Plasma membrane rafts of rainbow trout are subject to thermal acclimation
Biology Department, Arizona State University, Tempe, AZ 85287, USA
* Author for correspondence (e-mail: jzehmer{at}imap4.asu.edu)
Accepted 28 February 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: raft, plasma membrane, raft-depleted plasma membrane, temperature, cholesterol, detergent, thermal acclimation, rainbow trout, Oncorhynchus mykiss
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Favorable interactions among cholesterol and the saturated hydrocarbon
chains of lipids, especially sphingomyelin, result in patches of
liquid-ordered (Lo) phase membrane (raft) separating from the
remaining liquid-disordered (Ld) membrane
(Ahmed et al., 1997). The
Lo phase is characterized by a highly ordered hydrophobic region
compared with the Ld phase
(Brown and London, 1998
).
Specific proteins are targeted to, and concentrated in, rafts based on the
greater solubility of their lipid anchors in Lo over Ld
phase membrane (Wang et al.,
2000
). This localization is clearly important since the
dissolution of rafts, by stripping the membrane of cholesterol, results in the
loss of function of the signaling proteins normally localized there
(Pike and Miller, 1998
). Raft
structure is dependent on lipid phase behavior
(Brown and London, 1998
),
making it temperature sensitive. Therefore, acute increases in temperature
could result in raft dissolution while acute decreases could result in
inappropriate accretion of membrane components. The resulting disruption of
signaling activities would necessitate a homeostatic response to stabilize
raft structure and restore appropriate signaling.
The goal of this study was to determine if raft structural integrity is defended during thermal acclimation in the vertebrate poikilotherm rainbow trout (Oncorhynchus mykiss). We measured compositional changes in total PM as well as in the raft and raft-depleted PM (RDPM) sub-domains of hepatocytes from rainbow trout acclimated to 5°C and 20°C. Thermally induced adjustments in membrane lipid composition were greater in raft compared with PM. In addition, we developed a novel approach for measuring membrane molecular interaction strength (and thus the tendency to stabilize raft structure) based on the susceptibility of membranes to detergent. We present evidence that molecular interaction strength is regulated in raft but not in RDPM. These data provide the first evidence for the homeostatic regulation of raft structure and suggest the need to re-examine membrane acclimation from the perspective of a spatially heterogeneous PM.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals
Rainbow trout (Oncorhynchus mykiss Walbaum) were obtained from the
Alchesay National Fish Hatchery in Whiteriver, AZ, USA and were maintained at
the Animal Resource Center of Arizona State University. Fish were housed in
recirculating freshwater aquaculture systems consisting of circular fiberglass
tanks; water temperatures were controlled using flow-through chillers. Animals
were acclimated to 5°C or 20°C for at least three weeks before use in
experiments. Fish were held under a constant 12 h:12 h L:D cycle and were fed
trout food (Rangen Inc., Buhl, ID, USA) to satiation daily.
Plasma and raft membrane isolations
Plasma membranes (PM) were isolated from approximately 14 g of liver
according to a modification of the procedure of Armstrong and Newman
(1985), as described previously
(Hazel et al., 1992
). The PM
was resuspended in working buffer (WB; 0.25 mol l1 sucrose,
20 mmol l1 tricine, pH 7.8, 1 mmol l1
EDTA) and was separated into raft-depleted PM (RDPM) and raft-enriched PM
(raft) using a non-detergent-based method
(Smart et al., 1995
). The
membrane fractions were separated based on the lighter buoyant density of
raft, compared with RDPM. Briefly, the PM was sonicated and then adjusted to
23% OptiPrep before layering a 1020% OptiPrep gradient on top (for a
total of 11 ml in each tube). After centrifuging for 90 min at 72 800
g in a Beckman SW 41-Ti rotor (OP1), the top 5.5 ml (raft) was
removed, mixed with 4 ml of 50% OptiPrep in a fresh tube, capped with 250
µl of 5% OptiPrep and centrifuged for 90 min at 72 800 g
(OP2). The raft membrane concentrated at the top of the OP2 tube was collected
with a Pasteur pipette, diluted three times with buffered saline and
centrifuged to a pellet for 20 min at 20 800 g in a
refrigerated microcentrifuge (Eppendorf 5417 R). The bottom 5.5 ml from OP1
(RDPM) was diluted approximately four times with buffered saline and was
centrifuged for 1 h at 23 700 g in a Beckman JA 30.50
rotor.
Protein, phosphate and cholesterol assays
Total protein was determined colorimetrically by the method of Bradford
(1976). Phospholipid
concentration was measured by colorimetric phosphate analysis of total lipid
extracts after complete hydrolysis (Ames,
1966
). An average molecular mass of 750 was assumed for conversion
to mass. A coupled cholesterol oxidase fluorometric assay was used to measure
cholesterol (Crockett and Hazel,
1995
).
