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The Journal of Immunology, 2003, 171: 78-87.
Copyright © 2003 by The American Association of Immunologists

T Cell Glycolipid-Enriched Membrane Domains Are Constitutively Assembled as Membrane Patches That Translocate to Immune Synapses1

Stephen Jordan and William Rodgers2

Molecular Immunogenetics Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In T cells, glycolipid-enriched membrane (GEM) domains, or lipid rafts, are assembled into immune synapses in response to Ag presentation. However, the properties of T cell GEM domains in the absence of stimulatory signals, such as their size and distribution in the plasma membrane, are less clear. To address this question, we used confocal microscopy to measure GEM domains in unstimulated T cells expressing a GEM-targeted green fluorescent protein molecule. Our experiments showed that the GEM domains were assembled into membrane patches that were micrometers in size, as evidenced by a specific enrichment of GEM-associated molecules and resistance of the patches to extraction by Triton X-100. However, treatment of cells with latrunculin B disrupted the patching of the GEM domains and their resistance to Triton X-100. Similarly, the patches were coenriched with F-actin, and actin occurred in the detergent-resistant GEM fraction of T cells. Live-cell imaging showed that the patches were mobile and underwent translocation in the plasma membrane to immune synapses in stimulated T cells. Targeting of GEM domains to immune synapses was found to be actin-dependent, and required phosphatidylinositol 3-kinase activity and myosin motor proteins. We conclude from our results that T cell GEM domains are constitutively assembled by the actin cytoskeleton into micrometer-sized membrane patches, and that GEM domains and the GEM-enriched patches can function as a vehicle for targeting molecules to immune synapses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cell membranes are heterogeneous structures composed of discrete domains with a protein and lipid composition that is distinct from the bulk composition of the bilayer. One important type of membrane domain is the glycolipid-enriched membrane (GEM)3 domain, or glycolipid raft. Structurally, GEM domains are modeled to consist of a liquid-ordered phase of sphingolipid-cholesterol molecules with which specific proteins and other lipid species associate (1, 2, 3, 4). Furthermore, many of the proteins that are associated with GEM domains function in cell signaling, thus suggesting that the domains serve as a specialized signaling compartment in the plasma membrane (5). For T cells, this model has been corroborated by studies that have shown that T cell signaling proximal to the TCR requires intact GEM domains (6, 7, 8). GEM domains in T cells also function in regulating cell signaling, because disruption of these structures leads to an activation phenotype (9).

One important example of a GEM-associated signaling protein in T cells is the Src kinase p56lck (Lck) (10, 11). Lck is targeted to GEM domains by lipid modification of its N terminus, and this consists of myristoylation of Gly1 and palmitoylation of Cys2 and Cys3 (10, 12, 13). Association of Lck with GEM domains is necessary for efficient T cell activation (14), and this is consistent with the notion that GEM domains function as a signaling compartment in the plasma membrane. However, in unstimulated T cells, GEM-associated Lck is selectively down-regulated in activity (9, 15), and this occurs at least in part by exclusion of the membrane phosphatase CD45 from GEM domains (15). Importantly, the GEM-targeting signal of Lck will also target soluble proteins to the plasma membrane and GEM domains (16, 17, 18). Fusion genes encoding the N-terminal region of Lck kinases followed by green fluorescent protein (GFP) have been useful in studying GEM domains with fluorescence imaging techniques (17, 19).

Studies of GEM domains often begin with lysis of cells with nonionic detergents followed by sucrose gradient equilibrium centrifugation to separate the detergent-resistant GEM fraction from the detergent-soluble cell fraction (20). Accordingly, the properties of GEM domains in intact cell membranes are poorly defined, including their size and distribution in the plasma membrane. Thus far, biophysical studies have shown that GEM domains occur as microdomains that are ~100 nm in size (21, 22, 23, 24). However, other studies, such as those that used fluorescence microscopy to measure labeled GEM-associated molecules, have shown that GEM domains can occur as macrodomains that are micrometers in diameter (19, 25). Important examples of the latter in T cells include functionally distinct regions of the outer membrane that form as a result of extracellular signals, such as the immune synapse that forms in T cells stimulated through the TCR (19, 26, 27, 28, 29), and the uropod and leading edge of motile T cells (30). However, whether GEM macrodomains occur in T cells in the absence of the stimulatory signals that lead to either immune synapses or cell motility is not known. Given the enrichment of signaling proteins in GEM domains, their size and membrane distribution in the resting or unstimulated state represent an important question. For example, micrometer-sized patches of GEM domains, such as those evidenced in adherent cells (19, 25), would represent a significant reservoir of proteins that could be targeted to specific sites for cell signaling.

To better define the properties of GEM domains in T cells and the mechanism underlying their organization in the plasma membrane, confocal microscopy was used to measure GEM domains in T cells. The GEM domains were labeled by expressing a GEM-targeted GFP fusion protein that contained the unique N-terminal domain of Lck (LckNT) (19). Our experiments showed that GEM domains occurred in the T cell plasma membrane as micrometer-sized membrane patches, and these formed in an actin-dependent manner but independently of signals from Src kinases. Live-cell imaging showed that the GEM-enriched patches were dynamic, undergoing diffusion in the plasma membrane, and directed diffusion to the site of T cell activation. Our results have important implications regarding the localization of signaling proteins in the plasma membrane of T cells before cell stimulation, as well as the method of their delivery to the immune synapse for signal transduction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and reagents

Jurkat T cells (clone E6-1) were purchased from American Type Culture Collection (Manassas, VA). Cytochalasin D, 4-amino-5-(4-chorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), biotinylated cholera toxin B subunit (CTX), and rabbit polyclonal specific to actin were purchased from Sigma-Aldrich (St. Louis, MO). Wortmannin, latrunculin B, and 2,3-butanedioine 2-monoxime (BDM) were purchased from Calbiochem (La Jolla, CA). mAb to CD45 (clone HI30) was purchased from Upstate Cell Signaling Solutions (Charlottesville, VA). mAb to Lck (clone 28) was purchased from BD Transduction Laboratories (Lexington, KY). Streptavidin-conjugated Texas Red (TR) and TR-conjugated anti-mouse secondary Ab (from donkey) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). TR-labeled phalloidin (Phal) was purchased from Molecular Probes (Eugene, OR).

