Arabidopsis thaliana cryptochrome 2 (AtCRY2), a light-sensitive photosensory protein, was previously adapted for use controling protein-protein interactions through light-dependent binding to a partner protein, CIB1. While the existing CRY2/CIB dimerization system has been used extensively for optogenetic applications, some limitations exist. Here, we set out to optimize function of the CRY2/CIB system, to identify versions of CRY2/CIB that are smaller, show reduced dark interaction, and maintain longer or shorter signaling states in response to a pulse of light. We describe minimal functional CRY2 and CIB1 domains maintaining light-dependent interaction and new signaling mutations affecting AtCRY2 photocycle kinetics. The latter work implicates a α13-α14 turn motif within plant CRYs where perturbations alter signaling state lifetime. Using a long-lived L348F photocycle mutant, we engineered a second generation photoactivatable Cre recombinase, PA-Cre2.0, that shows five-fold improved dynamic range allowing robust recombination following exposure to a single, brief pulse of light.

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.

Over the past decades, there has been growing recognition that light can provide a powerful stimulus for biological interrogation. Light-actuated tools allow manipulation of molecular events with ultra-fine spatial and fast temporal resolution, as light can be rapidly delivered and focused with sub-micrometer precision within cells. While light-actuated chemicals such as photolabile “caged” compounds have been in existence for decades, the use of genetically-encoded natural photoreceptors for optical control of biological processes has recently emerged as a powerful new approach with several advantages over traditional methods. Here we review recent advances using light to control basic cellular functions and discuss the engineering challenges that lie ahead for improving and expanding the ever-growing optogenetic toolkit.

Optogenetic tools allow regulation of cellular processes with light, which can be delivered with spatiotemporal resolution. In previous work, we used cryptochrome 2 (CRY2) and CIB1, Arabidopsis proteins that interact upon light illumination, to regulate transcription with light in yeast. While adopting this approach to regulate transcription in mammalian cells, we observed light-dependent redistribution and clearing of CRY2-tethered proteins within the nucleus. The nuclear clearing phenotype was dependent on the presence of a dimerization domain contained within the CRY2-fused transcriptional activators. We used this knowledge to develop two different approaches to regulate cellular protein levels with light: a system using CRY2 and CIB1 to induce protein expression with light through stimulation of transcription, and a system using CRY2 and a LOV-fused degron to simultaneously block transcription and deplete protein levels with light. These tools will allow precise, bi-directional control of gene expression in a variety of cells and model systems.

NAD is a key metabolic redox cofactor that is regenerated from nicotinamide through the NAD salvage pathway. Here, we find that inhibiting the NAD salvage pathway depletes serine biosynthesis from glucose by impeding the NAD-dependent protein, 3-phosphoglycerate dehydrogenase (PHGDH). Importantly, we find that PHGDH breast cancer cell lines are exquisitely sensitive to inhibition of the NAD salvage pathway. Further, we find that PHGDH protein levels and those of the rate-limiting enzyme of NAD salvage, NAMPT, correlate in ER-negative, basal-like breast cancers. Although NAD salvage pathway inhibitors are actively being pursued in cancer treatment, their efficacy has been poor, and our findings suggest that they may be effective for PHGDH-dependent cancers.