, 1998). These results show that there are
strong interactions between lipid headgroups in the gel phase as well
as in the liquid crystalline phase, but they exhibit different
orienting properties. Table 2 shows that in DRM vesicles the order
parameter S of SD-Tempo at 20°C, which is 0.06, is smaller
in absolute value than for SM dispersions in either phase, indicating
that the orienting potential at the surface of the DRM vesicles is
weaker than in the bilayer surface of SM dispersions. In addition,
Table 2 shows that the R of SD-Tempo in DRM
vesicles of 4.15 × 107 s1 is faster
than that in the L phase of SM, 2.82 × 107 s1. That is, the headgroup region in the
DRMs is also more "fluid" than in the pure SM dispersions.
To summarize, the physical properties (ordering and dynamics of the
bilayers) in DRM vesicles are very similar to those of DPPC/Chol = 1 dispersions, which are known to have a liquid ordered phase, but they
are distinctly different from those of the pure lipid dispersions of
DPPC and SM. It would thus appear that the DRM vesicles also have a
liquid ordered phase. However, the chemical composition of the DRM
vesicles is much more complex than that of the model systems. Recently
it was shown by mass spectrometric analyses that DRM vesicles contain
over 90 different species of SM, PC, phosphatidylethanolamine,
phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and
phosphatidic acid, with each class of lipids exhibiting a variety of
different chain lengths and/or degrees of unsaturation (Fridriksson et
al., 1999). Moreover, DRM vesicles contain Lyn and other membrane
proteins. For such a complex system, one may ask to what extent its
dynamic structure is affected by phospholipid/phospholipid and
phospholipid/protein interactions, in addition to the phospholipid/Chol
interactions. It would appear from the present study that, as far as
the physical properties of the DRM vesicles are concerned, the
lipid/Chol interactions dominate over all other lipid/lipid and
lipid/protein interactions, such that the DRM vesicles and the
DPPC/Chol = 1 dispersions have the same phase structure. (This
statement applies to the DRM component that gives rise to the sharp
line spectrum.) To examine this conclusion further, we next compare the
properties that we found for the DRM with those of model membranes in
the presence of high concentrations of Chol, as examined in previous studies.
Why are high concentrations of Chol essential for DRM?
Ipsen et al. (1987) defined the phase structure of model membranes
containing high concentrations of Chol as a liquid ordered phase or,
equivalently, as a high-cholesterol liquid phase. This definition was
based on the two main effects of Chol on the structure and dynamics of
lipid bilayers.
Ordering effect
Chol increases the acyl chain orientational ordering, which was observed by a variety of techniques, including 2H NMR (Vist and Davis, 1990; Bloom et al., 1991), x-ray diffraction (McIntosh,
1978), fluorescence (Straume and Litman, 1987), and ESR (Kar et al.,
1985; Shin and Freed, 1989a,b; Ge et al., 1994). Chol condenses the
lipid monolayers at the air/water interface for both phosphoglycerol
and sphingolipids (Bittman et al., 1994; Smaby et al., 1994, 1996).
This effect is due to the strong van der Waals attractive interactions
between the acyl chains and the planar rings of Chol, which reduces the
number of gauche conformers of the acyl chains.
Fluidizing effect
Chol greatly increases both the acyl chain rotational mobility (Straume and Litman, 1987) and the lipid lateral mobility (Rubenstein et al., 1979) in the gel phase when the concentration of Chol is above
20-25 mol%. At high concentrations of Chol, the
gel-to-L phase transition of lipid is abolished (Lewis
and McElhaney, 1991), and the lipid bilayers remain in a fluid state
over a wide range of temperatures (Vist and Davis, 1990). Another
example of the fluidizing effect is that DMPC/Chol bilayers of molar
ratio 1:1 have a very small surface rigidity, even at temperatures well below the DMPC L-to-gel phase transition, i.e., the
bilayers behave like a surface liquid (Needham et al., 1988). The
combined ordering and fluidizing effects of Chol were shown by
2H NMR spectra obtained from chain deuterated lipids
containing high concentrations of Chol. They are characteristic of a
fluid lipid phase, yet the splittings are as large as in the gel phase (Bloom et al., 1991; Jacobs and Oldfield, 1979). Our ESR spectra, which
provide both the order parameter and the rotational diffusional rate,
show that even at 5°C DRM vesicles and DPPC/Chol = 1 dispersions have substantial R but possess a
solid-like ordering of the acyl chains. Furthermore, no phase
transition in DRM vesicles and in DPPC/Chol = 1 dispersions could
be detected.
