Lipid rafts present on the plasma membrane play an important role in spatiotemporal regulation of cell signaling. Physical and chemical characterization of lipid raft size and assessment of their composition before, and after cell stimulation will aid in developing a clear understanding of their regulatory role in cell signaling. We have used visual and biochemical methods and approaches for examining individual and lipid raft sub-populations isolated from a mouse CD4+ T cell line in the absence of detergents.
Detergent-free rafts were analyzed before and after their interaction with antigen presenting cells. We provide evidence that the average diameter of lipid rafts isolated from un-stimulated T cells, in the absence of detergents, is less than 100 nm. Lipid rafts on CD4+ T cell membranes coalesce to form larger structures, after interacting with antigen presenting cells even in the absence of a foreign antigen.
Findings presented here indicate that lipid raft coalescence occurs during cellular interactions prior to sensing a foreign antigen.
raft coalescenceCD4+ T cellsantigen presenting cellselectron microscopyraft-ELISA
Signals emanating from the plasma membrane have spatial and temporal components [1–5]. Spatial distribution and accessibility of signaling proteins on the plasma membrane can potentially have profound effects on the outcome of signaling. While knowledge of temporal signaling events has rapidly advanced, the spatial distribution of signaling proteins remains unclear. More so, how the spatial distribution of signaling molecules relates to temporal signaling is unknown. However, recently, re-organization on the plasma membrane of quiescent cells was recognized after triggering signaling from the membrane [6–11].
Lipid raft membrane domains are rich in cholesterol and sphingolipids and known to compartmentalize signaling proteins [12–17]. Heterogeneity of lipid rafts, with respect to protein composition, on the plasma membrane may provide an additional level of spatial segregation [18–26]. Ligand and receptor induced molecular interactions on the plasma membrane trigger a signaling cascade that culminates into specific gene expression. Compositional heterogeneity of lipid rafts on the surface of quiescent cells and their subsequent coalescence, when the receptors engage their ligands, might promote interactions between appropriate signaling proteins [14, 27]. However, this is only one of several proposed models to explain signal transduction from the plasma membrane to the interior of the cell [28–35].
Lipid rafts assemble to form an immunological synapse, a central structure at the contact site of CD4+ T cells and antigen presenting cells involved in regulating cell signaling [36–45]. These early signaling events are crucial in generating a response by T cells, especially since CD4+ T cells are capable of generating specific cellular responses after the engagement of the same antigen receptor, ranging from differentiation to Th1 or Th2 or Th17 (T helper cell subsets).
In light of the observation that lipid rafts are compositionally heterogeneous, it remains unclear whether distinct sub-populations of rafts assemble at or around the synapse and thus, contribute to signal transduction and distinct cellular responses. Methods allowing enumeration of lipid rafts as on a single raft and sub-population basis in quiescent, activated, and differentiating cells will aid in addressing the role of lipid rafts in signaling. To enumerate lipid rafts in T cells, we have used a published detergent-free isolation procedure . Lipid rafts isolated from a T cell line in the presence and absence of a specific antigen were visualized by transmission electron microscopy. It was surprising to find that lipid rafts isolated from co-cultures of CD4+ T cell and antigen presenting cells in the absence of antigen show raft coalescence/clustering.
Materials and methods
Mouse CD4+ T-T hybrid of Th1 phenotype YH16.33  and A20  cell lines (generous gifts from Dr. Ken Rock, University of Massachusetts Medical Ctr, MA) were grown in Dulbecco's modified eagle medium (DMEM) with 4.5 g/ml of glucose (Invitrogen, Carlsbad, CA) supplemented with 10% heat inactivated fetal bovine serum, L-glutamine (Atlanta Biologicals, Atlanta, GA), sodium pyruvate, penicillin/streptomycin, and fungizone (Invitrogen, Carlsbad, CA). Cell cultures were maintained at 37°C in a 10% CO2 incubator.
Detergent-Free Isolation Protocol
Lipid rafts were isolated using a previously published protocol . Briefly, 6 × 107 of total cells either YH16.33 alone or co-cultured with A20 (1:1 ratio) in the presence or absence of 1 mg/ml chicken ovalbumin (antigen) was cultured for 16-18 hrs. Cells were centrifuged for 5 minutes at 1000 × g at 4°C. The supernatant was decanted; the pellet was re-suspended in 10 ml of base buffer solution consisting of 20 mM Tris-HCl, 250 mM Sucrose (pH 7.8), supplemented with 1 mM CaCl2 and 1 mM MgCl2 followed by centrifugation for 2 minutes at 250 × g at 4°C. Then the supernatant was decanted, the pellet was re-suspended in 1 ml of the base buffer solution supplemented with, CaCl2 and MgCl2, a protease inhibitor cocktail set (EMD BioSciences, Darmstadt, Germany), and a calpain inhibitor (Sigma-Aldrich, St. Louis, MO), and then lysed by passaging through a ¾ inch 23 gauge needle, 20 times. The lysate was centrifuged at 1000 × g for 10 minutes at 4°C. The supernatant was collected and stored on ice. The pellet was re-suspended with 1 ml of the base buffer solution supplemented with CaCl2, MgCl2, and protease inhibitor and lysed again by passaging through a ¾ inch 23 gauge needle, 20 times. The lysate was centrifuged at 1000 × g for 10 minutes at 4°C. The supernatant was pooled with the previously collected supernatant. Two ml of the base buffer supplemented with an equal volume of 50% Optiprep solution (Sigma Aldrich, St. Louis, MO) was transferred to an ultracentrifuge tube (Beckman Instruments, Paolo Alto, CA). The solution was then overlaid with 1.6 ml each of 20%, 15%, 10%, 5% and 0% Optiprep solution, respectively, with a total final volume of 12 ml. The gradient was centrifuged for 90 minutes at 52,000 × g at 4°C in an ultracentrifuge (Beckman Instruments, Paolo Alto, CA). The sample was then fractionated in 1.3 ml aliquot from the top of the gradient and stored at -20°C. For detergent isolation experiments, lipid rafts were obtained in the presence of 1% Triton X-100 and subjected to sucrose density gradient as described previously [23, 49].
