Curated Optogenetic Publication Database

Abstract: Tools capable of modulating gene expression in living organisms are very useful for interrogating the gene regulatory network and controlling biological processes. The catalytically inactive CRISPR/Cas9 (dCas9), when fused with repressive or activating effectors, functions as a versatile platform to reprogram gene transcription at targeted genomic loci. However, without temporal control, the application of these reprogramming tools will likely cause off-target effects and lack strict reversibility. To overcome this limitation, we report herein the development of a chemical or light-inducible transcriptional reprogramming device that combines photoswitchable genetically encoded calcium actuators with dCas9 to control gene expression. By fusing an engineered Ca2+-responsive NFAT fragment with dCas9 and transcriptional coactivators, we harness the power of light to achieve photoinducible transcriptional reprogramming in mammalian cells. This synthetic system (designated CaRROT) can also be used to document calcium-dependent activity in mammals after exposure to ligands or chemicals that would elicit calcium response inside cells.

Abstract: Cellular signal transduction is often regulated at multiple steps in order to achieve more complex logic or precise control of a pathway. For instance, some signaling mechanisms couple allosteric activation with localization to achieve high signal to noise. Here, we create a system for light activated nuclear import that incorporates two levels of control. It consists of a nuclear import photoswitch, Light Activated Nuclear Shuttle (LANS), and a protein engineered to preferentially interact with LANS in the dark, Zdk2. First, Zdk2 is tethered to a location in the cytoplasm, which sequesters LANS in the dark. Second, LANS incorporates a nuclear localization signal (NLS) that is sterically blocked from binding to the nuclear import machinery in the dark. When activated with light, LANS both dissociates from its tethered location and exposes its NLS, which leads to nuclear accumulation. We demonstrate that this coupled system improves the dynamic range of LANS in mammalian cells, yeast, and C. elegans and provides tighter control of transcription factors that have been fused to LANS.

Abstract: End-binding proteins (EBs) are adaptors that recruit functionally diverse microtubule plus-end-tracking proteins (+TIPs) to growing microtubule plus ends. To test with high spatial and temporal accuracy how, when and where +TIP complexes contribute to dynamic cell biology, we developed a photo-inactivated EB1 variant (π-EB1) by inserting a blue-light-sensitive protein–protein interaction module between the microtubule-binding and +TIP-binding domains of EB1. π-EB1 replaces endogenous EB1 function in the absence of blue light. By contrast, blue-light-mediated π-EB1 photodissociation results in rapid +TIP complex disassembly, and acutely and reversibly attenuates microtubule growth independent of microtubule end association of the microtubule polymerase CKAP5 (also known as ch-TOG and XMAP215). Local π-EB1 photodissociation allows subcellular control of microtubule dynamics at the second and micrometre scale, and elicits aversive turning of migrating cancer cells. Importantly, light-mediated domain splitting can serve as a template to optically control other intracellular protein activities.

Abstract: Structures arising from actin-based cell membrane movements, including ruffles, lamellipodia, and filopodia, play important roles in a broad spectrum of cellular functions, such as cell motility, axon guidance in neurons, wound healing, and micropinocytosis. Previous studies investigating these cell membrane dynamics often relied on pharmacological inhibition, RNA interference, and constitutive active/dominant negative protein expression systems. However, such studies did not allow the modulation of protein activity at specific regions of cells, tissues, and organs in animals with high spatial and temporal precision. Recently, optogenetic tools for inducing cell membrane dynamics have been developed which address several of the disadvantages of previous techniques. In a recent study, we developed a powerful optogenetic tool, called the Magnet system, to change cell membrane dynamics through Tiam1 and PIP3 signal transductions with high spatial and temporal resolution. In this review, we summarize recent advances in optogenetic tools that allow us to induce actin-regulated cell membrane dynamics and unique membrane ruffles that we discovered using our Magnet system.

Abstract: The ability to study cellular physiology using photosensitive, genetically encoded molecules has profoundly transformed neuroscience. The modern optogenetic toolbox includes fluorescent sensors to visualize signaling events in living cells and optogenetic actuators enabling manipulation of numerous cellular activities. Most optogenetic tools are not targeted to specific subcellular compartments but are localized with limited discrimination throughout the cell. Therefore, optogenetic activation often does not reflect context-dependent effects of highly localized intracellular signaling events. Subcellular targeting is required to achieve more specific optogenetic readouts and photomanipulation. Here we first provide a detailed overview of the available optogenetic tools with a focus on optogenetic actuators. Second, we review established strategies for targeting these tools to specific subcellular compartments. Finally, we discuss useful tools and targeting strategies that are currently missing from the optogenetics repertoire and provide suggestions for novel subcellular optogenetic applications.

