Curated Optogenetic Publication Database

Abstract: An optogenetic Bax has been designed that facilitates light-induced apoptosis. We demonstrate that mitochondrial recruitment of a genetically encoded light-responsive Bax results in the release of mitochondrial proteins, downstream caspase-3 cleavage, changes in cellular morphology, and ultimately cell death. Mutagenesis of a key phosphorylatable residue or modification of the C-terminus mitigates background (dark) levels of apoptosis that result from Bax overexpression. The mechanism of optogenetic Bax-mediated apoptosis was explored using a series of small molecules known to interfere with various steps in programmed cell death. Optogenetic Bax appears to form a mitochondrial apoptosis-induced channel analogous to that of endogenous Bax.

Abstract: Light-activatable proteins allow precise spatial and temporal control of biological processes in living cells and animals. Several approaches have been developed for controlling protein localization with light, including the conditional inhibition of a nuclear localization signal (NLS) with the Light Oxygen Voltage (AsLOV2) domain of phototropin 1 from Avena sativa. In the dark, the switch adopts a closed conformation that sterically blocks the NLS motif. Upon activation with blue light the C-terminus of the protein unfolds, freeing the NLS to direct the protein to the nucleus. A previous study showed that this approach can be used to control the localization and activity of proteins in mammalian tissue culture cells. Here, we extend this result by characterizing the binding properties of a LOV/NLS switch and demonstrating that it can be used to control gene transcription in yeast. Additionally, we show that the switch, referred to as LANS (light-activated nuclear shuttle), functions in the C. elegans embryo and allows for control of nuclear localization in individual cells. By inserting LANS into the C. elegans lin-1 locus using Cas9-triggered homologous recombination, we demonstrated control of cell fate via light-dependent manipulation of a native transcription factor. We conclude that LANS can be a valuable experimental method for spatial and temporal control of nuclear localization in vivo.

Abstract: We describe an engineered photoactivatable Cas9 (paCas9) that enables optogenetic control of CRISPR-Cas9 genome editing in human cells. paCas9 consists of split Cas9 fragments and photoinducible dimerization domains named Magnets. In response to blue light irradiation, paCas9 expressed in human embryonic kidney 293T cells induces targeted genome sequence modifications through both nonhomologous end joining and homology-directed repair pathways. Genome editing activity can be switched off simply by extinguishing the light. We also demonstrate activation of paCas9 in spatial patterns determined by the sites of irradiation. Optogenetic control of targeted genome editing should facilitate improved understanding of complex gene networks and could prove useful in biomedical applications.

Abstract: Protein trafficking in and out of the nucleus represents a key step in controlling cell fate and function. Here we report the development of a red light-inducible and far-red light-reversible synthetic system for controlling nuclear localization of proteins in mammalian cells and zebrafish. First, we synthetically reconstructed and validated the red light-dependent Arabidopsis phytochrome B nuclear import mediated by phytochrome-interacting factor 3 in a nonplant environment and support current hypotheses on the import mechanism in planta. On the basis of this principle we next regulated nuclear import and activity of target proteins by the spatiotemporal projection of light patterns. A synthetic transcription factor was translocated into the nucleus of mammalian cells and zebrafish to drive transgene expression. These data demonstrate the first in vivo application of a plant phytochrome-based optogenetic tool in vertebrates and expand the repertoire of available light-regulated molecular devices.

Abstract: Targeted endogenous gene activation is necessary for understanding complex gene networks and has great potential in medical and industrial applications. The CRISPR-Cas system offers simple and powerful tools for this purpose. However, these CRISPR-Cas-based tools for activating user-defined genes are unable to offer precise temporal control of gene expression, despite the fact that many biological phenomena are regulated by highly dynamic patterns of gene expression. Here we created a light-inducible, user-defined, endogenous gene activation system based on CRISPR-Cas9. We demonstrated that this CRISPR-Cas9-based transcription system can allow rapid and reversible targeted gene activation by light. In addition, using this system, we have exemplified photoactivation of multiple user-defined endogenous genes in mammalian cells. The present CRISPR-Cas9-based transcription system offers simple and versatile approaches for precise endogenous gene activation in basic biological research and biotechnology applications.

