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Article |
Address correspondence to J. Lippincott-Schwartz, Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bldg. 18T, Rm. 101, 18 Library Dr., Bethesda, MD 20895. Tel.: (301) 402-1010. Fax: (301) 402-0078. email: jlippin{at}helix.nih.gov
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Abstract |
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Key Words: lipid rafts; membrane microdomains; lateral diffusion; fluorescence recovery after photobleaching; cholesterol
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Introduction |
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One area of widespread interest is how rafts affect a protein's ability to laterally diffuse across cell membranes. In model membranes, the lateral mobility of lipids in a liquid-ordered phase is slower than in a liquid-disordered phase (Brown and London, 1998b; Korlach et al., 1999; Dietrich et al., 2002; Kahya et al., 2003). Indeed, several studies suggest proteins and lipids undergo constrained and/or slowed diffusion within rafts (Sheets et al., 1997; Jacobson and Dietrich, 1999; Schütz et al., 2000; Niv et al., 2002; Shvartsman et al., 2003). Other measurements imply that raft proteins are stably associated over minutes with discrete domains, which themselves can either diffuse across the cell surface (Pralle et al., 2000) or are immobile, such as cell surface caveolae (Pelkmans et al., 2001; Thomsen et al., 2002). However, individual raft proteins do not appear to undergo correlated diffusion with one another (Vrljic et al., 2002), and the GPI-anchored protein CD59 shows similar behavior to a nonraft lipid in single molecule tracking studies (Subczynski and Kusumi, 2003). Thus, conflicting evidence exists as to whether raft domains are mobile or immobile structures, if protein associations with rafts are stable or transient, or how perturbations of raft structure affect the dynamics of individual proteins.
The relative importance of raft association compared with other factors known to modulate protein diffusional mobility is also uncertain. The lateral diffusion of proteins is typically 10100-fold slower in cell membranes than in model membrane systems, even for lipid-binding proteins such as cholera toxin B subunit (CTXB; Bacia et al., 2002). In addition to the presence of membrane microdomains, several factors may contribute to the slowing of protein diffusion within biological membranes, including cytoskeletal barriers, interactions between protein ectodomains, and molecular crowding (Sheets et al., 1995; Edidin, 1996; Kusumi and Sako, 1996; Saxton, 1999; Lippincott-Schwartz et al., 2001). However, many of the studies defining these barriers to diffusion were performed before the development of the lipid raft model, and the role of lipid rafts in regulating membrane protein diffusion has not been systematically investigated.
We have measured the cell surface diffusion of putative raft-associated proteins tagged with GFP using FRAP. FRAP has been used extensively to characterize protein and lipid diffusional mobility and the domain structure of the plasma membrane, typically by monitoring recoveries into small spots (1 µm; Edidin, 1994). In our experiments, we bleached and monitored protein recovery into an area of the membrane much larger than the proposed size of lipid raft domains. In these measurements, diffusional recovery would require either the diffusion of entire rafts or the diffusion of individual proteins outside of raft domains. We tested several models for the stability and organization of lipid raft domains by comparing the diffusional mobility of several kinds of raft proteins (glycolipid-binding, transmembrane, GPI-anchored, acylated, and prenylated proteins). We also examined the effects of cholesterol depletion, cholesterol loading, and reduced temperature on protein mobility. Our results indicate that putative raft-associated proteins are freely mobile and do not diffuse as part of discrete, stable domains across the cell surface. We also find that perturbations reported to affect lipid rafts have similar effects on the diffusional mobility of raft and nonraft proteins. This finding further indicates that raft association is not the dominant factor in determining protein mobility at the cell surface. Thus, if raft domains exist, raft proteins must rapidly partition into and out of them. Alternatively, raft domains may not be a significant feature of the cell surface under steady-state conditions.
