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The Journal of Immunology, 2002, 168: 2737-2744.
Copyright © 2002 by The American Association of Immunologists

Lipid Microdomain Clustering Induces a Redistribution of Antigen Recognition and Adhesion Molecules on Human T Lymphocytes1

Jason S. Mitchell, Oguz Kanca and Bradley W. McIntyre2

Department of Immunology, University of Texas, M. D. Anderson Cancer Center, Houston, TX 77030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The study of lipid microdomains in the plasma membrane is a topic of recent interest in leukocyte biology. Many T cell activation and signaling molecules are found to be associated with lipid microdomains and have been implicated in normal T cell function. It has been proposed that lipid microdomains with their associated molecules move by lateral diffusion to areas of cellular interactions to initiate signaling pathways. Using sucrose density gradients we have found that human T cell {beta}1 integrins are not normally associated with lipid microdomains. However, cross-linking of GM1 through cholera toxin B-subunit (CTB) causes an enrichment of {beta}1 integrins in microdomain fractions, suggesting that cross-linking lipid microdomains causes a reorganization of molecular associations. Fluorescent microscopy was used to examine the localization of various lymphocyte surface molecules before and after lipid microdomain cross-linking. Lymphocytes treated with FITC-CTB reveal an endocytic vesicle that is enriched in TCR and CD59, while {beta}1 integrin, CD43, and LFA-3 were not localized in the vesicle. However, when anti-CTB Abs are used to cross-link lipid microdomains, the microdomains are not internalized but are clustered on the cell surface. In this study, CD59, CD43, and {beta}1 integrin are all seen to colocalize in a new lipid microdomain from which LFA-3 remains excluded and the TCR is now dissociated. These findings show that cross-linking lipid microdomains can cause a dynamic rearrangement of the normal order of T lymphocyte microdomains into an organization where novel associations are created and signaling pathways may be initiated.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The lymphocyte cell surface membrane consists of microscopically and biochemically distinguishable lipid microdomains that result from the assembly of sphingolipids and cholesterol into laterally mobile rafts (1, 2). Sphingolipids contain predominately large, saturated acyl chains that allow them to pack tightly together. Furthermore, phase separations between lipid microdomains and loosely ordered membrane glycerophospholipids give lipid microdomains a high degree of lateral mobility within the membrane (3). These lipid microdomains are also known as lipid rafts or glycolipid-enriched membrane domains and, due to their insolubility in nonionic mild detergents, they are also called detergent-insoluble glycolipid-enriched domains (DIGs)3 or detergent-resistant membranes (4). Lipid microdomains have a low density, and sucrose gradient ultracentrifugation of mild detergent cell lysates can isolate these microdomains into low-density fractions along with the molecules that are associated with them. Western blot analysis of these low-density fractions has revealed the presence of many GPI-anchored proteins, such as Thy-1, Ly-6, and CD59, and intracellular signaling proteins such as Ras, Lck, Grb-2, phosphatidylinositol-3 kinase, and Fyn (2, 4, 5, 6, 7). Lipid rafts/microdomains are also believed to have a functional significance, because cells with cholesterol depleted from lipid microdomains have decreased levels of basal adhesion and decreased CD3-induced TCR{zeta} phosphorylation (8, 9). Furthermore, lipid raft/microdomain patching can induce Ca2+ flux, Z-chain associated-70 kDa protein/linker for activation of T cells/extracellular signal-regulated kinase-2 phosphorylation, and NFAT stimulation (10). The partitioning of these signaling molecules to laterally mobile sphingolipids has been proposed as a means for signaling molecules to specifically "traffic" to areas of receptor engagement and initiate a variety of different signaling pathways (1, 3).

Lipid microdomains can be detected with the B-subunit of cholera toxin (CTB), the membrane-binding subunit that binds a major component of lipid rafts, GM1 gangliosides (11, 12). Costimulation of naive T lymphocytes with anti-CD3 and anti-CD28 beads results in redistribution of GM1 lipids from a diffuse distribution to a concentration at the bead contact site, as visualized by FITC-CTB. It was also determined that cross-linking GPI-anchored CD59, a raft/microdomain-associated lymphocyte surface protein, or, more importantly, cross-linking of GM1 directly with immobilized CTB provided efficient T cell costimulation (13). The implication of these studies was that costimulation is mediated by lipid microdomains. Furthermore, the integrin LFA-1 was shown to colocalize to membrane rafts/microdomains, and high-avidity adhesion could be induced by clustering these membrane microdomains (8). Therefore, lipid microdomains may also regulate lymphocyte intercellular interactions needed for efficient adhesion to APCs during immune recognition or for adhesion to endothelial cells to mediate lymphocyte migration and recirculation. The biochemical evidence available so far has substantially implicated lipid rafts/microdomains in the normal function of T cells (14, 15, 16). However, the information from this type of analysis may be limited because the use of detergents in these experiments may partially solubilize some lipid microdomains even at low temperatures, disrupting low-affinity interactions and transient associations (3, 15). Because lipid microdomain trafficking is based on molecular compartmentalization, fluorescence microscopy has become an invaluable tool to visualize lipid microdomain associations and movements. In this paper we have investigated the spatial relationships of various T cell adhesion and activation molecules in the context of Ab cross-linking of GM1 lipid microdomains. Using CTB to follow lipid microdomains and specific fluorescent mAbs to follow surface molecules, we have found that clustering lipid microdomains at physiological temperatures causes a dynamic rearrangement of normal T cell microdomain organization and the formation of a novel supramolecular complex. The formation of new microdomains is significant in that it brings molecules together that were otherwise not associated, allowing for the initiation of signaling cascades and the production of novel extracellular receptor architecture.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

The human peripheral blood acute lymphocytic leukemia (HPB-ALL) T cell line was maintained in RPMI 1640 medium supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10% FBS at 37°C in 5% CO2.

Antibodies

The mAbs T40/25 (anti-TCR), 33B6 (anti-{beta}1 integrin), and IB7 (anti-CD43) were obtained from hybridomas produced in the laboratory. The mAbs anti-CD59, anti-CD45, and antitransferrin receptor were purchased from BD PharMingen (San Diego, CA), Caltag Laboratories (Burlingame, CA), and Zymed Laboratories (San Francisco, CA), respectively. The anti-LFA-3 mAb was obtained from the clone TS2/9 (17). FITC-conjugated isotype-specific IgG1 and IgG2a Abs were purchased from Cappel/ICN Pharmaceuticals (Costa Mesa, CA) and Caltag Laboratories, respectively. Goat anti-CTB was purchased from Calbiochem (La Jolla, CA). Donkey anti-goat AlexaFluor 488, rabbit anti-mouse AlexaFluor 594, and goat anti-mouse AlexaFluor 594 were purchased from Molecular Probes (Eugene, OR).