Abstract: The function of many eukaryotic proteins is regulated by highly dynamic changes in their nucleocytoplasmic distribution. The ability to precisely and reversibly control nuclear translocation would, therefore, allow dissecting and engineering cellular networks. Here we develop a genetically encoded, light-inducible nuclear localization signal (LINuS) based on the LOV2 domain of Avena sativa phototropin 1. LINuS is a small, versatile tag, customizable for different proteins and cell types. LINuS-mediated nuclear import is fast and reversible, and can be tuned at different levels, for instance, by introducing mutations that alter AsLOV2 domain photo-caging properties or by selecting nuclear localization signals (NLSs) of various strengths. We demonstrate the utility of LINuS in mammalian cells by controlling gene expression and entry into mitosis with blue light.

Abstract: Fibroblast growth factor receptors (FGFRs) regulate diverse cellular behaviors that should be exquisitely controlled in space and time. We engineered an optically controlled FGFR (optoFGFR1) by exploiting cryptochrome 2, which homointeracts upon blue light irradiation. OptoFGFR1 can rapidly and reversibly control intracellular FGFR1 signaling within seconds by illumination with blue light. At the subcellular level, localized activation of optoFGFR1 induced cytoskeletal reorganization. Utilizing the high spatiotemporal precision of optoFGFR1, we efficiently controlled cell polarity and induced directed cell migration. OptoFGFR1 provides an effective means to precisely control FGFR signaling and is an important optogenetic tool that can be used to study diverse biological processes both in vitro and in vivo.

Abstract: Cells sense gradients of extracellular cues and generate polarized responses such as cell migration and neurite initiation. There is static information on the intracellular signaling molecules involved in these responses, but how they dynamically orchestrate polarized cell behaviors is not well understood. A limitation has been the lack of methods to exert spatial and temporal control over specific signaling molecules inside a living cell. Here we introduce optogenetic tools that act downstream of native G protein-coupled receptor (GPCRs) and provide direct control over the activity of endogenous heterotrimeric G protein subunits. Light-triggered recruitment of a truncated regulator of G protein signaling (RGS) protein or a Gβγ-sequestering domain to a selected region on the plasma membrane results in localized inhibition of G protein signaling. In immune cells exposed to spatially uniform chemoattractants, these optogenetic tools allow us to create reversible gradients of signaling activity. Migratory responses generated by this approach show that a gradient of active G protein αi and βγ subunits is sufficient to generate directed cell migration. They also provide the most direct evidence so for a global inhibition pathway triggered by Gi signaling in directional sensing and adaptation. These optogenetic tools can be applied to interrogate the mechanistic basis of other GPCR-modulated cellular functions.

Abstract: Receptor tyrosine kinases (RTKs) are a family of cell-surface receptors that have a key role in regulating critical cellular processes. Here, to understand and precisely control RTK signalling, we report the development of a genetically encoded, photoactivatable Trk (tropomyosin-related kinase) family of RTKs using a light-responsive module based on Arabidopsis thaliana cryptochrome 2. Blue-light stimulation (488 nm) of mammalian cells harbouring these receptors robustly upregulates canonical Trk signalling. A single light stimulus triggers transient signalling activation, which is reversibly tuned by repetitive delivery of blue-light pulses. In addition, the light-provoked process is induced in a spatially restricted and cell-specific manner. A prolonged patterned illumination causes sustained activation of extracellular signal-regulated kinase and promotes neurite outgrowth in a neuronal cell line, and induces filopodia formation in rat hippocampal neurons. These light-controllable receptors are expected to create experimental opportunities to spatiotemporally manipulate many biological processes both in vitro and in vivo.

Abstract: We present a versatile platform to inactivate proteins in living cells using light, light-activated reversible inhibition by assembled trap (LARIAT), which sequesters target proteins into complexes formed by multimeric proteins and a blue light-mediated heterodimerization module. Using LARIAT, we inhibited diverse proteins that modulate cytoskeleton, lipid signaling and cell cycle with high spatiotemporal resolution. Use of single-domain antibodies extends the method to target proteins containing specific epitopes, including GFP.

Abstract: Calcium ion (Ca2+) is an important second messenger implicated in the control of many different cellular processes in living organisms. Ca2+ is typically studied by direct visualization using chemically or genetically encoded indicators. A complementary, and perhaps more useful, approach involves direct manipulation of Ca2+ concentration; tools for this exist but are rather poorly developed compared to the indicators at least. Here, we report a photoactivatable Ca2+-releasing protein, photoactivatable Ca2+ releaser (PACR), made by the insertion of a photosensitive protein domain (LOV2) into a Ca2+ binding protein (calmodulin fused with the M13 peptide). As the PACR is genetically encoded, and unlike conventional optical control tools (e.g., channel rhodopsin) not membrane bound, we are able to restrict expression within the cell, to allow subcellular perturbation of Ca2+ levels. In whole animals, we are able to control the behavior of Caenorhabditis elegans with light by expressing the PACR only in the touch neuron.

