预印本 / 版本 1

Monomerizing the ultrabright AausFP1 yields mBiyu for advanced imaging across challenging bacterial and mammalian cells

本文是预印本,尚未经过同行评审认证。

作者

    Yu Yan, 
    Yu Yan
    Yiqing Shen, 
    Yiqing Shen
    Chaoying Peng, 
    Chaoying Peng
    Xinyu Wang, 
    Xinyu Wang
    Xiao Lin, 
    Xiao Lin
    Xiaodong Ye, 
    Xiaodong Ye
    Jiajia Wang, 
    Jiajia Wang
    Sijing Zhang, 
    Sijing Zhang
    Hui Wang, 
    Hui Wang
    Xianghui Ou, 
    Xianghui Ou
    Ying Li, 
    Ying Li
    Shishen Du, 
    Shishen Du
    Quan Chen, 
    Quan Chen
    Xinxing Yang
    Xinxing Yang
分类
关键词
Fluorescent protein; high molecular brightness; new FPs scaffolds; monomerization; imaging in challenging bacteria; expansion microscopy

摘要

Fluorescent proteins (FPs) enable noninvasive visualization of cellular processes. For advanced imaging applications, high molecular brightness is a key determinant of FP performance. AausFP1 is a newly discovered, ultrabright green FP derived from Aequorea cf. australis that shares low sequence identity with widely used avGFP. It combines a near-unity quantum yield with a high extinction coefficient, yet its native oligomerization and poor solubility limits use as a fusion tag. Here we report mBiyu, a monomeric AausFP1 engineered by structure‑guided interface disruption followed by iterative directed evolution. mBiyu preserves AausFP1’s high quantum yield, matures rapidly, and tolerates demanding conditions such as aldehyde fixation and protease digestion, enabling its use in expansion microscopy. In mammalian cells and across diverse prokaryotes, mBiyu behaves as a monomer and delivers strong fusion performance, including challenging bacteria such as Staphylococcus aureus, Mycobacterium smegmatis, and Deinococcus radiodurans. These properties make mBiyu a robust tag for long time-lapse and high-resolution imaging in difficult organisms and demanding conditions.

参考文献

Shimomura, O., Johnson, F. H. & Saiga, Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J. Cell. Comp. Physiol.59, 223–239 (1962).

Cormack, B. P., Valdivia, R. H. & Falkow, S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene173, 33–38 (1996).

Pédelacq, J. D., Cabantous, S., Tran, T., Terwilliger, T. C. & Waldo, G. S. Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol.24, 79–88 (2006).

Shaner, N. C. et al.Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat. Methods5, 545–551 (2008).

Hirano, M. et al.A highly photostable and bright green fluorescent protein. Nat. Biotechnol.40, 1132–1142 (2022).

Lambert, G. G. et al.Aequorea’s secrets revealed: New fluorescent proteins with unique properties for bioimaging and biosensing. PLoS Biol.18, e3000936 (2020).

Shaner, N. C., Steinbach, P. A. & Tsien, R. Y. A guide to choosing fluorescent proteins. Nat. Methods2, 905–909 (2005).

Shaner, N. C. et al.A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat. Methods10, 407–409 (2013).

Zhang, H. et al.Bright and stable monomeric green fluorescent protein derived from StayGold. Nat. Methods21, 657–665 (2024).

Schrödinger, LLC. The PyMOL Molecular Graphics System, Version 4.6. PyMOL (2015).

Campbell, R. E. et al.A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. U.S.A.99, 7877–7882 (2002).

Wilson, D. S. & Keefe, A. D. Random mutagenesis by PCR. Curr. Protoc. Mol. Biol.Chapter 8, Unit 8.3 (2001).

Crameri, A., Whitehorn, E. A., Tate, E. & Stemmer, W. P. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat. Biotechnol.14, 315–319 (1996).

Ren, H. et al.Cysteine Sulfoxidation Increases the Photostability of Red Fluorescent Proteins. ACS Chem. Biol.11, 2679–2684 (2016).

Shaner, N. C. et al.Improved monomeric red, orange and yellow fluorescent proteins derived from Discosomasp. red fluorescent protein. Nat. Biotechnol.22, 1567–1572 (2004).

Costantini, L. M., Fossati, M., Francolini, M. & Snapp, E. L. Assessing the Tendency of Fluorescent Proteins to Oligomerize Under Physiologic Conditions. Traffic13, 643–649 (2012).

Cranfill, P. J. et al.Quantitative assessment of fluorescent proteins. Nat. Methods13, 557–562 (2016).

Landgraf, D., Okumus, B., Chien, P., Baker, T. A. & Paulsson, J. Segregation of molecules at cell division reveals native protein localization. Nat. Methods9, 480–482 (2012).

Fraikin, N., Couturier, A., Mercier, R. & Lesterlin, C. A palette of bright and photostable monomeric fluorescent proteins for bacterial time-lapse imaging. Sci. Adv.11, eads6201 (2025).

Orm, M. et al.Crystal structure of the Aequorea victoriagreen fluorescent protein. Science273, 1392–1395 (1996).

Teijeiro-Gonzalez, Y. et al.Time-Resolved Fluorescence Anisotropy and Molecular Dynamics Analysis of a Novel GFP Homo-FRET Dimer. Biophys. J.120, 254–269 (2021).

Liu, Z. et al.Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci. Rep.7, 2193 (2017).

Tan, Z. et al.Time-resolved fluorescent proteins expand fluorescent microscopy in temporal and spectral domains. Cell188, 6987–7005.e6928 (2025).

Goedhart, J. et al.Bright cyan fluorescent protein variants identified by fluorescence lifetime screening. Nat. Methods7, 137–139 (2010).

