Abstract
The high brightness and photostability of the green fluorescent protein StayGold make it a particularly attractive probe for long-term live-cell imaging; however, its dimeric nature precludes its application as a fluorescent tag for some proteins. Here, we report the development and crystal structures of a monomeric variant of StayGold, named mBaoJin, which preserves the beneficial properties of its precursor, while serving as a tag for structural proteins and membranes. Systematic benchmarking of mBaoJin against popular green fluorescent proteins and other recently introduced monomeric and pseudomonomeric derivatives of StayGold established mBaoJin as a bright and photostable fluorescent protein, exhibiting rapid maturation and high pH/chemical stability. mBaoJin was also demonstrated for super-resolution, long-term live-cell imaging and expansion microscopy. We further showed the applicability of mBaoJin for neuronal labeling in model organisms, including Caenorhabditis elegans and mice.
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Data availability
The PDB IDs are 8QBJ, 8Q79 and 8QDD for atomic structures of mBaoJin crystallized at pH 4.6, pH 6.5 and pH 8.5, respectively. The total size of the files acquired for this study exceeds the 20-GB limit of the FigShare repository, therefore only the most essential raw datasets, including source files for supplementary figures and raw unprocessed images, are available at figshare (https://doi.org/10.6084/m9.figshare.25001768). The remaining files are available from the corresponding author upon request. All plasmids used in this study are available from WeKwikGene (https://wekwikgene.wllsb.edu.cn/). Source data are provided with this paper.
Code availability
Custom code used in this study for BaLM imaging reconstruction is available at https://github.com/Perfus/mBaoJin.
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Acknowledgements
We are grateful to A. N. Asanova and A. N. Romantsova for their help with mammalian plasmids cloning. We thank G. G. Lambert and N. Shaner for providing the mChilada gene. We thank Guangzhou CSR Biotech Co. for assistance with live-cell imaging and image analysis using HIS-SIM. The work was carried out within the state assignment of NRC Kurchatov Institute (development of mBaoJin and characterization of mBaoJin in vitro) and thematic plan of NRC Kurchatov Institute (crystallization and X-ray analysis); by the Ministry of Science and Higher Education of the Russian Federation for the development of the Kurchatov Center for Genome Research 075-15-2019-1659 (protein purification); by start-up funding from the Foundation of Westlake University, Westlake Laboratory of Life Sciences and Biomedicine, National Natural Science Foundation of China grant 32171093 and ‘Pioneer’ and ‘Leading Goose’ R&D Program of Zhejiang 2024SSYS0031 to K.D.P. (characterization of mBaoJin in mammalian cells and super-resolution imaging). Assessment of the mBaoJin reporter in BaLM super-resolution microscopy was funded by the Russian Science Federation, project no. 22-14-00400 (https://rscf.ru/project/22-14-00400/). The work was also supported by the Resource Centers department of the National Research Center Kurchatov Institute (imaging of bacteria). The authors acknowledge the BL19U1 beamline of the National Facility for Protein Science Shanghai at Shanghai Synchrotron.
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Authors and Affiliations
Contributions
G.D.L., O.M.S. and F.V.S. developed mBaoJin and characterized it in vitro. F.V.S. and T.P.K. obtained mutants of mBaoJin and characterized their properties in vitro. A.V.V. and H.Z. purified mBaoJin. H.Z., K.D.P. and F.V.S. characterized mBaoJin in mammalian cells and performed live-cell imaging. M.D. measured QY of proteins. S.P. and H.Z. performed the OSER assay. H.Z. characterized proteins in vivo. H.Z. and W.Z. performed ExM. L.C., X.Y., H.Z. and K.D.P. performed SIM. M.M.P., A.S.G. and A.S.M. performed BaLM microscopy. A.G., V.B. and W.Q. performed X-ray data collection. A.Yu.N. performed crystallization. V.R.S. performed crystallization, structure solution, refinement and analysis. K.D.P., F.V.S, H.Z., M.M.P., V.R.S. and A.S.M. wrote the manuscript. K.D.P. and F.V.S. supervised all aspects of the project. All authors reviewed the manuscript.
