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The light-oxygen-voltage (LOV) domains of phototropins emerged as essential constituents of light-sensitive proteins, helping initiate blue light-triggered responses. Moreover, these domains have been identified across all kingdoms of life. LOV domains utilize flavin nucleotides as co-factors and undergo structural rearrangements upon exposure to blue light, which activates an effector domain that executes the final output of the photoreaction. LOV domains are versatile photoreceptors that play critical roles in cellular signaling and environmental adaptation; additionally, they can noninvasively sense and control intracellular processes with high spatiotemporal precision, making them ideal candidates for use in optogenetics, where a light signal is linked to a cellular process through a photoreceptor. The ongoing development of LOV-based optogenetic tools, driven by advances in structural biology, spectroscopy, computational methods, and synthetic biology, has the potential to revolutionize the study of biological systems and enable the development of novel therapeutic strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Photoswitchable proteins enable specific molecular events occurring in complex biological settings to be probed in a rapid and reversible fashion. Recent progress in the development of photoswitchable proteins as components of optogenetic tools has been greatly facilitated by directed evolution approaches in vitro, in bacteria, or in yeast. We review these developments and suggest future directions for this rapidly advancing field., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Three classes of flavoprotein photoreceptors, cryptochromes (CRYs), light-oxygen-voltage (LOV)-domain proteins, and blue light using FAD (BLUF)-domain proteins, have been identified that control various physiological processes in multiple organisms. Accordingly, signaling activities of photoreceptors have been intensively studied and the related mechanisms have been exploited in numerous optogenetic tools. Herein, we summarize the current understanding of photoactivation mechanisms of the flavoprotein photoreceptors and review their applications.

This chapter provides an overview of the technologies we have developed to control proteins with light. First, we focus on the LOV domain, a versatile building block with reversible photo-response, kinetics tunable through mutagenesis, and ready expression in a broad range of cells and animals. Incorporation of LOV into proteins produced a variety of approaches: simple steric block of the active site released when irradiation lengthened a linker (PA-GTPases), reversible release from sequestration at mitochondria (LOVTRAP), and Z-lock, a method in which a light-cleavable bridge is placed where it occludes the active site. The latter two methods make use of Zdk, small engineered proteins that bind selectively to the dark state of LOV. In order to control endogenous proteins, inhibitory peptides are embedded in the LOV domain where they are exposed only upon irradiation (PKA and MLCK inhibition). Similarly, controlled exposure of a nuclear localization sequence and nuclear export sequence is used to reversibly send proteins into the nucleus. Another avenue of engineering makes use of the heterodimerization of FKBP and FRB proteins, induced by the small molecule rapamycin. We control rapamycin with light or simply add it to target cells. Incorporation of fused FKBP-FRB into kinases, guanine exchange factors, or GTPases leads to rapamycin-induced protein activation. Kinases are engineered so that they can interact with only a specific substrate upon activation. Recombination of split proteins using rapamycin-induced conformational changes minimizes spontaneous reassembly. Finally, we explore the insertion of LOV or rapamycin-responsive domains into proteins such that light-induced conformational changes exert allosteric control of the active site. We hope these design ideas will inspire new applications and broaden our reach towards dynamic biological processes that unfold when studied in vivo.

Cells employ a plethora of signaling pathways to make their life-and-death decisions. Extensive genetic, biochemical, and physiological studies have led to the accumulation of knowledge about signaling components and their interactions within signaling networks. These conventional approaches, although useful, lack the ability to control the spatial and temporal aspects of signaling processes. The recently emerged optogenetic tools open exciting opportunities by enabling signaling regulation with superior temporal and spatial resolution, easy delivery, rapid reversibility, fewer off-target side effects, and the ability to dissect complex signaling networks. Here we review recent achievements in using light to control intracellular signaling pathways and discuss future prospects for the field, including integration of new genetic approaches into optogenetics., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Cells generate a highly diverse microtubule network to carry out different activities. This network is comprised of distinct tubulin isotypes, tubulins with different post-translational modifications, and many microtubule-based structures. Defects in this complex system cause numerous human disorders. However, how different microtubule subtypes in this network regulate cellular architectures and activities remains largely unexplored. Emerging tools such as photosensitive pharmaceuticals, chemogenetics, and optogenetics enable the spatiotemporal manipulation of structures, dynamics, post-translational modifications, and cross-linking with actin filaments in target microtubule subtypes. This review summarizes the design rationale and applications of these new approaches and aims to provide a roadmap for researchers navigating the intricacies of microtubule dynamics and their post-translational modifications in cellular contexts, thereby opening new avenues for therapeutic interventions. [ABSTRACT FROM AUTHOR]

The ability to manipulate expression of exogenous genes in particular regions of living organisms has profoundly transformed the way we study biomolecular processes involved in both normal development and disease. Unfortunately, most of the classical inducible systems lack fine spatial and temporal accuracy, thereby limiting the study of molecular events that strongly depend on time, duration of activation, or cellular localization. By exploiting genetically engineered photo sensing proteins that respond to specific wavelengths, we can now provide acute control of numerous molecular activities with unprecedented precision. In this review, we present a comprehensive breakdown of all of the current optogenetic systems adapted to regulate gene expression in both unicellular and multicellular organisms. We focus on the advantages and disadvantages of these different tools and discuss current and future challenges in the successful translation to more complex organisms.

