Correspondence to: J.K.H. Hörber, Cell Biology and Biophysics, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. Tel:49-6221-387569 Fax:49-6221-387306 E-mail:horber{at}embl-heidelberg.de.
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Abstract |
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To probe the dynamics and size of lipid rafts in the membrane of living cells, the local diffusion of single membrane proteins was measured. A laser trap was used to confine the motion of a bead bound to a raft protein to a small area (diam 100 nm) and to measure its local diffusion by high resolution single particle tracking. Using protein constructs with identical ectodomains and different membrane regions and vice versa, we demonstrate that this method provides the viscous damping of the membrane domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI) -anchored and transmembrane proteins are raft-associated, their diffusion becomes independent of the type of membrane anchor and is significantly reduced compared with that of nonraft transmembrane proteins. Cholesterol depletion accelerates the diffusion of raft-associated proteins for transmembrane raft proteins to the level of transmembrane nonraft proteins and for GPI-anchored proteins even further. Raft-associated GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 min. The measurements agree with lipid rafts being cholesterol-stabilized complexes of 26 ± 13 nm in size diffusing as one entity for minutes.
Key Words: laser trap, lipid raft, protein diffusion, single particle tracking, thermal position fluctuation analysis
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Introduction |
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Membrane microdomains enriched in glycosphingolipids and cholesterol and containing glycosylphosphatidylinositol (GPI)1 -anchored proteins have been proposed as lateral structural components of the plasma membrane (
Rafts are thought to form by self-association of sphingolipids because of their long and mostly saturated hydrocarbon chains. The interaction between glycosphingolipids can be enhanced by hydrogen bonds between their headgroups. The voids between the hydrocarbon chains caused by the rather bulky headgroups would be filled by cholesterol, which might also participate in the hydrogen bonding to the sphingolipids. Although cholesterol has been shown to be essential for raft formation, the precise nature of its interaction with sphingolipids remains unclear. A complementary view of sphingolipidcholesterol rafts is that they form separate liquid-ordered phases in the bilayer, which are dispersed in the liquid-disordered matrix formed by unsaturated glycerophospholipids (
Biochemically, the components of lipid rafts are characterized by their insolubility in the detergent Triton X-100 at 4°C, forming so-called detergent insoluble glycolipid-enriched complexes (DIGs) that are enriched in cholesterol, glycosphingolipids, sphingomyelin, and saturated glycerophospholipids (
The existence of lipid rafts in the plasma membrane of living cells has been demonstrated recently, but controversy persists and the exact structure of rafts remains unclear. on the sphere and the attached protein. The viscous drag determined with a temporal resolution of 0.3 s relates to the diffusion coefficient D of a freely diffusing protein via the Einstein relation: D = kBT/
(kBT being the thermal energy). The local diffusion coefficients obtained with this method for protein diffusion in the plasma membrane are the first to agree with the results from artificial lipid bilayers.
This novel microscopic technique allowed us to compare the diffusion of proteins with different membrane anchors in intact rafts to that of proteins in rafts disintegrated by cholesterol depletion and to the diffusion of a nonraft protein. The viscous drag of three raft-associated proteins, two with a GPI anchor and one transmembrane protein, is independent of the type of membrane anchor, and significantly larger than the one of nonraft proteins. After cholesterol depletion, the viscous drag of raft-associated proteins decreases to the level of the nonraft protein, whereas the diffusion of the nonraft protein remains unchanged. The mean radius of the raft assemblies obtained from these measurements is 26 ± 13 nm.
