We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.
The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer’s attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.
The immunological synapse (IS) lies at a critical junction for cell activation and function1. It is the primary medium by which antigen presentation and cell-mediated immunity are carried out. The earliest microscopic studies of synapse formation utilized a cell-cell conjugate system2. The major limitation with this approach is that most of the conjugates will be viewed ‘in profile’, as it were, thus restricting the observer's view of the synaptic structure itself. In 1999, the Dustin laboratory addressed this limitation by utilizing the glass-supported lipid bilayer (SLB) technique3, which had been pioneered earlier by the McConnel laboratory4,5. This approach disposed of antigen presenting cells (APCs) in favor of a glass-supported planar lipid surface, into which proteins could be attached and move freely in two dimensions. Using this method, Dustin and colleagues were able to peer directly up into the synapse using high resolution fluorescence microscopy, and for the first time get a “face-to-face” look at the structure of the IS.
With the use of SLB system, the detail with which the IS can be visualized has been restricted only by the limitations of current imaging techniques6-8. Using standard illumination techniques, the minimum resolution (i.e., minimum distance between two distinct objects wherein they can be distinguished) has been < 200 nm on the basis of Rayleigh’s criterion9. This limit hinders the imaging of very fine, molecular-scale structures that make up the synapse, and until the development of super-resolution imaging techniques10-12, visualization of these structures was confined to imaging of fixed cells using electron microscopy.
With the recent advent of a variety of super-resolution techniques, such as SIM (structured illumination microscopy), PALM (photoactivated localization microscopy), STORM (stochastical optical reconstruction microscopy), and STED10-12, investigators are now able to study these synaptic structures in unprecedented detail, which has in turn provided an increasingly clarified understanding of the IS. The advantages of STED microscopy have been described before13. Here we describe super-resolution imaging with STED microscopy equipped with the newly-developed 660 nm depletion laser. Compared to the conventional 592 nm depletion laser, the 660 nm laser allows for a broader selection of fluorescent dyes (see http://nanobiophotonics.mpibpc.mpg.de/old/dyes/), especially these red fluorophores.
Other publications have described the STED imaging of NK cell synapses on antibody-coated glass slides13,14. Here, the SLB system is combined with super-resolution STED microscopy to study the NK cell synapse. This technique has the advantage over antibody-coated slides of being a fluid mosaic, in which the embedded surface proteins can move freely in a flat two-dimensional surface (x-y plane). This more faithfully mimics the organic and mobile surface of a target cell, and consequently better recapitulates the formation of a physiologically relevant immune synapse.
The goal of this protocol is to provide the end-user with a detailed description of how to image the immunological synapse of NK cells by combining the SLB system and super-resolution STED microscopy. It will provide the end user with the steps necessary to: prepare the liposomes, construct protein-embedded bilayers, determine the protein density on the lipid bilayers, and acquire super-resolution images using STED microscopy. These techniques are not limited to the field of immunology, and can be broadly utilized across a variety of disciplines.
The novelty of the current study is that it combines the SLB technique with STED to study NK cell synapses. Previous studies have imaged the lipid bilayer with TIRF to study T cell synapse formation8 and signaling molecule trafficking on the plasma membrane6. Others have described STED imaging of NK cell synapses using antibody-coated glass slides13,14. The hybrid method described herein further builds upon these efforts by imaging the NK cell synapse with the enhanced clarity afforded by super-resolution imaging on the lipid bilayer surface, which better models the dynamic surface of an APC.
Although SLBs are artificial membranes lacking of cytoskeleton, lipid rafts, and other ligands that actual target cells or APCs possess, this technique can recapitulate important features such as the mobility and orientation of ligands. This allows the SLB system to serve as a reductionist approach in dissecting the contribution of individual receptors and ligands to formation of the IS and the dynamics of IS. The most important feature of SLBs is that researchers can combine this technique with high-resolution imaging approaches, such as confocal and TIRF microscopy. The introduction of STED microscopy even further increases this advantage, providing unprecedented insights into IS research and its clinical applications.
One potential criticism of this system is that the SLB does not adequately mimic the complex surface of an APC, thus giving rise to potentially non-physiological anatomical features in the resulting synapses. While it is true that the limited repertoire of surface molecules on the SLB does not fully recapitulate the heterogeneously populated surface of an APC, this limit can also be advantageous in that it allows investigators to determine the influence of individual receptor and ligand interactions on synapse formation.
There are several crucial steps in the process. Among the most critical is that oxidation of the liposomes be prevented through constantly using argon to displace the oxygen in the tube and solution, such as in steps 1.10, 2.6, and 2.11. Oxidation of the lipids will result in decreased lipid mobility, thus impeding the ability of surface proteins to move freely and participate in synaptic structuring. Likewise, it is also crucial to remove all the chloroform in the liposome by lyophilization (step 1.2). In the determination of protein density in the lipid bilayer, it is of importance to first disperse the silicon beads into a homogenous suspension free from clusters. If necessary, sonication of beads can be applied. In the assembling the SLB, the early steps (5.1-5.8) wherein the coverslips are cleaned, the drops are placed, and coverslip is affixed are vital. A mistake in any of these can necessitate starting the experiment over (from the beginning of section 5). For this reason, it is good practice to clean more coverslips than will be needed to save time in case of a mishap.
