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

<div><p><i>Schizosaccharomyces pombe</i> 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 <i>S</i>.<i>pombe</i>, Super-folder GFP, monomeric Kusabira Orange 2 and E2Crimson. Upon optimization and expression in <i>S</i>. <i>pombe</i>, 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 <i>in vivo</i>. We observed maturation kinetics in <i>S</i>. <i>pombe</i> that are expected from those described for these proteins <i>in vitro</i> and/or in other cell types, but also unpredicted behaviors. Our studies provide a kinetically-characterized, integrated three-color XFP toolbox for <i>S</i>. <i>pombe</i>.</p>