Membrane affinity of individual toxic protein oligomers determined at the single-molecule level

2020 ◽  
Vol 22 (26) ◽  
pp. 14613-14620
Author(s):  
Simli Dey ◽  
Anirban Das ◽  
Arpan Dey ◽  
Sudipta Maiti
Keyword(s):  
Single Molecule ◽  
Single Molecule Imaging ◽  
Toxic Protein ◽  
Single Molecule Level ◽  
Membrane Affinity ◽  
Amyloid Oligomers

Single molecule imaging addresses the “which oligomer” question in membrane mediated toxicity of amyloid oligomers.

Drug Discovery at the Single Molecule Level: Inhibition-in-Solution Assay of Membrane-Reconstituted β-Secretase Using Single-Molecule Imaging

2015 ◽  
Vol 87 (8) ◽  
pp. 4100-4103 ◽  
Author(s):  
Anders Gunnarsson ◽  
Arjan Snijder ◽  
Jennifer Hicks ◽  
Jenny Gunnarsson ◽  
Fredrik Höök ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Single Molecule ◽  
Single Molecule Imaging ◽  
Single Molecule Level

scholarly journals Single-molecule imaging of telomerase reverse transcriptase in human telomerase holoenzyme and minimal RNP complexes

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Robert Alexander Wu ◽  
Yavuz S Dagdas ◽  
S Tunc Yilmaz ◽  
Ahmet Yildiz ◽  
Kathleen Collins
Keyword(s):  
Catalytic Activity ◽  
Reverse Transcriptase ◽  
Single Molecule ◽  
Cell Extract ◽  
Telomerase Reverse Transcriptase ◽  
Single Molecule Imaging ◽  
Purification Method ◽  
Single Molecule Level ◽  
Synthesis Properties ◽  
Human Telomerase

Telomerase synthesizes chromosome-capping telomeric repeats using an active site in telomerase reverse transcriptase (TERT) and an integral RNA subunit template. The fundamental question of whether human telomerase catalytic activity requires cooperation across two TERT subunits remains under debate. In this study, we describe new approaches of subunit labeling for single-molecule imaging, applied to determine the TERT content of complexes assembled in cells or cell extract. Surprisingly, telomerase reconstitutions yielded heterogeneous DNA-bound TERT monomer and dimer complexes in relative amounts that varied with assembly and purification method. Among the complexes, cellular holoenzyme and minimal recombinant enzyme monomeric for TERT had catalytic activity. Dimerization was suppressed by removing a TERT domain linker with atypical sequence bias, which did not inhibit cellular or minimal enzyme assembly or activity. Overall, this work defines human telomerase DNA binding and synthesis properties at single-molecule level and establishes conserved telomerase subunit architecture from single-celled organisms to humans.

scholarly journals Single-molecule imaging and kinetic analysis of intermolecular polyoxometalate reactions

2021 ◽  
Author(s):  
Jack W. Jordan ◽  
Kayleigh L. Y. Fung ◽  
Stephen T. Skowron ◽  
Christopher S. Allen ◽  
Johannes Biskupek ◽  
...  
Keyword(s):  
Kinetic Analysis ◽  
Single Molecule ◽  
Analytical Tool ◽  
Single Molecule Imaging ◽  
Single Molecule Level

We induce and study reactions of polyoxometalate (POM) molecules, [PW12O40]3− (Keggin) and [P2W18O62]6− (Wells–Dawson), at the single-molecule level, utilising TEM as an analytical tool, and nanotubes as test tubes.

scholarly journals Stoichiometric analysis of protein complexes by cell fusion and single molecule imaging

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Avtar Singh ◽  
Alexander L. Van Slyke ◽  
Maria Sirenko ◽  
Alexander Song ◽  
Paul J. Kammermeier ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Complexes ◽  
Correlation Spectroscopy ◽  
Membrane Receptors ◽  
Single Molecule Imaging ◽  
Critical Determinant ◽  
Single Molecule Level ◽  
Cellular Environment ◽  
Alternative Approach ◽  
Subunit Stoichiometry

