Article |
Address correspondence to Yoav I. Henis, Dept. of Neurobiochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. Tel.: (972)-3-640-9053. Fax: (972)-3-640-7643. email: henis{at}post.tau.ac.il
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Abstract |
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Key Words: influenza hemagglutinin; rafts; fluorescence; lateral diffusion; photobleaching
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Introduction |
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Proteins that interact with lipid rafts are often identified by their ability to be floated in vitro on gradients with detergent-resistant membranes (DRMs) from cells lysed in nonionic detergents (typically Triton X-100) at 4°C (Simons and Ikonen, 1997; Brown and London, 2000). Although this method identifies proteins capable of a fairly stable interaction with lipid rafts, it is unclear how strong these interactions must be to allow isolation in DRMs. In addition, there is evidence for the existence of more than one type of lipid raft. Caveolae, morphologically distinct membrane invaginations containing caveolin/VIP21 (Kurzchalia et al., 1992; Rothberg et al., 1992), are a specialized form of raft, but rafts exist also in cells lacking caveolae (Kurzchalia and Parton, 1999; Smart et al., 1999; Brown and London, 2000; Simons and Toomre, 2000; Anderson and Jacobson, 2002). The immunological synapse in T cells may also be considered a specific type of lipid raft that binds to the actin cytoskeleton (Grakoui et al., 1999; Janes et al., 1999; Zhang and Samelson, 2000). The sensitivity of proteins in different types of rafts to detergent extraction has not been systematically compared. Adding complexity to this situation, membrane fractions soluble in Triton X-100, but not in another nonionic detergent, have been reported and equated with a subpopulation of lipid rafts of distinct composition existing in biological membranes (Roper et al., 2000). In intact cells, FRET studies of acylated GFP derivatives in the cytoplasmic leaflet of the plasma membrane suggest that they cluster in more than one lipid microdomain type (Zacharias et al., 2002). Determining the relationships between the isolation of specific proteins with DRMs, their affinities to lipid rafts in intact cells and their biological activities, requires measurement of lipid raft association in living cells.
Several biophysical methods have been employed to measure raft association in cells and yielded variable results (Kurzchalia and Parton, 1999; Simons and Toomre, 2000; Anderson and Jacobson, 2002), probably due to the different parameters measured by each method. Thus, FRET studies yielded results either in favor (Varma and Mayor, 1998; Zacharias et al., 2002) or against (Kenworthy and Edidin, 1998; Kenworthy et al., 2000) the existence of rafts in live cells. Other methods (chemical cross-linking, single particle tracking, and laser trap) detected clustering of raft proteins in submicron domains, but yielded highly variable estimates of domain sizes (Friedrichson and Kurzchalia, 1998; Pralle et al., 2000; Anderson and Jacobson, 2002). These studies did not investigate the dynamics of proteinraft interactions (stable or transient association). This issue is important, as some hypotheses of the biological functions of lipid rafts implicitly require stable interaction of signaling molecules with rafts and others require the opposite. Recent studies on the dynamics of raft association have been limited to proteins in highly organized specialized raft structures, such as caveolin-1 in caveolae and LAT (linker for activation of T cells) in the T cell immunological synapse; these proteins were found to be laterally immobile in these structures (Thomsen et al., 2002, Tanimura et al., 2003).
