Ex Vivo Single-molecule Analysis of Ryanodine Receptor 2 Assembly in Cardiac and Neuronal Tissue

Summary

This study presents a vesicle-based, single-molecule imaging method that preserves native receptor organization, enabling precise stoichiometry quantification and broad applications in receptor assembly, function, and disease research.

Abstract

Understanding receptor assembly is critical for elucidating the mechanisms underlying their function and regulation in physiological processes. While traditional in vitro single-molecule studies rely on isolating proteins from heterologous expression systems, they often fail to capture the in vivo physiological complexity involved in the organization and assembly of cell surface proteins. This protocol employs Total Internal Reflection Fluorescence Microscopy (TIRFM) to study GFP-tagged Ryanodine Receptor 2 (RyR2) molecules encapsulated within nanoscale vesicles. These vesicles, generated from organs rapidly extracted from the animal, effectively provide a snapshot of the receptor's assembly state at the time of extraction, enabling detailed analysis of subunit stoichiometry and receptor organization in response to changes in the animal's physiological environment. This approach utilizes TIRFM and stepwise photobleaching analysis to provide a readout of receptor stoichiometry. Imaging receptors at the single-molecule level facilitates the detection of heterogeneity within the receptor populations assembled in a live animal and enables monitoring of assembly changes associated with disease states. As a result, this method offers a powerful tool for determining the distribution of receptor assemblies and their correlation to changes in their physiological environment.

Introduction

Membrane receptors play crucial roles in physiological processes throughout the human body¹. A primary function is to mediate communication between cells through the transfer of extracellular stimuli to initiate intracellular signaling events. Because of their fundamental role in signaling, nearly 70% of existing therapeutics target membrane receptors². They are often oligomeric structures composed of multiple subunits assembled into heteromeric forms with multiple stoichiometries^3,4. Understanding how receptors organize into functional complexes is critical for elucidating their roles in health and disease^5,6. Traditional biochemical methods, such as immunoprecipitation and western blotting, provide averaged ensemble measurements, which obscure heterogeneity in receptor subpopulations⁷. In contrast, single-molecule techniques allow for the direct observation of individual receptors, enabling quantification of heterogeneity within receptor populations^8,9,10. Single-molecule techniques have become the standard to quantify membrane receptor assembly, stoichiometry, and oligomerization^11,12. This approach has been widely applied to many physiologically relevant membrane receptors using isolated protein or cell-culture conditions. Single-molecule studies primarily rely on in vitro expression systems, where proteins can be isolated from a cellular environment. However, the complexity within a living system is not fully reflected in these types of heterologous expression systems.

To overcome these limitations, we developed an approach that isolates and images membrane receptors in a physiologically relevant state by capturing them within nanovesicles derived directly from cardiac and neuronal tissues. This method effectively preserves the receptor assembly state at the time of vesicle formation, allowing for the direct visualization of receptor subunit stoichiometry, which is a pivotal indicator of the response of the receptor in different physiological environments. This approach allows us to examine the stoichiometric assembly of membrane receptors that occur with the complex biological environment within live animals without requiring heterologous expression or reconstitution. By employing Total Internal Reflection Fluorescence Microscopy (TIRFM), we achieve single-molecule resolution imaging of vesicle-encapsulated receptors and extract photobleaching traces to determine subunit composition.

TIRFM is well-suited for single-molecule membrane protein imaging because it selectively excites fluorophores within ~150 nm of the glass surface, reducing background fluorescence from molecules in solution^3,10. This confinement significantly enhances the signal-to-noise ratio, enabling accurate detection of individual membrane receptor complexes. By analyzing stepwise photobleaching of fluorescently tagged receptors, we can directly determine subunit stoichiometry and assess potential heterogeneity within receptor populations¹². This is particularly relevant for Ryanodine Receptor 2 (RyR2), a large intracellular Ca²⁺ release channel that plays a crucial role in excitation-contraction coupling in the heart, where it only assembles as a homo-tetramer, and in synaptic function in the brain, where its assembly has not been established^1,13,14. Given the presence of multiple RyR isoforms in neuronal tissues, it remains unclear whether RyR2 forms purely homomeric tetramer complexes in the brain, consisting of four identical subunits as it does in the heart, or whether heteromeric assemblies with RyR1 or RyR3 occur in vivo^15,16,17,18.