Labeling of Abs

Purified 33B6 was radioiodinated using 1,3,4,6-tetrachloro-3{alpha},6{alpha},diphenylglycoluril (IODO-GEN; Pierce, Rockford, IL) (18). In brief, 25 µg of 33B6 in 100 µl of PBS was added to an IODO-GEN-coated 12 x 75 mm glass test tube (10 µg of IODO-GEN per tube). The reaction was initiated by the addition of 1 mCi of Na125I (New England Nuclear, Boston, MA) and allowed to proceed for 20 min at room temperature. The reaction was stopped by removing the 33B6 from the reaction vessel. Radioiodinated 33B6 was separated from free iodine by gel filtration on Sephadex G-25 (Sigma-Aldrich, St. Louis, MO) equilibrated in PBS containing 1% BSA .

AlexaFluor 350 and 594 protein labeling kits were purchased from Molecular Probes. T40/25 and 33B6 Abs were concentrated to 2 mg/ml and incubated with the AlexaFluor 350 and 594 succinimidyl ester dyes, respectively, stirring at room temperature for 1 h. Previous labeling of 33B6 showed the 1-h incubation did not sufficiently label 33B6, so the reaction was continued overnight at 4°C. The reactions were stopped with hydroxylamine and unlabeled dye was separated from the labeled Abs with a sizing column supplied by the kit. The degree of labeling was then determined by taking the absorbances of the labeled Abs at 280/346 nm for AlexaFluor 350 and 280/590 nm for AlexaFluor 594. T40/25 labeled with AlexaFluor 350 was labeled at 5.74 mol of dye/mol of Ab, and 33B6 labeled with AlexaFluor 594 was labeled at 6.25 mol of dye/mol of Ab.

Iodination of cell surface proteins and immunoprecipitation

Cell surface proteins were labeled by lactoperoxidase-catalyzed radioiodination as described in a previous publication (18). Briefly, 3 x 108 cells were washed three times in PBS and once in PBS with 1 µM potassium iodide (KI). Cells were resuspended in 500 µl of PBS with 1 µM KI, and 10 µg of lactoperoxidase (Sigma-Aldrich) and 0.2 IU of glucose oxidase (Sigma-Aldrich) were added. Next, 3 mCi of Na125I in 500 µl of PBS with 1 µM KI and 10 mM glucose was added and the sample was incubated at room temperature. After 15 min the cells were washed three times in ice-cold PBS containing 5 mM KI. 125I-labeled cell lysates were processed in sucrose gradients and the collected fractions were immunoprecipitated with protein G-agarose beads (Pierce) precomplexed with the anti-{beta}1 integrin mAb 18D3. Polypeptides were eluted by boiling in reducing Laemmli sample buffer and separated by 7.5% SDS-PAGE. Dried gels were exposed to Kodak XR film (Kodak, Rochester, NY) at -80°C.

Fluorescence staining and image analysis

In the GM1 internalization experiments, HPB-ALL T cells were stained in complete RPMI 1640 medium with FITC-conjugated CTB (Sigma-Aldrich) at 25 µg/ml for 1 h at 37°C. The cells were then washed twice with complete medium, fixed for 45 min with 4% paraformaldehyde, permeabilized gently with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 5 min, and stained with specific monoclonal (10 µg/ml) and AlexaFluor 594 goat anti-mouse Abs. Finally, the cells were mounted to glass slides with Prolong Antifade reagents (Molecular Probes).

In the lipid microdomain cross-linking experiments, HPB-ALL T cells were incubated with specific mAbs and unconjugated CTB (Calbiochem) at 25 µg/ml. After washing twice with medium, the lipid microdomains were cross-linked with a goat anti-CTB polyclonal Ab at 1/100 for 1 h at 37°C. The cells were then washed twice in medium and the lipid microdomains and surface molecules were capped by staining with donkey anti-goat AlexaFluor 488 and rabbit anti-mouse AlexaFluor 594 at 1 µg/ml (Molecular Probes) at 37°C for 1 h. Finally, the cells were washed three times in medium, fixed with 4% paraformaldehyde for 20 min, and mounted to slides with Prolong Antifade reagents. Images of the cells were taken on an Olympus IX70 fluorescent microscope (Olympus, Melville, NY). Exposure times and settings were done within limits where no bleed-through was apparent between the red, green, and blue filters. Image analysis and merging of images was done with Adobe PhotoShop 5.5 software (Adobe Systems, Mountain View, CA).

Sucrose gradient ultracentrifugation

The sucrose gradient procedure was modified from previously published protocols (4, 7, 19, 20). Briefly, 3 x 108 HPB-ALL T cells/sample were treated with 125I-labeled mAb 33B6 with or without CTB for 1 h at 37°C. After washing twice with medium, the CTB-treated samples were cross-linked with a goat anti-CTB polyclonal Ab for 1 h at 37°C. The cells were then washed twice in medium and treated with rabbit anti-mouse IgG to cross-link {beta}1 integrins and donkey anti-goat IgG to cross-link lipid microdomains. After cross-linking, the cells were washed in PBS and lysed in ice-cold gradient buffer (25 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.5) containing 1% Brij-97 (Sigma-Aldrich). The lysed cells were kept on ice for 1 h, vortexed vigorously every 15 min, and spun at 800 x g at 4°C for 5 min. One milliliter of the postnuclear supernatant was then mixed with 1 ml of 80% sucrose in gradient buffer and placed at the bottom of an ultracentrifuge tube. Six milliliters of 34% sucrose and 4 ml of 5% sucrose was then carefully overlaid on top of the sample and the gradient was then spun in a SW41 centrifuge tube at 200,000 x g at 4°C for 20 h. One-milliliter fractions were then collected from the top of the gradient and each fraction and the pellet were counted in a gamma counter where the percentages of total counts were determined. Fractions 4 and 5 represent the 5/34% sucrose interface and contain the majority of the DIGs (7).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cross-linking GM1 microdomains enhances {beta}1 integrin DIG associations