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Results |
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Control experiments verified that fluorescence recovery had the characteristics of lateral diffusion. Recovery did not occur in samples fixed with 3.7% PFA (unpublished data). We also confirmed that the characteristic recovery time was proportional to the size of the bleached region, as expected for a protein undergoing lateral diffusion. To test this, we compared recoveries for a given protein into two different size bleach boxes (Fig. 3 B). Recovery was faster in the smaller bleach box, yet identical Ds were obtained for both and were well described by the simulation data for diffusive recoveries (unpublished data). Although dimerization of GFP-tagged membrane proteins has been observed by FRET (Zacharias et al., 2002), introduction of point mutations to eliminate dimerization had no detectable effect on lateral diffusion (unpublished data).
To compare the diffusional mobility of different proteins, we performed FRAP experiments using identically sized bleach boxes. Under these conditions, differences in the kinetics and extent of recovery will reflect the diffusional mobility of the individual proteins. Recovery curves for a population of cells expressing a given protein were similar, but differences in the kinetics of recovery were apparent between proteins (Fig. 3 C). Only a small fraction of proteins were immobile, as Mf was typically >85% (Fig. 3 C). In contrast, caveolin-1-GFP was nearly 100% immobile (unpublished data).
Diffusional mobility for raft and nonraft proteins under steady-state conditions
Next, we used the confocal FRAP assay to compare the diffusional mobility of raft proteins with one another. For all the proteins examined, we observed recoveries characteristic of lateral diffusion, with high Mf (Table I). CTXB is internalized over time, and the endosomal fraction of the protein is unable to recover rapidly after a photobleach, giving rise to an effective immobile fraction (Table I). D varied significantly for different raft proteins, ranging from 0.1 to 1.2 µm2/s at 37°C (Fig. 4 A). The slowest recoveries were observed for Cy3-CTXB and HA-GFP. D was higher for the three GPI-anchored proteins and LAT-GFP, but was significantly slower for YFP-GL-GPI than for GFP-CD59 and GFP-GPI (t test, P < 0.001). Raft proteins localized to the inner leaflet of the plasma membrane, Fyn-GFP and GFP-HRas, exhibited the most rapid diffusion, similar to values recently reported for GFP-HRas by spot photobleaching (Niv et al., 2002).
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Lower temperatures slow both raft and nonraft protein diffusion
Raft proteins partition into DRMs more efficiently at 4°C than 37°C (Brown and Rose, 1992; Cerneus et al., 1993), and the formation of raft domains is driven by lowered temperature in model membranes (Dietrich et al., 2001a,b; Samsonov et al., 2001). We tested whether or not at decreased temperatures, in which DRM association is enhanced (Fig. 5 A), we could observe any evidence for increased partitioning of raft proteins into domains by FRAP. D was lowered by approximately twofold at 20°C for both raft and nonraft proteins and showed further decreased mobility at 10°C (Fig. 5 B). A similar effect was observed for VSVG-GFP in the ER (Reits and Neefjes, 2001; unpublished data), suggesting that this phenomenon reflects a general effect on membrane viscosity throughout the cell. These results are consistent with previous measurements showing that protein and lipid diffusion at the plasma membrane is temperature dependent (Wey et al., 1981; Hillman and Schlessinger, 1982; Jacobson et al., 1984; Aroeti and Henis, 1988) but do not show a simple correlation with raft domains.
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Discussion |
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Diffusional mobility is more strongly correlated with membrane anchorage than with raft association
Our finding that Ds varied tenfold for different raft proteins is consistent with previous observations showing that a variety of barriers to protein diffusional mobility exist in cell membranes (Sheets et al., 1995; Edidin, 1996; Kusumi and Sako, 1996; Saxton, 1999; Lippincott-Schwartz et al., 2001). These results also indicate that raft association is not the dominant factor in determining the overall mobility of a particular protein, a possibility consistent with either the dynamic partitioning or no raft models (Fig. 1). Instead, we observed a correlation between protein diffusional mobility and the mode of membrane anchorage, with Dacylated or prenyalated > DGPI-anchored proteins > Dtransmembrane regardless of whether or not a particular protein is raft associated (Fig. 4).