Western Blot Analysis
Fifteen μl of each fraction was combined with 6.3 μl of lithium dodecyl sulfate (LDS) buffer (Invitrogen, Carlsbad, CA) and 2.3 μl DTT (Invitrogen, Carlsbad, CA). Twenty-two μl of the fraction solution was loaded into 4-15% gels (BioRad, Hercules, CA). The gel was electrophoresed using 2-(N-morpholino) ethanesulfonic acid (MES) buffer (Invitrogen, Carlsbad, CA) at 100 volts for approximately 45 minutes. The gel was then transferred to a polyvinylidene fluoride (PVDF) membrane for 1 hour at 45 volts. The membrane was blocked with 5% non-fat Carnation Instant milk prepared in phosphate buffer saline solution with Tween-20 (PBST) (Sigma Aldrich, St. Louis, MO) and incubated with appropriate primary antibodies against Linker of Activated T cells (LAT), β-COP (Santa Cruz Biotechnology Inc, CA), overnight at 4°C. The species specific, secondary antibodies conjugated to horseradish peroxidase (HRP) (Pierce, Rockford, IL) were added and incubated for 75 minutes at room temperature. The membrane was then exposed to substrate and chromogen solution, a mixture of equal volumes of H2O2 and a luminol solution (SuperSignal West Dura) (Pierce, Rockford, IL) for 2 minutes and then exposed using an image analyzer (Alpha-Innotech, San Leandro, CA).
Dot Blot Protocol
PVDF membranes were soaked in methanol for two minutes to moisten the membrane. Three μl dots of fraction samples were placed on the PVDF membrane. The samples were allowed to dry on the membrane, and blocked with 5% non-fat Carnation Instant milk prepared in PBST for 60 minutes at room temperature. The membrane was then incubated in cholera toxin β chain conjugated to HRP (BD Biosciences, San Jose, CA) for 60 minutes. The membrane was then exposed to SuperSignal West Dura (Pierce, Rockford, IL) substrate for 2 minutes and then exposed using an image analyzer (Alpha-Innotech, San Leandro, CA).
Raft ELISA Protocol
Lipid rafts were analyzed by raft-ELISA as reported in previous publications [23, 49], with one exception: detergent-free rafts were used instead of the detergent-resistant rafts. Briefly, 96 well flat bottom, high bonding, enzyme immuno-assay/radioimmuno assay (EIA/RIA) plates (Costar, New York, NY) were coated with 50 μl capture antibody (2 μg/ml) and covered with saran wrap and incubated at 4°C overnight. The microwells were then washed with 100 μl of wash buffer, PBST, 4 times. Wells were then blocked with blocking buffer PBST supplemented with 1% (w/v) fraction V bovine serum albumin (BSA) (PBST/BSA), (Fisher Scientific, Pittsburg, PA) for 30 minutes at room temperature. Excess of blocking reagents were removed with washing buffer, PBST; this step was repeated three times. Fifty μl samples (1:5 diluted raft fractions in PBST/BSA) were added to wells and incubated overnight at 4°C. Unbound lipid rafts were removed by washing with PBST 9 times. Biotinylated detection antibody (1 μg/ml) was added to each microtitre well and incubated for 1 hour at room temperature followed by washing unbound antibody 6 times with PBST. Avidin-HRP was added to each well and incubated for 30 minutes at room temperature. Unbound avidin-HRP conjugate was removed by washing 8 times with PBST. A 100 μl solution of a 1:1 mixture of 2,2'-azino-di[3-ethyl-benzthiazoline 6-sulphonate] (solution A) and 0.02% solution of H2O in citric acid buffer (solution B) were added to appropriate well. The absorbance was read at 405 nm with a Spectramax 190 plate reader (Molecular Devices, Sunnyvale, CA).
Formvar Coating EM Grids
Coating of nickel grids with formvar was carried out according to previous publications. Nickel grids (Electron Microscopy Sciences, Fort Washington, PA) were sonicated 3 times in ethanol prior to their use. Clean microscopic glass slides were dipped into a formvar solution in ethylene dichloride (Electron Microscopy Sciences, Fort Washington, PA) and chloroform (Fisher Scientific, Pittsburg, PA) for a few seconds to allow coating of formvar on the slide. The edges of the glass slides were scored and tilted to release the formvar in a clean bowl of double distilled water. Nickel grids were mounted on top of the floating formvar sheets. Using a different microscope slide wrapped in parafilm, the floating formvar, with the grids on top, was carefully scooped up from the water bowl and allowed time to dry and store at RT until further use.