Although not found in DIGs in one study with epithelial cells, {beta}1 integrins from fibroblasts and CD36-transfected melanoma cells have been shown to be insoluble in detergents such as Triton X-100, Brij 96, and Brij 97, and move to low-density fractions in a sucrose gradient, suggesting {beta}1 integrins could be associated with lipid microdomains (20, 21, 22). To examine {beta}1 integrin associations with lipid microdomains in human T cells at physiologic temperatures, HPB-ALL T cells were treated with radioiodinated anti-{beta}1 integrin Ab 33B6 with or without treatment with CTB. The cells were then lysed in 1% Brij 97 and centrifuged in a layered sucrose gradient to isolate the DIGs, and radiolabeled 33B6 was used to follow quantitatively where {beta}1 integrin moved in the sucrose gradient (19, 20). Without CTB treatment, cross-linked {beta}1 integrins in these T cells are not significantly found in the DIG fractions (Fig. 1Go, No CTB), and this is similar to the findings with the epithelial cells. When CTB is added to the HPB-ALL T cells, but not cross-linked, {beta}1 integrin localization to the DIG fractions is not significantly changed (Fig. 1Go, CTB). In contrast, when HPB-ALL T cells are treated with CTB and cross-linked with anti-CTB Abs at 37°C, the lipid microdomain fractions now contain almost 10% of the {beta}1 integrins (Fig. 1Go, CTB X-linked). This shift of {beta}1 integrins from the pellet to the DIG fractions suggests that cross-linking of CTB promotes the association of {beta}1 integrins with lipid microdomains. Furthermore, this is the first demonstration that {beta}1 integrins can be triggered to localize into DIGs in T lymphocytes.



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  		FIGURE 1. Cross-linking CTB enhances the association of {beta}1 integrin with DIGs. Human HPB-ALL T cells were treated with anti-{beta}1 integrin with or without CTB at 37°C for 1 h. Rabbit anti-mouse IgG with or without goat anti-CTB Abs were added at 37°C for 1 h to cross-link the integrin and CTB, respectively. Donkey anti-goat Abs were added to appropriate samples and incubated for 37°C for 1 h. The cells were lysed in 1% Brij 97 and DIGs were isolated by sucrose gradient ultracentrifugation. A, The anti-{beta}1 integrin mAb was radiolabeled and the fractions collected were counted in a gamma counter and percentages of counts were determined. The graph is representative of four separate experiments. B, HPB-ALL T cell lysates from radiolabeled cells are subjected to sucrose gradient ultracentrifugation, and then the {beta}1 integrin is immunoprecipitated from the different fractions and resultant polypeptides are resolved by SDS-PAGE.

 
To confirm that the DIG counts detected actually correspond to the presence of {beta}1 integrin, the Fig. 1GoA experiments were repeated using cell surface radioiodinated HPB-ALL T cell lysates. The fractions collected were then immunoprecipitated with the anti-{beta}1 integrin mAb 18D3 bound to protein G beads, and the eluted polypeptides were resolved by SDS-PAGE. As seen in Fig. 1GoB, only the cross-linked CTB cells show a significant enrichment of integrin {beta}1 and the associated {alpha}4 subunit in the DIG fractions. In the CTB alone-treated cells, there seemed to be a slight movement of integrins to the higher gradient fractions (fractions 7–11), as compared with cells with no CTB treatment. CTB exists as a pentamer and it binds the oligosaccharide portion of GM1 sphingolipids cooperatively (23). This pentavalency action of CTB may cross-link GM1, promoting a mild {beta}1 integrin-lipid microdomain interaction and increasing the buoyancy of {beta}1 integrin in the gradient. However, treatment with CTB alone does not seem to be significant enough to cause {beta}1 integrin enrichment in the DIG fractions (fractions 4–5).

Cross-linking GM1 lipid microdomains redistributes GM1 sphingolipids

Another way to observe lipid microdomains is by fluorescent microscopy. To visualize GM1 lipid microdomains HPB-ALL T cells were treated with FITC-labeled CTB. When the staining was done at 4°C the GM1 was localized diffusely over the cells with no clear localization pattern (Fig. 2GoA). But when the staining was done at 37°C to see GM1 sphingolipid localization at physiological temperatures, the GM1 appeared to cluster and was localized centrally on the cell (Fig. 2GoA). Previous studies have found that in resting T cells most of the GM1 is intracellular but in T cell blasts a higher proportion of the GM1 is found on the cell surface (13). Furthermore, hippocampal neuronal cells have been shown to endocytose CTB and target it to the Golgi complex, and COS-7 cells have been shown to continuously circulate GM1 sphingolipids between the plasma membrane and Golgi pools (24, 25). To determine whether the altered staining seen at 37°C is CTB in endocytic vesicles, HPB-ALL T cells were treated with FITC-CTB at 37°C for 1 h and then stained with anti-CTB and AlexaFluor 594 secondary Abs at 4°C for 1 h each (Fig. 2GoB, upper panels). The shift to 4°C stops further endocytosis and membrane movements and, if the large central cluster of GM1 was located on the cell surface, both the FITC-CTB and anti-CTB would bind and the overlays would colocalize. However, Fig. 2GoB, upper panels, shows that the anti-CTB did not bind the centrally clustered GM1 but rather bound the periphery of the cell, indicating that the centrally clustered GM1 is in fact inside the cell. The clustering of GM1 does not appear to be CTB dependent as HPB-ALL T cells fixed, permeabilized, and stained with FITC-CTB also showed the centrally clustered GM1 (Fig. 2GoA).



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  		FIGURE 2. The distribution of GM1 sphingolipids on T cells. A, HPB-ALL T cells stained with FITC-CTB at 4°C and 37°C show different distribution patterns of GM1 sphingolipids. Fixed and permeabilized cells show a similar distribution pattern to 37°C FITC-CTB-treated cells. B, T cells stained with FITC-CTB at 37°C and then cross-linked with goat anti-CTB and anti-goat Alexa 594 Abs at 4°C show a central GM1 sphingolipid endocytic vesicle (upper panels), while T cells stained with FITC-CTB and goat anti-CTB/anti-goat Alexa 594 at 37°C show the GM1 sphingolipids clustered at the cell surface (lower panels). C, Kinetic analysis of FITC-CTB being internalized (upper panels) or being clustered on the cell surface with anti-CTB Abs (lower panels). Fluorescent images taken through the appropriate filters and computer-generated overlays are shown.

 
To examine the localization of GM1 during cross-linking of GM1 lipid microdomains at physiologic temperatures, HPB-ALL cells were treated with FITC-CTB for 1 h at 37°C and cross-linked with an anti-CTB Ab and AlexaFluor 594 secondary Abs at 37°C for 1 h each (Fig. 2GoB, lower panels). Instead of seeing the characteristic CTB-defined GM1 endocytic vesicle, the Ab was able to capture and retain the GM1 at the cell surface and form distinct patches or caps of GM1 sphingolipids. We have concluded from this that CTB binding of GM1 allows us to follow the GM1 being recycled and that CTB cross-linking Abs can prevent this recycling by capturing and clustering the GM1 at the cell surface. The disappearance of the GM1 endocytic vesicle is not believed to be due to degradation of the CTB, because when HPB-ALL T cells are treated with FITC-CTB at 37°C for 1 h, washed of excess CTB, and then incubated at 37°C for an additional 3 h, the GM1 endocytic vesicle was still apparent (data not shown).

