scholarly journals Total workflows of the single-molecule imaging analysis in living cells: a tutorial guidance to the measurement of the drug effects on a GPCR

2020 ◽  
Author(s):  
Masataka Yanagawa ◽  
Yasushi Sako
Keyword(s):  
Single Molecule ◽  
Metabotropic Glutamate Receptor ◽  
Particle Density ◽  
Drug Effects ◽  
Quantitative Information ◽  
Single Molecule Imaging ◽  
Single Molecule Tracking ◽  
Metabotropic Glutamate ◽  
Protein Protein Interaction ◽  
Diffusion Dynamics

AbstractSingle-molecule imaging (SMI) is a powerful method to measure the dynamics of membrane proteins on the cell membrane. The single-molecule tracking (SMT) analysis provides information about the diffusion dynamics, the oligomer size distribution, and the particle density change. The affinity and on/off-rate of a protein—protein interaction can be estimated from the dual-color SMI analysis. However, it is difficult for trainees to determine quantitative information from the SMI movies. The present protocol guides the detailed workflows to measure the drug-activated dynamics of a G protein-coupled receptor (GPCR) and metabotropic glutamate receptor 3 (mGluR3), by using the total internal reflection fluorescence microscopy (TIRFM). This tutorial guidance comprises an open-source software named smDynamicsAnalyzer, with which one can easily analyze the SMT dataset by just following the workflows after building a designated folder structure (https://github.com/masataka-yanagawa/IgorPro8-smDynamicsAnalyzer).

scholarly journals Single-molecule imaging of telomerase RNA reveals a Recruitment – Retention model for telomere elongation

2020 ◽  
Author(s):  
Hadrien Laprade ◽  
Emmanuelle Querido ◽  
Michael J. Smith ◽  
David Guérit ◽  
Hannah Crimmins ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Single Molecule ◽  
Cellular Level ◽  
Telomerase Rna ◽  
Single Molecule Imaging ◽  
Retention Model ◽  
Single Molecule Tracking ◽  
Time Dynamics ◽  
Telomere Elongation ◽  
Spatio Temporal

AbstractExtension of telomeres is a critical step in the immortalization of cancer cells. This complex reaction requires proper spatio-temporal coordination of telomerase and telomeres, and remains poorly understood at the cellular level. To understand how cancer cells execute this process, we combined CRISPR genome editing and MS2 RNA-tagging to image single-molecules of telomerase RNA (hTR). Real-time dynamics and photoactivation experiments of hTR in Cajal bodies (CBs) reveal that hTERT controls the exit of hTR from CBs. Single-molecule tracking of hTR at telomeres shows that TPP1-mediated recruitment results in short telomere-telomerase scanning interactions, then base-pairing between hTR and telomere ssDNA promotes long interactions required for stable telomerase retention. Interestingly, POT1 OB-fold mutations that result in abnormally long telomeres in cancers act by enhancing this retention step. In summary, single-molecule imaging unveils the life-cycle of telomerase RNA and provides a framework to understand how cancer-associated mutations mechanistically drive defects in telomere homeostasis.

scholarly journals Single molecule analysis reveals positive allosteric modulators are required to stabilize the active state of a metabotropic glutamate receptor

2021 ◽  
Author(s):  
Anne-Marinette Cao ◽  
Robert B. Quast ◽  
Fataneh Fatemi ◽  
Philippe Rondard ◽  
Jean-Philippe Pin ◽  
...  
Keyword(s):  
Single Molecule ◽  
Metabotropic Glutamate Receptors ◽  
Metabotropic Glutamate Receptor ◽  
Brain Diseases ◽  
Allosteric Modulators ◽  
Fast Kinetics ◽  
Single Molecule Level ◽  
Metabotropic Glutamate ◽  
Subtype Selectivity ◽  
Positive Allosteric Modulators

AbstractMetabotropic glutamate receptors (mGlu) are G protein-coupled receptors that represent promising targets for brain diseases. Much hope in drug development come from the discovery of positive allosteric modulators that display subtype selectivity, and act by increasing agonist potency, as well as efficacy in most cases. How such compounds can influence agonist efficacy remains unclear. Here, we explore the structural dynamics of the full-length mGlu2 dimers at submillisecond timescales using single molecule FRET on diffusing receptors in optimized detergent micelles. We show that glutamate binding in the Venus flytrap extracellular domains does not stabilize fully the receptors in their active states. The full activation of all receptors can only be observed in the presence of either a positive allosteric modulator or the Gi protein. Our results provide important new insights on the fast kinetics and the action of the allosteric modulators on mGlu activation at the single molecule level.

scholarly journals Light Sheet Illumination for 3D Single-Molecule Super-Resolution Imaging of Neuronal Synapses

2021 ◽  
Vol 13 ◽  
Author(s):  
Gabriella Gagliano ◽  
Tyler Nelson ◽  
Nahima Saliba ◽  
Sofía Vargas-Hernández ◽  
Anna-Karin Gustavsson
Keyword(s):  
Single Molecule ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Super Resolution ◽  
Light Sheet ◽  
Three Dimensions ◽  
Single Molecule Imaging ◽  
Single Molecule Tracking ◽  
Resolution Imaging

