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Highly photostable fluorescent labeling of proteins in live cells using exchangeable coiled coils heterodimerization

Abstract

Fluorescent proteins are commonly used to label target proteins in live cells. However, the conventional approach based on covalent fusion of targeted proteins with fluorescent protein probes is limited by the slow rate of fluorophore maturation and irretrievable loss of fluorescence due to photobleaching. Here, we report a genetically encoded protein labeling system utilizing transient interactions of small, 21–28 residues-long helical protein tags (K/E coils, KEC). In this system, a protein of interest, covalently tagged with a single coil, is visualized through binding to a cytoplasmic fluorescent protein carrying a complementary coil. The reversible heterodimerization of KECs, whose affinity can be tuned in a broad concentration range from nanomolar to micromolar, allows continuous exchange and replenishment of the tag bound to a targeted protein with the entire cytosolic pool of soluble fluorescent coils. We found that, under conditions of partial illumination of living cells, the photostability of labeling with KECs exceeds that of covalently fused fluorescent probes by approximately one order of magnitude. Similarly, single-molecule localization microscopy with KECs provided higher labeling density and allowed a much longer duration of imaging than with conventional fusion to fluorescent proteins. We also demonstrated that this method is well suited for imaging newly synthesized proteins, because the labeling efficiency by KECs is not dependent on the rate of fluorescent protein maturation. In conclusion, KECs can be used to visualize various target proteins which are directly exposed to the cytosol, thereby enabling their advanced characterization in time and space.

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Acknowledgements

We thank Nikita Podkuychenko (Institute of Experimental Cardiology, National Medical Research Center for Cardiology, Moscow, Russia) for giving the C2C12 cell culture. We thank Dr. Irina Shagina (Pirogov Russian National Research Medical University, Moscow, Russia) for providing cDNA for caveolin-1 gene cloning.

Funding

This work was supported by the Russian Science Foundation grant 16-14-10364 and the NIH grants EY022959 (V.Y.A.), EY05722 (V.Y.A.), and EY029929 (T.R.L.). Experiments were partially carried out using equipment provided by the IBCH core facility (CKP IBCH, supported by Russian Ministry of Science and Higher Education, grant RFMEFI62117X0018).

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Authors

Contributions

KL and NG conceived the idea. KL, NG, and AM designed experiments and supervised the project. MP, NG, and SK did the cloning. MP and NG performed confocal and wide-field imaging. MP, PM, and VB performed TIRF imaging. ES, TL, and VA designed and performed experiments with somatostatin receptor. MP and AM analyzed the data. MP and AM wrote the manuscript. All co-authors discussed the results and exchanged comments on the manuscript.

Corresponding authors

Correspondence to Alexander S. Mishin or Konstantin A. Lukyanov.

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Perfilov, M.M., Gurskaya, N.G., Serebrovskaya, E.O. et al. Highly photostable fluorescent labeling of proteins in live cells using exchangeable coiled coils heterodimerization. Cell. Mol. Life Sci. 77, 4429–4440 (2020). https://doi.org/10.1007/s00018-019-03426-5

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Keywords

  • Exchangeable labels
  • Live-cell localization microscopy
  • Coiled coil tag
  • Protein-PAINT