Single-molecule imaging of replication fork conflicts at genomic DNA G4 structures in human cells

SUMMARYFaithful replication of chromatin domains during cell division is fundamental to eukaryotic development. During replication, nucleosomes are disrupted ahead of the replication fork, followed by their rapid reassembly on daughter strands from the pool of recycled parental and newly synthesized histones. Here, we use single-molecule imaging and replication assays in Xenopus laevis egg extracts to determine the outcome of replication fork encounters with nucleosomes. Contrary to current models, the majority of parental histones are evicted from the DNA, with histone recycling, nucleosome sliding and replication fork stalling also occurring but at lower frequencies. The anticipated local histone transfer only becomes dominant upon depletion of free histones from extracts. Our studies provide the first direct evidence that parental histones remain in close proximity to their original locus during recycling and reveal that provision of excess histones results in impaired histone recycling, which has the potential to affect epigenetic memory.

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Faculty Opinions recommendation of Single-molecule imaging reveals replication fork coupled formation of G-quadruplex structures hinders local replication stress signaling.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.740065452.793585364 ◽

2021 ◽

Author(s):

Sergei Mirkin

Keyword(s):

Single Molecule ◽

Replication Fork ◽

Replication Stress ◽

Stress Signaling ◽

Single Molecule Imaging ◽

G Quadruplex

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

Essays in Biochemistry ◽

10.1042/ebc20200023 ◽

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

10.26434/chemrxiv.9784799.v1 ◽

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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