title: Sensitive and Quantitative Three-Color Protein Imaging in Fission Yeast Using Spectrally Diverse, Recoded Fluorescent Proteins with Experimentally-Characterized In Vivo Maturation Kinetics.
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.
authors.affiliation: Department of Microbiology and Immunology, the GW Hooper Foundation, University of California San Francisco, San Francisco, California 94143, United States of America.
authors.forename: Bassem
authors.initials: B
authors.lastname: Al-Sady
authors.affiliation: 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.
authors.forename: Rachel A
authors.initials: RA
authors.lastname: Greenstein
authors.affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California 94143, United States of America.
authors.forename: Hana J
authors.initials: HJ
authors.lastname: El-Samad
authors.affiliation: Department of Physiological Chemistry, Biomedical Center, Ludwigs-Maximilians-University of Munich, 82152 Martinsried, Germany.
authors.forename: Sigurd
authors.initials: S
authors.lastname: Braun
authors.affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California 94143, United States of America.
authors.forename: Hiten D
authors.initials: HD
authors.lastname: Madhani
mesh_terms: D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D005838:Genotype; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D017510:Protein Folding; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D008164:Luminescent Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins