This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.
Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., β2-adrenergic and γ-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.
Receptors located on the cell surface sense the extracellular environment and respond to a variety of stimuli, such as odorants, ions, small neurotransmitters and large protein hormones. The fluid nature of cellular membranes allows movements of receptors and other membrane proteins. This is essential for the formation of protein complexes and the occurrence of transient protein-protein interactions, such as those used by receptors to assemble into functional units and transduce signals into the cell interior. For instance, G-protein-coupled receptors (GPCRs), which constitute the largest family of cell-surface receptors1, have been suggested to form di-/oligomers, which appears to be involved in the fine-tuned regulation of signal transduction and might have important physiological and pharmacological consequences2-5.
Single-molecule microscopy has the great potential of directly visualizing with high spatiotemporal resolution the dynamic behavior of individual receptors located on the surface of living cells, including their association to form dimers and higher order molecular complexes6-10. This offers several advantages compared to standard biochemical and microscopy methods, which usually report the average behavior of thousands or millions of molecules.
Protein labeling with a sufficiently bright and photostable fluorophore is essential for single-molecule microscopy. This protocol takes advantage of the recently introduced SNAP tag11 to covalently attach small and bright organic fluorophores to cell-surface receptors. SNAP is a 20 kD protein tag derived from the human DNA repair enzyme O6-alkylguanine-DNA alkyltransferase, which can be irreversibly labeled with fluorophore-conjugated benzylguanine (fluorophore-BG) derivatives. CLIP, a further engineered tag derived from SNAP, can be instead labeled with fluorophore-conjugated benzylcytosine derivatives12.
The protocol reported in this manuscript explains how to transfect and label SNAP-tagged11 receptors with small organic fluorophores and use total internal reflection fluorescence (TIRF) microscopy to visualize single receptors or receptor complexes on the surface of living cells10. The reported protocol results in >90% labeling efficiency of an extracellularly SNAP-tagged cell-surface protein10. Further information on how to use single-molecule data to analyze the size and mobility of receptor complexes, as well as to capture transient receptor-receptor interactions, is provided. A schematic workflow of the entire protocol is given in Figure 1. As an example, the transfection of Chinese Hamster Ovary (CHO) cells with SNAP-tagged G-protein-coupled receptors (GPCRs) followed by labeling with a fluorophore-BG derivative as well as its application to quantify and monitor receptor di-/oligomerization are described. This protocol can be extended to other cell-surface proteins and fluorescent tags (e.g., CLIP), as well as to other transfection and labeling methods.
1. Sample Preparation
2. Image Acquisition
NOTE: Use a total internal reflection fluorescence (TIRF) microscope, equipped with an oil-immersion high numerical aperture objective (e.g., 100X magnification/1.46 numerical aperture), suitable lasers (e.g., 405 nm, 488 nm, 561 nm and 645 nm diode lasers), an electron multiplying charge coupled device (EMCCD) camera, an incubator and a temperature control to visualize single fluorescent molecules.
3. Calibration (Single Fluorophores on Glass and Monomeric/dimeric Receptor Controls)
4. Image Analysis
The described protocol can be applied to a variety of different membrane proteins. As an example, representative results obtained with β2-adrenergic and GABAB receptors are reported10. Since fluorescent signals from single molecules are weak, minimization of background fluorescence is the first key step to successful results. Thus, it is important to use extensively cleaned coverslips (Figure 2A) as well as to minimize sample autofluorescence (e.g., by using phenol-red free media). The next step is to determine the fluorescence intensity of single fluorophore molecules. This can be done by imaging single fluorophores spotted on a clean coverslip (Figure 2B). Typically, a serial dilution of the fluorophore is used to select the best conditions for the analysis, i.e., the concentration that produces well-separated and uniformly distributed single fluorophores. Additional controls can be performed by imaging monomeric control proteins, e.g., NAP-tagged CD86 receptors labeled with a fluorophore-BG derivative. Once these preliminary controls and calibration have been performed, the real experiments can begin. Figure 3A shows the first frame of a typical TIRF image sequence of a cell transfected with SNAP-tagged β2-adrenergic receptors and labeled with a fluorophore-BG derivative. The spots represent single receptors or receptor complexes. This image also demonstrates a suitable particle density for automated detection and tracking – according to our experience, densities above 0.45 particles/µm2 result in poor tracking quality and should be avoided10. Figure 3B shows the results of the detection algorithm applied to the same image sequence. Each blue circle indicates a detected particle. The results of the tracking algorithm are reported in Figure 3C, where the blue splines represent the trajectories of the individual particles. The trajectories of each particle can then be used to calculate their diffusion coefficients. This method also allows capturing dynamic events as shown in Figure 3D, where two particles apparently undergo a transient interaction. Figure 4 shows the distribution of diffusion coefficients measured for two different GPCRs, i.e., β2-adrenergic and GABAB receptors. This type of analysis allowed us to show that a large fraction of GABAB receptors is immobile or has a very low mobility10. The mixed Gaussian fitting and step-fitting analyses provide a precise quantification of the size of receptor complexes on the surface of living cells (Figure 5). This analysis can also reveal complex distributions, e.g., the coexistence of monomers and dimers as shown in the example of Figure 5A. Figures 5B and 5C provide two examples of particles bleaching in one or two steps and the corresponding step-fitting analysis that correctly assigned them as monomeric and dimeric receptors, respectively.
Figure 1. Workflow of a typical single-molecule experiment as detailed in this protocol. Please click here to view a larger version of this figure.
Figure 2. Coverslip cleaning and preparation of calibration samples. (A) Comparison of the background fluorescence before and after extensive coverslip cleaning. Coverslips were imaged by TIRF microscopy. (B) TIRF images of increasing concentrations of a fluorophore-BG derivative spotted on cleaned coverslips. Scale bar: 10 µm. Please click here to view a larger version of this figure.
Figure 3. Typical single-molecule images and results of detection/tracking algorithms. (A) CHO cells transfected with SNAP-tagged β2-adrenergic receptors and labeled with a fluorophore-BG derivative were visualized by TIRF microscopy. Shown is the first frame of the acquired image sequence. Scale bar: 5 µm. (B) Detected particles are indicated by blue circles on top of the original image. (C) The trajectories resulting from the application of the tracking algorithm to the same image sequence are shown on a white background. The snapshot corresponds to the situation at frame no. 35. Green segments, merging events. Red segments, splitting events. (D) Representative trajectories obtained from two receptor particles (blue and green, respectively), undergoing an apparent transient interaction (red). The two particles merge, move together for several frames, and split again. Please click here to view a larger version of this figure.
Figure 4. Analysis of receptor mobility. The trajectories of individual particles are used to calculate their diffusion coefficients. In this example, the distributions of diffusion coefficients calculated for two different GPCRs are reported. β2-adrenergic receptors are characterized by fast lateral diffusion on the cell surface, whereas GABAB receptors have limited mobility. Modified from Calebiro, D. et al.10
Figure 5. Analysis of the size of receptor complexes. (A) The size of receptor complexes can be precisely estimated by fitting the distributions of particle intensities with a mixed Gaussian model. The reported example shows the application of this analysis to a cell expressing SNAP-tagged β2-drenergic receptors. The fitting reveals two components: one corresponding to the intensity of single fluorophores (largely superimposable to that of calibration samples as well as to the distribution obtained after partial bleaching of the sample, shown here) and one with approximately double intensity. The two components can be assigned as monomers and dimers, respectively, and the areas under the two components can be used to estimate the relative abundance of monomeric and dimeric receptors. (B) Example of a monomeric receptor particle bleaching in one step. (C) Example of a dimeric receptor particle with the characteristic two-step bleaching. The red lines in (B) and (C) are the result of the step-fitting algorithm. The figure was modified from Calebiro, D. et al.10 Please click here to view a larger version of this figure.
The described protocol allows analysis of the spatial arrangement, mobility and size of cell-surface receptor complexes at single-molecule level. Compared to the use of fluorescent proteins, labeling with small organic fluorophores, which are brighter and more photostable, has the advantage of permitting extended visualization of single receptor particles. Since extremely low expression levels are achieved (<0.45 receptor particles/µm2), the properties of receptors and other membrane proteins can be analyzed at densities that do not exceed physiological ones. Moreover, the effects of receptor stimulation with agonists10 or other manipulations, for instance aimed at reproducing a pathological situation, can be analyzed. In addition, due to the flexibility of the labeling strategy with SNAP/CLIP tags11,12, different fluorophores can be used according to one´s specific needs – Noticeably, the combination of SNAP and CLIP tags can be used to perform two-color experiments, e.g., to observe the colocalization between two interacting proteins. Finally, this protocol can be modified at several points, for instance by using different cells, transfection methods and labeling strategies.
