Cellular encoding of Cy dyes for single-molecule imaging

Leisle, Lilia; Chadda, Rahul; Lueck, John D.; Infield, Daniel T.; Galpin, Jason D.; Krishnamani, Venkatramanan; Robertson, Janice; Ahern, Christopher A.

doi:10.7554/elife.19088

Cited by 25 publications

(16 citation statements)

References 60 publications

(75 reference statements)

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“…A key step in achieving single-fluorophore detection in a solution is to decrease the background fluorescence in the bulk solution. TIRFM is among the most popular options for single-molecule imaging [19,23,24] and was the method of choice for these analyses. Using single-molecule imaging in combination with photobleaching, we found that only one photobleaching step was necessary for the GFP-A206K mutant, confirming that GFP-A206K remains monomeric at high concentrations.…”

Section: Discussionmentioning

confidence: 99%

Single-Molecule Imaging and Computational Microscopy Approaches Clarify the Mechanism of the Dimerization and Membrane Interactions of Green Fluorescent Protein

Wang

Song

et al. 2019

IJMS

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Green fluorescent protein (GFP) is widely used as a biomarker in living systems; however, GFP and its variants are prone to forming low-affinity dimers under physiological conditions. This undesirable tendency is exacerbated when fluorescent proteins (FP) are confined to membranes, fused to naturally-oligomeric proteins, or expressed at high levels in cells. Oligomerization of FPs introduces artifacts into the measurement of subunit stoichiometry, as well as interactions between proteins fused to FPs. Introduction of a single mutation, A206K, has been shown to disrupt hydrophobic interactions in the region responsible for GFP dimerization, thereby contributing to its monomerization. Nevertheless, a detailed understanding of how this single amino acid-dependent inhibition of dimerization in GFP occurs at the atomic level is still lacking. Single-molecule experiments combined with computational microscopy (atomistic molecular dynamics) revealed that the amino group of A206 contributes to GFP dimer formation via a multivalent electrostatic interaction. We further showed that myristoyl modification is an efficient mechanism to promote membrane attachment of GFP. Molecular dynamics-based site-directed mutagenesis has been used to identify the key functional residues in FPs. The data presented here have been utilized as a monomeric control in downstream single-molecule studies, facilitating more accurate stoichiometry quantification of functional protein complexes in living cells.

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Section: Discussionmentioning

confidence: 99%

Single-Molecule Imaging and Computational Microscopy Approaches Clarify the Mechanism of the Dimerization and Membrane Interactions of Green Fluorescent Protein

Wang

Song

et al. 2019

IJMS

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“…How this will be achieved is not yet clear, but promising starting points are noncanonical amino acids. Recently, Cy dyes were site-specifically incorporated into proteins in Xenopus laevis oocytes as noncanonical amino acids (Leisle et al 2016), so it is not a stretch to imagine this being performed with fluorescently tagged mRNA to monitor translation dynamics. The ultimate goal would be in situ sequencing of protein synthesis in vivo.…”