Western blots
Western blots were performed to determine the relative abundance of
specific proteins in the fractions. Samples (30 µg of total homogenate
protein and 15 µg of all other samples) were mixed with an equal volume of
2x Laemmli buffer [62.5 mmol l1 Tris, pH 6.8, 25%
glycerol, 2% sodium dodecyl sulfate (SDS), 5% ß-mercaptoethanol], boiled
for 3 min and subjected to SDSpolyacrylamide gel electrophoresis (12%
gel for caveolin, 7.5% gel for all others). The resolved proteins were then
transferred to 0.45-µm nitrocellulose membranes and blocked for 1 h in 5%
non-fat dry milk (NFDM) in phosphate-buffered saline (PBS). The membranes were
immunolabeled for 1 h in 5% NFDM/PBS with an anti-caveolin polyclonal antibody
(pAb; 1:2000), anti-ß2 adrenergic receptor pAb (1:250),
anti-adenylyl cyclase pAb (1:400), anti-insulin receptor ß-subunit pAb
(1:1600) or anti-clathrin pAb (1:250). The blots were washed three times in
PBS with 0.1% Tween-20 for 5 min and once in PBS for 5 min. The blots were
then incubated for 1 h in goat anti-rabbit horseradish peroxidase
(HRP)-conjugated secondary antibody (1:12 000 for caveolin, 1:10 000 for all
other westerns). The blots were then washed four times in PBS with 0.1%
Tween-20 for 5 min, followed by detection using enhanced chemiluminesence and
X-ray film. At least two blots were prepared for each experiment, and
representative films are shown.
Stable plurilamellar vesicle preparation
Stable plurilamellar vesicles of synthetic lipids were prepared according
to the method of Grüner et al.
(1985) for use in detergent
assays.
Detergent assays
Assay
Detergent assays were performed to measure the molecular interaction
strength of membrane samples. Vesicles or biological samples (in 200 µl WB)
were dispersed into 2.8 ml assay buffer (150 mmol l1 NaCl,
20 mmol l1 Hepes, pH 7.4) and maintained in thermally
controlled cuvettes. The quantities used for myristoylpalmitoyl
phosphatidylcholine (MPPC) vesicles, palmitoyloleoyl phosphatidylcholine
(POPC)/cholesterol vesicles and biological samples were 470 nmol of lipid, 270
nmol of lipid and 170 nmol of lipid, respectively. The turbidity of the
suspension was measured continuously as the average optical density between
394 nm and 404 nm in a diode array spectrophotometer (8452A; Hewlett Packard,
Palo Alto, CA, USA). Triton X-100 was added as serial additions of 12.5 µl
and allowed to stabilize for 1020 min
(Fig. 1B). The final five data
points before each new detergent addition were averaged as the effect of the
previous detergent addition (e.g. Fig.
1B, arrow). The resulting optical density values were normalized
as a percentage of the initial optical density and were plotted against
detergent/lipid (D/L) ratios, calculated as detergent concentration (% v/v)
divided by µmol total lipid (Fig.
1B, inset). The resulting curves were fit with a four-parameter
sigmoidal equation using Sigma Plot 2000 (most r2 values
were 0.996 or better, with the lowest r2=0.987).
|
Analysis
Detergentmembrane interactions proceed in two phases, both of which
are sensitive to molecular interaction strength. Initially, detergent is
incorporated into membrane structure until saturation, which is followed by
solubilization as the membrane components are incorporated into mixed micelles
(Fig. 1A; Lichtenberg et al., 1983).
Values reflecting each of these processes were derived from the data. The D/L
values corresponding to the peaks of the 2nd derivative of the fitted curve
(Fig. 1C, inset, points
a and b) define the onset and completion of the high slope
region of the curve (Fig. 1C,
points a and b). The D/L value corresponding to point
a was chosen as an index of the maximum capacity of the membrane to
accommodate detergent (saturation point;
Fig. 1C). Further additions of
detergent result in membrane dissolution. The slope of a linear regression of
the fitted data between points a and b provides an index of
the amount of detergent required for solubilization (solubilization slope;
Fig. 1C).
Fourier transform infrared spectroscopy
Infrared spectroscopy was performed to estimate the gelfluid melting
temperature of MPPC vesicles. The MPPC vesicle sample was loaded between two
CaFl2 crystals with a 50.8 µm Teflon spacer and placed in a
thermally controlled sample block. Using a Fourier transform infrared
spectrometer (Spectrum 2000; Perkin Elmer, Shellon, CT, USA), the mean of 75
scans between the wavenumbers of 370 and 7800 was taken at 2°C intervals
between 20°C and 50°C and were background subtracted. The instrument
software package was used to find the peaks of the 2nd derivative, identifying
the absorption peak corresponding to the methylene symmetric stretching
frequencies. This frequency was plotted as a function of temperature. The
points were fitted using TableCurve 2D 5.0 (Systat Software, Inc., Richmond,
CA, USA), and the gelfluid melting temperature was found as the peak of
the 1st derivative.