Immunogold labeling for TEM
Lipid rafts were captured and detected by the method we have previously used for detection of detergent isolated lipid rafts [23, 49]. A capture antibody, purified anti-mouse CD90 (Thy-1) (G7) (BD Biosciences, San Jose, CA) was coated on the nickel grid at 4 μg/ml antibody concentration in carbonate/bicarbonate buffer in a humid chamber. Antibody coating was carried out by placing the formvar coated side of the grid faces down on a drop of carbonate-bicarbonate buffer with capture antibody for an overnight period at 4°C. Nickel grids were washed 4 times with phosphate buffer saline (13.7 mM NaCl, 0.27 mM KCl, 0.43 mM Na2HPO4-7H20, 0.14 mM KH2PO4, pH 7.3) supplemented with 1% BSA-C (Aurion, Costerweg, Netherlands). For each washing step, grids were incubated with the washing buffer for 5 minutes at room temperature in a humid chamber. Non-specific sites on the grids were then blocked with a blocking buffer consisting of 1 × PBS supplemented with 0.05% (w/v) of fraction V bovine serum albumin for 20 minutes at room temperature. Grids were then washed with incubation buffer 2 times, 5 minutes each, at room temperature followed by incubation with 30 μl drops of lipid raft fraction samples for an overnight period at 4°C. Unbound lipid rafts were removed by washing with PBS/BSA buffer at room temperature, and this step was repeated 5 times. Grids were than incubated with biotin-conjugated detection antibody Ly6A/E (Sca-1) (D7) (BD Biosciences, San Jose, CA) at 3 μg/ml in PBS-BSA buffer for 60 minutes at room temperature. Grids were washed 4 times with PBS-BSA buffer at room temperature to remove excess detection antibody. Non-specific sites in the grids were blocked by incubating on top of 30 μl droplets of blocking buffer for 15 minutes at room temperature followed by incubation with goat anti-biotin antibody conjugated to 10 nm gold particles at a 1:250 dilution of the stock (Aurion, Costerweg, Netherlands) for 60 minutes at room temperature. Grids were washed 2 times with double distilled water (ddw) for 5 minutes each at room temperature and incubated on 30 μl drops of 1% gluteraldehyde (Electron Microscopy Sciences, Fort Washington, PA) in double distilled water for 5 minutes at room temperature. Grids were allowed time to dry, preparation side up, on Whatmann paper after washing with ddw. Lipid rafts on the grid were fixed with 1% osmium tetroxide (Electron Microscopy Sciences, Fort Washington, PA) in double distilled water for 10 minutes. This process was followed by counter staining with1% tannic acid (Electron Microscopy Sciences, Fort Washington, PA) and 2% uranyl acetate (Electron Microscopy Sciences, Fort Washington, PA) in double distilled water for 30 minutes at room temperature, under a cover to prevent light exposure. Grids were washed with double-distilled water 2 times, 5 minutes each, at room temperature and dried on Whatmann paper, specimen side up. Grids were then analyzed on an H-7600 Hitachi Transmission Electron Microscope (Tokyo, Japan). NIH ImageJ software was used to mark the boundaries of lipid rafts that were imaged. The longest distance on the boundary of the captured and detected rafts, including the counter stained part, was used to determine the Ferret's diameter.
Cholesterol Depletion. Cholesterol was depleted from cell-free lipid rafts (lipid rafts previously isolated from cells) by treatment with 10 mMol/L of methyl-beta-cyclodextrin (MβCD) (Sigma-Aldhrich Company, St Louis, MO, USA) for 18-24 hours at 4°C before their use in the raft ELISA according to previously published report . YH16.33 and A20 co-cultured cells, with or without chicken ovalbumin antigen were treated with 10 mM MβCD for 15 minutes at 37°C as per earlier published report  and isolated lipid rafts were examined by transmission electron microscopy.
Characterization of detergent-free rafts from a CD4+ T cell line
Detergents promote coalescence of lipid rafts that may undermine assessment of raft heterogeneity [51, 52]. To overcome the problems that are associated with the use of detergent in isolating lipid rafts from plasma membrane, we chose to isolate and characterize lipid rafts from a T-T hybrid CD4+ T cell line in the absence of a non-ionic detergent. To achieve this, we adopted an isolation procedure used for detergent-free lipid rafts from an epithelial cell line , as shown in Figure 1. To assess the success of the isolation procedure and identify which density gradient fractions were enriched in lipid rafts from YH16.33, a T-T hybrid cell line, following detergent-free isolation, we carried out raft-ELISA using monoclonal antibodies directed to Thy-1 and Ly-6A.2, two GPI-anchored proteins known to localize in lipid rafts [14, 15, 23]. Anti-Thy-1 mAb was coated on the ELISA plate and used to capture detergent-free lipid rafts and biotinylated anti-Ly-6A.2 followed by avidin-HRP was used for detection. Figure 2A shows that fractions 5 and 6 contained lipid rafts. Cholesterol is an essential component of lipid rafts and thus, cholesterol-depleting compounds destabilize these membrane structures . To assess the specificity of the captured membrane rafts, we treated the cell-free fractions with such a compound, methyl-beta-cyclodextrin (MβCD), at 10 mM for 18-24 hours. Incubation of lipid raft fractions with MβCD significantly decreased the capture and detection of Thy-1 and Ly-6 lipid raft subsets (Figure 2A). Through our binary approach of capture and detection of rafts we observed the presence of the antigen receptor, TCRαβ, in fraction numbers 5 and 6, as well (Figure 2B). Enrichment of TCRαβ in rafts has been observed by other investigators [53, 54]. However, TCRαβ is present in the heavy fraction (fraction 9) which perhaps reflects its representation in the non-raft fractions as previously reported . Alternately, the presence of TCRαβ in the heavy fraction reflects its presence in the cellular organelles (ER/Golgi etc), which is expected. To further analyze these fractions we carried out SDS-PAGE followed by Western blot analysis to detect Linker of activated T cells, LAT, another known component of lipid rafts  (Figure 2C). Ganglioside (GM1), another lipid raft marker, was detected in fractions 4-6 when dot blots of isolated detergent-free fractions were probed with HRP-conjugated Cholera toxin-β chain (Figure 2D). In contrast, β-COP, a Golgi-resident protein and a non-raft marker representing an internal cellular compartment was absent from these fractions (Figure 2C). Taken together, the raft ELISA and biochemical data show that detergent-free lipid rafts are present in fractions 4, 5 and 6 of the density gradient.