Section: Discussionmentioning

confidence: 99%

Quantifying Single mRNA Translation Kinetics in Living Cells

Morisaki

Stasevich

2018

Cold Spring Harb Perspect Biol

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One of the last hurdles to quantifying the full central dogma of molecular biology in living cells with single-molecule resolution has been the imaging of single messenger RNA (mRNA) translation. Here we describe how recent advances in protein tagging and imaging technologies are being used to precisely visualize and quantify the synthesis of nascent polypeptide chains from single mRNA in living cells. We focus on recent applications of repeat-epitope tags and describe how they enable quantification of single mRNA ribosomal densities, translation initiation and elongation rates, and translation site mobility and higher-order structure. Together with complementary live-cell assays to monitor translation using fast-maturing fluorophores and mRNA-binding protein knockoff, single-molecule studies are beginning to uncover striking and unexpected heterogeneity in gene expression at the level of translation. Outline 1 Introduction 2 Breaking the single-molecule barrier for live-cell imaging of translation 3 Amplifying live-cell single mRNA translation signals with repeat-epitope tags 4 Limitations of repeat-epitope tags for translation imaging 5 Accessory tags for imaging single mRNA translation in living cells 6 Tagging endogenous proteins with repeatepitope tags 7 Counting ribosomes in polysomes with repeat-epitope tags 8 Quantifying translation elongation with repeat-epitope tags 9 Quantifying translation initiation with repeatepitope tags 10 Quantifying polysome mobility and shape with repeat-epitope tags 11 Emerging consensus in the field of live-cell imaging of single RNA translation 12 Heterogeneity in single mRNA translation efficiency 13 Heterogeneity in ribosome movement along single mRNA 14 Heterogeneity in the localization and mobility of translation sites 15 Outlook References

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“…Leisle et al incorporated Cy-ncAA-based fluorescent probes (Cy3 and Cy5) into a chloride channel (CIC-0) and a voltage-gated sodium channel auxiliary subunit, opening the door to study proteins by single-molecule approaches in cellular environments ( Fig. 4C; Leisle et al 2016).…”

Section: Nonsense Suppression With Orthogonal Trnas In Xenopus Laevismentioning

confidence: 99%

The current chemical biology tool box for studying ion channels

Braun

Sheikh

Pless

2020

The Journal of Physiology

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Ion channels play important roles in human physiology and their dysfunction is linked to a variety of diseases. This has sparked considerable interest in their molecular function and pharmacology and generated a need to manipulate them with great precision. The use of high-sensitivity electrophysiological methods allows for the implementation of chemical biology manipulations, as even minute protein amounts can be studied. For example, modification of solvent-accessible cysteines is a powerful tool to site-selectively modify proteins through the introduction of charged moieties or those with fluorescent properties. This has been harnessed to study ion conduction pathways and monitor conformational dynamics. In ligand-directed chemistry, a high-affinity ligand is used to modify an ion channel with a chemical probe via a reactive linker. While these approaches are typically limited to extracellular positions, genetic code expansion provides a means to introduce non-canonical amino acids in any position of the protein. This enables the insertion of subtle analogues of naturally occurring side chains or the protein backbone, as well as amino acids with fluorescent, cross-linking or photo-switchable properties. Finally, protein semi-synthesis enables the simultaneous insertion of multiple modifications, including those that would not be tolerated by the ribosomal translation machinery. Collectively, these chemical biology tools have overcome various shortcomings of conventional mutagenesis and vastly expanded the scope of possible modifications and the type of ion channels they can be Nina Braun studied biochemistry at the University of Bayreuth and Stockholm University before discovering her interest in ion channels and non-canonical amino acids while working on her Masters thesis with Stephan A. Pless at the University of Copenhagen. She completed her PhD in the Pless lab and is currently working on high-throughput assessment of non-canonical amino acid incorporation using photocrosslinkers in acid-sensing ion channels, with the goal of studying protein-protein interactions. Zeshan P. Sheikh completed his MSc degree at the University of Copenhagen, where he discovered his passion for ion channels, mainly focusing on recombinant expression and purification. He embarked on a PhD in the Pless lab pursuing his interest in ion channel biophysics. Here, he gained particular interest in the application of chemical biological techniques for modifying ion channels. His current research priority is applying split inteins to modify acid-sensing ion channels to gain insights into their structural dynamics.

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Cellular encoding of Cy dyes for single-molecule imaging

Cited by 25 publications

References 60 publications

Single-Molecule Imaging and Computational Microscopy Approaches Clarify the Mechanism of the Dimerization and Membrane Interactions of Green Fluorescent Protein

Single-Molecule Imaging and Computational Microscopy Approaches Clarify the Mechanism of the Dimerization and Membrane Interactions of Green Fluorescent Protein

Quantifying Single mRNA Translation Kinetics in Living Cells

The current chemical biology tool box for studying ion channels

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