Statistics
The protein, phospholipid and cholesterol compositional data were expressed
as ratios (e.g. cholesterol/protein). The data were analyzed by two-way
analysis of variance (ANOVA) with acclimation group as a factor with two
levels (cold and warm) and membrane fraction as a factor with three levels
(PM, RDPM and raft). Post hoc testing was performed using Tukey's
test. The solubilization slopes and saturation points from the detergent
experiments were each analyzed using three-way ANOVAs with acclimation group
as factor with two levels (cold and warm), assay temperature as a factor with
two levels (5°C and 20°C), and fraction as a factor with three levels
(PM, RDPM and raft). Where appropriate (significant effect with no
interaction), the data were collapsed across one or two factors for multiple
comparisons using Tukey's test. The data were also analyzed separately by
fraction (PM, RDPM and raft) by one-way ANOVA followed by Tukey's test. In all
cases, a P value of 0.05 was accepted as indicating statistical
significance.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Hepatocyte plasma membranes (PMs) isolated from rainbow trout acclimated to 5°C and 20°C were separated into raft-enriched (raft) and raft-depleted PM (RDPM) by sonication followed by density gradient centrifugation. Based on the yield of protein, cholesterol and phospholipid (estimated as phosphate), and an assumption that the membrane consisted of only these components, we estimated that the fractions taken as raft consisted of 39% and 32% (by mass), respectively, of the total membrane for the cold- and warm-acclimated animals.
Microdomains (rafts and caveolae) have previously been described in
numerous systems to be enriched in proteins of many signal transduction
cascades, including the ß2 adrenergic receptor
(ß2AR; Xiang et al.,
2002) and adenylyl cyclase (AC;
Ostrom et al., 2001
;
Rybin et al., 2000
).
Therefore, we examined the distribution of ß2AR and AC in the
crude homogenate, PM, RDPM and raft fractions. Both proteins were depleted in
the RDPM fraction, compared with the PM, while they were substantially
enriched in the raft fraction (Fig.
2B,C). The insulin receptor has been reported as absent from
microdomains in the majority of the literature
(Mastick et al., 1995
;
Ohira et al., 2000
), but some
researchers have found it to be microdomain associated
(Vainio et al., 2002
). In our
system, the insulin receptor ß subunit was depleted in the raft, compared
with the PM or RDPM, fractions (Fig.
2D). Clathrin, a protein involved in receptor-mediated
endocytosis, is not associated with rafts or caveolae
(Razani and Lisanti, 2001
).
Surprisingly, we found clathrin to be evenly distributed among the PM, RDPM
and raft fractions (Fig.
2E).
Acclimation temperature effects on composition
To further characterize the fractions, we measured the concentrations of
cholesterol, phospholipid phosphate and protein in the fractions from cold-
and warm-acclimated trout to assess compositional changes associated with
thermal acclimation. The data were expressed as mass ratios
(Fig. 3AC) and as mass
percentages (Table 1; the table
was constructed with the assumption that the membrane consisted of only
protein, phospholipid and cholesterol). Whereas the cholesterol/protein
(Ch/Pr) ratios were similar in PM and RDPM in both acclimation groups, this
ratio was significantly higher in raft fractions, being elevated 66% and 104%
over PM in cold- and warm-acclimated fish, respectively
(Fig. 3A). Furthermore, the
Ch/Pr of rafts from warm-acclimated fish was significantly greater
(P<0.05, N=6) than that from cold-acclimated fish by
33%.
|
|
Rafts from cold-acclimated fish had significantly higher (P<0.05, N=6) phospholipid/protein (PL/Pr) ratios than all other fractions, which did not differ from one another (Fig. 3B). This represented a 65% increase over the RDPM from cold-acclimated fish. There was a non-significant 42% increase of PL/Pr in raft, compared with RDPM, from warm-acclimated fish.
The Ch/PL ratio was 32% greater in PM from warm-compared with cold-acclimated animals, while this increase was 63% in rafts (Fig. 3C). Furthermore, the increase in cholesterol concentration associated with acclimation to 20°C was 3.4 times greater in raft than in PM (Table 1).
Detergent assays vesicles
We developed a novel approach for measuring membrane molecular interaction
strength based on the susceptibility of membranes to detergent. We followed
both the capacity of a membrane to incorporate detergent into the bilayer
[saturation point (SP)] and the amount of detergent required to solubilize the
membrane (solubilization slope). More tightly packed membranes were expected
to saturate with less detergent (low SP) but to require more detergent to
solubilize them (shallow solubilization slope) compared with loosely packed
membranes (Fig. 1A; see
Discussion).
We first analyzed several model vesicle preparations to evaluate the efficacy of this approach. Molecular interaction strength was manipulated by varying assay temperature of myristoylpalmitoyl phosphatidylcholine (MPPC) vesicles and cholesterol content of palmitoyloleoyl phosphatidylcholine (POPC) vesicles.