By isolating vesicles from specific regions of the heart and brain, this method enables the investigation of potential tissue-specific differences in RyR2 organization. We take advantage of the properties of RyR2 in cardiac tissue where it can only form homotetramers to provide a statistically robust basis for ex vivo single-molecule subunit stoichiometry, and the known tetramer assembly serves as a control for investigating unknown assemblies in other organs. The ability to image receptors in their native state while preserving their physiological interactions provides a robust framework for studying receptor assembly in different organs and under different physiological conditions. This protocol describes the workflow for vesicle isolation, immobilization, and single-molecule imaging of GFP-tagged RyR2, allowing for precise characterization of receptor stoichiometry. This technique not only advances our understanding of RyR2 organization in cardiac and neuronal tissues but also offers broader applications for studying receptor assembly and regulation in various physiological and pathological contexts^19,20,21.

Protocol

1. Sample preparation

1. Transcardial perfusion and organ extraction
   1. Anesthetize the mouse using CO₂ until respiration ceases.
   2. Position the mouse in a supine position and secure the limbs.
   3. Make a midline incision along the abdomen and extend laterally toward the ribcage to expose the thoracic cavity.
   4. Carefully insert a needle into the left ventricle and transect the right atrium to allow perfusate drainage.
   5. For vesicle preparation, perfuse with cold 1x PBS until the circulatory system is cleared of blood.
   6. For cryosectioning, after PBS perfusion, continue perfusing with 4% paraformaldehyde (PFA) to fix tissues.
   7. Carefully extract the heart and place it immediately on ice.
   8. Using fine scissors, carefully cut along the midline of the scalp and peel back the skin to expose the skull.
   9. Make an incision at the base of the skull near the foramen magnum.
   10. Using fine forceps, carefully pry open the skull along the longitudinal fissure, removing bone fragments to expose the brain.
   11. Gently lift the brain using curved forceps, severing the cranial nerves to fully extract it intact.
   12. If used for cryosectioning, post-fix the brain in 4% PFA at 4 °C for 24 h.
   13. If used for vesicle preparation, immediately place the brain on ice without fixation for further processing.
2. Cryosectioning
   1. Remove the brain or heart from 4% PFA and blot dry.
   2. Embed the entire organ in OCT compound within a cryomold.
   3. Freeze the embedded tissue by placing the mold in a -80 °C freezer until fully solidified.
   4. Before sectioning, equilibrate the cryostat chamber and specimen head to -20 °C.
   5. Transfer the frozen OCT block from the -80 °C freezer to the cryostat chamber and allow it to equilibrate for 10 min.
   6. Clean the specimen chuck with 70% ethanol, dry it, and apply a thin layer of OCT as an adhesive.
   7. Secure the frozen OCT block onto the chuck, ensuring the correct orientation.
   8. Insert the chuck into the cryostat specimen holder and adjust the blade position.
   9. Trim away excess OCT until the tissue surface is exposed.
   10. Section the tissue at 10 µm thickness, collecting sections onto glass slides.
   11. After air-drying the sections at room temperature for 1 h, apply mounting media over the sections and place a coverslip to seal the sample.
   12. Allow the mounting media to set before storing the slides at 4 °C for imaging.
3. Vesicle preparation
   1. Cardiac vesicle preparation
      1. Extract the heart following transcardial perfusion with 1x PBS and place it on ice.
      2. Using a sterile scalpel, dissect approximately 30 mg of ventricular muscle from the bottom of the heart.