For the headgroup region, strong interactions between headgroups were
shown previously by nuclear Overhauser studies (Yeagle et al., 1975;
1977). In addition, it was shown (Yeagle et al., 1977) that Chol
disrupts the molecular interactions between the headgroups. The spacer
effect of Chol molecules was suggested as an explanation for the
weakening of the interactions between headgroups (McIntosh et al.,
1989). However, it seems that there is an additional effect of Chol.
There was a significant increase in the surface dipole potential of egg
PC monolayer when Chol was added until a PC-to-Chol molar ratio of 1:1
was reached (McIntosh et al., 1989). Furthermore, an increase in
ordering of a fluorescent probe in the headgroup/water interface of a
variety of PC vesicles was observed when 30 mol% of Chol was
incorporated (Straume and Litman, 1987). These observations show that
the structure of the lipid/water interface is altered by the
incorporation of Chol. It was reported that there is a significant
increase in the fluorescent probe depolarizing motion at the
lipid/water interface in the presence of Chol (Straume and Litman,
1987). ESR (Tanaka et al., 1997) and dielectric relaxation (Henze,
1980) studies have shown that the motion of headgroups in PC
dispersions is substantially increased upon incorporation of Chol. Our
ESR observations of increased R and reduced
absolute values of S of SD-Tempo in DRM vesicles compared to
the pure SM dispersions (see Table 2) are consistent with the enhanced
motion of headgroups and the disrupted interactions between headgroups
caused by the incorporation of Chol.
The above comparisons indicate that in both the acyl chain and
headgroup regions the ordering and dynamics of DRM vesicles derived
from RBL-2H3 cells are consistent with the characteristic properties of
lipid bilayers containing high concentrations of Chol. In other words,
lipid/Chol interactions do appear to dominate the molecular
interactions in the DRM vesicles, consistent with DRM vesicles having a
liquid ordered phase structure.
This conclusion can be further rationalized. As shown by Smaby et al.
(1994), the requirement for a maximum Chol-induced condensation effect
is that at least one hydrocarbon chain is long and capable of an
extended conformation, which would ensure strong van der Waals
attractive interactions with Chol's planar hard core. This requirement
is not stringent. The 18C sphingosine base of SM satisfies this
condition, because its 4,5 trans double bond is quite close to the interface region and does not disrupt the linear extension of
sphingosine base of SM. That may well explain why SM is enriched in
DRM. It is known that in biomembranes, PC lipids frequently contain a
saturated sn-1 acyl chain, which can have Chol-inducing condensation effects similar to those of SM (Smaby et al., 1994). Such
PC lipids are the most abundant species in DRM (Fridriksson et al,
1999), which is consistent with our ESR evidence that these membranes
contain liquid ordered structures. Chol has a stronger condensing
effect on SM monolayers than on PC monolayers (Lund-Katz et al., 1988).
However, this difference, compared with the strong interactions between
the saturated acyl chains and Chol, is a secondary effect. Therefore,
if the concentrations of Chol in biomembranes is high enough, strong
attractive interactions between Chol and lipids would organize them
into DRM domains, with the Chol playing an essential role. Thus we
infer that other cell membranes containing high concentrations of Chol
should also have a liquid ordered phase. Erythrocyte membranes might be
such a case. It was reported that erythrocyte membranes remain fluid at
5°C (Maraviglia et al., 1982), and no gel-to-liquid crystalline phase transition could be detected between 5° and 45°C (Davis et
al., 1979).