Fourier transform infrared spectroscopy indicated that MPPC vesicles melted from the tightly packed gel phase to the loosely packed fluid phase at 33°C (Tm; Fig. 4A, circles). SP values for MPPC decreased between 25°C and 30°C as Tm was approached, before increasing above Tm (Fig. 4A, bars). Substantially more detergent was required to reach saturation above the Tm, reflecting the looser packing of the fluid-phase lipids.
|
Vesicles of POPC alone or with 30 mol% cholesterol were analyzed at
25°C in the fluid phase. Cholesterol has a condensing effect on fluid
phase membranes, causing membrane lipids to pack more tightly
(Phillips, 1972). The addition
of 30 mol% cholesterol to the POPC vesicles caused a substantial reduction in
the SP, reflecting this tighter packing
(Fig. 4B).
In analyzing the solubilization slopes produced from these same vesicle preparations, we expected membranes with tightly packed lipids to produce more shallow slopes (more detergent required to complete solubilization). Contrary to our prediction, solubilization slopes for MPPC vesicles were substantially steeper at temperatures below the Tm compared with those above the Tm (Fig. 5A). However, as expected, POPC vesicles with 30 mol% cholesterol had a more shallow solubilization slope than did POPC vesicles lacking cholesterol (Fig. 5B). These results suggest that SP is a more reliable indicator of molecular interaction strength than is solubilization slope.
|
Detergent assays biological membranes
Saturation points
Representative detergent solubility curves of PM, RDPM and raft are
presented in Fig. 6. Note that
the RDPM curve has a more pronounced sigmoidal shape, reflecting a higher SP,
compared with the more flattened curve produced by raft. The SPs for all
samples were analyzed by three-way ANOVA (acclimation group x assay
temperature x fraction). There was a significant effect of fraction and
assay temperature, with no effect of acclimation group, and no significant
interactions. However, the fraction x acclimation group interaction term
was near significance (P=0.075, N=4). Therefore, the data
were collapsed across assay temperature for comparisons of fractions from the
two acclimation groups (Fig.
7A).
|
|
Within acclimation groups, the SPs of the RDPM samples were significantly greater (P<0.05, N=4) than both the PM and raft samples (Fig. 7A). The SP of PM and raft from warm-acclimated fish did not differ significantly. In the samples from cold-acclimated fish the SP of the PM was significantly greater than that for the raft. It should be noted that the SPs for the cold-acclimated raft samples were, on average, negative. The second-derivative peak was chosen as a convenient index of saturation; the negative values are a product of this approach and simply reflect a very low SP. The SPs of all of the fractions were sensitive to assay temperature. Increasing the assay temperature from 5°C to 20°C caused a significant (P=0.011, N=24) mean reduction in SP of 38% (Fig. 7B).
The uncollapsed SP data, grouped and analyzed by fraction, are presented in Fig. 8. The PM and RDPM displayed similar patterns. When assayed at a common temperature, samples had similar SPs, regardless of their acclimation group. As a result, warm-acclimated samples had lower SPs than cold-acclimated samples when assayed at their respective physiological temperatures (Fig. 8, dots). By contrast, the SPs of the raft samples from warm-acclimated fish were up-shifted relative to those from cold-acclimated fish, the source of the near-significant acclimation group x fraction interaction term in the three-way ANOVA. As a result, the SPs of the rafts from the two acclimation groups do not differ at their respective physiological temperatures.
|
Three of the cold-acclimated raft samples showed a reduced SP when assayed at 20°C compared with at 5°C, while one sample displayed a dramatically higher SP. Based on the data collected on the MPPC vesicles (Fig. 4A), this suggested that 20°C was near the LoLd phase transition temperature for these samples. Therefore, we repeated the assay at 28°C to determine if melting was detectable (Fig. 9). Neither cold- nor warm-acclimated RDPM showed a significant increase in SP when assayed at 28°C. By contrast, the SPs of rafts from both acclimation groups increased significantly at 28°C compared with 20°C.
|
Solubilization slopes
The region of the detergent solubility curves between points a and
b in Fig. 1C was
nearly linear and represented the shift from bilayer morphology to mixed
micelles (solubilization). The slopes from linear regressions fitted to these
regions were compared with a three-way ANOVA. The analysis found an effect of
acclimation group and fraction but no effect of assay temperature. There was a
significant interaction between acclimation group and fraction but no other
significant interactions. Therefore, the data were collapsed across assay
temperature (Fig. 10).
|
In both acclimation groups, the raft solubilization slopes were intermediate in value between those of the PM and RDPM. The RDPM slopes were significantly greater than those of the PM by 87% and 46% for the cold- and warm-acclimation groups, respectively. In the cold-acclimation group, the slope for the RDPM was significantly greater (P<0.05, N=8) than that for the raft by 59%, while these fractions did not differ in the warm-acclimation group.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We used sonication and density gradient centrifugation to separate the PM
into low and high buoyant density fractions. Liver from rainbow trout does not
express the protein caveolin (Fig.
