Deciphering the Receptor Repertoire Encoding Specific Odorants by Time-Lapse Single-Cell Array Cytometry.

Masato Suzuki,Nobuo Yoshimoto,Ken Shimono,Shun’Ichi Kuroda

doi:10.1038/srep19934

Masato Suzuki, Nobuo Yoshimoto + Show 2 more

Open Access

https://doi.org/10.1038/srep19934

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Journal: Scientific reports	Publication Date: Feb 2, 2016
Citations: 15	License type: CC BY 4.0

Affiliation: Osaka University, Panasonic (Japan)

Abstract

Mammals can recognize a vast number of odorants by using olfactory receptors (ORs) known as G protein-coupled receptors. The OR gene family is one of the most diverse gene families in mammalian genomes. Because of the vast combinations of ORs and odorants, few ORs have thus far been linked to specific odorants. Here, we established a functional screening method for OR genes by using a microchamber array containing >5,400 single olfactory epithelium-derived cells from mice applied to time-lapse single-cell array cytometry. This method facilitated the prompt isolation of single olfactory sensory neurons (OSNs) responding to the odorant of interest. Subsequent single-cell RT-PCR allowed us to isolate the genes encoding respective ORs. By using volatile molecules recognized as biomarkers for lung cancers, this method could deorphanize ORs and thereby reconstitute the OR-mediated signaling cascade in HEK293T cells. Thus, our system could be applied to identify any receptor by using specific ligands in the fields of physiopathology and pharmacology.

Highlights

When screening olfactory receptors (ORs) that respond to a specific odorant, native olfactory sensory neurons (OSNs) activated by odorants can be identified using Ca2+ imaging[9]
For screening OR genes from OSNs under physiological conditions, we planned to prepare the cell array of olfactory epithelium (OE)-derived cells, measure Ca2+ influx elicited by odorant stimulation, and retrieve responsive OSNs by an automated single-cell analysis and isolation system
The robot was vested with the following 3 key components to realize the time-lapse single-cell array cytometry analysis (Fig. 1): (i) A microchamber array chip containing 202,500 wells (10 μ m diameter, Fig. 1a) for aligning single OE-derived cells at high density. (ii) An open perfusion system (Fig. 1b) for exchanging solutions on 57,600 wells of the chip continuously; the upper surface of the system is open for access of the glass capillary. (iii) An automated single-cell analysis and isolation system (Fig. 1c) for the acquisition of fluorescent intensity of each OSN on the microchamber array in a time-resolved manner, identification of OSNs responding to a specific odorant and automated isolation of assigned OSNs by glass capillary equipped on the micromanipulator

Summary

Introduction

When screening ORs that respond to a specific odorant, native OSNs activated by odorants can be identified using Ca2+ imaging[9]. To isolate target OSNs expressing an odorant-specific OR from a cell library, after stimulation with an odorant, it is necessary to measure the transient increase of intracellular Ca2+ concentration in each cell using time-resolved analysis for at least 5 min[7]. We have developed an automated single-cell analysis and isolation system to facilitate high-throughput isolation of fluorescently labeled mammalian cells on a cell array in an undisruptive and single cell-based manner[17]. The time-lapse single-cell array cytometry could analyze the extracellular calcium influx of ~5,400 olfactory epithelium-derived cells in a single-cell and time-resolved manner, and could retrieve positive single cells in an undisruptive manner for further analyses (functional assays, omics analyses, DNA sequencing)

Methods

Results

Conclusion