      3. Transfer the tissue to a centrifuge tube containing 4-5 mL of 10 mM NaHCO₃ with 5 mM NaN₃ to maintain vesicle integrity.
      4. Homogenize the tissue using a homogenizer until fully lysed.
      5. Centrifuge the homogenate at 10,000 × g for 20 min to remove large debris and collect the supernatant.
      6. Perform ultracentrifugation at 37,000 × g for 65 min, then discard the supernatant and resuspend the pellet in 3-4 mL of 20 mM Tris-maleate buffer (pH 6.8) with 0.6 M KCl.
      7. Repeat ultracentrifugation at 37,000 × g for another 65 min to remove solubilized actomyosin.
      8. Resuspend the final pellet in 0.5 mL of 20 mM Tris-maleate buffer (pH 6.8) with 50 mM KCl to obtain the cardiac vesicle solution and store it at -80 °C.
   2. Brain vesicle preparation
      1. Extract the brain following transcardial perfusion with 1x PBS and place it on ice.
      2. Slice the brain into 1 mm sections using a 1 mm mouse brain coronal matrix.
      3. Identify the third and fourth sections from the olfactory bulb and carefully dissect: 1) the outermost cortical layer from both hemispheres; 2) the hippocampus, located in the central region of the section.
      4. From the posterior end, isolate the cerebellum by making a transverse cut at its base.
      5. Pool the same brain regions from five mice to ensure adequate vesicle yield.
      6. Homogenize the tissue immediately using a Dounce homogenizer in 2 mL of cold homogenization buffer (0.32 M sucrose, 10 mM HEPES, 2 mM EDTA, protease inhibitor, pH 7.4).
      7. Add 3 mL of additional homogenization buffer and centrifuge at 200 × g for 15 min to remove large tissue fragments.
      8. Collect the supernatant and centrifuge at 1,000 × g for 15 min at 4 °C to remove the nuclear fraction.
      9. Further centrifuge the supernatant at 10,000 × g for 20 min at 4 °C to remove mitochondria.
      10. Ultracentrifuge the resulting supernatant at 100,000 × g for 2 h at 4 °C to pellet the vesicles.
      11. Resuspend the final vesicle pellet in 1x PBS to obtain the brain vesicle solution and store it at -80 °C.
4. Nanovesicle characterization via Nanoparticle Tracking Analysis (NTA)
   1. Assemble the detection cell following the manufacturer's instructions.
   2. Turn on the instrument and launch the compatible software.
   3. Load a 1 mL syringe with deionized (DI) water and manually flush the system tubing until the background is clear of contaminants.
   4. Prepare the nanovesicle sample by diluting it in 1x PBS.
   5. Load ~1 mL of the diluted nanovesicle sample into a clean 1 mL syringe, ensuring no air bubbles.
   6. Manually inject the sample into the detection cell and adjust the focus until vesicles are clearly visible.
   7. Secure the syringe onto the syringe pump, and set the flow rate to 200 µL/s in the hardware panel.
   8. In the SOP panel, set the measurement duration to 60 s, the number of cycles to 5, and confirm temperature control is enabled and set to 25 °C. Save the settings as a new SOP by clicking Save SOP and naming the file appropriately (e.g., "nanovesicle_standard.sop").
   9. In the Capture panel, configure the instrument settings by selecting a camera level of 13 and an initial detection threshold of 3.
   10. Click Run and select the previously saved SOP. The software will automatically carry out the measurement according to the settings.
   11. Once completed, click Analyze to process each video. Adjust the detection threshold to ensure all particles are clearly visible, and click Finish after batch analysis is complete. Export the result by clicking Export Data | All Measurements, and save as a CSV file for later use.
   12. After completing the experiment, flush the system with ~ 2 mL of DI water until no residual vesicles remain.
   13. Inject air through the tubing to remove any remaining liquid, ensuring the system is dry for the next use.