2A). Nevertheless, the low density fraction was enriched in
molecules characteristic of lipid microdomains, including cholesterol
(Fig. 3A; Table 1) and both the
ß2 adrenergic receptor and adenylyl cyclase
(Fig. 2B,C). These three
molecules have been reported to be lipid microdomain enriched in other systems
(Ostrom et al., 2001;
Rybin et al., 2000
;
Xiang et al., 2002
).
Furthermore, the low density fraction was substantially depleted in the ß
subunit of the insulin receptor, which is also consistent with previous
observations (Mastick et al.,
1995
; Ohira et al.,
2000
). The even distribution of the protein clathrin among PM,
RDPM and raft in our system is surprising since it is not associated with raft
or caveolae microdomains (Razani and
Lisanti, 2001
). However, unlike all other proteins examined in
this study, clathrin is not an integral membrane protein and can be separated
from membranes by gentle dissociation
(Keen et al., 1979
).
Therefore, it is possible that the sonication step used in separating the
fractions acted to redistribute the protein among all membrane particles
present in the preparation. Regardless, the weight of evidence suggests that
this method separates trout liver PM into biochemically distinct fractions,
including a low buoyant density fraction consistent with lipid microdomains.
As this membrane fraction lacks caveolae, we will refer to it operationally as
raft-enriched PM (raft) and the high buoyant fraction as raft-depleted PM
(RDPM).
The discovery that the PM of most cells is not homogenous, but contains
microdomains with distinct compositions, presents an opportunity for the
re-examination of membrane thermal acclimation. In response to changing
environmental temperatures, poikilotherms substantially alter the lipid
composition of the PM (Hazel,
1997). Many studies have sought to rationalize these compositional
changes as mechanisms of homeostasis for specific physical properties, notably
hydrophobic region fluidity/order (homeoviscous adaptation), with the
implication that critical functions are sensitive to these properties
(Hazel, 1997
). However, these
studies have examined the PM as a whole. It is possible that the compositions
of different regions of the PM are altered in different ways during thermal
acclimation. Furthermore, microdomains form as a result of a separation of
liquid-ordered (Lo; raft) and liquid-disordered (Ld;
RDPM) phases within the membrane (Ahmed et
al., 1997
). Since lipid phase behavior is temperature sensitive,
it is reasonable to hypothesize that acute changes in temperature could
disrupt raft structure and the function of raft-targeted proteins.
Consequently, acclimatory compositional changes of the PM may be aimed at
stabilizing raft structure against thermal perturbation. Therefore, we sought
to determine what general compositional changes occur in raft and RDPM
portions of the PM during thermal acclimation. We measured the cholesterol,
phospholipid and total protein content of raft, RDPM and total PM of livers
from cold- and warm-acclimated trout.
Rafts from both acclimation groups are substantially depleted in total protein compared with PM or RDPM (Table 1). The protein is primarily replaced by an enrichment in phospholipids in the cold-acclimated raft (although cholesterol is also enriched). By contrast, cholesterol accounts for most of the lipid enrichment in warm-acclimated rafts (Fig. 3A,B; Table 1). As a result, the cholesterol/phospholipid (Ch/PL) ratio is highly elevated in warm-acclimated raft while the Ch/PL is not changed in cold-acclimated raft compared with its PM source (Fig. 3B). The Ch/PL ratio is probably the most important measure for raft stabilization since it is an interaction between cholesterol and specific lipids, and not cholesterol concentration per se, that favors raft formation. This suggests that there are important differences in how rafts are stabilized in cold- and warm-acclimated animals. The data also demonstrate that the increase in cholesterol concentration is larger in raft than in RDPM with acclimation to 20°C (Table 1). Collectively, these data suggest that the compositional changes associated with thermal acclimation are at least partially related to properties specific to raft membrane.
If thermal acclimatory compositional adjustments can be explained in terms
of raft stabilization, we would expect that molecular interaction strength
(and thus the tendency to form the Lo phase) would be regulated in
rafts to offset thermal perturbation. Membrane solubilization by detergent has
previously been used to measure this property
(Ahmed et al., 1997), but most
analyses have failed to appreciate or account for the different stages of
detergentmembrane interaction. Detergent molecules added to a membrane
suspension are initially incorporated into the bilayer structure, suppressing
the free detergent monomer concentration below the critical micelle
concentration (CMC; Fig. 1A,
plates 1, 2). As the membrane saturates, detergent monomer concentration in
solution exceeds the CMC, mixed micelles form and the membrane begins to be
solubilized (Fig. 1A, plates 3,
4) (Lichtenberg et al., 1983
).
The capacity of a membrane to incorporate detergent monomers is inversely
proportional to the lipidlipid interaction strength in the membrane
(Lichtenberg et al., 1979
).
For example, a Langmuir trough study demonstrated that small increases in
lateral pressure, with concomitant increases in lipid cohesion forces,
resulted in large decreases in the incorporation of Triton X-100
(Nyholm and Slotte, 2001
).
We found this interpretation to be well supported in model vesicle studies.
The membrane condensing effect of an addition of 30 mol% cholesterol to
palmitoyloleoyl phosphatidylcholine (POPC) vesicles was easily detected as a
dramatic reduction in the SP (Fig.
4B). Furthermore, myristoylpalmitoyl phosphatidylcholine (MPPC)
vesicles saturated with less detergent in the tightly packed gel phase than in
the fluid phase (Fig. 4A).
Similar results have previously been reported for sphingomyelin vesicles
(Patra et al., 1999). A
reduction in SP was observed with an increase in temperature up to, but
remaining below, the acyl chain melting temperature in MPPC vesicles
(Fig. 4A), sphingomyelin
vesicles (Patra et al., 1999
)
and biological membranes (Fig.
7B). Prior to the dramatic decreases in packing density associated
with a phase transition, increases in temperature within a phase cause
increased membrane lateral pressure (de
Kruijff, 1997
). This may explain the lowered SP observed in these
cases.
Membrane molecular interaction strength also influences the detergent/lipid
values necessary to disrupt bilayer morphology and incorporate membrane
molecules into mixed micelles (solubilization slopes). For example, a
relatively high concentration of detergent was required to fully dissolve the
vesicles made of POPC and 30 mol% cholesterol, producing a shallow
solubilization slope (Fig. 5B).
As predicted, the solubilization slope produced during dissolution of the more
loosely packed POPC vesicles was comparatively steep. However, this measure
tends to be less informative than SPs since this process is much more
sensitive to variables other than molecular interaction strength. For example,
the CMC of Triton X-100 is temperature sensitive
(Elworthy et al., 1968a) and,
contrary to prediction, the solubilization slopes for MPPC vesicles are much
steeper in the gel phase compared with the fluid phase
(Fig. 5A). Furthermore, the
ability of detergent micelles to incorporate lipids, a property known as
maximum additive concentration, is strongly dependent on the molecular species
to be solubilized (Elworthy et al.,
1968b
). This complicates the interpretation of these data
collected on biochemically distinct fractions.
Nevertheless, differences in solubilization slope between raft and RDPM are
probably responsible for the detergent resistance of rafts, which is widely
observed and commonly used in their isolation. The most commonly used
detergent-based raft isolation method exposes isolated PM to Triton X-100 at
4°C and floats the remaining undissolved raft-enriched membrane on a
sucrose density gradient (Brown and Rose,
1992). In the current study, raft membranes produced lower
normalized optical densities than either PM or RDPM at most detergent
concentrations (Fig. 6). This
reflects the large differences in the SPs of these membranes but seems
contradictory to the use of detergent as a means of raft isolation since it
suggests that raft would dissolve more readily than RDPM. However, this is
easily resolved when considering the solubilization process of PM as a mixture
of raft and RDPM membranes. The PM sample as a whole would become saturated
with detergent before the process of solubilization became dominant. This
would effectively remove the differences in SPs between raft and RDPM
membranes, and the solubilization process would be primarily defined by the
process measured by the solubilization slopes of the RDPM and raft. The RDPM
fractions had steeper solubilization slopes than the raft or PM in both
acclimation groups (Fig. 10).
Therefore, RDPM membrane would be more rapidly solubilized, leaving a
raft-enriched fraction behind.
The difference in packing strength between raft and RDPM indicated by the
solubilization slopes is also evident in the SP data
(Fig. 7A). The more shallow
solubilization slope and lower SP of raft, compared wiht RDPM, probably
reflects the Lo phase of this microdomain. Proteins can be targeted
to the Lo phase rafts by modification with saturated lipids that
are more soluble in the tightly packed Lo than in the Ld
phase (Melkonian et al.,
1999). Since the SP (and packing strength) of membrane in either
phase is acutely temperature sensitive
(Fig. 7B), it is possible that
perturbations of this property could influence the targeting of proteins to
rafts. Furthermore, differences in packing strength may have functional
consequences for the proteins embedded in the membrane. Integral membrane
proteins may require a specific range of lateral pressure to allow appropriate
flexing without allowing the protein to relax into non-functional
conformations (de Kruijff,
1997
). Finally, a sufficiently large increase in temperature could
drive a phase transition, effectively melting Lo phase raft and
resulting in mixing with the RDPM membrane. Conversely, a large decrease in
temperature could result in accretion of lipids and proteins normally excluded
from rafts.
Therefore, we examined the fractions from both acclimation groups at both assay temperatures to determine if membrane packing strength was conserved during thermal acclimation (Fig. 8). In both the PM and RDPM, samples assayed at a common temperature gave similar SPs. As a result, the warm-acclimated samples assayed at 20°C were different from the cold-acclimated samples assayed at 5°C (physiological comparisons; Fig. 8, dots). However, this perturbation was not seen in the raft membranes. The SPs of the warm-acclimated rafts were substantially up-shifted with respect to the cold-acclimated rafts such that there was little difference in this property when examined at physiological temperatures. To our knowledge, this is the first evidence of an apparent regulation of a physical property of a subdomain of the PM. Such regulation would be expected to maintain raft structural integrity and packing properties despite differing thermal environments experienced by the organism.
An increase of assay temperature above the gelfluid phase transition was detected as a dramatic increase in SP for the MPPC vesicles (Fig. 4). Our observation that an increase in assay temperature from 5°C to 20°C resulted in an increase in SP in one raft sample from cold-acclimated fish suggested that rafts from these animals were close to their LoLd melting temperature at 20°C. To test this hypothesis, all samples were assayed at 28°C (Fig. 9). Since RDPM is in the Ld phase, an increase in temperature should not cause an increase in SP. Indeed, there were only small and non-significant SP increases in RDPM from both acclimation groups. By contrast, the SP of the raft samples assayed at 28°C was significantly greater than those measured at 20°C in both acclimation groups. The dramatic increase in SP at 28°C suggests that the raft membranes from both acclimation groups were above their LoLd melting temperature at this temperature. In the plasma membrane, clustered raft proteins and lipids would be expected to mix randomly with RDPM molecules under these conditions.
In conclusion, there is apparent spatial heterogeneity in the response of the PM to thermal change. Compositional changes associated with thermal acclimation are different in the raft and RDPM portions of the PM. Specifically, the ratio of cholesterol to phospholipid is elevated in rafts from warm-acclimated, but not from cold-acclimated, animals compared with their PM sources. Furthermore, there is a conservation of membrane packing strength in the raft, but not the raft-depleted, regions of the PM. This is consistent with a regulation of this physical property, which may have functional consequences for phase-dependent protein targeting. Furthermore, the detergent data suggest that elevated temperature can abolish the Lo/Ld phase separation responsible for raft structure. The raft-associated compositional changes observed may also function to stabilize these microdomains.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahmed, S. N., Brown, D. A. and London, E. (1997). On the origin of sphingolipid/cholesterol-rich detergent-insoluble cell membranes: physiological concentrations of cholesterol and sphingolipid induce formation of a detergent-insoluble, liquid-ordered lipid phase in model membranes. Biochemistry 36,10944 -10953.[CrossRef][Medline]
Ames, B. N. (1966). Assay of inorganic phosphate, total phosphate and phosphatases. Methods Enzymol. 8,115 -118.
Armstrong, J. M. and Newman, J. D. (1985). A simple, rapid method for the preparation of plasma membranes from liver. Arch. Biochem. Biophys. 238,619 -628.[Medline]
Bradford, M. M. (1976). Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal. Biochem. 72,248 -254.[CrossRef][Medline]
Brown, D. A. and London, E. (1998). Functions of lipid rafts in biological membranes. Annu. Rev. Cell Dev. Biol. 14,111 -136.[CrossRef][Medline]
Brown, D. A. and Rose, J. K. (1992). Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68,533 -544.[Medline]
Crockett, E. L. and Hazel, J. R. (1995). Sensitive assay for cholesterol in biological membranes reveals membrane-specific differences in kinetics of cholesterol oxidase. J. Exp. Zool. 271,190 -195.[Medline]
de Kruijff, B. (1997). Lipid polymorphism and biomembrane function. Curr. Opin. Chem. Biol. 1, 564-569.[CrossRef][Medline]
Elworthy, P. H., Florence, A. T. and Macfarlane, C. B. (1968a). Micellization. In Solubilization by Surface Active Agents, pp. 61-116. London: Chapman and Hall.
Elworthy, P. H., Florence, A. T. and Macfarlane, C. B. (1968b). Solubilization. In Solubilization by Surface Active Agents, pp. 13-60. London: Chapman and Hall.
Grüner, S. M., Lenk, R. P., Janoff, A. S. and Ostro, M. J. (1985). Novel multilayered lipid vesicles: comparison of physical characteristics of multilamellar liposomes and stable plurilamellar vesicles. Biochemistry 24,2833 -2842.[Medline]
Hazel, J. R. (1997). Thermal adaptation in biological membranes: beyond homeoviscous adaptation. In Advances in Molecular and Cell Biology, vol. 19 (ed. J. S. Willis), pp. 57-101. New York: JAI Press.
Hazel, J. R., McKinley, S. J. and Williams, E. E. (1992). Thermal adaptation in biological membranes: interacting effects of temperature and pH. J. Comp. Physiol. B 162,593 -601.