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Materials and Methods |
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Instrumentation
Our instrument combines a laser trap implemented in an inverted microscope (Axiovert 35; Carl Zeiss) with fast three-dimensional position detection (see Fig 2). The trapping laser, a Nd:YVO4 laser ( = 1064 nm, model T20-B10-106Q; Spectra Physics), is directed by a scanning mirror mounted on a triple-axis piezo (PiezoSystems Jena) into the microscope and focused in the object plane by an oil immersion objective lens (Plan Neofluor 100x, NA 1.3; Carl Zeiss). The condenser back focal plane (BFP) is projected onto a quadrant photodiode (QPD) (model S5981; Hamamatsu) via a dichroic mirror. To reduce the mechanical noise, the microscope's stage is replaced by a custom made stage to which the objective lens and the condenser with the detection system are rigidly connected. The two-photon fluorescence (TPF) intensity is measured by a photomultiplier (model R2949; Hamamatsu). All signals recorded are amplified and low passfiltered (50 kHz) by amplifiers developed at EMBL. A transputer data acquisition board (model ADWin F5; Jäger Electronics) is used for recording and to provide the axial feedback controlling the interaction force between the sphere and the membrane. To move the trapping focus along the optical axis, the objective lens is mounted on a piezo drive (model PiFoc P-721; Physik Instrumente). In addition, the temperature-controlled sample chamber is placed on a piezo scan stage with capacitive position sensors and digital feedback (model NPS-XY-100A; Queensgate).
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To determine the average axial position of the sphere in the trap, the fluorescence intensity emitted by the fluorophores inside the spheres excited by the trapping laser is measured (
Because the response of the QPD depends on the properties of sphere and the laser focus, it is necessary to calibrate the signal with the sphere used for an experiment at a location near the actual measurement. The local detector sensitivity ß is determined from the thermal position fluctuations using the Stokes drag of the sphere. Analogous to the local viscosity measurements (see below), the autocorrelation time of the position movements
and the spring constant of the trap are calculated, being now an uncalibrated spring constant
(in units Nm/Volt2 instead of N/m). Because
=
and
=
ß2, the sensitivity ß is determined from ß2 = 6
r/
, which is valid for a sphere in a harmonic potential as long as the position fluctuations remain within the linear response range of the detector, and the calibration is performed in the solution. We calibrated the detector to be at least ten times the diameter of the sphere away from any surface, so the influence of the surface is smaller than 2% (
Local Viscosity Measurement
The motion of a Brownian particle in a harmonic potential is characterized by an exponentially decaying position autocorrelation function <<r(o)·r(t)>>=exp(
)with the mean square amplitude
=kB
o
o
µ
=
/
. Thus, the local viscous drag
and the diffusion coefficient D = kBT/
of a sphere in a harmonic potential are calculated from the measured correlation time
of the motion and the stiffness
of the potential (
The stiffness of a potential can be determined by measuring the position distribution of a trapped particle (P(r)dr = 1. Conversely, the trapping potential is given by the probability distribution as V(r) = -kBT · lnP(r) + kBT · ln(c), where c is an offset. This method allows to profile the trapping potential even below the thermal energy with a temporal and spatial resolution given by the strength of the potential and the bead size, while requiring only minimal knowledge about the system, i.e., the temperature. In our experiments, the lateral spring constant of the laser trap was adjusted to about k ~1 µN/m for a sphere of 0.2-µm diam. The sample chamber was maintained at 36 ± 1°C, which leads to lateral position fluctuations of ±60 nm RMS displacement.
To achieve diffusion coefficient measurements with errors <10%, the observation interval has to be ~1,000-fold longer than the correlation time (estimated using methods developed by
, overlapping intervals of 0.3 s were used. The autocorrelation function in each interval was calculated and fitted by an exponential decay from 0.15 to 25 ms. The potential
and the viscous drag
=
were computed for each interval and plotted against time.
Over the time course of an experiment, the viscous drag of the free bead, of the same bead near the membrane, and bound are determined (see Fig 3). Each value was measured for 210 s, so that mean (x) and SD () for each individual molecule is determined. These distributions were taken to compute the probability densities for all molecules measured: f(x) = n-1
ni = 1G(x,
) as shown in Fig 4 and Fig 5.
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Saffman-Delbrück Model of Protein Diffusion
The diffusion of a transmembrane protein moving in an obstacle-free lipid bilayer has been described by = 4
mh/(ln(
mh/
wr) -
), where
w denotes the viscosity of the surrounding fluid,
m the viscosity of the lipid bilayer, and
denotes Euler's constant (0.5772;
m >>
w, which is fulfilled for cellular membranes (
= (
w+
cr/
mh) to be <<1, with
c being the viscosity of the cytoplasm. Because
c ~1.5 ·
w (
raft ~ 0.08, the difference between the Saffman-Delbrück solution and the more elaborate solution of
1, is negligible.
