Al-Sady Bassem, Greenstein Rachel A, El-Samad Hana J, Braun Sigurd, Madhani Hiten D
Department of Microbiology and Immunology, the GW Hooper Foundation, University of California San Francisco, San Francisco, California 94143, United States of America.
TETRAD graduate program, University of California San Francisco, San Francisco, California 94143, United States of America.
PLoS One. 2016 Aug 1;11(8):e0159292. doi: 10.1371/journal.pone.0159292. eCollection 2016.
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.
粟酒裂殖酵母是细胞生物学研究中一种出色的模式生物，但有用且特征明确的荧光蛋白（XFPs）种类有限。我们生成并表征了三种用于粟酒裂殖酵母三色分析的重新编码荧光蛋白，即超折叠绿色荧光蛋白、单体橙色荧光蛋白2和E2深红色荧光蛋白。在粟酒裂殖酵母中进行优化和表达后，这三种蛋白实现了灵敏的同时三色检测能力。此外，我们描述了一种结合脉冲追踪方法和数学建模来量化这些蛋白在体内成熟动力学的策略。我们在粟酒裂殖酵母中观察到的成熟动力学与这些蛋白在体外和/或其他细胞类型中所描述的预期一致，但也有一些意外行为。我们的研究为粟酒裂殖酵母提供了一个具有动力学特征的集成三色XFP工具箱。