To assess the dynamics of the GM1 sphingolipid endocytosis and CTB cross-linking, the kinetics of these events were next examined. Cell surface GM1 sphingolipids on HPB-ALL T cells were labeled with FITC-CTB at 4°C for 1 h. The cells were then washed extensively at 4°C and then either placed at 37°C (CTB-treated cells) or incubated with anti-CTB Abs at 4°C for 1 h and then placed at 37°C (CTB cross-linked cells). The cells were then fixed in 3.2% paraformaldehyde at specific time intervals. Fig. 2GoC shows the general cell population phenotype at each time interval. For the CTB alone-treated cells (Fig. 2GoC, upper panels), at 0 and 1 min it can be clearly seen that GM1 sphingolipids are evenly dispersed over the cells. However, by 2 min traces of CTB are seen inside the cells as GM1 sphingolipid cycling resumes. By 3 min the endocytic GM1 pools are clearly evident in the cells, and this phenotype is also readily visible at 10 min. For the cross-linked CTB cells (Fig. 2GoC, lower panels) at 0 min the GM1 sphingolipids are evenly dispersed over the cells, similar to CTB alone-treated cells. As the cells warm, the GM1 sphingolipids are not endocytosed as seen in the CTB alone-treated cells, but rather they begin to cluster on the cell surface, forming multiple small clusters at the early time points (1–2 min), then forming larger clusters (3 min), and eventually forming large caps (10 min).

Exclusion of {beta}1 integrin, CD43, and LFA-3 from the TCR, CD59, and GM1 lipid microdomains

The endocytosis of GM1 could be viewed as the endocytosis of a specific lymphocyte microdomain containing GM1 and GM1-associated molecules. To determine which lymphocyte surface molecules are being internalized with the GM1, HPB-ALL T cells were stained with FITC-CTB at 37°C (Fig. 3Go, CTB), fixed and permeabilized with paraformaldehyde and Triton X-100, and stained with mAbs to TCR, {beta}1 integrin, CD43, CD59, and LFA-3 (Fig. 3Go, mAb). The GM1 endocytic vesicles are highly enriched with TCR and CD59 as clearly demonstrated in the overlay (Fig. 3Go, A and D). However, {beta}1 integrin, CD43, and LFA-3 (Fig. 3Go, B, C, and E) were not internalized with the GM1 endocytic vesicle, as seen by the diffuse and peripheral staining. These internalization experiments were also done with some classically defined non-raft-associated molecules such as transferrin receptor and CD45 (2, 4, 10, 26). We found that CD45 was not internalized with GM1 and, similar to the findings from the Lippincott-Schwartz laboratory (25), transferrin receptor was internalized with GM1 endocytic vesicles (data not shown).



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  		FIGURE 3. Cointernalization of T cell surface molecules with GM1 sphingolipids. HPB-ALL T cells were treated with FITC-CTB at 37°C for 1 h, fixed, permeabilized, and stained with specific mAbs and AlexaFluor 594 secondary Abs for the T cell surface molecules TCR (A), {beta}1 integrin (B), CD43 (C), CD59 (D), and LFA-3 (E). Brightfield, fluorescent images taken through the appropriate filters and computer-generated overlays are shown.

 
{beta}1 integrin, CD43, and LFA-3 colocalize in a microdomain independent of CD59/TCR/GM1

The results in Fig. 3Go indicate that {beta}1 integrin, CD43, and LFA-3 do not colocalize with the GM1 and, by inference, suggest no colocalization with TCR and CD59. Because the spatial relationships within the group of CD43, LFA-3, and {beta}1 integrin were not determined, {beta}1 integrin was visualized with a directly conjugated AlexaFluor 594 mAb, while CD43 and LFA-3 were visualized with mAbs and FITC-conjugated secondary Abs. Each Ab step was done at 37°C for 1 h. As shown in Fig. 4Go, CD43 and LFA-3 are associated with {beta}1 integrin in the same region of the cell. These results indicate that {beta}1 integrin, CD43, and LFA-3 have the capacity to colocalize to a region of the cell separate from the TCR/CD59/GM1 microdomain. This previously undescribed organization of surface proteins defines a novel microdomain.



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  		FIGURE 4. CD43 and LFA-3 colocalize with {beta}1 integrin. HPB-ALL T cells were stained at 37°C with {beta}1 integrin-specific mAb 33B6 directly conjugated with AlexaFluor 594, and with mAbs to CD43 (A) and LFA-3 (B), which were visualized with an isotype-specific rabbit anti-mouse FITC conjugate. Brightfield, fluorescent images taken through the appropriate filters and computer-generated overlays are shown.

 
Cross-linking the GM1 microdomain results in the redistribution of molecules from two different microdomains into a supramolecular complex

The Ab cross-linking of GM1 through CTB was shown in Fig. 2Go to redistribute the GM1 into defined caps or patches. It is possible that these dynamic events may be accompanied by the lateral movement of membrane molecules into novel associations. HPB-ALL cells were incubated with CTB and then cross-linked with a goat anti-CTB polyclonal Ab and then with a donkey anti-goat AlexaFluor 488, where each step is done for 1 h at 37°C. This causes {beta}1 integrin and CD43 to localize into the GM1 microdomain (Fig. 5Go, B and C), while CD59 remained in the GM1 microdomain (Fig. 5GoD). LFA-3 was always seen to localize to a close proximity of the GM1 microdomain, but the overlays show that LFA-3 and the GM1 microdomain never completely colocalize (Fig. 5GoE). The TCR was typically seen to also stay in the GM1 microdomain, as seen by the arrow in the overlay (Fig. 5GoA); however, there were some occasions where the TCR and GM1 microdomains were not seen to cocluster together as seen by the distinct localization of the TCR at the right periphery of the cell (Fig. 5GoA). Therefore, some of the components ({beta}1 integrin and CD43) from the previously defined novel microdomain have merged with the TCR/CD59/GM1 microdomain upon GM1 cross-linking. Transferrin receptor and CD45 were also examined under these cross-linking conditions. Upon CTB cross-linking, transferrin receptor is still seen to colocalize with GM1 sphingolipids at the cell surface while the majority of CD45 is not colocalized with GM1 sphingolipids (data not shown). The fidelity of the noncolocalization of CD45 with lipid rafts was not absolute, as small regions of colocalization are seen, but this is not surprising, as it has been reported that CD45 can localize to lipid rafts (27).



