Analysis of detergent-free lipid rafts isolated from CD4+ T cell line: interaction with antigen presenting cells promotes coalescing of lipid rafts
© Kennedy et al; licensee BioMed Central Ltd. 2011
Received: 29 June 2011
Accepted: 8 December 2011
Published: 8 December 2011
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.
Keywordsraft coalescence CD4+ T cells antigen presenting cells electron microscopy raft-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
Visualization and determination of size of lipid rafts using electron microscopy
Lipid rafts coalesce in the presence of antigen presenting cells
Analysis of detergent-extracted lipid rafts
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.
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