DRM vesicles have an inhomogeneous structure
One feature of the spectra from 16PC and 5PC in DRM vesicles is
that they consist of two components, a normal three hyperfine line
spectrum and a broad single-line spectrum. The latter is likely to be
caused by spin exchange interactions due to clustering of nitroxide
radicals (Fajer et al., 1992; Earle et al., 1994). Our NLLS analyses
showed that ~70% of 5PC and of 16PC molecules partition into an
environment of the bilayers where they are enriched. Using a simple
model analysis (Earle et al., 1994), we find that they collide with
each other at a frequency of ~3 × 108
s1. We are able to discount the possibility that these
are micelles (or vesicles) formed by pure 16PC molecules that have
separated from the DRM, because the solubility of 16PC in lipid/Chol
vesicles is very large: we have prepared a variety of model membrane
vesicles containing as much as 5 mol% 16PC (10 times greater than the
concentration in the model membranes used in the present experiments).
These show exchange broadening characteristic of a single component that is different from what is observed in the present study (see Figs.
2 and 5). Indeed, we find that concentrations of 16PC significantly greater than 5 mol% may be incorporated into model membranes. Our
results imply that the model membranes are homogeneous, whereas the DRM
vesicles are not.
On the other hand, the spectra from SD-Tempo in DRM vesicles between
5°C and 45°C and the CSL in DRM vesicles we studied (cf. Table 2)
can be satisfactorily simulated with only one component. This might
suggest that the SD-Tempo (related to SM) and CSL (related to Chol)
remain preferentially in the liquid ordered region of the membrane as
compared to the PC labels.
The inhomogeneity in DRM could originate from lipid/lipid as well as
from lipid/protein interactions. It was reported that heterogeneity of
bilayers was induced in bilayers containing gramicidin or
bacteriorhodopsin (Williams et al., 1990). The inhomogeneity in the DRM
structure could play an important role in the signaling mediated by the
IgE receptor and other receptors. Lateral heterogeneity in bilayers has
been shown to be important for the function of certain enzymes such as
phospholipase A2 (Hoenger et al., 1996) and protein kinase
C (Dibble et al., 1996), and these enzymes are important downstream
signaling molecules in IgE receptor-mediated cell activation (Beaven
and Metzger, 1993).
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CONCLUSIONS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
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Our ESR study demonstrated that DRM vesicles derived from RBL-2H3 cells have a liquid ordered phase, which is characterized by a liquid-like mobility in both the acyl chain and headgroup regions, and a gel-like ordering in the acyl chain region. This is the first direct evidence that DRMs isolated from live cells have a liquid ordered phase. We also found evidence that DRM vesicles are inhomogeneous, with a second region in which the acyl-chain spin labels are concentrated. This work shows the utility and power of spin-label ESR, especially when combined with quantification by NLLS fitting to the slow motional theory for ESR, in the study of the dynamic structure of biological membranes. It will be interesting to extend these investigations to the plasma membranes of RBL-2H3 cells, to further address the relationship between the structure of DRM domains derived from them and their functional role in the IgE receptor signaling process.
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ACKNOWLEDGMENTS |
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We appreciate helpful discussions with Dr. Erin Sheets. Computations were performed at the Cornell Theory Center and the Cornell Material Science Center.
This work was supported by National Institutes of Health grants GM25862 and AI18306.
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FOOTNOTES |
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Received for publication 8 December 1998 and in final form 30 April 1999.
Address reprint requests to Dr. Jack H. Freed, Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, NY 14853-1301. Tel.: 607-255-3647; Fax: 607-255-0595; E-mail: jhf{at}msc.cornell.edu.
Dr. Field's present address is G. W. Hooper Foundation, University of California, San Francisco, CA 94143-0552.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
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RI case.
Immunol. Today.
14:222-226[Medline].RI-mediated recruitment of p53/56lyn to detergent-resistant membrane domains accompanies cellular signaling.
Proc. Natl. Acad. Sci. USA.
92:9201-9205[Abstract].-dipalmitoylphosphatidylcholine water system.
Biophys. J.
48:569-595[Abstract].5°C.
Biochim. Biophys. Acta.
686:137-140[Medline].RI-mediated association of 6-µm beads with RBL-2H3 mast cells results in exclusion of signaling proteins from the forming phagosome and abrogation of normal downstream signaling.
J. Cell Biol.
134:1427-1439[Abstract].
Biophys J, August 1999, p. 925-933, Vol. 77, No. 2
© 1999 by the Biophysical Society 0006-3495/99/08/925/09 $2.00
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), DPPC/Chol = 1 (
),
and DRM vesicles (
). The specific values of
R