The high mobility of GPI-anchored proteins has been previously noted and is thought to reflect the lipid-based anchor of these proteins (Ishihara et al., 1987; Edidin and Stroynowski, 1991) but is modulated by the ectodomain (Zhang et al., 1991). This finding could explain why we observed significantly higher Ds for GPI-anchored proteins than a transmembrane form of HA, in contrast to a recent report (Shvartsman et al., 2003). Until now, the diffusional properties of acylated and prenylated proteins have remained largely unexplored because of their inaccessibility to labeling with exogenous fluorescent probes. The high Ds of these proteins suggest that their lipid anchors facilitate their relatively free diffusion and/or that the microenvironment of the inner leaflet contains fewer barriers to protein diffusion than the outer leaflet where GPI-anchored proteins are found.
LAT-GFP and Cy3-CTXB are two exceptions to the correlation between D and membrane anchorage (Fig. 4). We speculate that, given the effective absence of an ectodomain (Zhang et al., 1998a), LAT is more directly comparable to proteins localized to the inner leaflet of the plasma membrane than other transmembrane proteins. The slow diffusion of CTXB, a homo-pentamer, could potentially arise from cross-linking and trapping other cell surface proteins that can interact with the cytoskeleton, similar to that proposed for cross-linked GPI-anchored proteins (Suzuki and Sheetz, 2001). Association of CTXB with immobile caveolae before its eventual internalization may also slow its diffusion.
The effects of reduced temperature and cholesterol depletion on protein diffusion are not limited to alterations of raft dynamics
To distinguish between the dynamic partitioning (Fig. 1, Model 3) and no raft domain (Fig. 1, Model 4) models, we first investigated the sensitivity of protein mobility to two conditions previously shown to alter partitioning of raft proteins into DRMs, temperature reduction and cholesterol depletion. We found that both temperature reduction and cholesterol depletion decreased the diffusional mobility of both raft and nonraft proteins to a similar extent (Figs. 5 B and 6 B). This observation does not completely eliminate the possibility of altered partitioning of raft proteins into rafts in response to these treatments, but indicates that other factors play a more dominant role in determining a protein's lateral mobility under these conditions. Interestingly, an immobilizing effect of cholesterol depletion also has been noted in a recent study of MHC-class I lateral mobility (Kwik et al., 2003). This effect was linked to changes in the organization of the actin cytoskeleton in response to MßCD treatment, which have also been noted in other studies (Grimmer et al., 2002; Hering et al., 2003; see also the retraction of cells during this treatment in Fig. 6 A). Given the potential connection between lipid rafts and the actin cytoskeleton (Maxfield, 2002), it is unclear if these effects occur in addition to, or as a consequence of, disruption of raft domains. Nevertheless, these findings raise questions about the specificity of acute cholesterol depletion on lipid domain structure/function and highlight the possibility that cytoskeletal reorganization might account for some of the observed functional effects of MßCD treatment.
Effects of cholesterol loading, another method to explore membrane domain structure and function on raft protein dynamics
To further attempt to distinguish the effect of raft perturbations on protein mobility, we used cholesterol loading. Given that major cellular defects are associated with conditions of aberrant cholesterol accumulation, such as hypercholesterolemia and Niemann-Pick type C (NPC) disease, understanding the effects of excess cholesterol on lipid raft structure and function is of physiological importance. Based on work in model systems (Kahya et al., 2003; Lawrence et al., 2003), we hypothesized that cholesterol loading should lead to the formation of additional lipid rafts and/or to an increase in size of existing rafts. However, little change in the halftime of recovery was seen for any of the proteins studied in cholesterol-loaded cells (Fig. 7). These data suggest that if cholesterol loading indeed increases the fraction of raftlike membrane, diffusion of raft proteins within this environment is not significantly slowed under these conditions. However, we did observe decreased Mf in response to cholesterol loading. This effect appears to be due to enhanced endocytosis of proteins in response to cholesterol loading, potentially resulting from proteins being driven out of an expanded raft phase. The identity of these putative endocytic structures remains to be determined. Both clathrin-mediated and nonclathrin endocytic pathways are sensitive to membrane cholesterol levels (Rodal et al., 1999; Subtil et al., 1999; Nichols and Lippincott-Schwartz, 2001). Moreover, defects in the trafficking of glycolipids internalized by nonclathrin pathways have been correlated with the accumulation of excess cholesterol in multiple sphingolipid storage diseases including NPC (Puri et al., 1999). Our observations of continued CTXB uptake in cholesterol-loaded cells (Fig. 7 B) are in line with observations that CTXB is able to enter endocytic structures in NPC1-deficient cells (Sugimoto et al., 2001; Choudhury et al., 2002). It will be of interest to determine the nature of the trafficking defects induced by acute cholesterol loading and to discern if the mechanisms are similar to those observed in NPC-deficient cells.