Here we employed a combination of FRAP and immunofluorescence co-patching to investigate the raft association dynamics of influenza HA mutants bearing different (or no) raft-targeting signals. In epithelial cells, sorting of HA to the apical surface depends critically on its TM sequence, in correlation with its partitioning into Golgi-derived DRMs (Skibbens et al., 1989; Scheiffele et al., 1997; Lin et al., 1998). The availability of TM point mutants of HA that are excluded from DRMs (Scheiffele et al., 1997; Lin et al., 1998) makes the HA system ideal for studies on raft association, as it enables direct comparison between proteins that do or do not partition into rafts but are otherwise similar, excluding possible effects of other interactions. Moreover, comparison with a glycosylphosphatidylinositol (GPI)-anchored HA (BHA-PI; Kemble et al., 1994) allows us to measure differences between two mutants of the same protein that are targeted to rafts by different signals. Our studies demonstrate that the lateral diffusion rate of raft-associated HA proteins is retarded relative to a nonraft mutant in a cholesterol-dependent manner. Using a combination of patching and FRAP studies, we show that a GPI-anchored HA and wild-type (wt) HA reside in mutual rafts, but differ in their mode of interaction with the raft domains (stable association for GPI-anchored proteins, and transient interactions for wt HA). The high sensitivity of this in vivo method allowed us to detect interactions with lipid rafts that are too weak for partitioning in DRMs in vitro but are sufficient to enable sorting to the apical surface.
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Results |
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Apical sorting of membrane proteins in polarized epithelial cells is proposed to depend on partitioning into Golgi or TGN raft-like domains. As some apical proteins (e.g., HA) can be isolated in DRMs only upon reaching the sorting point in the biosynthetic pathway (Skibbens et al., 1989) while other apical proteins are never isolated in DRMs, it has been argued that two separate and parallel pathways from the TGN to the apical surface may exist (Sarnataro et al., 2000; Jacob and Naim, 2001). Alternatively, the affinity for Golgi lipid rafts required for sorting an apical protein may be lower than that required for isolation in DRMs. To address the question of how measurements of detergent insolubility compare to measurements of raft association in living cells, we examined the behavior of two HA mutants differing by a single amino acid in the TM domain, TM 11.0 and TM 12.0 (Table I). Both the apical sorting of HA and its partitioning into Golgi DRMs depend upon its TM sequence (Skibbens et al., 1989; Scheiffele et al., 1997; Lin et al., 1998). Recently we have identified the minimum TM sequence required for apical sorting of HA and in the process produced several mutants including TM 11.0 that were sorted apically but were not isolated in DRMs (Alonso, M.A., personal communication; unpublished data). HA TM 11.0 was sorted less efficiently than TM 12.0, which does partition into DRMs (Table I), suggesting weaker association with rafts. The cholesterol-sensitive retardation of the lateral diffusion of HA mutants at the two extremes of raft association (raft-resident wt HA vs. the nonraft HA-2A520) correlated with their ability to enter DRMs (Scheiffele et al., 1997; Lin et al., 1998). To examine whether the correlation holds for weaker interactions, we used FRAP to investigate the interactions of TM 12.0 and TM 11.0 with cholesterol-sensitive rafts (Fig. 4). On untreated cells, D of TM 11.0 was significantly higher (1.5-fold) than that of TM 12.0. Importantly, cholesterol depletion increased D of TM 12.0 to the level observed for the non-DRM TM 11.0, whose lateral diffusion was insensitive to cholesterol depletion (Fig. 4). Thus, the in vivo FRAP studies are in accord with the detergent insolubility experiments (Table I) also for these mutants, suggesting that the strength of the interactions with rafts that can be detected by the two methods is similar. However, the apical sorting of HA TM 11.0 could still depend on raft interactions that are too weak for detection by both methods; this possibility has been validated by studies based on a combination of IgG-mediated patching and FRAP (see Fig. 8).
