scholarly journals The Ciliary Lumen Accommodates Passive Diffusion and Vesicle Trafficking in Cytoplasmic-Ciliary Transport

2019 ◽  
Author(s):  
Andrew Ruba ◽  
Wangxi Luo ◽  
Jingjie Yu ◽  
Daisuke Takao ◽  
Athanasios Evangelou ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
G Protein ◽  
Single Molecule ◽  
Primary Cilium ◽  
Intraflagellar Transport ◽  
Vesicle Transport ◽  
Passive Diffusion ◽  
G Protein Coupled Receptors ◽  
Live Cells ◽  
G Protein Coupled

AbstractTransport of membrane and cytosolic proteins into the primary cilium is essential for its role in cellular signaling. Using single molecule microscopy, we mapped the movement of membrane and soluble proteins at the base of the primary cilium. In addition to the well-known intraflagellar transport (IFT) route, we identified two new pathways within the lumen of the primary cilium - passive diffusional and vesicle transport routes - that are adopted by proteins for cytoplasmic-cilium transport in live cells. Independent of the IFT path, approximately half of IFT motors (KIF3A) and cargo (α-tubulin) take the passive diffusion route and more than half of membrane-embedded G protein coupled receptors (SSTR3 and HTR6) use RAB8A-regulated vesicles to transport into and inside cilia. Furthermore, ciliary lumen transport is the preferred route for membrane proteins in the early stages of ciliogenesis and inhibition of SSTR3 vesicle transport completely blocks ciliogenesis. Furthermore, clathrin-mediated, signal-dependent internalization of SSTR3 also occurs through the ciliary lumen. These transport routes were also observed in Chlamydomonas reinhardtii flagella, suggesting their conserved roles in trafficking of ciliary proteins.

scholarly journals Mini G protein probes for active G protein–coupled receptors (GPCRs) in live cells

2018 ◽  
Vol 293 (19) ◽  
pp. 7466-7473 ◽  
Author(s):  
Qingwen Wan ◽  
Najeah Okashah ◽  
Asuka Inoue ◽  
Rony Nehmé ◽  
Byron Carpenter ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Live Cells ◽  
G Protein Coupled

Quantitative Single-Residue Bioorthogonal Labeling of G Protein-Coupled Receptors in Live Cells

2019 ◽  
Vol 14 (6) ◽  
pp. 1141-1149 ◽  
Author(s):  
Robert Serfling ◽  
Lisa Seidel ◽  
Andreas Bock ◽  
Martin J. Lohse ◽  
Paolo Annibale ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Live Cells ◽  
Bioorthogonal Labeling ◽  
G Protein Coupled

scholarly journals Imaging of G protein-coupled receptors in solid-supported planar membranes at the single molecule level

2008 ◽  
Author(s):  
Iwan Märki ◽  
Marcel Leutenegger ◽  
Matthias Geissbuehler ◽  
Rudolf Robelek ◽  
Eva-Kathrin Sinner ◽  
...  
Keyword(s):  
G Protein ◽  
Single Molecule ◽  
G Protein Coupled Receptors ◽  
Single Molecule Level ◽  
G Protein Coupled ◽  
Planar Membranes

scholarly journals Spirohexene-Tetrazine Ligation Enables Bioorthogonal Labeling of Class B G Protein-Coupled Receptors in Live Cells

2017 ◽  
Vol 139 (38) ◽  
pp. 13376-13386 ◽  
Author(s):  
Carlo P. Ramil ◽  
Maoqing Dong ◽  
Peng An ◽  
Tracey M. Lewandowski ◽  
Zhipeng Yu ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Live Cells ◽  
Bioorthogonal Labeling ◽  
Class B ◽  
G Protein Coupled ◽  
Tetrazine Ligation

scholarly journals Metastable GPCR dimers trigger the basal signal by recruiting G-proteins

2020 ◽  
Author(s):  
Rinshi S. Kasai ◽  
Takahiro K. Fujiwara ◽  
Akihiro Kusumi
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Single Molecule ◽  
Resting State ◽  
G Protein Coupled Receptors ◽  
Β2 Adrenergic Receptor ◽  
Low Levels ◽  
Protein Recruitment ◽  
Novel Drug ◽  
G Protein Coupled

G-protein-coupled receptors (GPCRs) constitute the largest family of integral membrane proteins in the human genome and are responsible for various important signaling pathways for vision, olfaction, gustation, emotion, cell migration, etc. A distinct feature of the GPCR-family proteins is that many GPCRs, including the prototypical GPCR, β2-adrenergic receptor (β2AR), elicit low levels of basal constitutive signals without agonist stimulation, which function in normal development and various diseases1–3. However, how the basal signals are induced is hardly known. Another general distinctive feature of GPCRs is to form metastable homo-dimers, with lifetimes on the order of 0.1 s, even in the resting state. Here, our single-molecule-based quantification4 determined the dissociation constant of β2AR homo-dimers in the PM (1.6 ± 0.29 copies/μm2) and their lifetimes (83.2 ± 6.4 ms), and furthermore found that, in the resting state, trimeric G-proteins were recruited to both β2AR monomers and homo-dimers. Importantly, inverse agonists, which suppress the GPCR’s basal constitutive activity, specifically blocked the G-protein recruitment to GPCR homo-dimers, without affecting that to monomers. These results indicate that the G-proteins recruited to transient GPCR homo-dimers are responsible for inducing their basic constitutive signals. These results suggest novel drug development strategies to enhance or suppress GPCR homo-dimer formation.

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.

Single-molecule force spectroscopy of G-protein-coupled receptors

2013 ◽  
Vol 42 (19) ◽  
pp. 7801 ◽  
Author(s):  
Michael Zocher ◽  
Christian A. Bippes ◽  
Cheng Zhang ◽  
Daniel J. Müller
Keyword(s):  
G Protein ◽  
Single Molecule ◽  
Force Spectroscopy ◽  
G Protein Coupled Receptors ◽  
Single Molecule Force Spectroscopy ◽  
G Protein Coupled
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