Sensitive and Quantitative Three-Color Protein Imaging in Fission Yeast Using Spectrally Diverse, Recoded Fluorescent Proteins with Experimentally-Characterized In Vivo Maturation Kinetics

Schizosaccharomyces pombe is an outstanding model organism for cell biological investigations, yet the range of useful and well-characterized fluorescent proteins (XFPs) is limited. We generated and characterized three recoded fluorescent proteins for 3-color analysis in S.pombe, Super-folder GFP, monomeric Kusabira Orange 2 and E2Crimson. Upon optimization and expression in S. pombe, the three proteins enabled sensitive simultaneous 3-color detection capability. Furthermore, we describe a strategy that combines a pulse-chase approach and mathematical modeling to quantify the maturation kinetics of these proteins in vivo. We observed maturation kinetics in S. pombe that are expected from those described for these proteins in vitro and/or in other cell types, but also unpredicted behaviors. Our studies provide a kinetically-characterized, integrated three-color XFP toolbox for S. pombe.Schizosaccharomyces pombe（粟酒裂殖酵母）是用于细胞生物学研究的优质模式生物，但目前可用且经过充分表征的荧光蛋白（XFPs）种类较为有限。我们构建并表征了三种适用于粟酒裂殖酵母三色分析的经密码子优化的荧光蛋白：超折叠绿色荧光蛋白（Super-folder GFP）、单体库巴橙2（monomeric Kusabira Orange 2）与E2Crimson。通过在粟酒裂殖酵母中优化表达条件并实现表达，这三种蛋白可实现灵敏的同步三色检测。此外，我们还描述了一种结合脉冲追踪（pulse-chase）实验与数学建模的策略，用于定量解析这些蛋白在体内（in vivo）的成熟动力学过程。我们在粟酒裂殖酵母中观测到的成熟动力学特征，既与这些蛋白在体外（in vitro）或其他细胞类型中的已有报道相符，同时也存在未被预测的行为模式。本研究为粟酒裂殖酵母提供了一套经过动力学表征的集成式三色荧光蛋白工具箱。