Abstract: Temporal kinetics and spatial coordination of signal transduction in cells are vital for cell fate determination. Tools that allow for precise modulation of spatiotemporal regulation of intracellular signaling in intact cells and multicellular organisms remain limited. The emerging optobiological approaches use light to control protein-protein interaction in live cells and multicellular organisms. Optobiology empowers light-mediated control of diverse cellular and organismal functions such as neuronal activity, intracellular signaling, gene expression, cell proliferation, differentiation, migration, and apoptosis. In this review, we highlight recent developments in optobiology, focusing on new features of second-generation optobiological tools. We cover applications of optobiological approaches in the study of cellular and organismal functions, discuss current challenges, and present our outlook. Taking advantage of the high spatial and temporal resolution of light control, optobiology promises to provide new insights into the coordination of signaling circuits in intact cells and multicellular organisms.

Abstract: Precise spatial and temporal control of cellular processes is in life sciences a highly sought-after capability. In the recent years, this goal has become progressively achievable through the field of optogenetics, which utilizes light as a non-invasive means to control genetically encoded light-responsive proteins. The latest optogenetic systems, such as those for control of subcellular localization or cellular decision-making and tissue morphogenesis provide us with insights to gain a deeper understanding of the cellular inner workings. Besides, they hold a potential for further development into biomedical applications, from in vitro optogenetics-assisted drug candidate screenings to light-controlled gene therapy and tissue engineering.

Abstract: A long-standing question in neurodevelopment is how neurons develop a single axon and multiple dendrites from common immature neurites. Long-range inhibitory signaling from the growing axon is hypothesized to prevent outgrowth of other immature neurites and to differentiate them into dendrites, but the existence and nature of this inhibitory signaling remains unknown. Here, we demonstrate that axonal growth triggered by neurotrophin-3 remotely inhibits neurite outgrowth through long-range Ca2+ waves, which are delivered from the growing axon to the cell body. These Ca2+ waves increase RhoA activity in the cell body through calcium/calmodulin-dependent protein kinase I. Optogenetic control of Rho-kinase combined with computational modeling reveals that active Rho-kinase diffuses to growing other immature neurites and inhibits their outgrowth. Mechanistically, calmodulin-dependent protein kinase I phosphorylates a RhoA-specific GEF, GEF-H1, whose phosphorylation enhances its GEF activity. Thus, our results reveal that long-range inhibitory signaling mediated by Ca2+ wave is responsible for neuronal polarization.Emerging evidence suggests that gut microbiota influences immune function in the brain and may play a role in neurological diseases. Here, the authors offer in vivo evidence from a Drosophila model that supports a role for gut microbiota in modulating the progression of Alzheimer's disease.

Abstract: We describe a detailed procedure for the use of LOVTRAP, an approach to reversibly sequester and release proteins from cellular membranes using light. In the application described here, proteins that act at the plasma membrane are held at mitochondria in the dark, and reversibly released by irradiation. The technique relies on binding of an engineered Zdk domain to a LOV2 domain, with affinity <30 nM in the dark and >500 nM upon irradiation between 400 and 500 nm. LOVTRAP can be applied to diverse proteins, as it requires attaching only one member of the Zdk/LOV2 pair to the target protein, and the other to the membrane where the target protein is to be sequestered. Light-induced protein release occurs in less than a second, and the half-life of return can be adjusted using LOV point mutations (∼2 to 500 sec). © 2016 by John Wiley & Sons, Inc.

Abstract: Photo-controlled or 'optogenetic' effectors interfacing with endogenous protein machinery allow the roles of endogenous proteins to be probed. There are two main approaches being used to develop optogenetic effectors: (i) caging strategies using photo-controlled conformational changes, and (ii) protein relocalization strategies using photo-controlled protein-protein interactions. Numerous specific examples of these approaches have been reported and efforts to develop general methods for photo-control of endogenous proteins are a current focus. The development of improved screening and selection methods for photo-switchable proteins would advance the field.

Abstract: LOVTRAP is an optogenetic approach for reversible light-induced protein dissociation using protein A fragments that bind to the LOV domain only in the dark, with tunable kinetics and a >150-fold change in the dissociation constant (Kd). By reversibly sequestering proteins at mitochondria, we precisely modulated the proteins' access to the cell edge, demonstrating a naturally occurring 3-mHz cell-edge oscillation driven by interactions of Vav2, Rac1, and PI3K proteins.