Cell Culture and Transfection
BHK-21 cells were grown in supplemented Glasgow (G) -MEM (including 5% FCS, 10% phosphate tryptose broth, 10 mM Hepes, pH 7.3, 2 mM glutamine, 100 units/ml penicillin, and 10 mg/ml streptomycin), and passaged every 3 d. For the studies, BHK cells were plated at low density on coverslips. PtK2 cells were grown in supplemented MEM (containing 10% FCS, 2 mM glutamine, 100 units/ml penicillin, and 10 mg/ml streptomycin). They were used at ~50% confluency. The experiments were carried out in cell culture medium supplemented with 5 mg/ml fish skin gelatin (FSG) to reduce the nonspecific adsorption and were filtered (0.1 µm SuporeAcrodisc; Gelman Sciences).
To express influenza virus hemagglutinin (HA) or placental alkaline phosphatase (PLAP), BHK cells were transiently transfected using Lipofectamine (GIBCO BRL) according to the instructions of the manufacturer. The cells were cotransfected with a soluble YFP (encoded for by plasmid pEYFP-N1; CLONTECH Laboratories) to facilitate the selection of expressing cells. Experiments were performed 1636 h after transfection.
The transiently transfected cells had normal morphology and were viable for several days. LYFPGT46 and YFPGLGPI were expressed in PtK2 cells by infection for 1 h at 37°C with recombinant adenoviruses encoding for the constructs, and experiments were performed 1236 h after transfection. For cholesterol depletion, the cells grown on coverslips were washed in serum-free culture medium and incubated with 10 mM methyl-ß-cyclodextrin for 30 min at 37°C. This procedure was reported to extract ~60% of total cellular cholesterol from BHK cells (
Protein Constructs and Generation of Recombinant Adenoviruses
The transient expression of PLAP in BHK and the BHK clone stably expressing PLAP have been described previously (
LFPGT46 is an artificial secretory protein containing the signal sequence of rabbit lactase-phlorizin hydrolase (
The cDNA for the EGFP used for the blocking of the antibodies was subcloned by PCR from pEGFP-N1 (CLONTECH Laboratories) into a derivative of pET9 (Stratagene, modified by Gunter Stier, EMBL). 6xHis-tagged EGFP was purified from Escherichia coli Bl21(DE3) using nickel affinity chromatography.
Bead Coating and Antibodies
Orange fluorescent (530 nm ex./560 nm em.) carboxyl-modified latex beads from Molecular Probes with a nominal diameter of 0.2 µm (radius = 108 ± 4 nm) were used. The beads were coated with the antibodies by adsorption, based on modified procedures of
mAbs against human PLAP were obtained from DAKO. Rabbit polyclonal antibodies were raised against human PLAP (Sigma Chemical Co.). mAbs HA were prepared as described (
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Results |
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The viscous drag of an individual membrane protein in the plasma membrane of living cells, maintained in a closed microscope chamber heated to 36 ± 1°C, was measured by observing the thermal position fluctuations of an attached microsphere (radius = 108 nm; Fig 1). The damping of the motion is expected to be dominated by the viscous drag on the membrane domain of the protein because of the 1,000-fold higher viscosity of the lipid bilayer compared with the aqueous medium. We used a laser trap to capture the sphere and place it onto the cell membrane while controlling the interaction force. The motion of the sphere was first recorded in the bulk solution, then near the membrane, and finally after binding to the membrane protein. The comparison of these three measurements allowed us to separate the influence of the sphere diffusing unbound near the membrane from the binding of the bead to the membrane protein. The continuous observation with high temporal resolution enabled us to directly observe the binding event. To demonstrate that the measurements were not influenced by the sphere size, some experiments were performed with smaller spheres (radius = 55 nm), yielding similar results (data not shown).