Gene construction

Generation of the GEM-associated GFP molecule LckNT-GFP has been described previously (19). In brief, LckNT-GFP encodes the N-terminal 64 aa of Lck, followed by enhanced GFP (Clontech, Palo Alto, CA) and a hemagglutinin epitope tag at the C terminus. The construct was subcloned into the mammalian expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA) under the control of the CMV promoter. Lck{Delta}10–32NT, which lacks residues 10 through 32, was subcloned from full-length Lck{Delta}10–32. The full-length construct has been described previously (31).

Cell culture and protein expression

Cells were grown in complete medium containing RPMI 1640 supplemented with antibiotics and 10% FBS and maintained at 37°C in the presence of 5% CO2. Medium for D10 T cells (32) was supplemented with glutamine and 2 U/ml IL-2. All cells were transfected by electroporation as described (17). Stable clones expressing LckNT-GFP were selected by limiting dilution using medium containing G418 at a concentration of 1.0 mg/ml. Following selection, clones were maintained in G418 at a concentration of 0.5 mg/ml.

Cell lysis, equilibrium centrifugation, immunoprecipitation, and immunoblotting

Cells (107) were lysed with 1.0 ml of a 1% Triton X-100 (TX-100) solution in 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 5 mM EDTA (TNE). For lysis, the samples were incubated with the detergent solution for 20 min at 4°C, and then dounced (eight strokes). Intact nuclei and large cell debris was removed by centrifuging (Eppendorf model 5417C; Brinkmann Instruments, Westbury, NY) at 4000 rpm at 4°C for 5 min. The supernatant was collected and diluted with an equal volume of an 85% sucrose solution in TNE. After gently mixing, the sample was overlaid in a SW50.1 rotor tune with 3.0 ml of a 30% and 1.5 ml of a 5% solution, each in TNE. The samples were centrifuged for 16–18 h at 200,000 x g. Following centrifugation, the gradients were harvested by fractionating from the top. A portion of each gradient fraction (~10% of each fraction) was separated by SDS-PAGE. LckNT-GFP was detected by immunoblotting using a mAb specific for the hemagglutinin tag (Roche, Indianapolis, IN). Endogenous Lck and actin were detected by immunoblotting with respective mAbs. The mAb to Lck did not recognize LckNT-GFP. The immunoblots were developed using ECL (Amersham, Rockford, IL) and detected using X-OMAT film (Eastman Kodak, Rochester, NY).

Fluorescence labeling of cells

Jurkat cells were seeded onto poly-L-lysine (Sigma-Aldrich)-coated coverslips by incubating 106 cells on a 22-mm coverslip for 5 min at room temperature. Next, the samples were washed with 150 mM NaCl and 20 mM sodium phosphate (pH 7.4) (PBS), and then fixed using 2% paraformaldehyde in PBS for 30 min at room temperature. Following fixation, the samples were washed with PBS containing 10 mM glycine (PBS-glycine). For experiments including staining of wild-type Lck and F-actin, the cells were permeabilized following fixation using 0.2% TX-100 in PBS-glycine for 10 min at room temperature. All labeling was performed by incubating the samples for 30 min at room temperature, followed by repeated washes with PBS-glycine. F-actin was stained using a 0.1 µg/ml solution of TR-conjugated Phal (Phal-TR). The glycolipid GM1 was labeled using 0.1 mg/ml biotinylated CTX followed by streptavidin-conjugated TR. Lck and CD45 were stained using a mAb and detected using fluorophore-conjugated secondary Ab that recognizes mouse Ig (anti-CD45-TR).

Stimulation of T cells

OKT3-coated latex beads were prepared as previously described (33). For stimulation, 1.2 x 106 cells were washed twice in prewarmed RPMI 1640, after which the cells were suspended in 300 µl of RPMI 1640, and 6 x 105 OKT3-coated beads were added. The mixture was immediately sedimented by centrifuging at 4000 rpm for 20 s in an Eppendorf centrifuge. The samples were then transferred to 37°C, incubated for 15 min, resuspended, and layered onto polylysine-coated coverslips. After 5 min, the cells were washed with PBS and fixed with 2% paraformaldehyde.

For samples receiving treatment before stimulation, incubations were done at 37°C with a cell density of 107/ml before addition of the OKT3-coated beads. The pretreatments consisted of 10 µM PP2 for 15 min, 10 µM cytochalasin D for 1 h, 100 nM wortmannin for 3 h, or 20 mM BDM for 5 min.

D10 T cells were stimulated using conalbumin-pulsed CH27 B cells. The CH27 cells were pulsed by incubating for 16 h at 37°C in DMEM containing 500 µg/ml conalbumin and supplemented with 15% FCS and 50 mM HEPES (pH 7.4). CH27 cells were seeded onto polylysine-coated coverslips and D10 cells expressing L10-GFP were gently pipetted onto the bound cells at the beginning of the experiment.

