The Arabidopsis photoreceptor cryptochrome 2 (CRY2) was previously used as an optogenetic module, allowing spatiotemporal control of cellular processes with light. Here we report the development of a new CRY2-derived optogenetic module, ‘CRY2olig’, which induces rapid, robust, and reversible protein oligomerization in response to light. Using this module, we developed a novel protein interaction assay, LINC (Light Induced Co-clustering) that can be used to interrogate protein interaction dynamics in live cells. In addition to use probing protein interactions, CRY2olig can also be used to induce and reversibly control diverse cellular processes with spatial and temporal resolution. Here, we demonstrate disrupting clathrin-mediated endocytosis and promoting Arp2/3-mediated actin polymerization with light. These new CRY2-based approaches expand the growing arsenal of optogenetic strategies to probe cellular function.
Natural genetic circuits enable cells to make sophisticated digital decisions. Building equally complex synthetic circuits in eukaryotes remains difficult, however, because commonly used components leak transcriptionally, do not arbitrarily interconnect or do not have digital responses. Here, we designed dCas9-Mxi1-based NOR gates in Saccharomyces cerevisiae that allow arbitrary connectivity and large genetic circuits. Because we used the chromatin remodeller Mxi1, our gates showed minimal leak and digital responses. We built a combinatorial library of NOR gates that directly convert guide RNA (gRNA) inputs into gRNA outputs, enabling the gates to be ‘wired' together. We constructed logic circuits with up to seven gRNAs, including repression cascades with up to seven layers. Modelling predicted the NOR gates have effectively zero transcriptional leak explaining the limited signal degradation in the circuits. Our approach enabled the largest, eukaryotic gene circuits to date and will form the basis for large, synthetic, cellular decision-making systems.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.