Viola Giulia, Ibrahim Yasmeen W, Jacobs Kyle A, Lemière Joël, Kutys Matthew L, Wittmann Torsten
Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA 94143, USA.
J Cell Sci. 2025 May 15;138(10). doi: 10.1242/jcs.263858. Epub 2025 May 21.
To standardize comparison of fluorescent protein performance on a molecule-by-molecule basis in a physiological intracellular environment, we constructed fluorescent protein-tagged I3-01 peptides that self-assemble into stable 60-subunit dodecahedrons inside live mammalian cells. We were especially interested in determining which of the recently published monomeric StayGold variants is best for live microscopy in mammalian cells. Combining nanocage brightness and photobleaching measurements into a single metric, mStayGold stood out as far superior to all other green and red fluorescent proteins we tested with a functional lifetime that is at least 8-10-fold longer compared with EGFP or mEmerald. Analysis of intracellular nanocage diffusion further confirmed the monomeric nature of mStayGold, and we demonstrate that mStayGold-tagged nanocages can serve as highly photostable nanoparticles to analyze intracellular biophysical properties. Analysis of frequently used red fluorescent proteins was less encouraging and recent mScarlet or mRuby variants did not perform substantially better than mCherry on a typical spinning disc confocal microscope system, highlighting the importance of a standardized method to benchmark fluorescent proteins to make optimal choices for specific experimental setups.
为了在生理细胞内环境中逐个分子地标准化荧光蛋白性能的比较，我们构建了荧光蛋白标记的I3-01肽，其在活的哺乳动物细胞内自组装成稳定的60亚基十二面体。我们特别感兴趣的是确定最近发表的单体StayGold变体中哪一种最适合用于哺乳动物细胞的活细胞显微镜检查。将纳米笼亮度和光漂白测量结合成一个单一指标，mStayGold表现突出，远远优于我们测试的所有其他绿色和红色荧光蛋白，其功能寿命比EGFP或mEmerald至少长8至10倍。细胞内纳米笼扩散分析进一步证实了mStayGold的单体性质，并且我们证明mStayGold标记的纳米笼可以作为高度光稳定的纳米颗粒来分析细胞内生物物理性质。对常用红色荧光蛋白的分析结果不那么令人鼓舞，并且在典型的转盘共聚焦显微镜系统上，最近的mScarlet或mRuby变体的表现并不比mCherry好很多，这突出了一种标准化方法对于基准荧光蛋白以针对特定实验设置做出最佳选择的重要性。