Implications for models for lipid rafts
Our data indicate that raft association is not the major determinant of plasma membrane protein diffusional mobility under steady-state conditions. Thus, we can rule out several models for raft dynamics, including stable immobile rafts and stable mobile rafts, on the basis of our findings. It is possible that a small fraction of proteins are associated with stable immobile domains, as we observed an immobile fraction of 1015% for the majority of the proteins examined (Table I). However, the absence of an effect of cholesterol depletion on the size of the immobile fractions that we and others (Lommerse et al., 2004) have detected, and the similar immobile fractions of raft and nonraft proteins, suggests that the immobile fraction is not generated by recruitment to lipid rafts. It is also possible that overexpression of our marker proteins may overwhelm and mask a small amount of behavior corresponding to models 1 or 2. However, diffusion of the raft marker GFP-HRas was previously shown to be independent of expression levels (Niv et al., 2002). Techniques with single molecule sensitivity such as fluorescence correlation spectroscopy could provide insight into this issue in the future.
Our findings also reveal that treatments shown to perturb the rafts detected in model membrane systems (cholesterol depletion, temperature reduction, and cholesterol addition) have effects on protein diffusion in cells that cannot be explained solely by predictions of the raft hypothesis. However, these treatments may give rise to pleiotropic effects that mask underlying changes in raft properties. Therefore, on the basis of our current data we cannot definitively distinguish between the dynamic partitioning model and the absence of detectable raft domains (Fig. 1). Interestingly, the predictions of the dynamic partitioning and no raft models converge under conditions where lipid rafts comprise a small fraction of the cell surface and proteins spend only a small amount of time visiting them, or if the fraction of raftlike membrane is normally large but protein diffusion within a raft is not very different than in nonraft regions of the membrane. Our understanding of how rafts function in vivo now needs to take into account the fact that incorporation into biochemically defined rafts is not indicative of incorporation into stable microdomains in the plasma membrane.
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Materials and methods |
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Cells were grown in DME (COS-7 and normal rat kidney [NRK] cells) or EMEM (BHK-21) supplemented with 10% FCS, glutamine, penicillin, and streptomycin (Biofluids). Transient transfections were performed using FuGENE 6 (Roche Molecular Biochemicals). CTXB was fluorescently labeled with Cy3 (Amersham Biosciences) as per the manufacturer's instructions and was used at a final concentration of 1 µg/ml.
Fluorescence microscopy and diffusional mobility measurements
Filipin fluorescence was imaged using a wide field microscope (model Axiophot; Carl Zeiss MicroImaging, Inc.). Fluorescence was excited with a mercury arc lamp, and emission was detected using a DAPI filter set. Images were collected using a 40x 1.3 NA Plan-Neofluar objective (Carl Zeiss MicroImaging, Inc.) and captured using a MicroMax CCD camera (Princeton Instruments) and MetaMorph acquisition software. Images were obtained using identical exposure times for cells subjected to various treatments in each experiment. Quantitation of filipin fluorescence was performed using NIH Image.
