Direct fluorescent-dye labeling of α-tubulin in mammalian cells for live cell and superresolution imaging

Schvartz, Tomer; Aloush, Noa; Goliand, Inna; Segal, Inbar; Nachmias, Dikla; Arbely, Eyal; Elia, Natalie

doi:10.1091/mbc.e17-03-0161

Cited by 31 publications

(50 citation statements)

References 29 publications

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“…1, G and H), or the reorganization of the actin cytoskeleton during cell fusion in fission yeast (Dudin et al, 2015). In custom-built systems, 2D and 3D SIM has been reportedly used at video frame rates and documented the organization and dynamics of microtubules (Kner et al, 2009;Schvartz et al, 2017) and mitochondria (Shao et al, 2011) in mammalian cells.…”

Section: Simmentioning

confidence: 99%

Advances in Imaging Plant Cell Dynamics

et al. 2017

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Section: Simmentioning

confidence: 99%

Advances in Imaging Plant Cell Dynamics

et al. 2017

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“…Single Molecule Localization Microscopy (SMLM) [6][7][8][9][10][11] . However, these studies focused on the proof of concept and therefore very little quantitative information on the spatiotemporal dynamics of proteins in cells has been obtained from them.…”

Section: Structured Illumination Microscopy (Sim) Stimulated Emissiomentioning

confidence: 99%

“…Transfection with the plasmids described in Figure S1 A-C, was carried out using Lipofectamine 2000 (Life Technologies, Carlsbad, CA) according to the manufacturer's guidelines. Genetic code expansion and biorthogonal labeling was carried out as described in Schvartz et al 11 . In short, cells were incubated for 48 h in the presence of BCNK (0.5 mM; Synaffix, Oss, Netherlands) and washed with fresh medium at 37°C.…”

Section: Genetic Code Expansion and Bioorthogonal Labeling In Mammalimentioning

confidence: 99%

Live cell single molecule tracking and localization microscopy of bioorthogonally labeled plasma membrane proteins

König

Sorkin

Alon

et al. 2019

Preprint

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ABSRACTTracking the localization and mobility of individual proteins in live cells is key for understanding how they mediate their function. Such information can be obtained from single molecule imaging techniques such as Single Particle Tracking (SPT) and Single Molecule Localization Microscopy (SMLM). Genetic code expansion (GCE) combined with bioorthogonal chemistry offers an elegant approach for direct labeling of proteins with fluorescent dyes, holding great potential for improving protein labeling in single molecule applications. Here we calibrated conditions for performing SPT and live-SMLM of bioorthogonally labeled plasma membrane proteins in live mammalian cells. Using SPT, the diffusion rates of bioorthogonally labeled EGF receptor and the prototypical Shaker voltage-activated potassium channel (Kv) were successfully measured. Applying live-SMLM to bioorthogonally labeled Shaker Kv channels enabled visualizing the plasma membrane distribution of the channel over time with ~30 nm accuracy. Finally, by competitive labeling with two Fl-dyes, SPT and live-SMLM were performed in a single cell and both the density and dynamics of the EGF receptor were measured at single molecule resolution in sub-regions of the cell. We conclude that GCE and bioorthogonal chemistry is a highly suitable, flexible approach for protein labeling in quantitative single molecule applications that outperforms current protein live cell labeling approaches.3 Genetic code expansion (GCE) together with biorthogonal labeling offers an elegant tool for labeling proteins with organic Fluorescent dyes (Fl-dyes) in live mammalian cells. In GCE-based labeling, a non-canonical amino acid (ncAA) carrying a functional group is incorporated into the protein sequence in response to an in-frame amber stop codon (TAG), via an orthogonal tRNA/tRNA-synthetase pair (reviewed in 1,2 ). Labeling is then carried out by a rapid and specific bioorthogonal reaction between the functional group and a Fl-dye. As a result, the protein of interest is fluorescently labeled on a specific residue, avoiding the need for conjugating protein tag fusions (i.e. fluorescent protein or self-labeling proteins) in live cell applications.Direct labeling of proteins in live cells with Fl-dyes has several advantages for quantitative, high-end live cell imaging. First, Fl-dyes are bright and photostable and therefore cells can be imaged for longer times and at reduced laser intensities 3 .Second, the label itself is relatively small (Fl-dye, ~0.5 nm; GFP, 4.2 nm; antibodies >10nm; quantum dots 2-60 nm) thereby allowing higher accuracy in localization measurements and better representation of the physiological properties of the protein 3-5 . Third, labeling does not involve amplification, and therefore the number of molecules can be quantified. Fourth, the Fl-dye is applied at the last step of the reaction providing flexibility in optimizing the Fl-dye to specific applications and the possibility to simultaneously label the protein population with more than one dye 6 . Indeed, ...

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“…Using genetic code expansion (GCE) and bioorthogonal chemistry, it is now possible to non-invasively attach fluorescent dyes (Fl-dyes) to specific protein residues, thereby allowing essentially "tag-free" labeling of proteins in live cells (1-3). Indeed, this approach has been applied, in recent years, for fluorescent labeling of extra and intra cellular proteins (4)(5)(6)(7)(8)(9)(10).…”

Section: Introductionmentioning

confidence: 99%

“…In practice, however, finding a suitable labeling site can be laborious and time-consuming for several reasons. First, prior knowledge or functional assays are necessary to ensure that insertion of the ncAA at a specific position does not affect protein structure and function (4)(5)(6)(7)10). Second, the efficiency of ncAA incorporation varies at different locations in the protein with no guidelines for the preferred sequence context having been reported (3)(4)(5)(6)(7)15).…”

Section: Introductionmentioning

confidence: 99%

A straightforward approach for bioorthogonal labeling of proteins and organelles in live mammalian cells, using a short peptide tag

Segal

Nachmias

Arbely

et al. 2019

Preprint

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In the high-resolution microscopy era, genetic code expansion (GCE)-based bioorthogonal labeling offers an elegant way for direct labeling of proteins in live cells with fluorescent dyes. This labeling approach is currently not broadly used live cell applications, partly because it needs to be adjusted to the specific protein under study. Here, we present a generic, 14-residues long, N-terminal tag for GCE-based labeling of proteins in live mammalian cells. Using this tag, we generated a library of GCE-based organelle markers, demonstrating the applicability of the tag for labeling a plethora of proteins and organelles. Finally, we show that the HA epitope, used as a backbone in our tag, can be substituted with other epitopes and, in some cases, can be completely removed, reducing the tag length to 5 residues. The GCE-tag presented here offers a powerful, easy-to-implement tool for live cell labeling of cellular proteins with small and bright probes. 3 BACKGROUND

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Direct fluorescent-dye labeling of α-tubulin in mammalian cells for live cell and superresolution imaging

Cited by 31 publications

References 29 publications

Advances in Imaging Plant Cell Dynamics

Advances in Imaging Plant Cell Dynamics

Live cell single molecule tracking and localization microscopy of bioorthogonally labeled plasma membrane proteins

A straightforward approach for bioorthogonal labeling of proteins and organelles in live mammalian cells, using a short peptide tag

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