Visualization and determination of size of lipid rafts using electron microscopy
Size of lipid rafts, reported in the literature using a variety of biophysical methods, has ranged from 10-100 nm in diameter [4, 56, 57]. Isolating the rafts from the plasma membrane in the absence of detergents and assessing their size will confirm their physical presence on the plasma membrane and will help clarify the disconnect between the biochemical and biophysical methods used to study these membrane entities. To examine the heterogeneity in size we used a clonal cell line, YH16.33, to generate detergent-free lipid rafts. Cell-free rafts from fraction 5 of the gradient were captured on nickel grids with anti-Thy-1 mAb and analyzed for the presence of Ly-6A.2 protein, another raft marker, with an anti-Ly-6A.2 mAb conjugated to biotin followed by anti-biotin antibody conjugated to 10 nm colloidal gold (see Materials and Methods section). Captured and detected rafts averaged 89.7 +/- 38.8 nm (n = 3721) in diameter (Figure 3A & 3B). Isolated lipid rafts are those structures that were both immune-stained (i.e. those containing gold particles) and showed counter staining with Tannic acid and Uranyl acetate, which stains lipids. These results highlight the innate heterogeneity of the size of rafts on the plasma membrane of a clonal cell line.
Lipid rafts coalesce in the presence of antigen presenting cells
We next sought to examine alterations in the lipid raft size and structure in CD4+ T cell after exposure to a specific antigen. Lipid rafts are known to contribute to the formation of the immunological synapse which is considered to be a large coalesced raft formed at the contact site of a CD4+ T cell and antigen presenting cell during T cell activation [4, 14, 27]. Previous studies have also suggested that lipid rafts take on the role of an anchoring platform for a number of signaling proteins [12–14]. To examine changes in the size of lipid rafts on the plasma membrane of T cells after engagement of their signaling receptors, we incubated YH16.33 T cells, with the antigen presenting cell, A20, in the presence and absence of a specific antigen, chicken ovalbumin. For each culture, detergent-free lipid rafts were isolated on an OptiPrep gradient after ultracentrifugation and lipid raft fractions were identified by raft ELISA (Without antigen, Figure 4A and with antigen, Figure 4B). Lipid rafts from fraction 5 were captured and detected with anti-Thy-1 and anti-Ly-6A.2 antibodies, respectively, for visualization under the electron microscope. Figure 5 shows that both in the absence (Figure 5A) and the presence (Figure 5C) of antigen, we frequently observed larger membrane entities (> 500 nm) composed of rafts attached to one another, and thus, appeared coalesced. These qualitatively distinct coalesced large rafts were not observed in the raft preparations from YH16.33 T cells alone. Moreover, the capture and detection of lipid rafts was T cell specific since we were unable to capture and detect lipid rafts generated from APCs (A20 cell line) alone with anti-T cell specific antibodies (Figure 5E). The average diameter of the rafts captured from T cells co-cultured with APCs in the absence of antigen was 116.327 +/- 52.112 nm (n = 2251) (Figure 5B). The average diameter of the rafts captured from T cells cultured with APCs in the presence of antigen was 114.430 +/- 46.748 nm (n = 2067) (Figure 5D). The diameter of both cultures of T cells with APCs, with or without antigen, produced similar sizes, although both were larger than un-stimulated YH16.33 cells (89.7+/-38.8 nm). About 72% of the rafts isolated, from the T cells in the absence of interaction with APC, ranged between 50-100 nm, and 22% ranged from 101-200 nm (Figure 5F). In contrast, the rafts isolated from YH16.33 with APC, either in the presence or the absence of antigen showed a shift towards higher size, with 38%-46% of total rafts showing 50-100 nm size distribution and 49% - 57% of 101-200 nm size (Figure 5F). When the co-cultures were treated with MβCD prior to lipid raft isolation there was a noticeable depletion in the formation of a circular shape of membrane rafts in YH16.33 and A20 co-cultures both in the absence (Figure 6A) and presence of antigen (Figure 6C). The average diameter of lipid rafts isolated from the co-cultures with and without antigen treated with MβCD was 83 +/- 39.9 nm (n = 499) and 95.2+/- 46.2 nm (n = 712), respectively (Figure 6B & 6D). Taken together, our data suggests that prior to co-culture with APCs the average size of the lipid rafts are relatively small (89.7+/- 38.8 nm) and that rafts coalesce on the plasma membrane of CD4+ T cells as they interact with APCs even in the absence of an antigen.