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  		FIGURE 5. Cross-linking GM1 microdomains causes redistribution of surface molecules. GM1 gangliosides were cross-linked on HPB-ALL T cells by CTB followed by a goat anti-CTB Ab and then a donkey anti-goat AlexaFluor 488 Ab. TCR (A), {beta}1 integrin (B), CD43 (C), CD59 (D), and LFA-3 (E) were each visualized with specific mAbs and a rabbit anti-mouse AlexaFluor 594 Ab. Brightfield, fluorescent images taken through the appropriate filters and computer-generated overlays are shown.

 
Cytochalasin D does not inhibit colocalization of {beta}1 integrin and GM1 sphingolipids

Actin polymerization in T cells has been shown to be important in the normal function of both lipid rafts/microdomains and integrins (9, 19, 28, 29, 30, 31). To investigate whether the colocalization of {beta}1 integrin with GM1 sphingolipids could be inhibited by disrupting actin polymerization, GM1 sphingolipids and {beta}1 integrins were cross-linked, as in Fig. 5GoB, in the presence of 0.1 mM cytochalasin D. This concentration of cytochalasin D totally inhibits HPB-ALL cellular spreading on fibronectin (data not shown). Inhibition of actin polymerization by cytochalasin D did not appear to affect the ability of {beta}1 integrin to colocalize with GM1 sphingolipids under cross-linking conditions, as can be seen in the overlay (Fig. 6GoA). However, cytochalasin D did appear to have an effect on the pattern of the colocalization, as cells treated with cytochalasin D stained with small patches, while cells stained in control medium generally showed single large caps (Fig. 6GoA). These different patterns were quantitated by classifying the colocalized patterns into three categories: one large cap, two or three medium-sized patches, and multiple small patches. The cells were stained in three different treatments: medium, medium and 1/100 ethanol/DMSO (cytochalasin D solvent), and in medium 1/100 ethanol/DMSO and 0.1 mM cytochalasin D, where the total number of cells counted for each treatment was 884, 388, and 921, respectively. As can be seen in Fig. 6GoB, the predominant phenotype was one large cap in the medium (51.9%) and medium/DMSO/ethanol (51.2%) treatments, while in the cytochalasin D-treated cells the predominant phenotype was small patches (70.9%). These data suggest that actin polymerization is necessary for the formation of large aggregated caps on T cells but may not be as important for the association of {beta}1 integrin with GM1.



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  		FIGURE 6. The effect of cytochalasin D treatment on GM1 sphingolipid and {beta}1 integrin colocalization. A, HPB-ALL T cells were treated with or without cytochalasin D and GM1 gangliosides were cross-linked by CTB followed by a goat anti-CTB Ab and then a donkey anti-goat AlexaFluor 488 Ab. {beta}1 integrin was visualized with a specific mAb and a rabbit anti-mouse AlexaFluor 594 Ab. B, The resultant patterns with and without cytochalasin D were quantitated as one large cap, two to three medium sized patches, or multiple small patches.

 
Triple-color stain shows lymphocyte microdomain reorganization

As presented in Fig. 5Go, cross-linking GM1 sphingolipids through CTB with secondary Abs results in a reorganization of microdomains such that {beta}1 integrins can become associated with the GM1-defined microdomain into a supramolecular cluster. To further visualize this rearrangement a triple-color stain using FITC-CTB and directly conjugated Abs was used to simultaneously follow the movements of GM1, TCR, and {beta}1 integrin under GM1 cross-linked and noncross-linked conditions. In Fig. 7GoA, HPB-ALL T cells were treated with FITC-CTB, anti-{beta}1 integrin mAb Alexa 594-33B6, and anti-TCR mAb Alexa 350-T40/25 for 1 h at 37°C. The overlay of the images clearly shows the CTB endocytic vesicle is enriched with TCR and the {beta}1 integrin is localized away in a separate microdomain. Upon cross-linking of GM1 sphingolipids, the overlay in Fig. 7GoB shows {beta}1 integrin localizing to the GM1 cap, but the TCR was always found away from the GM1 supramolecular cluster. Dissociation of the TCR from the GM1 supramolecular complex was also seen in Fig. 5GoA, but not to the extent that it is seen under the conditions in Fig. 7GoB. The difference between the two conditions is that the TCR in Fig. 5GoA was visualized with secondary Abs that may be clustering the TCR, while the TCR in Fig. 7GoB was visualized with a directly conjugated fluorescent mAb. This suggests that through cocapping the TCR can be held in the GM1 cap, while an unrestrained TCR is able to dissociate from the GM1 lipid microdomain after GM1 cross-linking.



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  		FIGURE 7. Dissociation of the TCR from the GM1 lipid microdomain. A, HPB-ALL T cells were stained with FITC-CTB to show the internalization of the GM1 microdomain. TCR was visualized with a TCR-specific mAb directly conjugated with AlexaFluor 350 and {beta}1 integrin localization was shown with a {beta}1 integrin mAb directly conjugated with AlexaFluor 594. B, GM1 gangliosides were stained and cross-linked on HPB-ALL T cells by adding CTB followed by a goat CTB Ab then a donkey anti-goat AlexaFluor 488 Ab. The TCR was visualized with a TCR-specific mAb directly conjugated with AlexaFluor 350 and {beta}1 integrin localization was shown with a {beta}1 integrin mAb directly conjugated with AlexaFluor 594. Brightfield, fluorescent images taken through the appropriate filters and computer-generated overlays are shown.

 
Quantitation of many of the events concerning integrin, TCR, and GM1 are presented in Fig. 8Go. Under conditions such as in Figs. 2Go and 3Go, where GM1 internalization is followed, in 78% of the cells {beta}1 integrins are not associated with GM1 and in none of these cells were these molecules found to be completely colocalized (Fig. 8GoA). In contrast, in >80% of the cells, the TCR completely colocalized with GM1 and <2% of the cells showed no colocalization of these two molecules (Fig. 8GoB). Under conditions such as in Fig. 5Go, where both molecules are cross-linked, most of the {beta}1 integrin (Fig. 8GoC) and the TCR (Fig. 8GoD) are found to be completely colocalized with GM1. Finally, under conditions where GM1, but not TCR, is cross-linked such as in Fig. 7GoB, the TCR and GM1 do not colocalize (Fig. 8GoE).



