Faculty Opinions recommendation of Single-Molecule Imaging Uncovers Rules Governing Nonsense-Mediated mRNA Decay.

The relationship between mRNA translation and decay is incompletely understood, with conflicting reports suggesting that translation can either promote decay or stabilize mRNAs. The effect of translation on mRNA decay has mainly been studied using ensemble measurements and global inhibitors of transcription and translation, which can mask the underlying mechanisms. We developed a single-molecule imaging approach to control the translation of a specific transcript that enabled simultaneous measurement of translation and mRNA decay. Our results demonstrate that mRNAs undergoing translation are degraded faster than non-translating ones, although with slower kinetics than translation-coupled degradation of transcripts targeted by NMD. Furthermore, our results indicate that miRNAs mediate efficient degradation of both translating and non-translating target mRNAs. Single-molecule measurements of translation and decay reveal a predominant role of mRNA decay in miRNA-mediated regulation. Simultaneous visualization of translation and decay on single mRNAs provides a framework to study how these processes are interconnected in cells.

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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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