The function of the neuronal synapse depends on the dynamics and interactions of individual molecules at the nanoscale. With the development of single-molecule super-resolution microscopy over the last decades, researchers now have a powerful and versatile imaging tool for mapping the molecular mechanisms behind the biological function. However, imaging of thicker samples, such as mammalian cells and tissue, in all three dimensions is still challenging due to increased fluorescence background and imaging volumes. The combination of single-molecule imaging with light sheet illumination is an emerging approach that allows for imaging of biological samples with reduced fluorescence background, photobleaching, and photodamage. In this review, we first present a brief overview of light sheet illumination and previous super-resolution techniques used for imaging of neurons and synapses. We then provide an in-depth technical review of the fundamental concepts and the current state of the art in the fields of three-dimensional single-molecule tracking and super-resolution imaging with light sheet illumination. We review how light sheet illumination can improve single-molecule tracking and super-resolution imaging in individual neurons and synapses, and we discuss emerging perspectives and new innovations that have the potential to enable and improve single-molecule imaging in brain tissue.

scholarly journals Investigating the Heteromerization of Metabotropic Glutamate Receptors using a Novel Single Molecule Imaging Method

2018 ◽  
Vol 114 (3) ◽  
pp. 531a
Author(s):  
Alexander L. Van Slyke ◽  
Avtar Singth ◽  
Nitya Deshmukh ◽  
Paul J. Kammermeier ◽  
Warren R. Zipfel
Keyword(s):  
Glutamate Receptors ◽  
Single Molecule ◽  
Metabotropic Glutamate Receptors ◽  
Imaging Method ◽  
Single Molecule Imaging ◽  
Metabotropic Glutamate

scholarly journals Single-molecule imaging and tracking of molecular dynamics in living cells

2017 ◽  
Vol 4 (5) ◽  
pp. 739-760 ◽  
Author(s):  
Nan Li ◽  
Rong Zhao ◽  
Yahong Sun ◽  
Zi Ye ◽  
Kangmin He ◽  
...  
Keyword(s):  
Single Molecule ◽  
Quantitative Information ◽  
Living Cells ◽  
Cellular Systems ◽  
Single Molecule Imaging ◽  
Biomolecular Interaction ◽  
Ensemble Averaging ◽  
Spatiotemporal Resolution ◽  
Detection And Tracking ◽  
Single Molecule Studies

Abstract Unlike the ensemble-averaging measurements, the single-molecule imaging and tracking (SMIT) in living cells provides the real-time quantitative information about the locations, kinetics, dynamics and interactions of individual molecules in their native environments with high spatiotemporal resolution and minimal perturbation. The past decade has witnessed a transforming development in the methods of SMIT with living cells, including fluorescent probes, labeling strategies, fluorescence microscopy, and detection and tracking algorithms. In this review, we will discuss these aspects with a particular focus on their recent advancements. We will then describe representative single-molecule studies to illustrate how the single-molecule approaches can be applied to monitor biomolecular interaction/reaction dynamics, and extract the molecular mechanistic information for different cellular systems.

scholarly journals Conformational dynamics between transmembrane domains and allosteric modulation of a metabotropic glutamate receptor

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Vanessa A Gutzeit ◽  
Jordana Thibado ◽  
Daniel Starer Stor ◽  
Zhou Zhou ◽  
Scott C Blanchard ◽  
...  
Keyword(s):  
Single Molecule ◽  
Metabotropic Glutamate Receptors ◽  
Metabotropic Glutamate Receptor ◽  
Conformational Dynamics ◽  
Resonance Energy ◽  
Transmembrane Domains ◽  
Metabotropic Glutamate ◽  
Receptor Modulation ◽  
Gpcr Activation ◽  
Class C

Metabotropic glutamate receptors (mGluRs) are class C, synaptic G-protein-coupled receptors (GPCRs) that contain large extracellular ligand binding domains (LBDs) and form constitutive dimers. Despite the existence of a detailed picture of inter-LBD conformational dynamics and structural snapshots of both isolated domains and full-length receptors, it remains unclear how mGluR activation proceeds at the level of the transmembrane domains (TMDs) and how TMD-targeting allosteric drugs exert their effects. Here, we use time-resolved functional and conformational assays to dissect the mechanisms by which allosteric drugs activate and modulate mGluR2. Single-molecule subunit counting and inter-TMD fluorescence resonance energy transfer measurements in living cells reveal LBD-independent conformational rearrangements between TMD dimers during receptor modulation. Using these assays along with functional readouts, we uncover heterogeneity in the magnitude, direction, and the timing of the action of both positive and negative allosteric drugs. Together our experiments lead to a three-state model of TMD activation, which provides a framework for understanding how inter-subunit rearrangements drive class C GPCR activation.

scholarly journals Single-molecule imaging of cholesterol-dependent cytolysin assembly

2021 ◽  
Author(s):  
Michael J Senior ◽  
Carina Monico ◽  
Eve E Weatherill ◽  
Robert Gilbert ◽  
Alejandro Heuck ◽  
...  
Keyword(s):  
Temporal Resolution ◽  
Single Molecule ◽  
Single Channel ◽  
Single Molecule Imaging ◽  
Single Channel Recording ◽  
Single Molecule Tracking ◽  
Perfringolysin O ◽  
Assembly Pathway

We exploit single-molecule tracking and optical single channel recording in droplet interface bilayers to resolve the assembly pathway of the Cholesterol-Dependent Cytolysin, Perfringolysin O. This enables quantification of the stoichiometry of PFO complexes during assembly with millisecond temporal resolution and 20 nanometre spatial precision. Our results support a model of overall stepwise irreversible assembly, dominated by monomer addition, but with infrequent assembly from larger partial complexes. Furthermore, our results suggest a dominant proportion of inserted, but non-conductive intermediates in assembly.

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