Fluorescence microscopy and image acquisition and analysis

Confocal imaging was performed using a Leica (Deerfield, IL) TCS laser scanning confocal microscope (William K. Warren Medical Research Institute, Oklahoma City, OK). GFP was excited at 488 nm, and TR was excited at 568 nm. Emission wavelengths between 530 and 560 nm were collected for GFP imaging, and emission wavelengths between 600 and 660 nm were collected for TR imaging. In double-labeling experiments, GFP and TR fluorescence was collected simultaneously in separate channels. Size calibrations were performed using calibrated fluorescent beads (Polysciences, Warrington, PA). Live-cell imaging was performed using a Zeiss (Oberkochen, Germany) LSM510 laser scanning confocal microscope (Cell Imaging Core Facility, Oklahoma Medical Research Foundation). During live-cell imaging, the sample was maintained at 37°C in complete medium plus 50 mM HEPES (pH 7.4).

Image processing was performed using IP Lab Spectrum software (Signal Analytics, Vienna, VA). Three-dimensional reconstruction of image stacks was performed using VolView (Kitware, Clifton Park, NY). Measurement of protein enrichment in caps was performed by identifying the location of the OKT3-coated beads relative to each cell using a differential interference contrast image of each field. Following subtraction of the background in the fluorescence images, the areas of the plasma membrane in contact with a bead and outside the region of contact were outlined separately, and their average fluorescence intensity was measured. The relative enrichment of LckNT-GFP at the bead contact site was then calculated using the ratio of average fluorescence intensity at the bead contact site divided by the average fluorescence intensity of the remaining plasma membrane.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GEM domains occur as membrane patches in Jurkat T cells

To label GEM domains for detection by confocal microscopy, we expressed in Jurkat T cells a GFP fusion protein that contained the unique LckNT (residues 1–64). To first measure association of LckNT-GFP with T cell GEM domains, cells expressing LckNT-GFP were lysed with TX-100, and the low-density GEM and TX-100-soluble (TXS) fractions were separated by sucrose gradient equilibrium centrifugation. This experiment showed that LckNT-GFP was targeted to the GEM fraction with an efficiency similar to that of endogenous wild-type Lck (Fig. 1A).



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  		FIGURE 1. Visualization and measurement of LckNT-GFP-enriched membrane patches in Jurkat T cells. A, Measurement of targeting of LckNT-GFP to the detergent-resistant GEM fraction of Jurkat cells. Jurkat cells expressing LckNT-GFP were lysed with 1% TX-100, and the GEM and TXS fractions were separated by sucrose gradient equilibrium centrifugation. Following centrifugation, the gradient was fractionated from the top. The gradient fractions were then separated by SDS-PAGE and immunoblotted with Ab to Lck (top) or to a hemagglutinin epitope in LckNT-GFP. The GEM fraction localized in gradient fractions 1–3, and the TXS fraction remained at the bottom of the gradient in fractions 7–10. Fifty-two percent of the total Lck and 48% of the LckNT-GFP occurred in the GEM fraction. B, Top, Confocal images of two separate live Jurkat cells expressing LckNT-GFP. The white arrowheads indicate LckNT-GFP-enriched patches in the plasma membrane. The outlined arrow indicates LckNT-GFP-labeled endosomes (34 ). Bottom, The fluorescence intensity of the plasma membrane was measured around the perimeter of each cell and plotted in the corresponding graphs. The y-axis of the plots represents relative fluorescence intensity of the pixels and has arbitrary units that range in value between 0 and 255. The x-axis represents the distance around the periphery of the cell. The arrows in the graphs mark the patches that are indicated with a white arrowhead in the corresponding image. C, Same as B, except that the cells were fixed with paraformaldehyde before seeding onto the coverslip. Each white bar represents 5 µm. D, Optical sectioning of an LckNT-GFP-enriched patch. Jurkat cells expressing LckNT-GFP were fixed before seeding onto a coverslip and optically sectioned by confocal microscopy. Top panel, Five optical sections of a region of a cell containing an LckNT-GFP-enriched patch in the outer membrane. The optical sections were collected 1.0 µm apart and are in the direction of top of the cell (left) to bottom. Bottom panel, A maximum intensity projection image of the same cell shown in the top panel. The white arrow indicates the patch designated by the white arrows in the optical sections. The cell was 10 µm in height. The projection image was generated from a stack with a step size of 0.5 µm and following cropping of the stack to remove intracellular labeling.

 
LckNT-GFP allowed us to study GEM domains in live cells and thereby avoid artifacts associated with fixation of the cells and fluorescence staining of proteins and lipids. Confocal images of live T cells that expressed LckNT-GFP showed that the fluorescence was enriched in patches in the outer membrane, and examples of the patches are indicated by the white arrows in Fig. 1B. Membrane patches enriched with LckNT-GFP were also observed in Jurkat cells that were fixed before seeding onto the coverslips (Fig. 1C). Thus, the patches were not due to nonspecific aggregation of proteins by polylysine. Note the intracellular membranes that were labeled with LckNT-GFP (Fig. 1, B and C, outlined arrow), and these represent endosomes (34).

To quantitate the distribution of LckNT-GFP in the plasma membrane, the fluorescence intensity of the plasma membrane was measured around the perimeter of each cell shown in Fig. 1, B and C. The measurements were performed by starting at the six o’clock position of the cell and then tracing in a clockwise direction. The resulting intensity measurements are plotted in the graphs below the corresponding confocal images. The arrows in the intensity plots indicate the same LckNT-GFP-enriched patch that is marked by the white arrowhead in the corresponding image. In each example, the fluorescence intensity of the outer membrane ranged from an average of ~50 to a maximum value of 175–200. Thus, the patches contained between a 3- and 4-fold enrichment of LckNT-GFP. Furthermore, based on the half-width of the peaks, the patches ranged between 3 and 5 µm in diameter.

