Format

Send to

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

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

Author information

1
Department of Microbiology and Immunology, the GW Hooper Foundation, University of California San Francisco, San Francisco, California 94143, United States of America.
2
TETRAD graduate program, University of California San Francisco, San Francisco, California 94143, United States of America.
3
Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California 94143, United States of America.
4
Department of Physiological Chemistry, Biomedical Center, Ludwigs-Maximilians-University of Munich, 82152 Martinsried, Germany.

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.

PMID:
27479698
PMCID:
PMC4968791
DOI:
10.1371/journal.pone.0159292
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Public Library of Science Icon for PubMed Central
Loading ...
Support Center