Xiong, H. et al.A highly stable monomeric red fluorescent protein for advanced microscopy. Nat. Methods22, 1288–1298 (2025).

Pulschen, A. A. et al.Live Imaging of a Hyperthermophilic Archaeon Reveals Distinct Roles for Two ESCRT-III Homologs in Ensuring a Robust and Symmetric Division. Curr. Biol.30, 2852–2859.e2854 (2020).

Campbell, B. C., Paez-Segala, M. G., Looger, L. L., Petsko, G. A. & Liu, C. F. Chemically stable fluorescent proteins for advanced microscopy. Nat. Methods19, 1612–1621 (2022).

Gadella, T. W. J. et al.mScarlet3: a brilliant and fast-maturing red fluorescent protein. Nat. Methods20, 541–545 (2023).

Balleza, E., Kim, J. M. & Cluzel, P. Systematic characterization of maturation time of fluorescent proteins in living cells. Nat. Methods15, 47–51 (2018).

Ivorra-Molla, E. et al.A monomeric StayGold fluorescent protein. Nat. Biotechnol.42, 1368–1371 (2024).

Rondthaler, S. N., Sarker, B., Howitz, N., Shah, I. & Andrews, L. B. Toolbox of Characterized Genetic Parts for Staphylococcus aureus. ACS Synth. Biol.13, 103–118 (2024).

Chen, F., Tillberg, P. W. & Boyden, E. S. Expansion microscopy. Science347, 543–548 (2015).

Tillberg, P. W. et al.Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies. Nat. Biotechnol.34, 987–992 (2016).

Yang, X. et al.A two-track model for the spatiotemporal coordination of bacterial septal cell wall synthesis revealed by single-molecule imaging of FtsW. Nat. Microbiol.6, 584–593 (2021).

Buss, J. et al.A multi-layered protein network stabilizes the Escherichia coliFtsZ-ring and modulates constriction dynamics. PLoS Genet.11, e1005128 (2015).

Hensel, Z. A plasmid-based Escherichia coligene expression system with cell-to-cell variation below the extrinsic noise limit. PLoS One12, e0187259 (2017).

Monteiro, J. M. et al.Cell shape dynamics during the staphylococcal cell cycle. Nat. Commun.6, 8055 (2015).

Wang, J. et al.Class A PBPs reinforce the septal cell wall following initial synthesis by SEDS-bPBP pairs during bacterial cytokinesis. bioRxiv2025.01.01.123456 (2025).

Hu, X. et al.Using Protein Design and Directed Evolution to Monomerize a Bright Near-Infrared Fluorescent Protein. ACS Synth. Biol.13, 1177–1190 (2024).

(此条在文档中编号存在跳跃或合并,按原文结构列出) Wilson, D. S. & Keefe, A. D. Random mutagenesis by PCR. (同12,此处为文档重复编号或子条目,保留原格式。)

Stemmer, W. P. Rapid evolution of a protein in vitro by DNA shuffling. Nature370, 389–391 (1994).

Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227, 680–685 (1970).

Schuck, P., Zhao, H., Brautigam, C. A. & Ghirlando, R. Basic principles of analytical ultracentrifugation. (CRC Press, Boca Raton, FL, 2016).

Schuck, P. Size-Distribution Analysis of Macromolecules by Sedimentation Velocity Ultracentrifugation and Lamm Equation Modeling. Biophys. J.78, 1606–1619 (2000).

Hayes, D., Laue, T. & Philo, J. Sedimentation interpretation program, version 1.09. (University of Connecticut, Storrs, CT, 2014).

Lakowicz, J. R. Principles of Fluorescence Spectroscopy. 3rd edn (Springer, 2006).

Ward, W. W. & Bokman, S. H. Reversible denaturation of Aequoreagreen-fluorescent protein: physical separation and characterization of the renatured protein. Biochemistry21, 4535–4540 (1982).

Ward, W. W., Prentice, H. J., Roth, A. F., Cody, C. W. & Reeves, S. C. Spectral perturbations of the Aequoreagreen-fluorescent protein. Photochem. Photobiol.35, 803–808 (1982).

Stringer, C. et al.Cellpose: a generalist algorithm for cellular segmentation. Nat. Methods18, 100–106 (2021).

Sarfatis, A., Wang, Y., Twumasi-Ankrah, N. & Moffitt, J. R. Highly multiplexed spatial transcriptomics in bacteria. Science387, eadr0932 (2025).

Yan, D., Xue, J., Xiao, J., Lyu, Z. & Yang, X. Protocol for single-molecule labeling and tracking of bacterial cell division proteins. STAR Protoc.5, 102766 (2024).

Tokunaga, M., Imamoto, N. & Sakata-Sogawa, K. Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat. Methods5, 159–161 (2008).

Zhao, W. et al.Sparse deconvolution improves the resolution of live-cell super-resolution fluorescence microscopy. Nat. Biotechnol.40, 606–617 (2022).

指标

收藏: 1
查看次数: 313
下载次数: 79

下载次数

已发布

2026-05-06

版本

如何引用

Yan, Y., Shen, Y., Peng, C., Wang, X., Lin, X., Ye, X., Wang, J., Zhang, S., Wang, H., Ou, X., Li, Y., Du, S., Chen, Q., & Yang, X. (2026). Monomerizing the ultrabright AausFP1 yields mBiyu for advanced imaging across challenging bacterial and mammalian cells. 浪淘沙预印本平台. https://doi.org/10.65215/LTSpreprints.2026.05.06.000227

利益冲突声明

作者声明无任何需要披露的利益冲突。