To further measure the distribution of the LckNT-GFP-enriched patches in the plasma membrane, Jurkat cells were optically sectioned by confocal microscopy. One such optical sectioning experiment is shown in the top panel of Fig. 1D. The frames in the panel were collected 1.0 µm apart in the z direction. Thus, the indicated LckNT-GFP-enriched patch (white arrows) extended several micrometers in the direction of the z-axis of the cell. Similarly, the bottom panel in Fig. 1D shows a maximum intensity projection image of the same cell, and this demonstrates that the patch occurred between the top and the middle of the cell and not where the cell contacted the coverslip. Thus, the patch was not due to nonspecific aggregation of proteins formed by the polylysine. The data in Fig. 1D also show that the patch is not an optical artifact arising from flattening of the cell during adhesion.

The results in Fig. 1 show LckNT-GFP is enriched in micrometer-sized membrane patches in unstimulated T cells, and that the patches are heterogeneous in size and enrichment. Furthermore, a separate construct that lacked residues 10–32 of LckNT-GFP (Lck{Delta}10–32NT-GFP), and therefore could not associate with CD4 (31), also occurred in membrane patches (data not shown). Therefore, enrichment of LckNT-GFP in membrane patches occurred independently of its association with CD4. Furthermore, staining of the samples with anti-tubulin showed that the position of the patches in the plasma membrane was independent of that of the microtubule-organizing center (data not shown). Thus, the LckNT-GFP-enriched patches were not due to polarization of T cells in the absence of stimulatory signals. Importantly, staining of Jurkat cells with a mAb that is specific for Lck and a secondary FITC-conjugated anti-mouse Ig (anti-Lck-FL) showed that endogenous Lck was also enriched in membrane patches that were similar to those that were detected with LckNT-GFP (Fig. 2A). Furthermore, confocal images of D10 T cells that expressed a GFP fusion protein that was targeted to GEM domains using only the first 10 aa of Lck (L10-GFP) (17) also showed patching of GFP fluorescence in the plasma membrane (Fig. 2B). Thus, the membrane patches were not an artifact due to overexpression of LckNT-GFP in Jurkat cells, and enrichment of GEM-associated molecules in membrane patches was not restricted to Jurkat T cells. We also observed enrichment of LckNT-GFP in membrane patches in both Lck-deficient JCaM1.6 cells (35) (Fig. 2C) and in Jurkat cells that were treated with 10 µM PP2 (D). Therefore, patching of LckNT-GFP does not require signals from Src kinases.



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  		FIGURE 2. Patching of LckNT-GFP is reflective of endogenous Lck in Jurkat cells and L10-GFP in D10 T cells, and it does not require Src kinases. Confocal images and intensity profiles of Jurkat cells that were immunostained with a primary mAb to Lck and FITC-conjugated secondary Ab (anti-Lck-FL) (A), D10 T cells expressing L10-GFP, which contains the first 10 aa of Lck followed by GFP (B), JCaM1.6 cells expressing LckNT-GFP (C), and Jurkat cells expressing LckNT-GFP and pretreated with 10 µM PP2 (D). Each white bar represents 5 µm.

 
To determine the specificity in targeting to the LckNT-GFP-enriched patches, Jurkat cells expressing LckNT-GFP were double-labeled with either biotinylated CTX to detect the glycolipid GM1, or immunostained with a mAb that is specific for CD45. CTX was detected using streptavidin-conjugated TR (CTX-TR) (Fig. 3A), and staining of CD45 was detected using a TR-conjugated Ab to mouse IgG (anti-CD45-TR) (B). Fig. 3A shows an example where LckNT-GFP and GM1 are coenriched in a membrane patch (arrowhead). Conversely, Fig. 3B shows an LckNT-GFP-enriched patch that lacks CD45. The distinct membrane distributions of GM1 and CD45 relative to LckNT-GFP are further illustrated by the fluorescence intensity measurements of the outer membrane that accompany each image in Fig. 3, A and B. For example, the graphs show that LckNT and CTX-TR exhibit a similar amount of enrichment in the indicated membrane patch (arrowhead), but that LckNT-GFP and CD45 are enriched in discrete but separate regions of the plasma membrane.



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  		FIGURE 3. LckNT-GFP-enriched patches represent GEM domains. Confocal images and fluorescence intensity profiles of Jurkat cells that were double-labeled with LckNT-GFP and either cholera toxin and TR-conjugated streptavidin (CTX-TR) (A), or Ab to CD45 and TR-conjugated anti-mouse Ig (anti-CD45-TR) (B). The white arrowheads indicate LckNT-GFP-enriched patches. In the fluorescence intensity plots, green and red represent the fluorescence in the GFP and TR channels, respectively. C, Confocal images of detergent-resistant LckNT-GFP in Jurkat cells. Cells expressing LckNT-GFP were extracted with 1% TX-100 at 4°C before fixing and staining with either CTX-TR (top) or anti-CD45-TR (bottom).

 
To further quantitate the membrane distribution of GM1 and CD45 in the outer membrane relative to LckNT-GFP, a cross-correlation analysis was performed on populations of double-labeled cells (36, 37, 38, 39). In this analysis, the degree of colocalization of separate fluorophores is represented by the correlation coefficient ({rho}). The value of {rho} can range from -1.0 to +1.0, with 1.0 being exact colocalization and 0.0 being random colocalization. {rho} had an average value of 0.63 ± 0.10 (n = 27) in cells that were double-labeled with LckNT-GFP and CTX. This value is similar to what has been measured for colocalization of GEM-associated molecules in other studies (19, 37). However, in experiments with staining of CD45, the average value of {rho} was 0.32 ± 0.14 (for n = 30). The results from the cross-correlation analysis are therefore consistent with the data in Fig. 3, A and B, showing that the LckNT-GFP-enriched patches are specifically coenriched with GM1.

