High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Summary

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.

Abstract

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 (GABA_B) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

Introduction

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-/ol....

Protocol

1. Sample Preparation

1. Coverslip cleaning
   NOTE: Work under a fume hood.
   1. Use clean tweezers to place glass coverslips (24 mm diameter) into a coverslip holder that separates individual coverslips.
   2. Put the holder with coverslips into a beaker, and add chloroform until the coverslips are covered. Cover the beaker with aluminum foil to reduce evaporation and sonicate in a bath sonicator for 1 hr at RT. Take the coverslip holder out of the beaker and let the coverslips dry.
   3. Repeat step 1.1.2 with 5M NaOH solution instead of chloroform.
   4. Take the coverslip holder into a new beaker and wash three ti....

Results

The described protocol can be applied to a variety of different membrane proteins. As an example, representative results obtained with β₂-adrenergic and GABA_B 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 phen.......

Discussion

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 a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 plate	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