Abstract The composition, stoichiometry and interactions of supramolecular protein complexes are a critical determinant of biological function. Several techniques have been developed to study molecular interactions and quantify subunit stoichiometry at the single molecule level. However, these typically require artificially low expression levels or detergent isolation to achieve the low fluorophore concentrations required for single molecule imaging, both of which may bias native subunit interactions. Here we present an alternative approach where protein complexes are assembled at physiological concentrations and subsequently diluted in situ for single-molecule level observations while preserving them in a near-native cellular environment. We show that coupling this dilution strategy with fluorescence correlation spectroscopy permits quantitative assessment of cytoplasmic oligomerization, while stepwise photobleaching and single molecule colocalization may be used to study the subunit stoichiometry of membrane receptors. Single protein recovery after dilution (SPReAD) is a simple and versatile means of extending the concentration range of single molecule measurements into the cellular regime while minimizing potential artifacts and perturbations of protein complex stoichiometry.

scholarly journals Dissecting the Membrane Dynamics of Amyloid Oligomers at a Single Molecule Level

2010 ◽  
Vol 98 (3) ◽  
pp. 424a
Author(s):  
Martino Calamai ◽  
Martin Zanni ◽  
Francesco Pavone
Keyword(s):  
Single Molecule ◽  
Membrane Dynamics ◽  
Single Molecule Level ◽  
Amyloid Oligomers

scholarly journals Label-free tracking and mass measurement of single proteins on lipid bilayers

2021 ◽  
Author(s):  
Eric Dylan Benjamin Foley ◽  
Manish Kushwah ◽  
Gavin Young ◽  
Philipp Kukura
Keyword(s):  
Lipid Bilayers ◽  
Single Molecule ◽  
Mass Measurement ◽  
Label Free ◽  
Single Molecule Level ◽  
Dynamic Mass ◽  
Membrane Affinity ◽  
Fundamental Building Block ◽  
Quantitative Studies ◽  
Associated Proteins

We introduce dynamic mass photometry, a method for label-free imaging, tracking and mass measurement of membrane-associated proteins. Our method enables quantitative studies of their mobility, membrane affinity and interactions at the single molecule level. Application to the membrane remodelling GTPase dynamin1 reveals heterogeneous mixtures of oligomers suggesting that the fundamental building block for oligomerisation is a dimer, challenging current tetramer-centric models. Dynamic mass photometry has the ability to transform our approach to studying biomolecular mechanisms in and on lipid bilayers.

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.

A Molecular Logic Gate Enables Super-Resolved Imaging of Intracellular Lipid Droplets

2019 ◽  
Author(s):  
Adam Eördögh ◽  
Carolina Paganini ◽  
Dorothea Pinotsi ◽  
Paolo Arosio ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Single Molecule ◽  
Lipid Droplets ◽  
Neutral Lipids ◽  
Logic Gate ◽  
Living Cells ◽  
Three Dimensions ◽  
Single Molecule Imaging ◽  
Molecular Logic Gate ◽  
Molecular Logic ◽  
Cellular Compartments

<div>Photoactivatable dyes enable single-molecule imaging in biology. Despite progress in the development of new fluorophores and labeling strategies, many cellular compartments remain difficult to image beyond the limit of diffraction in living cells. For example, lipid droplets, which are organelles that contain mostly neutral lipids, have eluded single-molecule imaging. To visualize these challenging subcellular targets, it is necessary to develop new fluorescent molecular devices beyond simple on/off switches. Here, we report a fluorogenic molecular logic gate that can be used to image single molecules associated with lipid droplets with excellent specificity. This probe requires the subsequent action of light, a lipophilic environment and a competent nucleophile to produce a fluorescent product. The combination of these requirements results in a probe that can be used to image the boundary of lipid droplets in three dimensions with resolutions beyond the limit of diffraction. Moreover, this probe enables single-molecule tracking of lipids within and between droplets in living cells.</div>

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