A characteristic feature of GEM domains is their insolubility in nonionic detergents such as TX-100 (20). To determine whether the LckNT-enriched patches share this property, Jurkat cells expressing LckNT-GFP were extracted with a chilled solution of 1% TX-100 before fixing and staining with either CTX-TR or anti-CD45-TR. The confocal images in Fig. 3C show that a significant fraction of the LckNT-GFP remained following extraction with TX-100, and this material included discrete patches of protein enrichment (arrowheads). Furthermore, GM1 colocalized with LckNT-GFP in the TX-100-resistant patches (top panel). However, no CD45 was detected following treatment with TX-100 (bottom panel). Together, the results in Fig. 3 show the LckNT-GFP-enriched membrane patches have properties that are representative of GEM domains, and therefore we conclude that T cell GEM domains are assembled into micrometer-sized membrane patches in the absence of stimulatory signals.

The actin cytoskeleton mediates assembly of the GEM-enriched membrane patches

Capping of GEM domains to form immune synapses occurs in an actin-dependent manner (40, 41, 42, 43). To determine whether the GEM-enriched patches also contained F-actin, Jurkat cells expressing LckNT-GFP were stained with Phal-TR. This experiment showed the LckNT-GFP-enriched patches were coenriched with F-actin (Fig. 4A). Similarly, the average correlation coefficient for cells that were double-labeled with LckNT-GFP and Phal-TR was 0.70 ± 0.15 (n = 24). Thus, LckNT-GFP and F-actin colocalized in the plasma membrane with similar efficiency as LckNT-GFP and CTX.



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  		FIGURE 4. Jurkat GEM-enriched patches form by association of GEM domains with actin cytoskeleton. A, Confocal images and fluorescence intensity profiles of Jurkat cells double-labeled with LckNT-GFP and Phal-TR. The green and red lines represent the fluorescence intensity of GFP and TR, respectively. The white bar represents 5 µm. B, Jurkat cells were lysed with 1% TX-100, and the GEM and TXS fractions were separated by sucrose gradient equilibrium centrifugation. Following centrifugation, the gradient was fractionated and then separated by SDS-PAGE. The samples were immunoblotted with Ab to either Lck (top) or actin (bottom). C, Jurkat cells expressing LckNT-GFP were treated with 5 µM latrunculin B for 30 min at 37°C before fixing. The distribution of LckNT-GFP in the plasma membrane was measured by confocal microscopy and quantitated based on the area of the largest LckNT-GFP-enriched patch in each cell. The patches were defined as those regions of the plasma membrane with intensity values 2 SDs above the average intensity of the entire outer membrane. Shown here is the distribution of the maximum area values for latrunculin B-treated () and untreated ({blacksquare}) cells. The unit for the areas is square pixels. The sample sizes were 38 and 39 for the untreated and latrunculin B-treated cells, respectively. D, Confocal images of TX-100-extracted Jurkat cells that were double-labeled with LckNT-GFP and Phal-TR. The cells were extracted with TX-100 before fixing and staining with Phal-TR. E, Same as D, except that the cells were pretreated with 5 µM latrunculin B before extraction with TX-100. The right panel in E represents a 5-fold enhancement of the image in the left panel. No Phal-TR was detectable in the cells treated with latrunculin B.

 
To further measure the association of F-actin with GEM domains, we performed a membrane fractionation experiment to determine whether actin occurred in the TX-100-resistant GEM fraction. Immunoblots of the sucrose gradient fractions following equilibrium centrifugation showed that endogenous Lck and the GEM fraction occurred in gradient fractions 2–4 (Fig. 4B, top), and these gradient fractions also contained actin (B, bottom). Note that most of the actin occurred in the TXS fraction, and this would be anticipated due to the high cellular content of soluble G-actin.

One hypothesis is that the GEM-enriched membrane patches form by actin-mediated clustering of GEM microdomains (19, 44). To test this hypothesis, Jurkat cells expressing LckNT-GFP were treated with latrunculin B, which disrupts actin filaments (45). The distribution of LckNT-GFP in the plasma membrane was quantitated by determining the area of the largest patch in the plasma membrane of the cells. The histograms in Fig. 4C show the distributions of the LckNT-GFP maximum patch area sizes that were measured for untreated and latrunculin B-treated cells. This result demonstrates that disruption of actin cytoskeleton significantly reduced the size of the membrane patches. For example, a ~50% reduction in the average maximum area value occurred with treatment with latrunculin B. In cells where GEM domains were labeled by immunostaining with Ab to Lck, a similar 50% reduction in the average maximum area size occurred with treatment with latrunculin B. Thus, the effect of latrunculin B on patch size and clustering of GEM domains was not restricted to LckNT-GFP-enriched GEM domains. Together, these results with latrunculin B are consistent with the model that the patches are formed by an actin-dependent process.

To determine whether F-actin associated with LckNT-GFP in the TX-100-resistant membranes that are detectable with confocal microscopy, such as shown in Fig. 3C, Jurkat cells expressing LckNT-GFP were extracted with TX-100 before fixing and staining with Phal-TR. Consistent with the data in Fig. 4B, F-actin occurred in the TX-100-resistant membrane (D). However, the degree of colocalization of F-actin with LckNT-GFP decreased with extraction of the samples with TX-100. For example, although many of the patches in the extracted cells showed nearly equal coenrichment of LckNT-GFP and F-actin (Fig. 4D, white arrowheads), membrane patches that contained an unequal enrichment of LckNT-GFP and F-actin were also evident (yellow arrowheads). The TX-100-induced changes in the relative enrichment of LckNT-GFP and F-actin in membrane patches was also reflected by the average correlation coefficient of the samples, which decreased from 0.70 ± 0.15 (n = 24) in the untreated sample to 0.47 ± 0.14 (n = 33) in the TX-100-extracted cells. This decrease in the colocalization of LckNT-GFP and F-actin could be due to the disruptive effects of TX-100 on cell membranes and GEM domains (46). Importantly, the average correlation coefficient of the detergent-extracted sample was still significantly higher than that measured for cells that were double-labeled with LckNT-GFP and the non-GEM marker CD45.

