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

PLoS One. 2016 Aug 1;11(8):e0159292. doi: 10.1371/journal.pone.0159292. eCollection 2016.

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.

MeSH terms

  • Flow Cytometry
  • Genetic Vectors / genetics
  • Genetic Vectors / metabolism
  • Genotype
  • Image Processing, Computer-Assisted
  • Kinetics
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism*
  • Microscopy, Fluorescence
  • Protein Folding
  • RNA, Messenger / metabolism
  • Real-Time Polymerase Chain Reaction
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Schizosaccharomyces / genetics
  • Schizosaccharomyces / metabolism*
  • Schizosaccharomyces pombe Proteins / genetics
  • Schizosaccharomyces pombe Proteins / metabolism*

Substances

  • Luminescent Proteins
  • RNA, Messenger
  • Recombinant Fusion Proteins
  • Schizosaccharomyces pombe Proteins