2. Imaging

1. Confocal imaging of tissue cryosections
   1. Power on the laser scanning confocal microscope and open the linked software.
   2. Select the appropriate objective (20x air or 60x oil) and load the prepared cryosection slide onto the microscope stage.
   3. Configure the laser settings for GFP excitation using a 488 nm laser line with a 535/70 nm emission filter.
   4. Optimize imaging parameters, including pinhole size ( 28.9 µm), laser intensity (17%), and detector gain (15.3), to enhance the signal-to-noise ratio while minimizing photobleaching.
   5. If necessary, acquire a Z-stack by setting the top and bottom focal planes and defining the appropriate step size (0.275 µm).
   6. Enable automated image stitching with a 15% image overlap when acquiring large-area images.
   7. Adjust image resolution and apply frame averaging if needed to reduce background noise.
   8. Capture images and save them in '.nd2' format for further analysis.
2. Total Internal Reflection Fluorescence (TIRF) imaging
   1. Vesicle immobilization via biocompatible anchor for membrane (BAM) method²²
      1. Clean 35 mm glass-bottom dishes by sonicating them in 5 M NaOH for 1 h at 45 °C, followed by three thorough DI water rinses.
      2. Sonicate the dishes in 0.1 M HCl for another 1 h at 45 °C, followed by three additional DI water rinses.
      3. Treat the cleaned glass surface with 2% 3-aminopropyltriethoxysilane (APTES) in ethanol for 30 min at room temperature to introduce amine functional groups.
      4. Rinse the dish 3x with DI water and ethanol to remove excess APTES and allow it to dry.
      5. Incubate the dish with 100 µM Oleyl-O(CH₂CH₂)nCO-CH₂CH₂-COO-NHS (Sunbright OE-020CS) in 1x PBS for 10 min.
      6. Wash the dish with PBS to remove unreacted reagent.
      7. Introduce vesicles at the desired concentration and incubate for 15 min to allow vesicle binding.
      8. Rinse the dish gently with PBS to remove unbound vesicles while ensuring the immobilized vesicles remain attached.
   2. TIRF imaging setup and acquisition
      1. Power on the inverted fluorescence microscope 488 nm laser source, and EMCCD camera.
      2. Launch Metamorph and CellSens software to initialize imaging control.
      3. Clean the 60x oil immersion objective (1.49 NA APO) with methanol to remove dust and residual oil.
      4. Apply a small drop of refractive index (RI) matching immersion oil onto the objective lens.
      5. Place the prepared vesicle dish onto the microscope stage, ensuring it is centered for optimal alignment.
      6. Adjust the laser beam focus by centering the excitation beam in the middle of the objective's back aperture in epifluorescence mode.
      7. Use the stepper motor to adjust the laser incident angle until the critical angle is reached, ensuring total internal reflection at the glass surface.
      8. Identify the focal plane by scanning through the Z-axis, distinguishing: 1) the first focus plane, corresponding to the glass-bottom surface; 2) the second focus plane, corresponding to the vesicle-immobilized surface.
      9. Once vesicles are in focus, apply auto-focus in CellSens to lock the objective position at this plane, preventing drift during imaging.
      10. Temporarily turn off the laser and move the microscope stage to a fresh field of view that has not yet been exposed to excitation light.
      11. Start recording while simultaneously turning on the laser, ensuring the capture of the full photobleaching process without prior exposure.
      12. Acquire images with the following parameters: Exposure time: 100 ms per frame, Acquisition mode: 900 continuous frames per field of view, Gain setting: 1,400.
      13. Repeat the process for multiple fields of view to ensure sufficient data collection.
      14. Save image stacks in TIFF format for further processing.