The ability to manipulate expression of exogenous genes in particular regions of living organisms has profoundly transformed the way we study biomolecular processes involved in both normal development and disease. Unfortunately, most of the classical inducible systems lack fine spatial and temporal accuracy, thereby limiting the study of molecular events that strongly depend on time, duration of activation, or cellular localization. By exploiting genetically engineered photo sensing proteins that respond to specific wavelengths, we can now provide acute control of numerous molecular activities with unprecedented precision. In this review, we present a comprehensive breakdown of all of the current optogenetic systems adapted to regulate gene expression in both unicellular and multicellular organisms. We focus on the advantages and disadvantages of these different tools and discuss current and future challenges in the successful translation to more complex organisms. [ABSTRACT FROM AUTHOR]

We report natural light-oxygen-voltage (LOV) photoreceptors with a blue light-switched, high-affinity (KD ~ 10-7 M), and direct electrostatic interaction with anionic phospholipids. Membrane localization of one such photoreceptor, BcLOV4 from Botrytis cinerea, is directly coupled to its flavin photocycle, and is mediated by a polybasic amphipathic helix in the linker region between the LOV sensor and its C-terminal domain of unknown function (DUF), as revealed through a combination of bioinformatics, computational protein modeling, structure-function studies, and optogenetic assays in yeast and mammalian cell line expression systems. In model systems, BcLOV4 rapidly translocates from the cytosol to plasma membrane (~1 second). The reversible electrostatic interaction is nonselective among anionic phospholipids, exhibiting binding strengths dependent on the total anionic content of the membrane without preference for a specific headgroup. The in vitro and cellular responses were also observed with a BcLOV4 homolog and thus are likely to be general across the dikarya LOV class, whose members are associated with regulator of G-protein signaling (RGS) domains. Natural photoreceptors are not previously known to directly associate with membrane phospholipids in a light-dependent manner, and thus this work establishes both a photosensory signal transmission mode and a single-component optogenetic tool with rapid membrane localization kinetics that approaches the diffusion limit. [ABSTRACT FROM AUTHOR]

Rational design of optogenetic tools is inherently linked to the understanding of photoreceptor function. Structural analysis of elements involved in signal integration in individual sensor domains provides an initial idea of their mode of operation, but understanding how local structural rearrangements eventually affect signal transmission to output domains requires inclusion of the effector regions in the characterization. However, the dynamic nature of these assemblies renders their structural analysis challenging and therefore a combination of high- and low-resolution techniques is required to appreciate functional aspects of photoreceptors.This review focuses on the potential of Hydrogen-Deuterium exchange coupled to mass spectrometry (HDX-MS) for complementing the structural characterization of photoreceptors. In this respect, the ability of HDX-MS to provide information on the conformational dynamics and the possibility to address multiple functionally relevant states in solution render this methodology ideally suitable. We highlight recent examples demonstrating the potential of HDX-MS and discuss how these results can help to improve existing optogenetic systems or guide the design of novel optogenetic tools.

Light-oxygen-voltage sensitive (LOV) flavoproteins are ubiquitous photoreceptors that mediate responses to environmental cues. Photosensory inputs are transduced into signaling outputs via structural rearrangements in sensor domains that consequently modulate the activity of an effector domain or multidomain clusters. Establishing the diversity in effector function and sensor-effector topology will inform what signaling mechanisms govern light-responsive behaviors across multiple kingdoms of life and how these signals are transduced. Here,we report the bioinformatics identification of over 6,700 candidate LOV domains (including over 4,000 previously unidentified sequences from plants and protists), and insights from their annotations for ontological function and structural arrangements. Motif analysis identified the sensors from ~42 million ORFs, with strong statistical separation from other flavoproteins and non-LOV members of the structurally related Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim family. Conserved-domain analysis determined putative light-regulated function and multidomain topologies. We found that for certain effectors, sensor-effector linker length is discretized based on both phylogeny and the preservation of α-helical heptad repeats within an extended coiled-coil linker structure. This finding suggests that preserving sensor-effector orientation is a key determinant of linker length, in addition to ancestry, in LOV signaling structure-function. We found a surprisingly high prevalence of effectors with functions previously thought to be rare among LOV proteins, such as regulators of G protein signaling, and discovered several previously unidentified effectors, such as lipases. This work highlights the value of applying genomic and transcriptomic technologies to diverse organisms to capture the structural and functional variation in photosensory proteins that are vastly important in adaptation, photobiology, and optogenetics. [ABSTRACT FROM AUTHOR]

Aureochromes in stramenopiles contain a basic leucine zipper (bZIP) domain in the central region and a light-oxygen-voltage-sensing (LOV) domain, and are thought to function as light-regulated transcription factors. To understand the molecular mechanism, we investigated the photoreactions, oligomeric structures, and DNA binding of recombinant aureochrome-1 (AUREO1). The results suggest that monomeric AUREO1 is present in reduced conditions and undergoes dimerization upon illumination. Blue light-induced dimerization enhances the affinity for the target sequence. Our N-terminally truncated mutants may be useful molecular tools as a photoactivatable- bZIP module (Photozipper) for optogenetics and biophysical analyses. [ABSTRACT FROM AUTHOR]

Optogenetics arises from the innovative application of microbial opsins in mammalian neurons and has since been a powerful technology that fuels the advance of our knowledge in neuroscience. In recent years, there has been growing interest in designing optogenetic tools extendable to broader cell types and biochemical signals. To date, a variety of photoactivatable proteins (refers to induction of protein activity in contrast to fluorescence) have been developed based on the understanding of plant and microbial photoreceptors including phototropins, blue light sensors using flavin adenine dinucleotide proteins, cryptochromes, and phytochromes. Such tools offered researchers reversible, quantitative, and precise spatiotemporal control of enzymatic activity, protein-protein interaction, protein translocation, as well as gene transcription in cells and in whole animals. In this review, we will briefly introduce these photosensory proteins, describe recent developments in optogenetics, and compare and contrast different methods based on their advantages and limitations. [ABSTRACT FROM AUTHOR]