Analysis of detergent-extracted lipid rafts
Use of non-ionic detergents for the extraction of lipid rafts from cells in a variety of cells has been controversial [51, 52]. It has been suggested that these detergents promote coalescence of lipid rafts that may undermine assessment of raft heterogeneity. To address this issue, we wanted to compare detergent isolated rafts with those isolated using the detergent-free methodology proposed by McDonald and Pike . We compared the size of lipid rafts from YH16.33 and A20 co-cultures that were isolated in the presence of the detergent, Triton X-100 (1% concentration) and examined their size by EM. These rafts were captured with an anti-Thy-1 mAb and detected with an anti-Ly-6A.2 antibody. Similar to the detergent-free rafts, lipid rafts isolated from YH16.33 alone with detergent-based isolation protocols yielded membrane entities less than 100 nm in diameter (Figure 7B & 7F), as reported before . In contrast to rafts isolated in a detergent-free environment, lipid rafts isolated in the presence of detergent from co-cultures of YH16.33 and A20 cells in presence or absence of specific antigen showed a higher frequency of macrodomains, some of which were up to micrometers in size (Figure 7C-E). However, like in the detergent-free case, frequency of macrodomain formation did not depend on the presence of antigen (Figure 7F). To examine if the presence of cholesterol was critical for formation of these large membrane domains we exposed the YH16.33 and A20 co-cultures to MβCD for 15 minutes prior to detergent isolation of lipid rafts. The isolated lipid rafts were captured and detected with anti-Thy-1 and anti-Ly-6A.2 monoclonal antibodies respectively. MβCD treated cultures showed considerably lower numbers of macrodomains than the untreated cultures (Figure 7F). These data suggest that cholesterol is necessary for formation and/or stability of the macrodomains we observed. Taken together, these results indicate that detergent does not affect the average size of rafts during isolation. However, in the presence of APCs, rafts isolated from T cells by detergent-based methods are considerably larger than the detergent-free rafts, supporting, perhaps, the hypothesis of detergent-dependent coalescence of lipid rafts that has been reported by other investigators . Regardless of the size of these detergent-resistant domains their macro structure is dependent on the presence of cholesterol and independent of the antigen.
Heterogeneity of lipid rafts in the plasma membrane and their re-organization during ligand-receptor interactions plays an important role in cell signaling . Resonance energy transfer (FRET) [15, 56, 58], super-resolution microscopy, [57, 59] and other biophysical methods, have provided significant insights into establishing the existence and heterogeneity of these nano-size membrane domains. Analysis of lipid rafts, after immune-EM staining of intact plasma membrane, has also been useful in providing insights into the size and heterogeneity of lipid rafts . Use of these methods in examining complex signaling cascades is challenging. Multitude of signaling proteins, participating in signal transduction, in native form or after post-translational modifications (phosphorylation) requires their visual detection simultaneously. Development of sensors allowing detection of several signaling molecules is currently underway. In here, we have examined alterations in size and composition of these membrane nano-domains following cellular interaction, on a single raft and raft-subpopulation basis. Use of biochemical approach to assess trafficking of native and post-translationally modified signaling receptors, moving in and out of lipid rafts isolated in the absence of detergent will be robust and without confounding issues with the use of detergents. Deciphering changes in size and composition in the same set of immune-isolated lipid raft populations is critical. While the biochemical approaches for examining the role of lipid rafts in spatiotemporal signaling in CD4+ T cells can be remarkably robust, in as much as it has potential for analysis of a complex series of a multitude interacting molecules in the signal transduction cascade. However, this reductionist approach has inherent limitations and needs to be complimented by dynamic cell imaging showing interactions of multitude signaling proteins on the plasma membrane. The biochemical approaches using detergent-free lipid rafts, as well as the biophysical/dynamic cell imaging approaches currently underway are essential for developing a thorough understanding of spatial and temporal regulation of cell signaling.
The data presented here suggest that antigen and its recognition by TCRαβ are not the primary mechanism for the creation of macrodomains on the membrane, since we find them to be formed in the absence of specific antigen recognition. It is long been recognized that T cells interact with antigen-presenting cells in two phases. The first step requires nonspecific adhesion involving interactions between a β1 integrin, LFA-1 on T cells with the ligand, ICAM-1, expressed on antigen presenting cells . In the second phase, the antigen receptor senses the antigen presented by the APC. The initial nonspecific interactions help launch the second phase, where the antigen receptor (TCRαβ) senses an antigen presented by the antigen-presenting cells. Detachment of T cells from APC occurs in the absence of recognition of an antigen. This opens up the opportunity to bind and sense the antigen on another APC. The data presented here suggest that during the first set of interactions between CD4+ T cells and APC the lipid rafts on T cells are spatially organized and coalesce. Previous reports have described antigen-independent immunological synapses between naïve CD4+ T cells and dendritic cells . Functional consequence of the antigen-independent interaction range from tyrosine phosphorylation, little calcium response and survival signals. It appears that these interactions allow survival of naïve T cell in vivo. However, the relationship between the antigen-independent synapse formation and coalescence of lipid rafts during T cell APC interactions needs to be elucidated. Further investigation needs to be carried out to understand the mechanism, and functional importance of this early spatial reorganization of the plasma membrane. Extent of raft coalescence and molecules that accumulate in it may depend on the source of interacting CD4+ T cell and degree of ligation of the antigen receptor and co-receptor . In addition, a functional role of lipid rafts may not be the same in distinct subsets of differentiated CD4+ T cells. For example, activated Th1 and Th2 cells behave differently in their re-organization of lipid rafts. While the antigen receptor is easily recruited in the lipid rafts in Th1 cells, similar recruitment is not observed in activated Th2 cells . Furthermore, it will be crucial to ascertain whether this re-organization reflects the underlying properties of the nanoscale assemblies that show additional interconnections when CD4+ T cells interact with antigen-presenting cells as suggested by a recent report . While antibodies to T cell surface proteins were used in our experiments to capture and detect isolated lipid rafts, it is possible that the captured coalesced rafts have some membranes belonging to APC. We have not directly tested this idea. Future experiments, where antibodies directed against MHC class II proteins and anti-TCRαβ used to capture and detect coalesced lipid rafts will be able to address this issue.