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Immunofluorescence studies based on antibody-mediated patching can identify putative raft association, if the association is stable enough to persist during patching (Mayor et al., 1994; Harder et al., 1998; Janes et al., 1999; Simons and Toomre, 2000). For coexpressed pairs of a GPI-anchored protein and HA, antibody-mediated patching was reported to bring together raft domains and cause their coalescence (Harder et al., 1998; Simons and Toomre, 2000). However, under the conditions used in those experiments, both vesicular stomatitis virus G protein and HA-Y543, proteins that are not isolated in DRMs, showed significant co-patching with the GPI-anchored placental alkaline phosphatase (Harder et al., 1998). We determined conditions that result in small distinct patches that increase the resolution for detecting co-patching and employed these in three sets of co-patching experiments (Fig. 5): (1) between two TM-anchored HA proteins, Japan wt HA and X:31 wt HA; (2) between Japan wt HA and GPI-anchored X:31 BHA-PI; and (3) between X:31 wt HA or BHA-PI and the nonraft mutant Japan HA-2A520 as controls. In the co-patching method (detailed in Keren et al., 2001), two antigenically distinct membrane proteins are coexpressed at the surface of live cells. One is forced into micropatches (in the cold, to avoid endocytosis) by a double layer of bivalent IgGs, using a fluorescent secondary IgG. The coexpressed protein is patched/labeled by IgG from another species and a secondary IgG coupled to another fluorophore. If the two proteins reside in mutual clusters or domains and do not dissociate from them appreciably during patching, they are swept into mutual patches. Using secondary IgGs coupled to red and green fluorophores, mutual patches appear yellow when the two images are overlapped. This holds also when the proteins are oligomeric; for this matter, a trimeric HA is a single unit, and one measures the association between several such units in clusters/domains. As HA is trimeric, studies on its clustering require coexpression of two antigenically distinct HAs that form only separate trimers. This condition is met by the HAs of the Japan and X:31 strains (Boulay et al., 1988; Keren et al., 2001). Typical co-patching results are shown in Fig. 5 (A and B); the averaged results of many such experiments are depicted in Fig. 5 C. The co-patching between Japan and X:31 wt HA proteins was very low (14%) and was not affected by cholesterol depletion. This low value is similar to that measured between the various HAs and the nonraft mutant HA-2A520 (Fig. 5 C), suggesting that this background co-patching level is not due to residual localization in rafts. It most likely represents the cumulative contribution of factors other than coresidence in rafts (e.g., occasional overlap of randomly distributed green and red patches, mutual targeting to other cellular structures, or nonspecific interactions). Interestingly, the pair of X:31 BHA-PI/Japan wt HA exhibited a small but significant cholesterol-sensitive increase in the co-patching level (Fig. 5 C). The low levels of co-patching between pairs of raft-associated HAs suggest that at least one HA in a coexpressed pair interacts transiently with the raft domains, enabling its dissociation during the patching step. The higher level of co-patching between BHA-PI and wt HA is in accord with the proposal that antibody cross-linking of GPI-anchored proteins stabilizes the raft domains (Harder et al., 1998; Simons and Toomre, 2000).
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Discussion |
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In the current study, we combined FRAP and co-patching studies to investigate these issues in live cells expressing a series of HA mutants that differ in their ability to be isolated in DRMs and to undergo apical sorting. The HA cytoplasmic tail is short and lacks signals known to interact with intracellular structures (Lazarovits and Roth, 1988; Kusumi and Sako, 1996), providing an experimental system that is not complicated by possible interactions with the cytoskeleton. Furthermore, the availability of HA mutants that vary in DRM affinity enables a direct comparison between the effects of mutations introduced into the same protein background. Our results (Figs. 2 and 3) demonstrate that the lateral diffusion rates of DRM-associating HA proteins (X:31 BHA-PI and wt HA) are significantly slower than that of an HA mutant (HA-2A520) that is excluded from DRMs and not sorted apically. This suggests that the lateral diffusion of the DRM-associating HAs is retarded by interactions with lipid rafts, a conclusion supported by the selective effects of cholesterol depletion on the HA proteins. Only D of DRM-associating HA proteins was elevated, reaching the same values measured for HA-2A520, whose mobility is insensitive to cholesterol (Fig. 3). As no IgG cross-linking is applied here, these results suggest that the raft domains with which wt HA and BHA-PI interact exist prior to IgG cross-linking. Our findings are in line with a study by single particle tracking (Pralle et al., 2000), which showed a cholesterol-sensitive increase in viscous drag (equivalent to a reduction in D) for some DRM-resident proteins. This retardation was about twofold higher than in our measurements of raft-resident HA proteins, probably reflecting the average nature of FRAP studies, where HA populations in and out of rafts both contribute to the measurements. Another possibility (not mutually exclusive) is that the association of some HA proteins with rafts may be transient, in which case their lateral diffusion would be retarded only during the association cycle, resulting in an apparent D value between that of a raft and of a nonraft HA protein.
The measurements of the association of HA mutants with raft domains by the sensitivity of their lateral diffusion to cholesterol depletion (Fig. 3) are in good agreement with their detergent insolubility (Scheiffele et al., 1997; Lin et al., 1998; Fig. 1 and Table I), suggesting comparable sensitivities of the two methods. However, the Triton X-100insoluble fraction of BHA-PI was less than that of wt HA (unpublished data), although BHA-PI exhibited stable interaction with rafts in FRAP co-patching experiments while wt HA displayed transient interactions. This suggests that the fraction of BHA-PI in rafts is not higher than that of wt HA. Yet, BHA-PI dissociation from raft patches is much slower (Figs. 6 and 7), raising the possibility that the dissociation rate of a protein from raft domains does not necessarily correlate with the equilibrium constant.
Interestingly, both detergent insolubility (Table I) and FRAP studies (Fig. 4) failed to detect interactions of HA TM 11.0 with lipid rafts, although this HA mutant still undergoes significant apical sorting (Alonso, M.A., personal communication; unpublished data). Because the apical sorting of HA proteins depends critically on partitioning into Golgi raft domains (Skibbens et al., 1989; Scheiffele et al., 1997; Lin et al., 1998; Melkonian et al., 1999), this finding suggests that the affinity to Golgi raft domains required for apical sorting may be weaker than that required to produce interactions that are measurable by the commonly employed methods (e.g., in vitro isolation in DRMs). This conclusion is strongly supported by the demonstration (Fig. 8) that the more sensitive method based on combining IgG-mediated patching and FRAP (see next paragraph) was capable of detecting association of HA TM 11.0 with rafts.
To investigate the dynamics of HAraft interactions, we employed immunofluorescence co-patching in combination with FRAP. A former study examined co-patching between a GPI-anchored protein and HA, but not between two different proteins targeted to rafts by their TM regions (such as two antigenically distinct wt HA proteins), and was also complicated by the detection of co-patching with nonraft TM proteins (Harder et al., 1998). We improved the resolution by defining conditions that lead to small and distinct patches and examined both the co-patching of GPI-anchored HA with wt HA and of one wt HA with another (Japan wt HA with X:31 wt HA). An important control is provided by the very low co-patching level obtained between X:31 wt HA and the nonraft mutant HA-2A520 (Fig. 5). A similar low co-patching level was measured for the pair of Japan wt HA/X:31 wt HA, suggesting either that they reside in separate rafts or that they interact transiently with raft domains and dissociate from them during the patching step. A significantly higher level of co-patching was found only when GPI-anchored HA was co-patched with wt HA, and this higher level was sensitive to cholesterol depletion (Fig. 5). This is in line with the suggestion that antibody-mediated aggregation of GPI-anchored proteins can stabilize and mediate coalescence of raft domains (Harder et al., 1998; Simons and Toomre, 2000). However, even in this case, the level of co-patching is not very high (2223%, on a background level of 14%), suggesting that although IgG cross-linking stabilizes to some extent the association with rafts, at least one of the two HA proteins examined can still dissociate from rafts to some degree during patching. As shown by the combined IgG patching/FRAP studies (Figs. 6 and 7), this protein is the transiently interacting wt HA.