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  		FIGURE 8. Quantitation of molecular reorganization. HPB-ALL cells were stained under conditions as described in previous figures. These conditions are as follows: 1) GM1 endocytosis is allowed to proceed and then cells are fixed, permeabilized, and stained for either the integrin (A) or TCR (B); 2) GM1 and integrin (C) or TCR (D) are treated with primary reagents, cross-linked with secondary Abs at physiologic temperatures and then fixed; or 3) at physiologic temperature, GM1 is treated with cholera toxin and cross-linked with Abs but the primary anti-TCR mAb alone is added (E). Results are presented as the percentage of cells with no colocalization, partial colocalization, or complete colocalization of the integrin or TCR with GM1. Partial colocalization indicates that the molecules in the caps are >50% colocalized even though parts of an individual cap could be segregated into GM1-, TCR-, or integrin-enriched zones. In contrast, complete colocalization means >95% of the molecules in the cap are colocalized. The number of cells analyzed for each condition are as follows: A, 107; B, 106; C, 108; D, 105; and E, 1030.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The physical relationship of lipid microdomains with other surface proteins has been explored in a variety of experimental conditions. One of these is the use of sucrose gradients to isolate detergent insoluble glycolipids and their associated molecules from low-density fractions. The tight packing from the long acyl chains of sphingolipids in the liquid-ordered (lo) and the gel-like phases leads to the detergent insolubility of the lipid rafts/microdomains because these lipid-lipid interactions are more stable than lipid-detergent interactions (3). {beta}1 integrin is a type I transmembrane molecule; therefore, it is predicted to be poorly associated with lipid microdomains in intact cells and in DIGs in cell lysates, as it is lacking a GPI anchor or closely spaced myristate and palmitate chains, which are found on classical DIG-associated molecules such as CD59, linker for activation of T cells, p56Lck, and p59Fyn (3, 32, 33, 34, 35, 36).

In other investigations of {beta}1 integrin associations with lipid microdomains there have been conflicting reports on the degree of DIG/{beta}1 integrin associations, which could be due to differences in cell types, detergents, and cell treatments (20, 21, 22). The function of {beta}1 integrin in T cells is notably different from nonhematopoietic cells. Besides serving roles in adhesion and migration, T lymphocyte {beta}1 integrins have been found to be potent costimulatory molecules (37). In our analysis to determine whether T cell {beta}1 integrins associated with lipid microdomains in DIGs, we clustered {beta}1 integrin and GM1 sphingolipids with Abs at 37°C. The cells were then lysed in 1% Brij 97, a detergent that is less hydrophobic than Triton X-100, which would help maintain lower-affinity interactions, which are nevertheless still highly specific (20). As the results showed, clustering {beta}1 integrin alone did little to stabilize {beta}1 integrin-DIG associations. However, by simultaneous cross-linking of {beta}1 integrins and GM1 lipid microdomains, the {beta}1 integrin content in DIG fractions increased 5-fold. The enrichment of {beta}1 integrin in DIG fractions through simultaneous cross-linking of {beta}1 integrins and lipid microdomains may be viewed as either a stabilization of existing molecular associations or a dynamic activation event where {beta}1 integrin is recruited into lipid microdomains. We have also found that CD43 from detergent-solubilized cells subjected to ultracentrifugation on sucrose gradients has a similar DIG distribution pattern to {beta}1 integrin. Under conditions where only CD43 is cross-linked with Abs, 2% of CD43 is DIG associated, but upon cross-linking both CD43 and GM1, ~13% of CD43 becomes DIG associated (data not shown). Because cross-linking T cell GM1 lipid microdomains at physiologic temperatures has been previously described to stimulate various T cell activation pathways, it will be interesting to follow the time course of {beta}1 integrin, CD43, and GM1 colocalization in relationship to the formation of an immunological synapse (8, 10).

In the study of microdomain organization many different models have been used to determine where molecules spatially position themselves on T cells. Some of these focus on T cell contact sites with other cells, microbeads, or planar membranes, while other studies have used models that rely on the addition of soluble proteins such as specific Abs to induce the capping of surface proteins (13, 38, 39, 40, 41, 42). We have chosen this latter approach to explore the dynamics of microdomains because we wanted to eliminate the constraints imposed by an opposing surface, thus allowing the examination of the inherent propensity of surface molecules to redistribute and colocalize into domains when triggered with soluble reagents.

The results of our localization experiments demonstrate that initially at least two distinct molecular regions or microdomains can be distinguished on the T cell surface. One is defined by the presence of the TCR, CD59, and GM1 sphingolipids and the other is a previously undefined microdomain consisting of {beta}1 integrin, CD43, and LFA-3. When GM1 sphingolipids are cross-linked with secondary Abs, a different novel microdomain is detected that contains GM1 sphingolipids, {beta}1 integrin, CD43, and CD59. Thus, it appears that {beta}1 integrin and CD43 can assemble free from the LFA-3 region and become stabilized with the GM1/CD59 microdomain, which in turn is dissociated from the TCR, forming a separate microdomain. These observed rearrangements imply a highly dynamic set of microdomains whose molecular composition is greatly influenced by the ligation of surface molecules. An understanding of the dynamics of these microdomain rearrangements will make clear which molecules are anchoring and which may be laterally mobile during critical events in lymphocyte function.

The functional significance of the distribution of surface proteins in microdomains is only now becoming realized. Many different signaling molecules have been shown to be associated with lipid microdomains such as Ras, Lck, Grb-2, phosphatidylinositol-3 kinase, and Fyn (2, 4, 5, 6, 7). Because different surface proteins are associated with characteristic intracellular signaling molecules, it is intriguing to speculate that novel combinations of signaling molecules could activate or inhibit a wide array of biological processes. When the novel supramolecular complex of TCR/GM1/{beta}1 integrin/CD43/CD59 is formed, proteins such as focal adhesion kinase, vinculin, paxillin, Ras, and c-Src may be brought in by {beta}1 integrin, which could create a new molecular configuration of cytoskeletal proteins, molecular scaffolds, and signaling proteins and allow novel signaling to occur (43, 44). Because p56Lck has been found to be enriched in lipid microdomains, and Lck has been shown to be a key regulator in the affinity of {alpha}4{beta}1 integrins for VCAM-1 (45), an intriguing possibility is that the movement of {beta}1 integrins into lipid microdomains may be a way of directing integrin affinity state or controlling cellular avidity (2, 6, 45). Thus, a mechanism of selective redistribution of surface receptors into different functional domains could provide a system for regulating lymphocyte activation, homing, and recirculation. Furthermore, when the TCR leaves this GM1/{beta}1 integrin/CD43/CD59 complex, the TCR contribution to this signaling complex would be terminated. Immune responses could be ended or enter a new phase as the TCR moves from its original location to other areas of the cell.