3. Image processing and data analysis

1. Confocal image processing
   1. Open the acquired confocal images in the linked software.
   2. Adjust the Look-Up Table (LUT) settings to optimize brightness and contrast.
   3. Apply the same LUT adjustments uniformly across all images to maintain visual consistency.
2. TIRF video processing and fluorescence intensity analysis
   1. Video processing for visualization
      1. Open the acquired TIRF image stacks (TIFF format) in ImageJ for visualization.
      2. Adjust brightness and contrast to enhance signal clarity, ensuring fluorescent vesicles are easily distinguishable from the background.
      3. Export adjusted images for presentation or further qualitative analysis.
   2. Photobleaching step analysis
      1. Extracting time traces
         1. Open the MATLAB script (see Supplemental File 1) named "getTimeTraces.m" and run it. In the graphical user interface (GUI), click Load Movie and select the TIRF imaging .tiff file to import the vesicle movie stack.
         2. Click Find Peaks to automatically identify single-molecule fluorescence signals. Signal regions will be marked with circles in the preview window.
         3. Click Populate Data. This will export the time-intensity trace for each spot into an .xlsx file for downstream analysis.
      2. Manual step counting
         1. Run the "simpleGraph.m" script (see Supplemental File 1) to launch the GUI, click Load Data Files and select the .xlsx file generated in step 3.2.2.1.3.
         2. Inspect the bleaching steps of each time trace displayed in the left panel. Ensure that only traces meeting the following criteria are accepted for analysis: 1) the time trace contains at least one distinct bleaching step. 2) The duration of one step is at least 1 s. 3) The intensity difference between step levels is at least 3x the standard deviation of the post-bleaching intensity.
         3. On the right, locate the Step # field and use the + or - buttons to input the number of photobleaching steps for each trace.
         4. After completing each entry, click Next to move to the following trace. Once all traces are processed, the software will automatically generate and save a .txt file recording the step count of each trace.
      3. Step count summary
         1. Run the "CombinedFiles.m" script (see Supplemental File 1) to open the GUI.
         2. Type the desired name for the summary output in the textbox.
         3. Click Combine multiple simpleGraph Results to one file. In the file browser, select one or more desired .txt files generated from step 3.2.2.2.3. The script will automatically compile all step count data and generate a combined .xlsx file summarizing the frequency of 1-step, 2-step,… traces.

Results

To confirm the applicability of this method, we first verified the expression and localization of GFP-RyR2 by confocal imaging of cardiac and neuronal tissue cryosections (Figure 1). The fluorescence signal in cardiac tissue displayed a transverse striated pattern consistent with RyR2's expected localization in cardiomyocytes but exhibited high intensity in certain regions in the brain, namely cortex, hippocampus, and cerebellum, indicating region-specific ex...

Discussion

This study establishes a detailed and reproducible protocol for ex vivo encapsulation and single-molecule imaging of membrane receptors using GFP-tagged RyR2 as a model system. The protocol significantly advances current methods by enabling stoichiometric analysis of receptor complexes directly from native tissue, preserving the physiological context of membrane proteins. Compared to traditional in vitro expression systems, this approach minimizes artifacts introduced by overexpression or detergent solubilizatio...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35 mm Dish | No. 1.5 Coverslip | 14 mm Glass Diameter	Mattek	P35G-1.5-14-C
Andwin Scientific Tissue-Tek Cryomold Molds/Adapters	Fisher Scientific	NC9542860
APTES	Millipore Sigma	440140-100ML	(3-Aminopropyl)triethoxysilane
Bio-Gen PRO200 Laboratory Homogenizer	Pro Scientific	1204B59
cellSENS	Olympus Scientific Solutions	n/a
EMCCD camera	Andor	n/a	iXon Ultra 897
Ethanol absolute ≥100% (v/v) USP for molecular biology (200 Proof)	VWR	71006-012
Leica CM1860	Leica	n/a	cryostat
MetaMorph Advanced	Molecular Devices	n/a
Nanosight NS300 (with Nanosight NTA software)	Malvern Panalytical	n/a
Nikon AXR Inverted Confocal Microscope	Nikon	n/a
Olympus IX83 Motorized Autofocus Inverted Fluorescence Microscope	Olympus	n/a	TIRFM
Paraformaldehyde 4% in PBS ready to use fixative reagent	VWR	76221-378
Phosphate Buffered Saline (PBS) 20x, Ultra Pure Grade	VWR	97062-950
Sodium hydroxide, beads, Reagent Grade	VWR	97064-526
SUNBRIGHT OE-020CS +	NOF America Corporation	n/a	Oleyl-O(CH2CH2)nCO-CH2CH2-COO-NHS
Tissue-Tek O.C.T. Compound	Sakura	M71484
VectaMount AQ Aqueous Mounting Medium	Vector Laboratories	H-5501-60