Faculty Opinions recommendation of Single-molecule imaging reveals receptor-G protein interactions at cell surface hot spots.

2017 ◽  
Author(s):  
Arun Shukla
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Single Molecule ◽  
Protein Interactions ◽  
Hot Spots ◽  
Single Molecule Imaging

scholarly journals Single-molecule imaging reveals receptor–G protein interactions at cell surface hot spots

Nature ◽  
2017 ◽  
Vol 550 (7677) ◽  
pp. 543-547 ◽  
Author(s):  
Titiwat Sungkaworn ◽  
Marie-Lise Jobin ◽  
Krzysztof Burnecki ◽  
Aleksander Weron ◽  
Martin J. Lohse ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Single Molecule ◽  
Protein Interactions ◽  
Hot Spots ◽  
Single Molecule Imaging

scholarly journals Title: Single-molecule diffusion-based estimation of ligand effects on G protein-coupled receptors

2017 ◽  
Author(s):  
Masataka Yanagawa ◽  
Michio Hiroshima ◽  
Yuichi Togashi ◽  
Mitsuhiro Abe ◽  
Takahiro Yamashita ◽  
...  
Keyword(s):  
Diffusion Coefficient ◽  
Cell Surface ◽  
G Protein ◽  
Single Molecule ◽  
G Protein Coupled Receptors ◽  
Single Molecule Imaging ◽  
Imaging Analysis ◽  
Coated Pits ◽  
Ligand Effects ◽  
G Protein Coupled

AbstractG protein-coupled receptors (GPCRs) are major drug targets and have high potential for drug discovery. The development of a method for measuring the activities of GPCRs is essential for pharmacology and drug screening. However, it is difficult to measure the effects of a drug by monitoring the receptor on the cell surface, and changes in the concentrations of downstream signaling molecules, which depend on signaling pathway selectivity of the receptor, are used as an index of the receptor activity. Here, we show that single-molecule imaging analysis provides an alternative method for assessing ligand effects on GPCR. We monitored the dynamics of the diffusion of metabotropic glutamate receptor 3 (mGluR3), a class C GPCR, under various ligand conditions by using total internal reflection fluorescence microscopy (TIRFM). The single-molecule tracking analysis demonstrates that changes in the average diffusion coefficient of mGluR3 quantitatively reflect the ligand-dependent activity. Then, we reveal that the diffusion of receptor molecules is altered by the common physiological events associated with GPCRs, including G protein binding or accumulation in clathrin-coated pits, by inhibition experiments and dual-color single-molecule imaging analysis. We also confirm the generality of agonist-induced diffusion change in class A and B GPCRs, demonstrating that the diffusion coefficient is a good index for estimating the ligand effects on many GPCRs regardless of the phylogenetic groups, chemical properties of the ligands, and G protein-coupling selectivity.One Sentence Summary: Single-molecule imaging for evaluating ligand effects on GPCRs by monitoring the diffusion dynamics on the cell surface.

scholarly journals Revealing the Stoichiometry of G Protein-Coupled Receptors (GPCRs) at the Cell Surface using Single Molecule Imaging

2013 ◽  
Vol 104 (2) ◽  
pp. 525a ◽  
Author(s):  
Laura Weimann ◽  
Steven F. Lee ◽  
James H. Felce ◽  
Simon J. Davis ◽  
David Klenerman
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Single Molecule ◽  
G Protein Coupled Receptors ◽  
Single Molecule Imaging ◽  
G Protein Coupled

scholarly journals Optimised insert design for improved single-molecule imaging and quantification through CRISPR-Cas9 mediated knock-in

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Abdullah O. Khan ◽  
Carl W. White ◽  
Jeremy A. Pike ◽  
Jack Yule ◽  
Alexandre Slater ◽  
...  
Keyword(s):  
Genome Editing ◽  
G Protein ◽  
Single Molecule ◽  
Protein Function ◽  
Single Molecule Imaging ◽  
Dependent Effect ◽  
Systematic Comparison ◽  
Selection Strategies ◽  
Localisation Microscopy ◽  
G Protein Coupled

Abstract The use of CRISPR-Cas9 genome editing to introduce endogenously expressed tags has the potential to address a number of the classical limitations of single molecule localisation microscopy. In this work we present the first systematic comparison of inserts introduced through CRISPR-knock in, with the aim of optimising this approach for single molecule imaging. We show that more highly monomeric and codon optimised variants of mEos result in improved expression at the TubA1B locus, despite the use of identical guides, homology templates, and selection strategies. We apply this approach to target the G protein-coupled receptor (GPCR) CXCR4 and show a further insert dependent effect on expression and protein function. Finally, we show that compared to over-expressed CXCR4, endogenously labelled samples allow for accurate single molecule quantification on ligand treatment. This suggests that despite the complications evident in CRISPR mediated labelling, the development of CRISPR-PALM has substantial quantitative benefits.

scholarly journals Apelin receptor homodimer-oligomers revealed by single-molecule imaging and novel G protein-dependent signaling

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Xin Cai ◽  
Bo Bai ◽  
Rumin Zhang ◽  
Chunmei Wang ◽  
Jing Chen
Keyword(s):  
G Protein ◽  
Single Molecule ◽  
Single Molecule Imaging ◽  
Apelin Receptor

scholarly journals Single molecule imaging of small G protein Ras

2000 ◽  
Vol 40 (supplement) ◽  
pp. S32
Author(s):  
H. Matsuura ◽  
Y. Arai ◽  
H. Yokota ◽  
A. Iwane ◽  
Y. Ishii ◽  
...  
Keyword(s):  
G Protein ◽  
Single Molecule ◽  
Single Molecule Imaging ◽  
Small G Protein
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