Benchmarking of Optical Dimerizer Systems

TLDR

Optical dimerizers enable light‑inducible protein–protein interactions and have been used to regulate cellular pathways with high spatiotemporal resolution, but the diversity of systems and heterogeneous characterizations make direct comparison difficult. This study systematically benchmarks four optical dimerizer systems—CRY2/CIB1, TULIPs, phyB/PIF3, and phyB/PIF6—to evaluate their properties. The authors employed a yeast transcriptional assay to measure light sensitivity and fold‑activation, compared CRY2/CIB1 and TULIP in a yeast MAPK signaling pathway, and developed an improved blue‑light‑regulated transcriptional system and a membrane‑tethered CRY2 variant. Red‑light systems differed markedly in sensitivity and activation, whereas CRY2/CIB and TULIP showed similar responses with CRY2/CIB exhibiting slightly lower dark background; the new blue‑light system and membrane‑tethered CRY2 demonstrate enhanced control of transcription and signaling in yeast, providing a clearer understanding of dimerizer capacities.

Abstract

Optical dimerizers are a powerful new class of optogenetic tools that allow light-inducible control of protein–protein interactions. Such tools have been useful for regulating cellular pathways and processes with high spatiotemporal resolution in live cells, and a growing number of dimerizer systems are available. As these systems have been characterized by different groups using different methods, it has been difficult for users to compare their properties. Here, we set about to systematically benchmark the properties of four optical dimerizer systems, CRY2/CIB1, TULIPs, phyB/PIF3, and phyB/PIF6. Using a yeast transcriptional assay, we find significant differences in light sensitivity and fold-activation levels between the red light regulated systems but similar responses between the CRY2/CIB and TULIP systems. Further comparison of the ability of the CRY2/CIB1 and TULIP systems to regulate a yeast MAPK signaling pathway also showed similar responses, with slightly less background activity in the dark observed with CRY2/CIB. In the process of developing this work, we also generated an improved blue-light-regulated transcriptional system using CRY2/CIB in yeast. In addition, we demonstrate successful application of the CRY2/CIB dimerizers using a membrane-tethered CRY2, which may allow for better local control of protein interactions. Taken together, this work allows for a better understanding of the capacities of these different dimerization systems and demonstrates new uses of these dimerizers to control signaling and transcription in yeast.