Critical steps include the minimization of background and autofluorescence (by using extensively cleaned coverslips, phenol-red free media and filtered solutions), the optimization of transfection conditions (e.g., amount of plasmid DNA and time after transfection) to achieve extremely low expression levels, efficient labeling and the choice of a bright and sufficiently photostable fluorophore. Concerning the choice of the fluorophore, red/far-red ones usually give better results because cell autofluorescence is higher in the blue/green part of the visible spectrum and usually almost negligible above 550 nm. Particular attention should be paid to avoiding photobleaching of the fluorophores during the search for a suitable cell and focus adjustment. Samples with fluorophores spotted on glass coverslips as well as monomeric and dimeric receptor controls (e.g., CD86 with either one or two SNAP tags)10 should be considered to calibrate the analysis and check labeling efficiency.
The limits of this approach are largely dependent on the spatiotemporal resolution that can be currently achieved. This is mostly dictated by the number of photons collected from one fluorophore as well as by the sensitivity and acquisition speed of the camera used. Typical values for the localization precision of a single, detected particle are 20 – 30 nm (for comparison the spatial resolution of conventional fluorescence microscopy is about 200 – 300 nm). These values are still larger than the real size of a typical membrane protein (about 2 – 8 nm). Since receptors falling at a distance below the diffraction limit of the microscope (about 200 – 300 nm) are detected as a single particle, appropriate controls and statistical analyses should be used to subtract random colocalizations (false positives) from the number of true receptor-receptor interactions8-10.The maximal acquisition rate achievable with current EMCCD cameras can exceed 1 KHz, at least in crop mode (i.e., only a part of the sensor is used). However, the acquisition speed is also limited by the number of photons emitted by a single fluorophore that reach the camera. In practice, exposure times of at least 10 – 20 msec are generally needed. For this reason, typical acquisition speeds vary between 10 and 50 Hz, i.e., one frame every 20 to 100 msec. Future developments in fluorophore design, optical components and detection technology might allow to further increase the spatiotemporal resolution of single-molecule methods.
The authors have nothing to disclose.
The development of this protocol was supported by grants from the European Research Council (Advanced Grant TOPAS to M.J.L.) and the Deutsche Forschungsgemeinschaft (Grants CA 1014/1-1 to D.C. and SFB487 to M.J.L.). T.S. was supported by the Alexander von Humboldt Foundation.
Chloroform | AppliChem GmbH | A1585 | CAUTION: toxic and irritating substance as well as a possible carcinogen |
NaOH | Sigma-Aldrich | S8045 | CAUTION: strong base and highly corrosive reagent |
Absolute ethanol | Sigma-Aldrich | 32205 | |
Glass coverslip | Marienfeld-Superior | 111640 | 24 mm diameter, 0.13-0.16 mm thickness |
0.2 mm sterile filter | Sarstedt | 83.1826.001 | |
CHO cells | ATCC, USA | ATCC CCL-61 | Chinese hamster ovary cell line |
6-well cell culture plare | Nunc | 140675 | |
DMEM/F-12 medium | GIBCO, Life Technologies | 11039-021 | Phenol-red free medium |
Fetal bovine serum | Biochrom | S 0115 | |
Penicillin – streptomycin | Pan Biotech GmbH | P06-07 100 | |
Trypsin-EDTA | Pan Biotech GmbH | P10-23100 | |
Lipofectamine 2000 | Invitrogen, Life Technologies | 11668-019 | |
Opti-MEM I Reduced Serum Medium | Invitrogen, Life Technologies | 31985-047 | |
Fluorophore-conjugated benzylguanine | New England BioLabs | S9136S | SNAP-Surface Alexa Fluor 647. Make a 1 mM stock solution in DMSO. Store at -20°C. |
DMSO | AppliChem GmbH | A1584 | |
Imaging buffer: | 137 mM NaCl, 5.4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.3, sterile-filtered | ||
NaCl | AppliChem GmbH | A1371 | |
KCl | AppliChem GmbH | A3582 | |
CaCl2 | AppliChem GmbH | A2303 | |
MgCl2 | AppliChem GmbH | A3618 | |
HEPES | AppliChem GmbH | A3724 | |
Imaging chamber | Molecular Probes, Life Technologies | A-7816 | Attofluor Cell Chamber, for microscopy |
TIRF-M | Leica | Model: DMI6000B | |
TIRF objective | Leica | 11 506 249 | HCX PL Apo 100x/1.46 Oil CORR |
EM-CCD camera | Roper Scientific | Photometrics Cascade 512B | |
Temperature controller | Pecon | Tempcontrol 37-2 digital | |
ImageJ software | NIH, USA | http://rsbweb.nih.gov/ij | |
u-track software | Laboratory for computational cell biology, Dept. of Cell Biology, Harvard Medical School, USA | http://lccb.hms.harvard.edu/software.html | |
Matlab software | The MathWorks, USA |