scholarly journals Actin filaments partition primary cilia membranes into distinct fluid corrals

2018 ◽  
Vol 217 (8) ◽  
pp. 2831-2849 ◽  
Author(s):  
Sungsu Lee ◽  
Han Yen Tan ◽  
Ivayla I. Geneva ◽  
Aleksandr Kruglov ◽  
Peter D. Calvert
Keyword(s):  
Diffusion Coefficient ◽  
G Protein ◽  
Primary Cilia ◽  
Super Resolution ◽  
G Protein Coupled Receptors ◽  
Membrane Diffusion ◽  
Filamentous Actin ◽  
Spatiotemporal Resolution ◽  
Fast Tracking ◽  
G Protein Coupled

Physical properties of primary cilia membranes in living cells were examined using two independent, high-spatiotemporal-resolution approaches: fast tracking of single quantum dot–labeled G protein–coupled receptors and a novel two-photon super-resolution fluorescence recovery after photobleaching of protein ensemble. Both approaches demonstrated the cilium membrane to be partitioned into corralled domains spanning 274 ± 20 nm, within which the receptors are transiently confined for 0.71 ± 0.09 s. The mean membrane diffusion coefficient within the corrals, Dm1 = 2.9 ± 0.41 µm2/s, showed that the ciliary membranes were among the most fluid encountered. At longer times, the apparent membrane diffusion coefficient, Dm2 = 0.23 ± 0.05 µm2/s, showed that corral boundaries impeded receptor diffusion 13-fold. Mathematical simulations predict the probability of G protein–coupled receptors crossing corral boundaries to be 1 in 472. Remarkably, latrunculin A, cytochalasin D, and jasplakinolide treatments altered the corral permeability. Ciliary membranes are thus partitioned into highly fluid membrane nanodomains that are delimited by filamentous actin.

scholarly journals Organized cannabinoid receptor distribution in neurons revealed by super-resolution fluorescence imaging

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Hui Li ◽  
Jie Yang ◽  
Cuiping Tian ◽  
Min Diao ◽  
Quan Wang ◽  
...  
Keyword(s):  
G Protein ◽  
Fluorescence Imaging ◽  
Cannabinoid Receptor ◽  
Super Resolution ◽  
G Protein Coupled Receptors ◽  
Cellular Functions ◽  
Cannabinoid Receptor 1 ◽  
Intracellular Organization ◽  
Dynamic Movement ◽  
G Protein Coupled

Abstract G-protein-coupled receptors (GPCRs) play important roles in cellular functions. However, their intracellular organization is largely unknown. Through investigation of the cannabinoid receptor 1 (CB1), we discovered periodically repeating clusters of CB1 hotspots within the axons of neurons. We observed these CB1 hotspots interact with the membrane-associated periodic skeleton (MPS) forming a complex crucial in the regulation of CB1 signaling. Furthermore, we found that CB1 hotspot periodicity increased upon CB1 agonist application, and these activated CB1 displayed less dynamic movement compared to non-activated CB1. Our results suggest that CB1 forms periodic hotspots organized by the MPS as a mechanism to increase signaling efficacy upon activation.

Functional membrane diffusion of G-protein coupled receptors

2007 ◽  
Vol 36 (8) ◽  
pp. 849-860 ◽  
Author(s):  
Aurélie Baker ◽  
Aude Saulière ◽  
Fabrice Dumas ◽  
Claire Millot ◽  
Serge Mazères ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Membrane Diffusion ◽  
Functional Membrane ◽  
G Protein Coupled

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 Discovery of ciliary G protein-coupled receptors regulating pancreatic islet insulin and glucagon secretion

2021 ◽  
Author(s):  
Chien-Ting Wu ◽  
Keren I. Hilgendorf ◽  
Romina J. Bevacqua ◽  
Yan Hang ◽  
Janos Demeter ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
G Protein ◽  
Pancreatic Islet ◽  
Primary Cilia ◽  
G Protein Coupled Receptors ◽  
Endocrine Function ◽  
Prostaglandin E ◽  
Β Cells ◽  
Β Cell ◽  
G Protein Coupled

Multiple G protein-coupled receptors (GPCRs) are expressed in pancreatic islet cells, but the majority have unknown functions. We observed specific GPCRs localized to primary cilia, a prominent signaling organelle, in pancreatic α and β cells. Loss of cilia disrupts β-cell endocrine function, but the molecular drivers are unknown. Using functional expression, we identified multiple GPCRs localized to cilia in mouse and human islet α and β cells, including FFAR4, PTGER4, ADRB2, KISS1R, and P2RY14. Free fatty acid receptor 4 (FFAR4) and prostaglandin E receptor 4 (PTGER4) agonists stimulate ciliary cAMP signaling and promote glucagon and insulin secretion by α- and β-cell lines and by mouse and human islets. Transport of GPCRs to primary cilia requires TULP3, whose knockdown in primary human and mouse islets relocalized ciliary FFAR4 and PTGER4 and impaired regulated glucagon or insulin secretion, without affecting ciliary structure. Our findings provide index evidence that regulated hormone secretion by islet α and β cells is controlled by ciliary GPCRs providing new targets for diabetes.

scholarly journals EJE AWARD 2020: Signalling by G protein-coupled receptors: why space and time matter

2021 ◽  
Vol 184 (2) ◽  
pp. R41-R49
Author(s):  
Davide Calebiro
Keyword(s):  
G Protein ◽  
Single Molecule ◽  
Drug Targets ◽  
Endocrine System ◽  
G Protein Coupled Receptors ◽  
Membrane Receptors ◽  
Optical Methods ◽  
Endocrine Disorders ◽  
Spatiotemporal Resolution ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors and major drug targets. They play a fundamental role in the endocrine system, where they mediate the effects of several hormones and neurotransmitters. As a result, alterations of GPCR signalling are a major cause of endocrine disorders such as congenital hypothyroidism or Cushing’s syndrome. My group develops innovative optical methods such as fluorescence resonance energy transfer (FRET) and single-molecule microscopy, which allow us to investigate GPCR signalling in living cells with unprecedented spatiotemporal resolution. Using this innovative approach, we have contributed to elucidate some long-debated questions about the mechanisms of GPCR signalling and their involvement in human disease. Among other findings, these studies have led to the unexpected discovery that GPCRs are not only signalling at the cell surface, as previously assumed, but also at various intracellular sites. This has important implications to understand how hormones and neurotransmitters produce specific responses in our cells and might pave the way to innovative treatments for common diseases like diabetes or heart failure.

scholarly journals TULP3 bridges the IFT-A complex and membrane phosphoinositides to promote trafficking of G protein-coupled receptors into primary cilia

2010 ◽  
Vol 24 (19) ◽  
pp. 2180-2193 ◽  
Author(s):  
S. Mukhopadhyay ◽  
X. Wen ◽  
B. Chih ◽  
C. D. Nelson ◽  
W. S. Lane ◽  
...  
Keyword(s):  
G Protein ◽  
Primary Cilia ◽  
G Protein Coupled Receptors ◽  
G Protein Coupled
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