All other fluorescence images were obtained using a confocal microscope (model LSM 510; Carl Zeiss MicroImaging, Inc.). Fluorescence emission resulting from 488-nm excitation for GFP, 488- or 514-nm excitation for YFP, and 543-nm excitation for Cy3 was detected using filter sets supplied by the manufacturer. Cells were held at 37°C on the microscope stage unless otherwise indicated. For FRAP measurements, a 40x 1.3 NA Plan-Neofluar objective or 100x 1.4 NA Plan-Apochromat objective (Carl Zeiss MicroImaging, Inc.) was used with the confocal pinhole set at 12 Airy units. Photobleaching of GFP, YFP, or Cy3 was performed using 520 scans with the 488-nm laser line at full power in a rectangular region of interest 4 µm wide (COS-7 cells) or 1.4 µm wide (BHK-21 and NRK cells). Pre- and postbleach images were monitored at low laser intensity. Fluorescence recoveries in the bleached region and overall photobleaching in the whole cell during the time series were quantitated using the LSM software (Carl Zeiss MicroImaging, Inc.). Photobleaching of Cy3 did not induce photodamage to the membrane, as evidenced by control experiments showing identical recoveries for GFP-HRas in cells labeled with Cy3-CTXB versus unlabeled CTXB. For presentation purposes, LSM 510 images were exported in TIFF format and their contrast and brightness optimized in Photoshop.
Ds were calculated from the photobleaching data using Diffuse, a program that simulates diffusive recovery into the bleached region using the series of images collected after the photobleaching episode (Ellenberg et al., 1997; Zaal et al., 1999; Nehls et al., 2000; Siggia et al., 2000; program available upon request). LSM 510 images were exported in TIFF format and converted to PPM format for analysis with the Diffuse program. Statistical differences were evaluated using the t test. Mf was calculated as described previously (Ellenberg et al., 1997).
TX-100 extraction
TX-100 extractions were performed as described previously (Nichols et al., 2001) except that cells were incubated in TX-100 for 15 min and after fixation subsequently labeled with Cy3-CTXB before imaging. Where indicated, TX-100 extractions were performed at 12°, 22°, or 37°C instead of 4°C.
Filipin staining
Fixed cells were stained with 5 µg/ml filipin as described previously (Neufeld et al., 1999). Filipin fluorescence was quantified from regions of interest containing plasma membrane and underlying cytoplasmic structures using NIH Image.
Cholesterol depletion, loading, and repletion
Solutions for cholesterol depletion, repletion, and loading were made in DME or RPMI supplemented with 25 mM Hepes and 1 mg/ml BSA. For cholesterol depletion, cells were briefly washed, and then incubated in 10 mM MßCD for 30 min at 37°C before TX-100 extraction or FRAP. Cholesterol loading was performed using water-soluble cholesterol (MßCDcholesterol complexes at 6:1 molar ratio; Sigma-Aldrich). Previous work has established that efficient loading of cells occurs when incubated with MßCDcholesterol complexes prepared at this ratio (Christian et al., 1997; Sheets et al., 1999). MßCDcholesterol complexes were prepared as a stock solution, 300 mM in MßCD, diluted directly onto the cells to 10 mM in MßCD, and incubated for 30 min at 37°C. Incubation for longer times (12 h) led to increased sensitivity to TX-100 extraction in DRM experiments. Cholesterol-loaded cells imaged live for FRAP experiments did not initially exhibit any gross morphological differences from control cells, but over time cells showed a tendency to round up.
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Acknowledgments |
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Experiments were performed in part through the use of the Vanderbilt University Medical Center Cell Imaging Core Resource (supported by National Institutes of Health grants CA68485, DK20593, and DK58404). A.K. Kenworthy was supported by a fellowship from the National Research Council, and B.J. Nichols was funded by an International Prize Travelling Research Fellowship from the Wellcome Trust.
Submitted: 24 December 2003
Accepted: 26 April 2004
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