Abstract: The coupling of light-inducible protein-protein interactions with gene regulation systems has enabled the control of gene expression with light. In particular, heterodimer protein pairs from plants can be used to engineer a gene regulation system in mammalian cells that is reversible, repeatable, tunable, controllable in a spatiotemporal manner, and targetable to any DNA sequence. This system, Light-Inducible Transcription using Engineered Zinc finger proteins (LITEZ), is based on the blue light-induced interaction of GIGANTEA and the LOV domain of FKF1 that drives the localization of a transcriptional activator to the DNA-binding site of a highly customizable engineered zinc finger protein. This chapter provides methods for modifying LITEZ to target new DNA sequences, engineering a programmable LED array to illuminate cell cultures, and using the modified LITEZ system to achieve spatiotemporal control of transgene expression in mammalian cells.

Abstract: We have developed an optogenetic technique for the activation of diaphanous-related formins. Our approach is based on fusion of the light-oxygen-voltage 2 domain of Avena sativa Phototrophin1 to an isolated Diaphanous Autoregulatory Domain from mDia1. This "caged" diaphanous auto-regulatory domain was inactive in the dark but in the presence of blue light rapidly activated endogenous diaphanous-related formins. Using an F-actin reporter, we observed filopodia and lamellipodia formation as well as a steady increase in F-actin along existing stress fibers, starting within minutes of photo-activation. Interestingly, we did not observe the formation of new stress fibers. Remarkably, a 1.9-fold increase in F-actin was not paralleled by an increase in myosin II along stress fibers and the amount of tension generated by the fibers, as judged by focal adhesion size, appeared unchanged. Our results suggest a decoupling between F-actin accumulation and contractility in stress fibers and demonstrate the utility of photoactivatable diaphanous autoregulatory domain for the study of diaphanous-related formin function in cells.

Abstract: Formins are potent activators of actin filament assembly in the cytoplasm. In turn, cytoplasmic actin polymerization can promote release of actin from megakaryocytic acute leukemia (MAL) protein for serum response factor (SRF) transcriptional activity. We found that formins polymerized actin inside the mammalian nucleus to drive serum-dependent MAL-SRF activity. Serum stimulated rapid assembly of actin filaments within the nucleus in a formin-dependent manner. The endogenous formin mDia was regulated with an optogenetic tool, which allowed for photoreactive release of nuclear formin autoinhibition. Activated mDia promoted rapid and reversible nuclear actin network assembly, subsequent MAL nuclear accumulation, and SRF activity. Thus, a dynamic polymeric actin structure within the nucleus is part of the serum response.

Abstract: Fluorescent proteins (FPs) are widely used as optical sensors, whereas other light-absorbing domains have been used for optical control of protein localization or activity. Here, we describe light-dependent dissociation and association in a mutant of the photochromic FP Dronpa, and we used it to control protein activities with light. We created a fluorescent light-inducible protein design in which Dronpa domains are fused to both termini of an enzyme domain. In the dark, the Dronpa domains associate and cage the protein, but light induces Dronpa dissociation and activates the protein. This method enabled optical control over guanine nucleotide exchange factor and protease domains without extensive screening. Our findings extend the applications of FPs from exclusively sensing functions to also encompass optogenetic control.

Abstract: Advanced gene regulatory systems are necessary for scientific research, synthetic biology, and gene-based medicine. An ideal system would allow facile spatiotemporal manipulation of gene expression within a cell population that is tunable, reversible, repeatable, and can be targeted to diverse DNA sequences. To meet these criteria, a gene regulation system was engineered that combines light-sensitive proteins and programmable zinc finger transcription factors. This system, light-inducible transcription using engineered zinc finger proteins (LITEZ), uses two light-inducible dimerizing proteins from Arabidopsis thaliana, GIGANTEA and the LOV domain of FKF1, to control synthetic zinc finger transcription factor activity in human cells. Activation of gene expression in human cells engineered with LITEZ was reversible and repeatable by modulating the duration of illumination. The level of gene expression could also be controlled by modulating light intensity. Finally, gene expression could be activated in a spatially defined pattern by illuminating the human cell culture through a photomask of arbitrary geometry. LITEZ enables new approaches for precisely regulating gene expression in biotechnology and medicine, as well as studying gene function, cell-cell interactions, and tissue morphogenesis.