High Resolution Particle Tracking in a Trapping Potential
We implemented an optical trap in an inverted microscope using a near infrared laser that simultaneously excites the fluorophores inside the trapped microsphere via a two-photon process (Fig 2). The dependence of the TPF intensity on the sphere's position relative to the focus of the trapping laser provides an axial displacement sensor used to control the force exerted by the sphere on the membrane below 0.1 pN. To record the thermal position fluctuations, we developed a fast three-dimensional position sensor based on the interference of the transmitted laser light with the light scattered by the trapped particle (
Local Viscous Drag of Single Membrane Proteins
The particle tracking in a trapping potential allowed us to calculate the viscous drag and the trapping potential at a temporal resolution of 0.3 s and for an area of 100 nm in diameter. The viscous drag of the particle was computed as the product of the autocorrelation time
of the position fluctuations and the spring constant
of the trapping potential (see Materials and Methods).
Fig 3 depicts a typical measurement of the lateral viscous drag , spring constant
of the trapping potential, and autocorrelation time
of the movement plotted against time. The measured viscous drag
is the sum of the Stokes drag of the sphere
s = 6
wr and the viscous drag of the protein in the lipid bilayer
p. The Stokes drag of the sphere was measured in the bulk solution, ~2 µm above the plasma membrane (Fig 3 A, I). Near a surface, like the membrane, diffusion is reduced because of the spatial confinement (
= b ·
s +
p was measured and used to calculate the viscous drag of the protein
p. We only included measurements in our analysis in which the strength of the lateral potential was not increased by binding to the membrane protein to ensure that the proteins measured were freely diffusing and not tethered to the cytoskeleton (Fig 3 B).
To determine the stability and size of the rafts, viscous drag measurements were performed on a series of proteins that are known to be components of rafts, as defined by association with DIGs and floatation to low density in a density gradient (
To facilitate the comparison to previous work in BHK fibroblasts (
To ensure binding of the bead to a single membrane protein, we have reduced the number of proteins present in the cells by selecting for weakly expressing ones using the YFP fluorescence. The number of active antibody sites per bead was minimized in the case of anti-PLAP and anti-GFP beads by adding free ligand, a high concentration of which would block binding completely and, for HA, by replacing the major part of the antibodies on the bead with an unrelated antibody. The concentration of the free ligand was chosen to reduce the active binding sites per bead to below 10. The probability for single binding to surface proteins is very high due to the geometric constrictions. To verify this assumption, the fraction of beads bound and the time passed in contact with the membrane before binding were analyzed using Poisson statistics. Under the blocking conditions used, only a fraction of these beads was bound after being in contact with the membrane for 50 s (25% for PLAP, 30% for YFPGLGPI and HA, and 20% for LYFPGT46), whereas 95% of the unblocked beads bound within a few seconds. A Poisson statistic for single binding fitting this behavior provides an estimate of <25 possible binding sites to ensure single binding during the observation interval, and predicts that under the conditions used, >80% of the beads would bind to a single surface protein.
Viscous Drag of Raft-associated Proteins
Fig 4 displays the distribution of viscous drag measurements for the raft-associated proteins PLAP, YFPGLGPI, and HA. Except for PLAP in untreated cells and YFPGLGPI in cholesterol extracted cells, the distributions of single molecule viscous drag measurements have two peaks. For the transmembranous HA expressed in BHK cells, we measured = (2.9 ± 0.7) x 10-9 Pa·s·m (67%) and
= (1.4 ± 0.4) x 10-9 Pa·s·m (33%) (n = 12, P < 0.01, Fig 4 A). The GPI-anchored protein PLAP had
= (3.2 ± 0.8) x 10-9 Pa·s·m (n = 20, 100% raft-associated, Fig 4 B), which is the summary of measurements performed on a stably expressing BHK cell line and on transiently transfected BHK cells. 76% of YFPGLGPI expressed in PtK2 cells had
= (3.0 ± 0.9) · 10-9 Pa·s·m, whereas 24% of YFPGLGPI had
= (0.7 ± 0.3) x 10-9 Pa·s·m (n = 29, P < 0.001, Fig 4 C). Thus, for all three proteins, the majority of molecules had a viscous drag ~3 x 10-9 Pa·s·m. This value was independent of the type of membrane anchor of the protein and remained unchanged for ~1 min, which was the maximal time period continuously accessible in these experiments. For YFPGLGPI and HA, a fraction of the proteins was found to have a lower viscous drag, which was dependent on the type of membrane anchor (P < 0.1).