Hazel, J. R., Williams, E. E., Livermore, R. and Mozingo, N. (1991). Thermal adaptation in biological membranes: functional significance of changes in phospholipid molecular species composition. Lipids 26,277 -282.[Medline]
Keen, J. H., Willingham, M. C. and Pastan, I. H. (1979). Clathrin-coated vesicles: isolation, dissociation and factor-dependent reassociation of clathrin baskets. Cell 16,303 -312.[Medline]
Lichtenberg, D., Robson, R. J. and Dennis, E. A. (1983). Solubilization of phospholipids by detergents. Structural and kinetic aspects. Biochim. Biophys. Acta 737,285 -304.[Medline]
Lichtenberg, D., Yedgar, S., Cooper, G. and Gatt, S. (1979). Studies on the molecular packing of mixed dispersions of Triton X-100 and sphingomyelin and its dependence on temperature and cloud point. Biochemistry 18,2574 -2582.[Medline]
Mastick, C. C., Brady, M. J. and Saltiel, A. R. (1995). Insulin stimulates the tyrosine phosphorylation of caveolin. J. Cell Biol. 129,1523 -1531.[Abstract]
Melkonian, K. A., Ostermeyer, A. G., Chen, J. Z., Roth, M. G.
and Brown, D. A. (1999). Role of lipid modifications in
targeting proteins to detergent-resistant membrane rafts. Many raft proteins
are acylated, while few are prenylated. J. Biol. Chem.
274,3910
-3917.
Nyholm, T. and Slotte, J. P. (2001). Comparison of triton x-100 penetration into phosphatidylcholine and sphingomyelin mono- and bilayers. Langmuir 17,4724 -4730.[CrossRef]
Ohira, K., Maekawa, S. and Hayashi, M. (2000). Absence of TrkB and insulin receptor beta in the Triton insoluble low-density fraction (raft). Neuroreport 11,1307 -1311.[Medline]
Ostrom, R. S., Gregorian, C., Drenan, R. M., Xiang, Y., Regan,
J. W. and Insel, P. A. (2001). Receptor number and caveolar
co-localization determine receptor coupling efficiency to adenylyl cyclase.
J. Biol. Chem. 276,42063
-42069.
Patra, S. K., Alonso, A., Arrondo, J. L. R. and Goñi, F. M. (1999). Liposomes containing sphingomyelin and cholesterol: detergent solubilisation and infrared spectroscopic studies. J. Liposome Res. 9,247 -260.
Phillips, M. C. (1972). The physical state of phospholipids and cholesterol in monolayers, bilayers, and membranes. In Progress in Surface and Membrane Science, vol.5 (ed. J. F. Danielli, M. D. Rosenberg and D. A. Cadenhead), pp. 139-221. New York: Academic Press.
Pike, L. J. and Miller, J. M. (1998).
Cholesterol depletion delocalizes phosphatidylinositol bisphosphate and
inhibits hormone-stimulated phosphatidylinositol turnover. J. Biol.
Chem. 273,22298
-22304.
Razani, B. and Lisanti, M. P. (2001). Caveolins and caveolae: molecular and functional relationships. Exp. Cell Res. 271,36 -44.[CrossRef][Medline]
Rybin, V. O., Xu, X., Lisanti, M. P. and Steinberg, S. F.
(2000). Differential targeting of beta-adrenergic receptor
subtypes and adenylyl cyclase to cardiomyocyte caveolae. A mechanism to
functionally regulate the cAMP signaling pathway. J. Biol.
Chem. 275,41447
-41457.
Sinensky, M. (1974). Homeoviscous adaptationa homeostatic process that regulates the viscosity of membrane lipids in Escherichia coli. Proc. Natl. Acad. Sci. USA 71,522 -525.[Abstract]
Smart, E. J., Ying, Y. S., Mineo, C. and Anderson, R. G. (1995). A detergent-free method for purifying caveolae membrane from tissue culture cells. Proc. Natl. Acad. Sci. USA 92,10104 -10108.[Abstract]
Vainio, S., Heino, S., Mansson, J. E., Fredman, P., Kuismanen,
E., Vaarala, O. and Ikonen, E. (2002). Dynamic association of
human insulin receptor with lipid rafts in cells lacking caveolae.
EMBO Rep. 3,95
-100.
Wang, T. Y., Leventis, R. and Silvius, J. R.
(2000). Fluorescence-based evaluation of the partitioning of
lipids and lipidated peptides into liquid-ordered lipid microdomains: a model
for molecular partitioning into "lipid rafts". Biophys.
J. 79,919
-933.
Xiang, Y., Rybin, V. O., Steinberg, S. F. and Kobilka, B.
(2002). Caveolar localization dictates physiologic signaling of
beta 2- adrenoceptors in neonatal cardiac myocytes. J. Biol.
Chem. 277,34280
-34286.