We conclude that lipid rafts on CD4+ T cell membranes coalesce to form larger structures, after interacting with antigen presenting cells even in the absence of a foreign antigen. Findings presented here indicate that lipid raft coalescence occurs during cellular interactions prior to sensing a foreign antigen.
List of Abbreviations
cluster differentiation antigen 4
antigen presenting cells
T cell receptor
This work was supported by SRFG & SRG grants from Office of Research and Sponsored Projects (ORSP), Villanova University and Intramural funding from Department of Biology, Villanova Universiy. CK and MDN were supported by Graduate Program, Department of Biology, Villanova University. The authors would like to thank Dr. Norman Dollahon, and Mrs. Sally Shrom for their expertise in electron microscopy and Dr. Sultan Jenkins for anti-transferrin receptor western blots.
Department of Biology, Villanova University
Physician Assistant Program, Cornell Medical College
Department of Neurology, University of Pennsylvania
Miyawaki A: Visualization of the spatial and temporal dynamics of intracellular signaling.Dev Cell 2003, 4:295–305.PubMedView Article
Singleton KL, Roybal KT, Sun Y, Fu G, Gascoigne NR, van Oers NS, Wülfing C: Spatiotemporal patterning during T cell activation is highly diverse.Sci Signal 2009, 2:ra15.PubMedView Article
Bamezai A: Lipid rafts and signaling.Immunol Endo and Met Agents in Med Chem 2008, 8:325–326.View Article
Kholodenko BN, Hancock JF, Kolch W: Signaling ballet in space and time.Nat Rev Mol Cell Biol 2010, 11:414–426.PubMedView Article
Dehmelt L, Bastiaens PI: Spatial organization of intracellular communication: insights from imaging.Nat Rev Mol Cell Biol 2010, 11:440–452.PubMedView Article
Alexander RT, Furuya W, Szászi K, Orlowski J, Grinstein S: Rho GTPases dictate the mobility of the Na/H exchanger NHE3 in epithelia: Role in apical retention and targeting.Proc Natl Acad Sci USA 2005, 102:12253–12258.PubMedView Article
Chang JT, Palanivel VR, Kinjyo I, Schambach F, Intlekofer AM, Banerjee A, Longworth SA, Vinup KE, Mrass P, Oliaro J, Killeen N, Orange JS, Russell SM, Weninger W, Reiner SL: Asymmetric T lymphocyte division in the initiation of adaptive immune responses.Science 2007, 315:1687–1691.PubMedView Article
Martin-Belmonte F, Gassama A, Datta A, Yu W, Rescher U, Gerke V, Mostov K: PTEN-mediated apical segregation of phosphoinositides controls epithelial morphogenesis through Cdc42.Cell 2007, 128:383–397.PubMedView Article
Osmani N, Vitale N, Borg JP, Etienne-Manneville S: Scrib controls Cdc42 localization and activity to promote cell polarization during astrocyte migration.Curr Biol 2006, 16:2395–2405.PubMedView Article
Van Keymeulen A, Wong K, Knight ZA, Govaerts C, Hahn KM, Shokat KM, Bourne HR: To stabilize neutrophil polarity, PIP3 and Cdc42 augment RhoA activity at the back as well as signals at the front.J Cell Biol 2006, 174:437–445.PubMedView Article
Wu KY, Hengst U, Cox LJ, Macosko EZ, Jeromin A, Urquhart ER, Jaffrey SR: Local translation of RhoA regulates growth cone collapse.Nature 2005, 436:1020–1024.PubMedView Article
Xavier R, Brennan T, Li Q, McCormack C, Seed B: Membrane compartmentation is required for efficient T cell activation.Immunity 1998, 88:723–732.View Article
Montixi C, Langlet C, Bernard AM, Thimonier J, Dubois C, Wurbel MA, Chauvin JP, Pierres M, He HT: Engagement of T cell receptor triggers its recruitment to low-density detergent-insoluble membrane domains.EMBO J 1998, 17:5334–5348.PubMedView Article
Lingwood D, Simons K: Lipid rafts as a membrane-organizing principle.Science 2010, 327:46–50.PubMedView Article
Kenworthy AK, Edidin M: Distribution of a glycosylphosphatidylinositol-anchored protein at the apical surface of MDCK cells examined at a resolution of < 100 A using imaging fluorescence resonance energy transfer.J Cell Biol 1998, 142:69–84.PubMedView Article
Brown A, London E: Functions of lipid rafts in biological membranes.Annu Rev Cell Dev Biol 1998, 14:111–136.PubMedView Article
Sohn HW, Tolar P, Pierce SK: Membrane heterogeneities in the formation of B cell receptor-Lyn kinase microclusters and the immune synapse.J Cell Biol 2008, 182:367–379.PubMedView Article
Karnovsky MJ, Kleinfeld AM, Hoover RL, Klausner RD: The concept of lipid domains in membranes.J Cell Biol 1982, 94:1–6.PubMedView Article
