Two coral fluorescent proteins of distinct colors for sharp visualization of cell-cycle progression

Abstract

Received for publication, April 12, 2023, accepted, June 26, 2023, and published online, June 30, 2023 We cloned and characterized two new coral fluorescent proteins: h2-3 and 1-41. h2-3 formed an obligate dimeric complex and exhibited bright green fluorescence. On the other hand, 1-41 formed a highly multimeric complex and exhibited dim red fluorescence. We engineered 1-41 into AzaleaB5, a practically useful red-emitting fluorescent protein for cellular labeling applications. We fused h2-3 and AzaleaB5 to the ubiquitination domains of human Geminin and Cdt1, respectively, to generate a new color variant of Fucci (Fluorescent Ubiquitination-based Cell-Cycle Indicator): Fucci5. We found Fucci5 provided more reliable nuclear labeling for monitoring cell-cycle progression than the 1st and 2nd generations that used mAG/mKO2 and mVenus/mCherry, respectively.