The identification of different organized regions on the T lymphocyte surface further illustrates the complexity of cellular behavior. The spatial and temporal dynamics of these microdomains indicate their formation probably depends on a variety of factors including proximity, lateral diffusion, molecular recognition and exclusion, cytoskeleton association, and activation modality. Future studies should help unravel the possible combinations of surface molecules that are driven by different activation processes or associated with various differentiation states. When compiled, a descriptive catalog of possible microdomains will be a useful tool in studies to identify the physical, molecular, and biological forces that establish microdomain composition and the rules that control molecular segregation between domains. These studies will be essential in our understanding of T lymphocyte biology.


   Acknowledgments
 
We thank Dr. Bernard Andruss for critically reviewing the manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant CA62596 and Predoctoral Cancer Immunobiology Training Program Grant CA09598. Back

2 Address correspondence and reprint requests to Dr. Bradley W. McIntyre, Department of Immunology University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 180, Houston, TX 77030. E-mail address: bmcintyr{at}mail.mdanderson.org Back

3 Abbreviations used in this paper: DIG, detergent-insoluble glycolipid-enriched domain; CTB, cholera toxin B-subunit; HPB-ALL, human peripheral blood acute lymphocytic leukemia; IODO-GEN, 1,3,4,6-tetrachloro-3{alpha},6{alpha},diphenylglycoluril. Back