To determine the effect of disrupting the actin cytoskeleton on the detergent-resistant membranes, Jurkat cells were treated with latrunculin B before extraction with TX-100. Interestingly, the effect of latrunculin B was to disrupt the detergent-resistant residue such that the LckNT-GFP was largely removed by TX-100. For example, in the latrunculin B-treated sample in Fig. 4E, LckNT-GFP was detectable only after significant (5-fold) enhancement (right panel) of an original image (left panel) that was collected at the same settings as the LckNT-GFP image in D. Thus, disruption of the actin cytoskeleton with latrunculin B causes LckNT-GFP to be dispersed by the TX-100, and this is consistent with the notion that the actin cytoskeleton serves as a scaffold for assembly and maintenance of the GEM-enriched membrane patches.

GEM-enriched patches diffuse in the plasma membrane and translocate to immune synapses

Cell membranes are dynamic structures, and this may be reflected in the properties of GEM domains and the GEM-enriched membrane patches. To measure the properties of the membrane patches over time, live-cell imaging was performed using Jurkat cells expressing LckNT-GFP. As demonstrated in Fig. 5A and supplemental Fig. 1,4 the time-resolved measurements showed that the LckNT-GFP-enriched patches migrated in the plasma membrane. For example, Fig. 5A shows two separate optical sections of the same cell, the frames of which were collected 20 s apart. The indicated patch (white arrowhead) is visible in both optical sections, and it diffused nearly one-half of the way around the circumference of the cell in <2 min.



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  		FIGURE 5. GEM-enriched membrane patches are mobile, and they diffuse to and within immune synapses. A, Time-lapse imaging of LckNT-GFP in Jurkat cells. Each frame was acquired 20 s apart. B and C, Time-lapse images of LckNT-GFP in Jurkat cells stimulated with OKT3-coated beads. The asterisks indicate the position of the beads. Each frame was acquired 1 min apart. The top and bottom rows in A and B are two separate optical sections that were 1.5 µm apart. D, Time-lapse confocal images of a D10 T cell stimulated with an Ag-pulsed CH27 B cell. The D10 cell expressed L10-GFP and the asterisk indicates the position of the CH27 cell. The two frames were collected 40 s apart. The brackets indicate an L10-GFP-enriched patch that was measured at the beginning and the end of the experiment.

 
Stimulation of Jurkat cells by cross-linking the TCR generates immune synapses by capping of GEM domains at the site of receptor cross-linking (19, 28). To measure the GEM-enriched patches during T cell stimulation, time-lapse measurements were performed using Jurkat cells that were stimulated with 6.0-µm latex beads coated with the CD3-specific mAb OKT3. The GEM-enriched patches were again detected by expressing LckNT-GFP. Fig. 5B (supplemental Fig. 2) shows that the GEM-enriched patches translocated in the plasma membrane to the site of TCR cross-linking. For example, an LckNT-GFP-enriched patch that is visible in both optical sections of the sample (white arrowhead) migrated in the plasma membrane from a position that was distal to where the bead contacted the cell (asterisk) to where capping is occurring at the bead contact site. Interestingly, a separate experiment showed that the patches could diffuse to distinct regions within the immune synapses. Specifically, the indicated patch in Fig. 5C began in one area of a cap that had formed where an OKT3-coated bead contacted the cell (asterisk), and it migrated through the bead-cell interface to a separate and distinct region of the cap. In Fig. 5, both B and C, the frames were collected 1 min apart so that each migration took between 5 and 6 min to occur. Migration of GEM-enriched patches was also evident in D10 cells stimulated with Ag-pulsed CH27 B cells (Fig. 5D and supplemental Fig. 4). Thus, migration of GEM domains was not restricted to Jurkat T cells.

The results in Fig. 5 showing migration of the GEM-enriched patches during T cell stimulation are representative of measurements of ~50 separate Jurkat cells. For example, migration of LckNT-GFP-enriched patches to immune synapses was evidenced in 78% of the cells that were measured (n = 45). Migration of patches within synapses was detected in 87% of the cells that contained membrane caps (n = 44). Similarly, 80% of the D10 cells (n = 30) showed evidence of patch migration to and within immune synapses.

Actin-dependent changes in cell shape or movement may also effect migration of the patches to the TCR. For example, as has been documented earlier for Jurkat cells, we found that capping of LckNT-GFP was frequently followed by phagocytosis of the beads (33). However, the differential interference contrast images in supplemental Figs. 2 and 3 show that phagocytosis did not occur during the capping and migration of GEM domains in these cases. Thus, the observed migration of GEM domains was not due to changes in cell shape that accompany phagocytosis. Furthermore, the migration of the patch in the stationary cell in Fig. 5C shows that migration was not dependent on cell movement. Thus, our results are consistent with the model that the GEM-enriched patches diffuse in the plasma membrane as a discrete structure that can be targeted to immune synapses and regions within synapses.