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Competing interests
K.D.P. is the co-founder of a company that pursues commercial applications of ExM and is listed as an inventor on several patent applications concerning the development of new ExM methods. The other authors declare no competing interests.
Peer review
Peer review information
Nature Methods thanks Dominique Bourgeois, Benjamin Campbell and Yi Yang for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team.
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Supplementary information
Supplementary Information
Supplementary Tables 1–11 and Supplementary Figs. 1–21
Supplementary Video 1
Three-dimensional view of expanded HeLa cell coexpressing H2B-mTurquoise2 and Mito (COX8Ax2)-mBaoJin acquired in the CFP and GFP channels using a Nikon spinning disk microscope (linear expansion factor 3.4×).
Supplementary Video 2
Long-term super-resolution imaging of tubulin dynamics in HeLa cells using CSU-W1 SoRa imaging setup (×100 NA 1.41, sampling rate 0.2 Hz, total duration 60 min).
Supplementary Video 3
Long-term super-resolution imaging of actin dynamics in HeLa cells using CSU-W1 SoRa imaging setup (×100 NA 1.41, sampling rate 0.2 Hz, total duration 30 min).
Supplementary Video 4
Long-term super-resolution imaging of EB3 dynamics in HeLa cells using CSU-W1 SoRa imaging setup (×100 NA 1.41, sampling rate 1 Hz, total duration 2 :30 min).
Supplementary Video 5
Long-term super-resolution imaging of actin dynamics with mBaoJin in HeLa cells using HIS-SIM imaging setup (×100 NA 1.41, sampling rate 0.1 Hz, total duration 15 min).
Supplementary Video 6
Long-term super-resolution imaging of actin dynamics with mNeonGreen in HeLa cells using HIS-SIM imaging setup (×100 NA 1.41, sampling rate 0.1 Hz, total duration 15 min).
Supplementary Video 7
Video showing 500 frames of vimentin-mBaoJin in live HeLa cells. Plays at 20 f.p.s. (acquisition speed 20 f.p.s.).
Supplementary Video 8
Video showing 1,000 frames of vimentin-mBaoJin in live HeLa cells. Plays at 100 f.p.s. (acquisition speed 100 f.p.s.).
Supplementary Video 9
Video showing 1,000 frames of vimentin-mBaoJin in live HeLa cells. Plays at 200 f.p.s. (acquisition speed 200 f.p.s.).
Supplementary Video 10
Video showing 500 frames of vimentin-mNeonGreen in live HeLa cells. Plays at 20 f.p.s. (acquisition speed 20 f.p.s.).
Supplementary Video 11
Video showing 1,000 frames of vimentin-mNeonGreen in live HeLa cells. Plays at 100 f.p.s. (acquisition speed 100 f.p.s.).
Supplementary Video 12
Video showing 1,000 frames of vimentin-mNeonGreen in live HeLa cells. Plays at 200 f.p.s. (acquisition speed 200 f.p.s.).
Supplementary Video 13
The 21 reconstructed frames of vimentin-mBaoJin in live HeLa cell. Acquisition speed 200 f.p.s. Each frame was reconstructed from 10,000 raw frames (50 s of acquisition). Time shift between reconstructed frames was 5 s. Video plays at three reconstructed f.p.s. Scale bar, 500 nm.
Supplementary Video 14
The 16 reconstructed frames of vimentin-mNeonGreen in live HeLa cell. Acquisition speed 200 f.p.s. Each frame was reconstructed from 10,000 raw frames (50 s of acquisition). Time shift between reconstructed frames was 5 s. Video plays at three reconstructed f.p.s. Scale bar, 500 nm.
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Zhang, H., Lesnov, G.D., Subach, O.M. et al. Bright and stable monomeric green fluorescent protein derived from StayGold. Nat Methods 21, 657–665 (2024). https://doi.org/10.1038/s41592-024-02203-y
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DOI: https://doi.org/10.1038/s41592-024-02203-y
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