Received for publication July 10, 2001. Accepted for publication January 14, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Simons, K., E. Ikonen. 1997. Functional rafts in cell membranes. Nature 38:569.
  2. Rogers, W., J. K. Rose. 1996. Exclusion of CD45 inhibits activity of p56lck associated with glycolipid-enriched membrane domain. J. Cell Biol. 135:1515.[Abstract/Free Full Text]
  3. Brown, D. A., E. London. 2000. Structure and function of sphingolipid-and cholesterol-rich membrane rafts. J. Biol. Chem. 275:17221.[Free Full Text]
  4. Montixi, C., C. Langlet, A. M. Bernard, J. Thimonier, C. Dubois, M. A. Wurbel, J. P. Chauvin, M. Pierres, H. T. He. 1998. Engagement of T cell receptor triggers its recruitment to low-density detergent-insoluble membrane domains. EMBO J. 17:5334.[Medline]
  5. Stulnig, T. M., M. Berger, T. Sigmund, D. Raederstorff, H. Stockinger, W. Waldhausl. 1998. Polyunsaturated fatty acids inhibit T cell signal transduction by modification of detergent-insoluble membrane domains. J. Cell Biol. 143:63.
  6. Bohuslav, J., T. Cinek, V. Horesji. 1993. Large, detergent-resistant complexes containing murine antigens Thy-1 and Ly-6 and protein tyrosine kinase p56lck. Eur. J. Immunol. 23:825.[Medline]
  7. Xavier, R., T. Brennan, Q. Li, C. McCormack, B. Seed. 1998. Membrane compartmentation is required for efficient T cell activation. Immunity 8:723.[Medline]
  8. Krauss, K., P. Altevogt. 1999. Integrin leukocyte function-associated antigen-1 mediated cell binding can be activated by clustering of membrane rafts. J. Biol. Chem. 274:36921.[Abstract/Free Full Text]
  9. Moran, M., M. C. Miceli. 1998. Engagement of GPI-liked CD48 contributes to TCR signals and cytoskeletal reorganization: a role for lipid rafts in T cell activation. Immunity 9:787.[Medline]
  10. Janes, P. W., S. C. Ley, A. I. Magee. 1999. Aggregation of lipid rafts accompanies signaling via the T cell antigen receptor. J. Cell Biol. 147:447.[Abstract/Free Full Text]
  11. Heyningen, S.. 1974. Cholera toxin: interaction of subunits with ganglioside GM1. Science 183:656.[Abstract/Free Full Text]
  12. Mulhern, S. A., P. H. Fishman, S. Spiegel. 1989. Interaction of the {beta} subunit of cholera toxin with endogenous ganglioside GM1 causes changes in membrane potential of rat thymocytes. J. Membrane Biol. 109:21.[Medline]
  13. Viola, A., S. Schroeder, Y. Sakakibara, A. Lanzavecchia. 1999. T lymphocyte costimulation mediated by reorganization of membrane microdomains. Science 283:680.[Abstract/Free Full Text]
  14. Cherukuri, A., M. Dykstra, S. K. Pierce. 2001. Floating the raft hypothesis: lipid rafts play a role in immune cell activation. Immunity 14:657.[Medline]
  15. Langlet, C., A. M. Bernard, P. Drevot, H. T. He. 2000. Membrane rafts and signaling by the multichain immune recognition receptors. Curr. Opin. Immunol. 12:250.[Medline]
  16. Galbiati, F. B. Razani, M. P. Lisanti. 2001. Emerging themes in lipid rafts and caveolae. Cell 106:403.[Medline]
  17. Sanchez-Madrid, F., A. M. Krensky, C. F. Ware, E. Robbins, J. L. Strominger, S. J. Burakoff, T. A. Springer. 1982. Three distinct antigens associated with human T-lymphocyte-mediated cytolysis: LFA-1, LFA-2, and LFA-3. Proc. Natl. Acad. Sci. USA 79:7489.[Abstract/Free Full Text]
  18. McIntyre, B. W., E. L. Evans, J. L. Bednarczyk. 1989. Lymphocyte surface antigen L25 is a member of the integrin receptor superfamily. J. Immunol. 264:13745.
  19. Rebes, R. A., J. M. Green, M. I. Reinhold, M. Ticchioni, E. J. Brown. 2001. Membrane raft association of CD47 is necessary for actin polymerization and protein kinase C {theta} translocation in its synergistic activation of T cells. J. Biol. Chem. 276:7672.[Abstract/Free Full Text]
  20. Claas, C., C. S. Stipp, M. E. Hemler. 2001. Evaluation of prototype transmembrane 4 superfamily protein complexes and their relation to lipid rafts. J. Biol. Chem. 276:7974.[Abstract/Free Full Text]
  21. Thorne, R. F., J. F. Marshall, D. R. Shafren, P. G. Gibson, I. R. Hart, G. F. Burns. 2000. The integrins {alpha}3{beta}1 and {alpha}6{beta}1 physically and functionally associate with CD36 human melanoma cells. J. Biol. Chem. 275:35264.[Abstract/Free Full Text]
  22. Nursat, A., C. A. Parkos, P. Verkade, C. S. Foley, T. W. Liang, W. Innis-Whitehouse, K. K. Eastburn, J. L. Madara. 2000. Tight junctions are membrane microdomains. J. Cell Sci. 113:1771.[Abstract]
  23. Schon, A., E. Freire. 1989. Thermodynamics of intersubunit interactions in cholera toxin upon binding to the oligosaccharide portion of its cell surface receptor, ganglioside GM1. Biochemistry 28:5019.[Medline]
  24. Sofer, A., A. H. Futerman. 1995. Cationic amphiphilic drugs inhibit the internalization of cholera toxin to the Golgi apparatus and the subsequent elevation of cyclic AMP. J. Biol. Chem. 270:12117.[Abstract/Free Full Text]
  25. Nichols, B. J., A. K. Kenworthy, R. S. Polishchuk, R. Lodge, T. H. Roberts, K. Hirschberg, R. D. Phair, J. Lippincott-Schwartz. 2001. Rapid cycling of lipid raft markers between the cell surface and Golgi complex. J. Cell Biol. 153:529.[Abstract/Free Full Text]
  26. Harder, T., P. Scheiffele, P. Verkade, K. Simons. 1998. Lipid domain structure of the plasma membrane revealed by patching of membrane components. J. Cell Biol. 141:929.[Abstract/Free Full Text]
  27. Ishii, T., K. Ohnuma, A. Murakami, N. Takasawa, S. Kobayashi, N. H. Dang, S. F. Schlossman, C. Morimoto. 2001. CD26-mediated signaling for T cell activation occurs in lipid rafts through its association with CD45RO. Proc. Natl. Acad. Sci. USA 98:12138.[Abstract/Free Full Text]
  28. Gomez-Mouton, C., J. L. Abad, E. Mira, R. A. Lacalle, E. Gallardo, S. Jimenez-Baranda, I. Illa, A. Bernad, S. Manes, C. Martinez-A. 2001. Segregation of leading-edge and uropod components into specific lipid rafts during T cell polarization. Proc. Natl. Acad. Sci. USA 98:9642.[Abstract/Free Full Text]
  29. Marcel, D., T. Michel, B. Alain. 1996. Endocytosis of GPI-anchored proteins in human lymphocytes: role of glycolipid-based domains, actin cytoskeleton, and protein kinases. J. Cell Biol. 133:791.[Abstract/Free Full Text]
  30. Chan, J. R., S. J. Hyduk, M. I. Cybulsky. 2000. {alpha}4{beta}1 integrin/VCAM-1 interaction activates {alpha}L{beta}2 integrin-mediated adhesion to intercellular adhesion molecule-1 in human T cells. J. Immunol. 164:746.[Abstract/Free Full Text]
  31. Bunnell, S. C., V. Kapoor, R. P. Trible, W. Zhang, L. E. Samelson. 2001. Dynamic actin polymerization drives T cell receptor-induced spreading: a role for the signal transduction adaptor LAT. Immunity 14:315.[Medline]
  32. Moffett, S., D. A. Brown, M. E. Linder. 2000. Lipid-dependent targeting of G proteins into rafts. J. Biol. Chem. 275:2191.[Abstract/Free Full Text]
  33. Melkonian, K. A., A. G. Ostermeyer, J. Z. Chen, M. G. Roth, D. A. Brown. 1999. Role of lipid modifications in targeting proteins to detergent-resistant membrane rafts. J. Biol. Chem. 274:3910.[Abstract/Free Full Text]
  34. Millan, J., M. Qaidi, M. A. Alonso. 2001. Segregation of co-stimulatory components into specific T cell surface lipid rafts. Eur. J. Immunol. 31:467.[Medline]
  35. Shenoy-Scaria, A. M., L. K. Gauen, J. Kwong, A. S. Shaw, D. M. Lublin. 1993. Palmitylation of an amino-terminal cysteine motif of protein tyrosine kinases p56lck and p59fyn mediates interaction with glycosyl-phosphatidylinositol-anchored proteins. Mol. Cell. Biol. 13:6385.[Abstract/Free Full Text]
  36. Brown, D. A., J. K. Rose. 1992. Sorting of GPI-anchored proteins to glycolipid-enriched membranes subdomains during transport to the apical cell surface. Cell 68:533.[Medline]
  37. Udagwa, T., D. G. Woodside, B. W. McIntyre. 1996. {alpha}4{beta}1 (CD49d/CD29) integrin costimulation of human T cells enhances transcription factor and cytokine induction in the absence of altered sensitivity to anti-CD3 stimulation. J. Immunol. 157:1965.[Abstract]
  38. Anderson, H. A., E. M. Hiltbold, P. A. Roche. 2000. Concentration of MHC class II molecules in lipid rafts facilitates antigen presentation. Nat. Immunol. 1:156.[Medline]
  39. Monks, C. R. F., B. A. Freiberg, H. Kupfer, N. Sciaky, A. Kupfer. 1998. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395:82.[Medline]
  40. Yashiro-Ohtani, Y., X. Y. Zhou, K. Toyo-Oka, X. G. Tai, C. S. Parks, T. Hamaoka, R. Miyake, H. Fujiwara. 2000. Non-CD28 costimulatory molecules present in T cell rafts induce T cell costimulation by enhancing the association of TCR with rafts. J. Immunol. 164:1251.[Abstract/Free Full Text]
  41. Ebert, P. J. R., J. F. Baker, J. A. Punt. 2000. Immature CD4+CD8+ thymocytes do not polarize lipid rafts in response to TCR-mediated signals. J. Immunol. 165:5435.[Abstract/Free Full Text]
  42. Grakoui, A., S. K. Bromley, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Allen, M. L. Dustin. 1999. The immunological synapse: a molecular machine controlling T cell activation. Science 285:221.[Abstract/Free Full Text]
  43. Burridge, K., M. Chrzanowska-Wodnicka, C. Zhong. 1997. Focal adhesion assembly. Trends Cell Biol. 7:342.[Medline]
  44. Schlaepfer, D. D., T. Hunter. 1998. Integrin signaling and tyrosine phosphorylation: just the FAKs?. Trends Cell Biol. 8:151.[Medline]
  45. Feigelson, S. W., V. Grabovsky, E. Winter, L. L. Chen, R. B. Pepinsky, T. Yednock, D. Yablonski, R. Lobb, R. Alon. 2001. The Src kinase p56Lck upregulates VLA-4 integrin affinity: implications for rapid spontaneous and chemokine-triggered T cell adhesion to VCAM-1 and fibronectin. J. Biol. Chem. 276:13891.[Abstract/Free Full Text]



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