Pike LJ: Lipid rafts: heterogeneity on the high seas.Biochem J 2004, 378:281–292.PubMedView Article
Vyas KA, Patel HV, Vyas AA, Schnaar RL: Segregation of gangliosides GM1 and GD3 on cell membranes, isolated membrane rafts, and defined supported lipid monolayers.Biol Chem 2001, 382:241–250.PubMedView Article
Schade AE, Levine AD: Lipid raft heterogeneity in human peripheral blood T lymphoblasts: a mechanism for regulating the initiation of TCR signal transduction.J Immunol 2002, 168:2233–2239.PubMed
George S, Nelson MD, Dollahon N, Bamezai A: A novel approach to examining compositional heterogeneity of detergent-resistant lipid rafts.Immunol Cell Biol 2006, 84:192–202.PubMedView Article
Sengupta P, Baird B, Holowka D: Lipid rafts, fluid/fluid phase separation, and their relevance to plasma membrane structure and function.Semin Cell Dev Biol 2007, 18:583–590.PubMedView Article
Harder T, Scheiffele P, Verkade P, Simons K: Lipid domain structure of the plasma membrane revealed by patching of membrane components.J Cell Biol 1998, 141:929–942.PubMedView Article
Ike H, Nakada C, Murase K, Fujiwara T: Lipid domain structure of the plasma membrane revealed by patching of membrane components.Semin Immunol 2005, 17:3–21.PubMedView Article
Zech T, Ejsing CS, Gaus K, de Wet B, Shevchenko A, Simons K, Harder T: Accumulation of raft lipids in T-cell plasma membrane domains engaged in TCR signalling.EMBO J 2009, 28:466–476.PubMedView Article
Burack WR, Lee KH, Holdorf AD, Dustin ML, Shaw AS: Cutting edge: quantitative imaging of raft accumulation in the immunological synapse.J Immunol 2002, 169:2837–2841.PubMed
Douglass AD, Vale RD: Single-molecule tracking of membrane molecules: plasma membrane compartmentalization and dynamic assembly of raft-philic signaling molecules.Cell 2005, 121:937–950.PubMedView Article
Lillemeier BF, Pfeiffer JR, Surviladze Z, Wilson BS, Davis MM: Plasma membrane-associated proteins are clustered into islands attached to the cytoskeleton.Proc Natl Acad Sci USA 2006, 103:18992–18997.PubMedView Article
Dopfer EP, Swamy M, Siegers GM, Molnar E, Yang J, Schamel WW: Segregation models.Adv Exp Med Biol 2008, 640:74–81.PubMedView Article
Balagopalan L, Barr VA, Samelson LE: Endocytic events in TCR signaling: focus on adapters in microclusters.Immunol Rev 2009, 232:84–98.PubMedView Article
Purbhoo MA, Liu H, Oddos S, Owen DM, Neil MA, Pageon SV, French PM, Rudd CE, Davis DM: Dynamics of subsynaptic vesicles and surface microclusters at the immunological synapse.Sci Signal 2010, 3:ra36.PubMedView Article
Hashimoto-Tane A, Yokosuka T, Ishihara C, Sakuma M, Kobayashi W, Saito T: T-cell receptor microclusters critical for T-cell activation are formed independently of lipid raft clustering.Mol Cell Biol 2010, 30:3421–3429.PubMedView Article
Owen DM, Oddos S, Kumar S, Davis DM, Neil MA, French PM, Dustin ML, Magee AI, Cebecauer M: High plasma membrane lipid order imaged at the immunological synapse periphery in live T cells.Mol Membr Biol 2010, 27:178–189.PubMedView Article
Bi K, Altman A: Membrane lipid microdomains and the role of PKCθ in T cell activation.Semin Immunol 2001, 13:139–146.PubMedView Article
Van Komen JS, Mishra S, Byrum J, Chichili GR, Yaciuk JC, Farris AD, Rodgers W: Early and dynamic polarization of T cell membrane rafts and constituents prior to TCR stop signals.J Immunol 2007, 179:6845–6855.PubMed
Geyeregger R, Zeyda M, Zlabinger GJ, Waldhäusl W, Stulnig TM: Polyunsaturated fatty acids interfere with formation of the immunological synapse.J Leukoc Biol 2005, 77:680–688.PubMedView Article
Tavano R, Gri G, Molon B, Marinari B, Rudd CE, Tuosto L, Viola A: CD28 and lipid rafts coordinate recruitment of Lck to the immunological synapse of human T lymphocytes.J Immunol 2004, 173:5392–5397.PubMed
Marwali MR, MacLeod MA, Muzia DN, Takei F: Lipid rafts mediate association of LFA-1 and CD3 and formation of the immunological synapse of CTL.J Immunol 2004, 173:2960–2967.PubMed
Balamuth F, Brogdon JL, Bottomly K: CD4 raft association and signaling regulate molecular clustering at the immunological synapse site.J Immunol 2004, 172:5887–5892.PubMed
Sanui T, Inayoshi A, Noda M, Iwata E, Oike M, Sasazuki T, Fukui Y: DOCK2 is essential for antigen-induced translocation of TCR and lipid rafts, but not PKC-theta and LFA-1, in T cells.Immunity 2003, 19:119–129.PubMedView Article