Effect of Cholesterol Extraction
To investigate the effect of cholesterol depletion on rafts, cholesterol was extracted from the cells by incubation with methyl-ß-cyclodextrin. Extraction of cholesterol from cells results in the dissociation of proteins from rafts and the disappearance of the proteins from the floating fraction in density gradient centrifugation (
The distribution of viscous drag measurements of the transmembrane raft protein HA in BHK cells was shifted slightly after cholesterol depletion: 50% of HA molecules had = (1.2 ± 0.6) x 10-9 Pa·s·m, whereas 50% of HA remained unchanged at
= (3.1 ± 0.6) x 10-9 Pa·s·m (n = 10, Fig 4 D). The effect on the GPI-anchored proteins was clearer: 40% of PLAP in BHK cells had a greatly reduced
= (0.6 ± 0.4) x 10-9 Pa·s·m (n = 15, P < 0.01, Fig 4 E). 60% of the measurements yielded values up to the result obtained for PLAP molecules before cholesterol extraction (Fig 4 B, the multiple peaks are not statistically significant [P > 0.1]). The change in the PtK2 cells was even more pronounced. The viscous drag of the raft-associated protein YFPGLGPI was (0.7 ± 0.6) x 10-9 Pa·s·m for all molecules measured (100%, n = 9, Fig 4 F). This is more than a fourfold reduction from the value obtained for 75% of YFPGLGPI before cholesterol extraction and agrees with the value obtained for the remaining 25% before extraction (Fig 4 C).
Viscous Drag of Nonraft Transmembrane Proteins
To determine the viscosity of the plasma membrane outside of rafts, the local viscous drag of the nonraft transmembrane protein LYFPGT46 was measured. This construct comprised YFP as ectodomain, the transmembrane domain of the LDL receptor and the cytoplasmic tail of CD46 containing a basolateral targeting signal, which, however, does not function as an endocytosis signal ( = (1.1 ± 0.5) x 10-9 Pa·s·m was determined for LYFPGT46 (n = 13, Fig 5 A). Using a construct of the human transferrin receptor, we measured comparable viscous drags in BHK cells (data not shown).
Effect of Cholesterol Extraction
To study whether cholesterol extraction has an effect on the diffusive behavior of nonraft proteins, we measured the viscous drag of LYFPGT46 after depletion of cholesterol from the cells. Their viscous drag did not change, remaining at = (1.1 ± 0.4) x 10-9 Pa·s·m for 100% of the molecules (n = 8, Fig 5 B).
Stability of Raft Association
The viscous drag was measured continuously over a period of ~1 min. Depending on the time that the antibody-coated bead bound to the membrane protein, this provides an observation interval for the protein diffusion of 1050 s. During this interval, we have never observed that a raft-associated protein has dissociated from the raft (n = 50).
To address the question whether raft proteins would stay associated with these domains on longer time scales and would diffuse over the cell surface together with the rafts, spheres bound to GPI-anchored raft proteins were released from the laser trap after the measurement (Fig 6 A, II). The spheres were allowed to diffuse for 2, 5, or 10 min over several micrometers of cell surface (510 µm), and they were captured again. The new measurement yielded comparable values (n = 6, Fig 6 A, III).
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To probe the raft association on intermediate length scales (~1 µm) the raft-associated proteins were translocated on the cell. After binding to the raft protein, the laser trap was used to move the sphere at a speed of ~250 nm/s for ~1 µm while continuously monitoring the position fluctuations. Fig 6 depicts a trace of the viscous drag of a YFPGLGPI molecule during such motion: after binding to the membrane protein, the trap and sphere were moved laterally (Fig 6 B). The viscous drag remained unchanged, indicating that the raft was dragged along as the bead was moved (n = 4).
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Discussion |
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To understand the role of rafts in cell signaling and protein sorting, knowledge about raft dynamics will be needed. Therefore, we have characterized the stability and size of rafts in cell membranes on a time scale from seconds to minutes by measuring the local viscous drag of single membrane proteins. The size dependence of the viscous drag of a protein diffusing in a lipid bilayer was described by 100 nm). The latter approach provided extended observation periods of obstacle-free diffusion of single membrane proteins as verified by profiling the confining potential from the thermal position fluctuations. The obtained local diffusion coefficients of proteins in the plasma membrane in intact cells agree for the first time well with the Saffman-Delbrück model, e.g., for LYFGT46 the viscous drag measurement translates into a diffusion coefficient D = 3.9 x 10-8 cm2/s at 36°C. The precision afforded by the excellent spatial and temporal resolution of this new method allowed us to go one step further and compare the behavior of proteins associated with lipid rafts with the one of nonraft proteins.
The distributions of the raft-associated proteins show a minority of molecules with low viscous drag, but most of the molecules have a large viscous drag. This higher value is the same for all three constructs independent of their type of membrane anchor or ectodomain. Therefore, we think that this value is correlated to the diffusion of the raft itself. However, the smaller viscous drag value measured for a few molecules depends on the type of membrane anchor of the protein. These molecules are assumed to be currently in a nonraft environment. The degree of raft association determined by our measurements (PLAP 100%, YFPGLGPI 76%) agrees with the raft association observed by the detergent extraction method: PLAP 90% (
Fig 7 summarizes the means of the majority of molecules for each raft and nonraft protein studied. Important to note is that two GPI-anchored proteins with different ectodomains (PLAP and YFP) yield similar results, whereas GPI-anchored YFP diffuses differently from transmembrane-anchored YFP, showing that the local viscous drag is determined by the membrane domain of the protein. Why do our data indicate that rafts diffuse as a small unit? First, some of the raft proteins (PLAP and YFPGLGPI) are attached to the membrane by a lipid anchor that just penetrates the outer leaflet of the bilayer, whereas HA is a transmembrane protein traversing both leaflets. Nevertheless, all three proteins diffuse as if connected to a stable membrane domain of similar size with a viscous drag significantly larger than that of the nonraft transmembrane protein LYFPGT46 (P < 0.01). Second, after depleting cholesterol from the cell membrane, the viscous drag of raft proteins is greatly reduced: the transmembrane protein HA shows a viscous drag similar to the nonraft transmembrane protein LYFPGT46, which is equivalent to a threefold reduction. However, the viscous drag of the GPI-anchored proteins is reduced about fivefold and is lower than that exhibited by the nonraft transmembrane proteins (P < 0.1). After cholesterol depletion, GPI-anchored raft-proteins diffuse faster than transmembrane raft proteins, even though in untreated cells they behave the same. The cholesterol depletion has negligible effect on the nonraft transmembrane proteins, indicating that the viscosity of the cell membrane outside of the raft domains remains unchanged. We interpret this difference in behavior as dissociation of the raft assembly or as segregation of the proteins from the lipid raft. After cholesterol depletion, the raft proteins diffuse like nonraft proteins, with the diffusion coefficient becoming dependent on the type of the membrane anchor. An alternative explanation for this reduction of the viscous drag would be that the diffusion observed was actually within a large raft, and that the viscosity of the raft domain was changed by cholesterol extraction. However, protein diffusion in a lipid bilayer depends approximately linearly on the membrane viscosity. Therefore, the latter interpretation is not compatible with the observation that cholesterol depletion reduces the viscous drags for GPI-anchored proteins fivefold, whereas the one of transmembrane raft proteins only threefold. The larger viscous drag of raft compared with nonraft proteins indicates that they are anchored to a membrane patch with a larger diameter than the transmembrane domain of the LDL receptor. Third, the absence of any change after translocating spheres bound to a raft protein laterally over the cell shows that the lipid rafts are moved together with the protein. The other explanation for these data would be that rafts are larger than 1 µm, which would contradict the observations published previously using fluorescence methods (
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Consequences for the Raft Model
Our data agree with the model suggested by 0.05 x 10-8 cm2/s. Even at this reduced mobility raft proteins would reach the edges of their raft at least once every millisecond. Although we have not observed proteins to leave the rafts, little is known yet about the exchange of lipid components between rafts and the surrounding membrane. Our observation that the GPI-anchored raft proteins and the transmembrane raft protein diffuse alike indicates that the linkage between the outer and inner leaflet of the lipid bilayer is in both cases similar. Thus, we assume a strong coupling between the two halves of the bilayer. Nevertheless, we cannot exclude that this coupling is being strengthened by transmembrane raft proteins.