Due to the weak (logarithmic) dependence of the lateral diffusion rate of membrane proteins on size (Saffman and Delbruck, 1975), raft domains may diffuse only somewhat slower than a nonraft membrane protein (Pralle et al., 2000). This makes it difficult to distinguish between a protein with a subpopulation that diffuses slower while stably associated with rafts, and a protein whose diffusion is retarded due to transient interactions with rafts. Therefore, we employed IgG cross-linking to immobilize one HA protein and measured by FRAP the effects on the lateral diffusion of a coexpressed HA mutant labeled with Fab'. Stable association of a protein with the immobilized patches of a coexpressed protein reduces its mobile fraction (Rf), while transient interactions reduce its lateral diffusion rate (D). If the two proteins do not interact or do not share the same raft domains, immobilization of one should not affect either Rf or D of the other. The results of this series of experiments (Figs. 68) lead to several important conclusions. First, the finding that patching/immobilization of BHA-PI, GFP-GPI, or wt HA does affect the lateral diffusion of the coexpressed protein demonstrates the coexistence of the two proteins in the same rafts. Second, the reduction in D of wt HA upon immobilization of BHA-PI (or GFP-GPI) suggests that wt HA interacts transiently with raft domains containing the GPI-anchored protein, even when the latter have been cross-linked by IgGs. Third, the reduction in Rf of BHA-PI (or GFP-GPI) upon immobilization of coexpressed wt HA indicates that the GPI-anchored proteins are stably associated with rafts containing IgGcross-linked wt HA. Thus, the same raft domains can exhibit stable association with some proteins and transient interactions with others. The identification of both types of interactions has implications for potential models of rafts as scaffolds that regulate biological functions. Some models would require stable association with rafts, e.g., activation by dimerization/oligomerization of two proteins already concentrated in the same raft, or activation of two proteins in different rafts by clustering the rafts together, allowing their coalescence (Kurzchalia and Parton, 1999; Brown and London, 2000; Simons and Toomre, 2000). Other models may require transient association with rafts to allow dynamic exchange into and out of these domains. Such is the case for activation due to altered partitioning of specific proteins in the raft domains after ligand binding and/or dimerization (Simons and Toomre, 2000). Another example is signaling by H-Ras, proposed to occur in two steps: activation of H-Ras within rafts, followed by its obligatory exit from the raft domains (Prior et al., 2001). Obviously, this would require the interactions of H-Ras with rafts to be transient. Our demonstration that different raft-associated proteins can display either stable or transient interactions with mutual raft domains suggests that both possibilities exist. The dynamics of the association with rafts may depend on the raft-targeting signal of the specific protein, as different moieties (e.g., a GPI anchor vs. a TM sequence) can interact differently with lipid and/or protein constituents of rafts.
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Materials and methods |
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Plasmids and recombinant virus vectors
The studies used several expression vectors (pkSVE for infection with recombinant SV40 viruses, all other vectors for transient transfection) encoding HA proteins. X:31 HA (Gething et al., 1980) in pkSVE (Keren et al., 2001) was also subcloned into pCB4 (Brewer, 1994). BHA-PI (the TM and cytoplasmic domains of X:31 HA replaced by nine amino acids from the GPI anchor addition signal of DAF, with a serine-for-lysine substitution) (Kemble et al., 1994) in the pEE14 vector (Celltech) was a gift from J.M. White. Expression plasmid for GFP-GPI (EGFP attached to the folate receptor GPI anchor signal) (Sabharanjak et al., 2002) was a gift from R.G. Parton (University of Queensland, Brisbane, Australia). Several mutants derived from the cDNA encoding A/Japan/305/57 HA were used (Lin et al., 1998; Table I). These cDNAs were introduced into the SV40-based vector pkSVE and also subcloned into transient expression vectors (pSVT7 for Japan wt HA; pCB6 for HA-2A520, TM 12.0, and TM 11.0).
Cell transfection and infection
CV-1 cells were infected with recombinant SV40 virus vectors; alternatively, COS-7 or CV-1 cells were subjected to direct transfection using Fugene 6 (Roche Chemicals). Infection of CV-1 cells was carried out as previously described (Naim and Roth, 1994), using second or third passage recombinant SV40 virus stocks prepared from the HA mutants cloned in pkSVE vectors. The infected cells were plated on glass coverslips for immunofluorescence-based studies. Experiments were conducted 3638 h after infection. Direct transfection using HAs cloned in expression vectors containing SV40 origin (pSVT7, pCB6, pEE14, and pEGFP) was carried out with Fugene 6 on subconfluent cells grown on glass coverslips. They were taken for co-patching or FRAP studies either after 48 h (CV-1 cells) or 24 h (the higher-expressing COS-7 cells).