Non-clustering is the most frequent issue when working with this system. If, when visualizing the cells in the final step, one fails to find any fluorescent synapses, there are a few steps that can be taken. Another stain for the cognate cell-surface receptor can be added to the chamber to verify that the cell has not formed a synapse with the bilayer, While cells can adhere non-specifically to the glass coverslip or bilayer surface, synaptically-involved surface proteins should appear as distinct clusters at the plane of the cell-bilayer interface, whereas unengaged surface proteins should appear as diffuse staining around the perimeter of the cell. Should this method fail, one should check their cells via flow cytometry to ensure that the particular marker one is hoping to study is expressed in adequate abundance on the cell surface. Certain surface proteins are known to be down-regulated over long term in vivo culture.
While this protocol details specifically how to visualize NK cell synapse formation, the SLB system can be used to study synapse formation in any immunocyte imaginable simply by substitution of the primary ligand in step 5.11. Multiple ligands may also be added simultaneously. One also need not use a streptavidin-biotin system for adhering the surface proteins within the bilayer. Nickel-NTA:histidine interactions are also viable. However, due to the high strength and specificity of the streptavidin:biotin interaction, our lab prefers this system. One may also vary the concentration of cells added onto the bilayer from the prescribed density in step 5.13, as well as the duration of the subsequent incubation period in order to observe synapses at different stages of maturation. This can even be done live, although this of course excludes the possibility of visualizing intracellular structures (unless they are already labeled with a fused fluorescent tag; our lab uses a few such altered cell lines). Due to the high degree of customization possible in this protocol, one can use the basic SLB technique, along with STED imaging, to address an incredibly diverse range of questions in immunology, cell biology, and biochemistry, including basic lipid dynamics15, synapse formation16, intracellular signaling17, and tumor cell metastasis18.
The authors have nothing to disclose.
We thank Emily Mace and Malini Mukherjee for imaging and deconvolution analysis. Work in the Liu laboratory was supported in part by the Baylor-UTHouston Center for AIDS Research Core Support Grant number AI36211 from the National Institute of Allergy and Infectious Diseases, the Caroline Wiess Law Fund for Research in Molecular Medicine, Texas Children’s Hospital Pediatric Pilot Research Fund, and the Lymphoma SPORE Developmental Research Program from Baylor College of Medicine and the Methodist Research Institute.
Name of Reagent/ Equipment | Company | Catalog Number | Purpose |
18:1 (Δ9-Cis) PC (DOPC) 1,2-dioleoyl-sn-glycero-3-phosphocholine |
Avanti | 850375C | Liposome preparation |
18:1 Biotinyl Cap PE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt) |
Avanti | 870273C | Liposome preparation |
Argon gas, compressed | Airgas | UN1006 | Liposome preparation |
A lyophilizer | Labconco | Freezone 7740020 | Liposome preparation |
Lyophilizer tubes | Labconco | 7540200 | Liposome preparation |
Chromatography Columns | Santa Cruz | sc-205558 | Liposome preparation |
1 M Tris pH 8.0 | Ambion | AM9856 | Liposome preparation |
5 M NaCl | Ambion | AM9759 | Liposome preparation |
Octyl-β-D-glucopyranoside | Sigma-Aldrich | O1008 | Liposome preparation |
Dialysis tubing | Spectrum Labs | 132676 | Liposome preparation |
96-well V-bottom polystyrene plate (untreated) | Corning | 3896 | antibody density determination |
Non-functionalized silica beads | Bangs Laboratories | SS06N | antibody density determination |
FACS tubes | Fisher Scientific | 14-959-2A | antibody density determination |
QuantumTM MESF beads | Bangs Laboratories | Variable | antibody density determination |
Casein | Sigma-Aldrich | C0875 | Lipid bilayer preparation |
HEPES buffered saline + 1% HSA | Homemade | Lipid bilayer preparation | |
Streptavidin | Life technologies | 434301 | Lipid bilayer preparation |
Fluorescently-labeled biotinylated protein | Homemade | Lipid bilayer preparation | |
ibidi Sticky-Slide VI 0.4 | ibidi | 80608 | Lipid bilayer preparation |
25×75 mm glass coverslip | ibidi | 10812 | Lipid bilayer preparation |
Hydrogen peroxide (30%) | Fisher Scientic | BP2633 | Lipid bilayer preparation |
Sulfuric acid | Sigma-Aldrich | 258105 | Lipid bilayer preparation |
D-Biotin | Invitrogen | B20656 | Lipid bilayer preparation |
Lens paper | VWR | 54826-001 | For imaging |
Type F Immersion Oil | Leica | 11 513 859 | For imaging |
A Leica TCS STED microscope | Leica | For imaging |