Capping of GEM domains to immune synapses is actin-dependent and requires myosin motor proteins

To identify the signals that function in targeting GEM domains to immune synapses, Jurkat cells expressing LckNT-GFP were stimulated with OKT3-coated beads either with no pretreatment (Fig. 6A) or following treatment with either 10 µM cytochalasin D to disrupt F-actin (B), 100 nM wortmannin for 3 h to inhibit phosphatidylinositol 3-kinase (PI3K) activity (C) (47), or 20 mM BDM to inhibit myosin motor proteins (D) (48). Capping of LckNT-GFP in each experiment was quantitated by determining the average fluorescence intensity of LckNT-GFP in the immune synapses and the remaining plasma membrane. Cells that exhibited >50% enrichment of LckNT-GFP in immune synapses were counted, and the percentage of total cells that exhibited capping of LckNT-GFP is shown in the plot in Fig. 6E. Our experiments showed that capping of LckNT-GFP was inhibited by cytochalasin D, and this is consistent with the documented role of the actin cytoskeleton in forming membrane caps and immune synapses (40). Furthermore, both wortmannin and BDM also inhibited capping of the GEM domains. Therefore, we conclude that trafficking of GEM domains to immune synapses occurs downstream of signals from PI3K. From our results with BDM, we also conclude that myosin-driven movement of actin filaments also functions in the diffusion of GEM domains to immune synapses.



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  		FIGURE 6. Targeting of GEM domains to the immune synapse is inhibited by cytochalasin D, and wortmannin and BDM. Jurkat cells expressing LckNT-GFP were stimulated with OKT3-coated beads for 15 min before fixing. Each row contains a confocal image of three separate cells that were untreated (A), treated with 10 µM cytochalasin D (B), treated with 100 nM wortmannin for 3 h at 37°C (C), or treated with 20 mM BDM for 5 min (D). E, Enrichment of LckNT-GFP in immune synapses of Jurkat cells stimulated with OKT3-coated beads was quantified by dividing the average fluorescence intensity of the membrane at the bead contact site by the average intensity of the remaining plasma membrane. Cells exhibiting >50% enrichment of LckNT-GFP in a membrane cap were scored as exhibiting capping. Seventy-five, 60, 46, and 66 cells were measured for the untreated, cytochalasin D-, wortmannin-, and BDM-treated samples, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using confocal microscopy, we have shown that T cell GEM domains are constitutively assembled into micrometer-sized patches in the outer membrane of the cell. Furthermore, it was determined that assembly of the patches occurred in absence of the stimulatory signals that are necessary for generation of immune synapses. For example, patches were evident in Lck-deficient JCaM1.6 cells and in Jurkat cells that were treated with the Src kinase inhibitor PP2. Furthermore, during T cell stimulation, the GEM-enriched patches were observed to translocate to immune synapses. Thus, GEM domains and GEM-enriched patches serve as a vehicle for targeting molecules to the site of cell signaling. This model is consistent with other reports that have shown that immune synapses are specifically enriched with GEM-associated molecules (28), and that association of GFP with GEM domains is necessary for its targeting to immune synapses (19).

Earlier biophysical studies have demonstrated that GEM domains occur as microdomains that are ~100 nm in size (21, 22, 23). Hence, one conclusion from our observations is that clustering of GEM microdomains assembles the GEM-enriched membrane patches. Based on our results that showed F-actin is enriched in the membrane patches and that latrunculin B inhibited patching of GEM domains, we conclude that assembly of GEM-enriched patches occurs in an actin-dependent manner. Our finding that latrunculin B disrupts the detergent-resistant membranes following extraction of cells with TX-100 is consistent with the hypothesis that GEM domains and patches are anchored to the actin cytoskeleton.

One model for the assembly of membrane domains by actin filaments is by nonspecific corralling of membrane proteins by actin fences that underlie the plasma membrane (49, 50, 51). However, our data are more consistent with a direct association between GEM domains and the actin cytoskeleton. For example, we found that actin was associated with the detergent-resistant GEM fraction (Fig. 4). Also, the GEM-enriched membrane patches have a specific composition that includes exclusion of the large, non-GEM protein CD45 (Fig. 2), and this is not consistent with a nonspecific corralling of proteins by actin fences. Although we are currently investigating the mechanism underlying the association between GEM domains and F-actin, we predict that it is related to the documented enrichment of molecules in GEM domains that signal actin polymerization (6, 8, 29, 52, 53). Thus, GEM domains may serve as a platform for actin polymerization and attachment of actin filaments to the plasma membrane, thus giving rise to the observed association of F-actin with GEM domains.

Investigation of the signals responsible for patching of GEM domains shows that they are distinct from that those that cause their capping following stimulation. For example, the GEM-enriched patches occurred both in JCaM1.6 cells and Jurkat cells treated with PP2, both of which are conditions where capping is inhibited (33, 42). Capping also requires Vav and Rac (41, 54), but we observed LckNT-GFP-enriched patches in Jurkat cells that expressed a dominant-negative Rac molecule (N17) (data not shown). The signals that lead to assembly of the patches in T cells may be similar to those that cause patching of GEM domains in adherent cells (19) and the adhesion-related glycosynapses that have been described (55).

As reported earlier using 5C.C7 T cells coated with latex beads, migration of proteins to engaged TCR molecules occurs in an actin- and myosin-dependent manner (56). Similarly, we determined that migration of GEM domains to membrane caps was also actin- and myosin-dependent. For example, capping of GEM domains was efficiently inhibited by cytochalasin D and BDM (Fig. 6). Thus, the translocation of proteins that was measured using latex beads in activated 5C.C7 T cells may reflect GEM-dependent migration of the molecules to the site of TCR signaling.

Our finding that targeting of GEM domains to the TCR is inhibited with wortmannin is consistent with a recent study showing immediate and sustained production of phosphatidylinositol 3,4,5-triphosphate (PIP3) during T cell stimulation (57). Interestingly, enrichment of phosphatidylinositides in the detergent-resistant GEM fraction has been documented (58), and this has led to the suggestion that PI3K activity selectively occurs in GEM domains (53). Importantly, the guanine nucleotide exchange factor Vav, which signals actin polymerization and clustering of GEM domains (41, 54, 59), binds to PIP3 through its pleckstrin homology domain, and Vav is enriched in GEM domains (6). Thus, GEM-associated PIP3 generated by PI3K may be instrumental in recruiting downstream molecules to GEM domains, which could in turn function in assembling the domains into membrane patches and caps.

