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 ....

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 ....

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