Estimation of the Raft Size
If the raft diffuses as a stable structure as our results suggest, then the size of the raft can be estimated from the local viscous drag measurements. The Saffman-Delbrück model provides the simplest description of the viscous drag of a protein with radius r diffusing in a lipid bilayer with the membrane viscosity m and thickness h (
Therefore, the Saffman-Delbrück model was applied to estimate the size of rafts. To obtain the membrane viscosity, we used the result of the nonraft protein LYFPGT46, which has a single transmembrane domain. Recently, m are coupled as h ·
m in the Saffman-Delbrück relation. Our data for the nonraft transmembrane protein are best fit by h ·
m = (6.3 ± 1.2) x 10-10 Pa·s·m. Therefore, the viscosity of a membrane with h = 5 nm would be
m = 0.13 ± 0.03 Pa·s. This value agrees well with the viscosity of a DMPC bilayer (
m = 0.11 Pa·s;
m obtained here. Also, the fact that HA in cholesterol-depleted cells diffused just like LYFPGT46 dismisses any effect from an extracellular coating. In addition, an extracellular coating would not influence the diffusion in a protein-radiusdependent manner. Under these conditions, the estimated radius of the rafts is r = 26 ± 13 nm. This estimate is also obtained with the formula for protein diffusion of
According to our study, rafts in the plasma membrane of fibroblast-like cells diffuse as a rather stable platform with an average area of 2,100 nm2. The size estimate allows an assessment of the maximal contents of one raft. If these were composed purely of lipid molecules, having a radius comparable to phosphoethanolamine (r = 0.44 nm), one raft would contain almost 3,500 lipid molecules. How many proteins a raft contains depends on how densely packed the proteins would be. If they were as densely packed as rhodopsin molecules are in frog rods (
Implications for Raft Function
The consequences of a small raft size and stable association is that proteins within rafts would be restricted in their interactions with other proteins. To use rafts as platforms in membrane trafficking (e.g., in apical transport from the Golgi complex) would imply that these rafts have to be clustered together by an apical sorting machinery to form a container comprising several rafts. Also, the formation of caveolae at the plasma membrane would involve the invagination of several rafts to form one caveola. Often caveolae are found like grapes in large clusters, thus comprising a reservoir of rafts with associated proteins at the cell surface.
A growing body of evidence implicates lipid rafts in signal transduction. Well-studied examples include IgE receptor signaling and T and B cell activation. If rafts normally contain only a limited set of proteins, then clustering of rafts would be necessary to achieve the concentration of interacting molecules required to elicit a signal above the activating threshold. There is ample indication that clustering is an essential feature of signal transduction processes involving rafts.
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Footnotes |
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1 Abbreviations used in this paper: DIG, detergent insoluble glycolipid-enriched complex; GFP, green fluorescent protein; GPI, glycosylphosphatidylinositol; HA, influenza virus hemagglutinin; LFPGT46, artificial transmembrane YFP; PLAP, placental alkaline phosphatase; SPT, single particle tracking; TfR, transferrin receptor; TPF, two-photon fluorescence; YFP, yellow color variant of green fluorescent protein; YFPGLGPI, artificial GPI-anchored YFP.
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Acknowledgements |
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We thank T. Harder, A. Rietfeld, P. Scheiffele and D. Toomre for many insightful discussions. We acknowledge T. Harder, N. LeBot, P. Scheiffele and J. White for protein constructs and antibodies.
The project was partially supported by the German Science Foundation.
Submitted: 29 October 1999
Revised: 22 December 1999
Accepted: 19 January 2000
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References |
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