T cells undergo dramatic changes in shape following their initial contact with the APC and during formation of the immune synapse (60). Hence, one interpretation of our results regarding migration of GEM domains to immune synapses may be that they represent a consequence of membrane movements associated with changes in cell shape. However, our data in Fig. 5 that showed migration of GEM-enriched patches in stationary cells provide evidence that migration of GEM domains is not dependent on cell movement. Furthermore, our results corroborate data from previous studies that have shown that GEM domains migrate or diffuse within the plasma membrane (23)

Membrane fractionation studies have shown the TCR is restricted to the non-GEM fraction before stimulation, after which it occurs in the GEM fraction (6). This result has often been interpreted as the TCR undergoing partitioning from the non-GEM regions of the plasma membrane to GEM domains. Another interpretation, based on our results, is that partitioning of TCR into GEM domains is due largely to trafficking of GEM domains to the site of receptor engagement. Therefore, our model predicts that targeting of TCR to GEM domains occurs downstream of initial signals that cause actin polymerization and translocation of GEM domains to immune synapses, and this is consistent with the reported actin-dependent association of TCR with GEM domains (8). These initial signals for actin polymerization may be initiated by the Fyn and Vav that is constitutively associated with the TCR (8).

Immune synapses are dynamic structures, and proteins within the synapses undergo considerable movement during maturation of the immune synapse (27, 61, 62, 63). Interestingly, we observed a similar movement of a GEM-enriched patch within a synapse that was formed with an OKT3-coated bead (Fig. 5C). This result suggests that GEM domains not only function in targeting proteins to synapses where the TCR has been engaged, but may also function as a vehicle for rearrangement of molecules within the synapses.

Fluorescence imaging experiments of T cells labeled with CTX and stimulated with planar membranes containing Ag-loaded MHC molecules and ICAM-1 showed that GEM domains are enriched in the central supramolecular activation complex (c-SMAC) region of the synapse (64). Furthermore, based on measurements of the area of the synapse and the amount of accumulation of GM1 in the c-SMAC, the authors concluded that GEM domains are clustered in the c-SMAC only from the peripheral supramolecular activation complex region of the immune synapse. This finding contrasts with measurements showing that proteins translocate to the synapse in an actin- and myosin-dependent manner (56) and with our data reported in this study showing a similar role of actin and myosin in the translocation of LckNT-GFP-enriched patches. An alternative explanation for the measured correlation between synapse area and domain enrichment is that the size of the synapse governs the strength of the signals that leads to actin polymerization and activity of myosin motor proteins. These signals would then lead to the observed migration of GEM domains and membrane proteins to the immune synapse from other regions of the plasma membrane. In this regard, it is interesting to note that one effect of inhibited or inefficient signaling from the TCR is to decrease the area of the immune synapse (60, 65).

Other studies of Jurkat cells using confocal microscopy have reported that GEM-associated molecules are evenly distributed in the plasma membrane (28). These studies may have discounted membrane patches as nonspecific aggregations. However, we have eliminated this possibility by showing that the patches do not represent nonspecific aggregations from cells adhering to the glass or polylysine, and they are not due to overexpression of our GFP reporter. Furthermore, the patches are not an artifact of fixation, because they were observed in live cells as well.

A recent study by Zacharias et al. (66) suggested that GFP undergoes dimerization when targeted to GEM domains due to the elevated concentration of protein in these structures. One effect of GFP dimerization would be to overreport the extent of protein enrichment in membrane patches. However, immunofluorescence staining of Jurkat cells with Ab to Lck showed patches with a similar size and enrichment as measured with LckNT-GFP (Fig. 3). Therefore, we conclude that the contribution of GFP dimerization in the measured enrichment of protein in GEM domains is nominal.

In summary, we have identified micrometer-sized assemblies of GEM domains in T cells that occur before stimulatory signals that lead to either immune synapses or cell motility. Furthermore, results from live-cell imaging experiments show GEM domains undergo trafficking to immune synapses by translocation in the plasma membrane. GEM domains can therefore serve as a vehicle for targeting signaling proteins to the immune synapse, as well as migration of proteins within the synapse itself. Therefore, our results demonstrate an important and novel function of GEM domains in T cell signaling.


   Acknowledgments
 
We thank J. Byrum and J. Zavzavadjian for technical assistance.


   Footnotes
 
1 This work was supported by American Heart Association Beginning Grant-in-Aid 9960341Z and Oklahoma Center for the Advancement of Science and Technology Grant HR02-009 to W.R. Back

2 Address correspondence and reprint requests to Dr. William Rodgers, Molecular Immunogenetics Program, Oklahoma Medical Research Foundation, 825 Northeast 13th Street, Mail Stop 17, Oklahoma City, OK 73104. E-mail address: william-rodgers{at}omrf.ouhsc.edu Back

3 Abbreviations used in this paper: GEM, glycolipid-enriched membrane; GFP, green fluorescent protein; LckNT, N-terminal domain of Lck; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; CTX, cholera toxin B subunit; BDM, 2,3-butanedioine 2-monoxime; TX-100, Triton X-100; TXS, TX-100 soluble; TR, Texas Red; Phal, phalloidin; PI3K, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol 3,4,5-triphosphate; c-SMAC, central supramolecular activation complex. Back

4 The on-line version of this article contains supplemental material. Back

Received for publication August 19, 2002. Accepted for publication April 25, 2003.


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 Discussion
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