Cholesterol depletion
After transfection or infection with HA-expressing vectors, the cells were preincubated at 37°C for 46 h (transfected COS-7 cells), 1618 h (infected CV-1 cells), or 2830 h (transfected CV-1 cells), and subjected to cholesterol depletion by incubation (1820 h) with 50 µM compactin and 50 µM mevalonate in DME supplemented with 10% lipoprotein-deficient serum following established procedures (Hua et al., 1996; Lin et al., 1998).
Triton X-100 insolubility
COS-7 cells (1.25 x 105) were replated from a subconfluent plate and transfected with expression plasmids for wt Japan HA or HA-2A520. After 24 h, cholesterol was depleted from the cells for 24 h as described in the previous section. The cells were washed four times in PBS plus 1 mM MgCl2 and 0.1 mM CaCl2, and incubated in DME lacking methionine and cysteine for 30 min at 37°C. The cells were radiolabeled with EXPRE[35S][35S] protein labeling mix at a concentration of 0.5 mCi/ml for 30 min at 37°C, followed by a chase in DME for 100 min at 37°C. Samples were lysed on ice in 1% Triton X-100 buffer, and soluble and insoluble fractions were prepared, immunoprecipitated, and analyzed by PAGE as previously described (Lin et al., 1998).
Immunofluorescence co-patching
Antibody-mediated co-patching (Gilboa et al., 2000) of antigenically distinct HA mutants was measured as previously described (Keren et al., 2001). In brief, CV-1 or COS-7 cells were infected or transfected with pairs of vectors encoding X:31 and Japan HA proteins, and subjected in some experiments to cholesterol depletion. All further incubations (at 4°C, to allow only cell surface labeling and eliminate internalization) were in HBSS supplemented with 20 mM Hepes, pH 7.2, and 1% BSA (HBSS/Hepes/BSA). The cells were incubated successively with the following IgGs (30 µg/ml, 45 min): (1) Fc125 mouse -Japan and rabbit
-X:31, together with 200 µg/ml normal goat IgG for blocking; and (2) G
R Alexa®594-IgG and G
M FITC-IgG. The cells were fixed as previously described (Harder et al., 1998) and mounted with Prolong Antifade (Molecular Probes). Images were recorded with a CCD camera as previously described (Gilboa et al., 2000). Superposition of the green and red channels and determination of the numbers of red, green, and overlapping (yellow) patches were performed using Image-Pro Plus software (Media Cybernetics), defining pairs of green/red patches as overlapping if the peaks of intensity of two nearest-neighbor patches were separated by <0.2 µm.
FRAP
FRAP studies were conducted on cells singly or doubly transfected with HA expression vectors exactly as described for co-patching. HA proteins at the cell surface were labeled with TRITC-tagged monovalent Fab' fragments, either alone or together with cross-linking IgGs against the antigenically distinct coexpressed HA protein (for details, see figure legends). Lateral diffusion coefficients (D) and mobile fractions were measured by FRAP (Axelrod et al., 1976; Koppel et al., 1976) using previously described instrumentation (Fire et al., 1991). The experiments were conducted at 22°C in HBSS/Hepes/BSA. The monitoring argon ion laser beam (529.5 nm, 1 µW) was focused through the microscope (Carl Zeiss MicroImaging, Inc.) to a Gaussian radius of 0.85 ± 0.02 µm (63x oil immersion objective). A brief pulse (5 mW, 1015 ms) bleached 5070% of the fluorescence in the illuminated region. Fluorescence recovery was followed by the attenuated monitoring beam. D and Rf were determined by nonlinear regression analysis, fitting to the lateral diffusion equation of a single species (single D value) (Fire et al., 1995).
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Acknowledgments |
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Submitted: 26 August 2003
Accepted: 29 September 2003
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References |
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