Abstract: Naturally photoswitchable proteins offer a means of directly manipulating the formation of protein complexes that drive a diversity of cellular processes. We developed tunable light-inducible dimerization tags (TULIPs) based on a synthetic interaction between the LOV2 domain of Avena sativa phototropin 1 (AsLOV2) and an engineered PDZ domain (ePDZ). TULIPs can recruit proteins to diverse structures in living yeast and mammalian cells, either globally or with precise spatial control using a steerable laser. The equilibrium binding and kinetic parameters of the interaction are tunable by mutation, making TULIPs readily adaptable to signaling pathways with varying sensitivities and response times. We demonstrate the utility of TULIPs by conferring light sensitivity to functionally distinct components of the yeast mating pathway and by directing the site of cell polarization.

Abstract: Apoptosis is a cell death program involved in the development of multicellular organisms, immunity, and pathologies ranging from cancer to HIV/AIDS. We present an engineered protein that causes rapid apoptosis of targeted cells in monolayer culture after stimulation with blue light. Cells transfected with the protein switch L57V, a tandem fusion of the light-sensing LOV2 domain and the apoptosis-executing domain from caspase-7, rapidly undergo apoptosis within 60 min after light stimulation. Constant illumination of under 5 min or oscillating with 1 min exposure had no effect, suggesting that cells have natural tolerance to a short duration of caspase-7 activity. Furthermore, the overexpression of Bcl-2 prevented L57V-mediated apoptosis, suggesting that although caspase-7 activation is sufficient to start apoptosis, it requires mitochondrial contribution to fully commit.

Abstract: Ca(2+) signals regulate diverse physiological processes through tightly regulated fluxes varying in location, time, frequency, and amplitude. Here, we developed LOVS1K, a genetically encoded and photoactivated synthetic protein to generate local or global Ca(2+) signals. With 300 ms blue light exposure, LOVS1K translocated to Orai1, a plasma membrane Ca(2+) channel, within seconds, generating a local Ca(2+) signal on the plasma membrane, and returning to the cytoplasm after tens of seconds. With repeated photoactivation, global Ca(2+) signals in the cytoplasm were generated to modulate engineered Ca(2+)-inducible proteins. Although Orai1 is typically associated with global store-operated Ca(2+) entry, we demonstrate that Orai1 can also generate local Ca(2+) influx on the plasma membrane. Our photoactivation system can be used to generate spatially and temporally precise Ca(2+) signals and to engineer synthetic proteins that respond to specific Ca(2+) signals.

Abstract: The precise spatio-temporal dynamics of protein activity are often critical in determining cell behaviour, yet for most proteins they remain poorly understood; it remains difficult to manipulate protein activity at precise times and places within living cells. Protein activity has been controlled by light, through protein derivatization with photocleavable moieties or using photoreactive small-molecule ligands. However, this requires use of toxic ultraviolet wavelengths, activation is irreversible, and/or cell loading is accomplished via disruption of the cell membrane (for example, through microinjection). Here we have developed a new approach to produce genetically encoded photoactivatable derivatives of Rac1, a key GTPase regulating actin cytoskeletal dynamics in metazoan cells. Rac1 mutants were fused to the photoreactive LOV (light oxygen voltage) domain from phototropin, sterically blocking Rac1 interactions until irradiation unwound a helix linking LOV to Rac1. Photoactivatable Rac1 (PA-Rac1) could be reversibly and repeatedly activated using 458- or 473-nm light to generate precisely localized cell protrusions and ruffling. Localized Rac activation or inactivation was sufficient to produce cell motility and control the direction of cell movement. Myosin was involved in Rac control of directionality but not in Rac-induced protrusion, whereas PAK was required for Rac-induced protrusion. PA-Rac1 was used to elucidate Rac regulation of RhoA in cell motility. Rac and Rho coordinate cytoskeletal behaviours with seconds and submicrometre precision. Their mutual regulation remains controversial, with data indicating that Rac inhibits and/or activates Rho. Rac was shown to inhibit RhoA in mouse embryonic fibroblasts, with inhibition modulated at protrusions and ruffles. A PA-Rac crystal structure and modelling revealed LOV-Rac interactions that will facilitate extension of this photoactivation approach to other proteins.