Abstract
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.
Highlights
Visualizing the relationships between cellular structures in vivo requires the ability to tag and image multiple proteins at once
We report here a recoded, optimized set of XFPs for S. pombe, that offers sensitive detection, simultaneous three-color detection, and for which we have determined the in vivo folding kinetics using a novel approach
MTagBFP2, turbo GFP and, surprisingly, YFP-Venus gave no detectable signal by flow cytometry. mKate2 and mOrange2 gave only weak signal, while SF-GFP, TagRFP-T, mKO2, E2Crimson and mCherry have low to intermediate signals (Fig 2B)
Summary
Visualizing the relationships between cellular structures in vivo requires the ability to tag and image multiple proteins at once. This is generally accomplished using engineered fluorescent proteins (XFPs)[1]. The fission yeast S. pombe is a premier system for investigations of cell biology. Only a few XFPs have been fully validated for dynamic analysis in S.pombe. The choice for characterized and optimized fluorescent proteins that are suitable for multicolor analysis is limited compared to the arsenal available for Saccharomyces cerevisiae [2]. Most studies in S.pombe focus on protein fusions to either GFP or YFP. There are few PLOS ONE | DOI:10.1371/journal.pone.0159292. There are few PLOS ONE | DOI:10.1371/journal.pone.0159292 August 1, 2016
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