Hiltbold EM, Poloso NJ, Roche PA: MHC class II-peptide complexes and APC lipid rafts accumulate at the immunological synapse.J Immunol 2003, 170:1329–1338.PubMed
Tavano R, Contento RL, Baranda SJ, Soligo M, Tuosto L, Manes S, Viola A: CD28 interaction with filamin-A controls lipid raft accumulation at the T-cell immunological synapse.Nat Cell Biol 2006, 8:1270–1276.PubMedView Article
Kim W, Fan YY, Barhoumi R, Smith R, McMurray DN, Chapkin RS: n-3 polyunsaturated fatty acids suppress the localization and activation of signaling proteins at the immunological synapse in murine CD4+ T cells by affecting lipid raft formation.J Immunol 2008, 181:6236–6243.PubMed
Macdonald JL, Pike LJ: A simplified method for the preparation of detergent-free lipid rafts.J Lipid Res 2005, 46:1061–1067.PubMedView Article
Yeh ETH, Reiser H, Bamezai A, Rock K: TAP transcription and phosphatidylinositol linkage mutants are defective in activation through the T cell receptor.Cell 1988, 52:665–674.PubMedView Article
Shimonkevitz R, Kappler J, Marrack P, Grey H: Antigen recognition by H-2-restricted T cells. I. Cell-free antigen processing.J Exp Med 1983, 158:303–316.PubMedView Article
Bamezai A, Kennedy C: Cell-free antibody capture method for analysis of detergent-resistant membrane rafts.Methods Mol Biol 2008, 477:137–147.PubMedView Article
Ilangumaran S, Hoessli DC: Effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane.Biochem J 1998, 335:433–440.PubMed
Shogomori H, Brown D: Use of Detergents to Study Membrane Rafts: The Good, the Bad and the Ugly.Biol Chem 2003, 384:1259–1263.PubMedView Article
Janes PW, Ley SC, Magee AI, Kabouridis PS: The role of lipid rafts in T cell antigen receptor (TCR) signalling.Semin Immunol 2000, 12:23–34.PubMedView Article
Drevot P, Langlet C, Guo XJ, Bernard AM, Colard O, Chauvin JP, Lasserre R, He HT: TCR signal initiation machinery is pre-assembled and activated in a subset of membrane rafts.EMBO J 2002, 21:1899–1908.PubMedView Article
Zhang W, Trible RP, Samelson LE: LAT palmitoylation: its essential role in membrane microdomain targeting and tyrosine phosphorylation during T cell activation.Immunity 1998, 9:239–246.PubMedView Article
Varma R, Mayor S: GPI-anchored proteins are organized in submicron domains at the cell surface.Nature 1998, 394:798–801.PubMedView Article
van Zanten TS, Gómez J, Manzo C, Cambi A, Buceta J, Reigada R, Garcia-Parajo MF: Direct mapping of nanoscale compositional connectivity on intact cell membranes.Proc Natl Acad Sci (USA) 2010, 107:15437–15442.View Article
Glebov OO, Nichols BJ: Lipid raft proteins have a random distribution during localized activation of the T-cell receptor.Nat Cell Biol 2004, 6:238–243.PubMedView Article
Eggeling C, Ringemann C, Medda R, Schwarzmann G, Sandhoff K, Polyakova S, Belov VN, Hein B, von Middendorff C, Schönle A, Hell SW: Direct observation of the nanoscale dynamics of membrane lipids in a living cell.Nature 2009, 457:1159–1162.PubMedView Article
Wilson BS, Steinberg SL, Liederman K, Pfeiffer JR, Surviladze Z, Zhang J, Samelson LE, Yang LH, Kotula PG, Oliver JM: Markers for detergent-resistant lipid rafts occupy distinct and dynamic domains in native membranes.Mol Biol Cell 2004, 15:2580–92.PubMedView Article
Donnadieu E, Bismuth G, Trautmann A: Antigen recognition by helper T cells elicits a sequence of distinct changes of their shape and intracellular calcium.Curr Biol 1994, 4:584–595.PubMedView Article
Revy P, Sospedra M, Barbour B, Trautmann A: Functional antigen-independent synapses formed between T cells and dendritic cells.Nat Immunol 2001, 2:925–931.PubMedView Article
Gubina E, Chen T, Zhang L, Lizzio EF, Kozlowski S: CD43 polarization in unprimed T cells can be dissociated from raft coalescence by inhibition of HMG CoA reductase.Blood 2002, 99:2518–2525.PubMedView Article
Balamuth F, Leitenberg D, Unternaehrer J, Mellman I, Bottomly K: Distinct patterns of membrane microdomain partitioning in Th1 and Th2 cells.Immunity 2001, 15:729–738.PubMedView Article
Lillemeier BF, Mörtelmaier MA, Forstner MB, Huppa JB, Groves JT, Davis MM: TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation.Nat Immunol 2010, 11:90–96.PubMedView Article
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