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

Abstract

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, ...

Using unnatural amino acids to selectively label proteins for cellular imaging: a cell biologist viewpoint

“…Genetic code expansion-based labeling has proven successful for the imaging of several receptors and channels in live cells [4,20,24,32,34] (Fig. 2).…”

“…Genetic code expansion-based intra-and extracellular labeling was also used in photomanipulation techniques, such as FRET and FRAP [4,30,33,34,36,38] ( Fig. 2A).…”

“…More recently, GCE-based labeling was applied to live-cell single-molecule applications, such as singleparticle tracking (SPT) and live STORM [32,34] ( Fig. 2B).…”

“…Substituting fluorescent proteins for Fl-dyes using self-labeling tags was shown to improve the temporal resolution of SMLM, enabling live-SMLM imaging [43][44][45]. Similarly, live SMLM of GCE-based labeled PM proteins was recently performed at a 30-nm spatial resolution and a 2.5-s temporal resolution [34]. Although the temporal resolution should be further improved, combining site-specific labeling with STORM in live-cell applications will surely advance our understanding of the dynamics and function of proteins in cells.…”

“…The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies microscopy (SMLM; STORM and GSDIM), and stimulated emission depletion (STED), were performed in fixed cells on proteins labeled via GCE and click chemistry [3,23,30,[33][34][35]37,[40][41][42] (Fig. 3).…”