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.

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 analysis of the entire perfringolysin O pore formation pathway

2021 ◽  
Author(s):  
Conall Mc Guinness ◽  
James Walsh ◽  
Charles Bayly-Jones ◽  
Michelle Dunstone ◽  
Craig Morton ◽  
...  
Keyword(s):  
Single Molecule ◽  
Pore Formation ◽  
Membrane Insertion ◽  
Formation Pathway ◽  
Perfringolysin O ◽  
Assembly Pathway ◽  
Bacterial Virulence Factor ◽  
Membrane Bound ◽  
Single Molecule Analysis ◽  
Kinetics Of

The cholesterol-dependent cytolysin perfringolysin O (PFO) is secreted by Clostridium perfringens as a bacterial virulence factor able to form giant ring-shaped pores that perforate and ultimately lyse mammalian cell membranes. To resolve the kinetics of all steps in the assembly pathway, we have used single-molecule fluorescence imaging to follow the dynamics of PFO on dye-loaded liposomes that lead to opening of a pore and release of the encapsulated dye. Formation of a long-lived membrane-bound PFO dimer nucleates the growth of an irreversible oligomer. The growing oligomer can insert into the membrane and open a pore at stoichiometries ranging from tetramers to full rings (~35-mers), whereby the rate of insertion increases linearly with the number of subunits. Oligomers that insert before the ring is complete continue to grow by monomer addition post insertion. Overall, our observations suggest that PFO membrane insertion is kinetically controlled.

scholarly journals Microsecond Single Molecule Tracking: Probing Protein Diffusion at High Spatial and Temporal Resolution

2011 ◽  
Vol 100 (3) ◽  
pp. 350a
Author(s):  
Anna Pezzarossa ◽  
Fabien Pinaud ◽  
Stefan Semrau ◽  
Thomas Schmidt
Keyword(s):  
Temporal Resolution ◽  
Single Molecule ◽  
Protein Diffusion ◽  
Single Molecule Tracking

scholarly journals Single-Molecule Imaging of Perfringolysin O Binding and Assembly on Model Membranes

2016 ◽  
Vol 110 (3) ◽  
pp. 415a
Author(s):  
Michael J. Senior ◽  
Carina Monico ◽  
Alejandro P. Heuck ◽  
Robert J.C. Gilbert ◽  
Mark I. Wallace
Keyword(s):  
Single Molecule ◽  
Model Membranes ◽  
Single Molecule Imaging ◽  
Perfringolysin O

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 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.

Label-free, mass-sensitive single-molecule imaging using interferometric scattering microscopy

2020 ◽  
Author(s):  
Nikolas Hundt
Keyword(s):  
Single Molecule ◽  
Cellular Systems ◽  
Single Molecule Imaging ◽  
Label Free ◽  
Measurement Sensitivity ◽  
Mass Distributions ◽  
Quantitative Measurements ◽  
Contrast Mechanism ◽  
Cellular Components ◽  
Scattering Signal

Abstract Single-molecule imaging has mostly been restricted to the use of fluorescence labelling as a contrast mechanism due to its superior ability to visualise molecules of interest on top of an overwhelming background of other molecules. Recently, interferometric scattering (iSCAT) microscopy has demonstrated the detection and imaging of single biomolecules based on light scattering without the need for fluorescent labels. Significant improvements in measurement sensitivity combined with a dependence of scattering signal on object size have led to the development of mass photometry, a technique that measures the mass of individual molecules and thereby determines mass distributions of biomolecule samples in solution. The experimental simplicity of mass photometry makes it a powerful tool to analyse biomolecular equilibria quantitatively with low sample consumption within minutes. When used for label-free imaging of reconstituted or cellular systems, the strict size-dependence of the iSCAT signal enables quantitative measurements of processes at size scales reaching from single-molecule observations during complex assembly up to mesoscopic dynamics of cellular components and extracellular protrusions. In this review, I would like to introduce the principles of this emerging imaging technology and discuss examples that show how mass-sensitive iSCAT can be used as a strong complement to other routine techniques in biochemistry.

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