Curated Optogenetic Publication Database

Abstract: Virotherapy using oncolytic adenovirus is an effective anticancer strategy. However, the tumor selectivity of oncolytic adenoviruses is not enough high. To develop oncolytic adenovirus with a low risk of off-tumor toxicity, we constructed a photoactivatable oncolytic adenovirus (paOAd). In response to blue light irradiation, the expression of adenoviral E1 genes, which are necessary for adenoviral replication, is induced and replication of this adenovirus occurs. In vitro, efficient lysis of various human cancer cell lines was observed by paOAd infection followed by blue light irradiation. Importantly, there was no off-tumor toxicity unless the cells were irradiated by blue light. In vivo, tumor growth in a subcutaneous tumor model and a mouse model of liver cancer was significantly inhibited by paOAd infection followed by blue light irradiation. In addition, paOAd also showed a therapeutic effect on cancer stem cells. These results suggest that paOAd is useful as a safe and therapeutically effective cancer therapy.

Abstract: The self-assembly of different cell types into multicellular structures and their organization into spatiotemporally controlled patterns are both challenging and extremely powerful to understand how cells function within tissues and for bottom-up tissue engineering. Here, we not only independently control the self-assembly of two cell types into multicellular architectures with blue and red light, but also achieve their self-sorting into distinct assemblies. This required developing two cell types that form selective and homophilic cell-cell interactions either under blue or red light using photoswitchable proteins as artificial adhesion molecules. The interactions were individually triggerable with different colors of light, reversible in the dark, and provide noninvasive and temporal control over the cell-cell adhesions. In mixtures of the two cells, each cell type self-assembled independently upon orthogonal photoactivation, and cells sorted out into separate assemblies based on specific self-recognition. These self-sorted multicellular architectures provide us with a powerful tool for producing tissue-like structures from multiple cell types and investigate principles that govern them.

Abstract: Methods that allow labeling and tracking of proteins have been instrumental for understanding their function. Traditional methods for labeling proteins include fusion to fluorescent proteins or self-labeling chemical tagging systems such as SNAP-tag or Halo-tag. These latter approaches allow bright fluorophores or other chemical moieties to be attached to a protein of interest through a small fusion tag. In this work, we sought to improve the versatility of self-labeling chemical-tagging systems by regulating their activity with light. We used light-inducible dimerizers to reconstitute a split SNAP-tag (modified human O6-alkylguanine-DNA-alkyltransferase, hAGT) protein, allowing tight light-dependent control of chemical labeling. In addition, we generated a small split SNAP-tag fragment that can efficiently self-assemble with its complement fragment, allowing high labeling efficacy with a small tag. We envision these tools will extend the versatility and utility of the SNAP-tag chemical system for protein labeling applications.

Abstract: Membrane fusion during synaptic transmission mediates the trafficking of chemical signals and neuronal communication. The fast kinetics of membrane fusion on the order of millisecond is precisely regulated by the assembly of SNAREs and accessory proteins. It is believed that the formation of the SNARE complex is a key step during membrane fusion. Little is known, however, about the molecular machinery that mediates the formation of a large pre-fusion complex, including multiple SNAREs and accessory proteins. Syntaxin, a transmembrane protein on the plasma membrane, has been observed to undergo oligomerization to form clusters. Whether this clustering plays a critical role in membrane fusion is poorly understood in live cells. Optogenetics is an emerging biotechnology armed with the capacity to precisely modulate protein-protein interaction in time and space. Here, we propose an experimental scheme that combines optogenetics with single-vesicle membrane fusion, aiming to gain a better understanding of the molecular mechanism by which the syntaxin cluster regulates membrane fusion. We envision that newly developed optogenetic tools could facilitate the mechanistic understanding of synaptic transmission in live cells and animals.

Abstract: Cell-free systems, as part of the synthetic biology field, have become a critical platform in biological studies. However, there is a lack of research into developing a switch for a dynamical control of the transcriptional and translational process. The optogenetic tool has been widely proven as an ideal control switch for protein synthesis due to its nontoxicity and excellent time-space conversion. Hence, in this study, a blue light-regulated two-component system named YF1/FixJ was incorporated into an Escherichia coli-based cell-free system to control protein synthesis. The corresponding cell-free system successfully achieved a 5-fold dynamic protein expression by blue light repression and 3-fold dynamic expression by blue light activation. With the aim of expanding the applications of cell-free synthetic biology, the cell-free blue light-sensing system was used to perform imaging, light-controlled antibody synthesis, and light-triggered artificial cell assembly. This study can provide a guide for further research into the field of cell-free optical sensing. Moreover, it will also promote the development of cell-free synthetic biology and optogenetics through applying the cell-free optical sensing system to synthetic biology education, biopharmaceutical research, and artificial cell construction.

Abstract: Stem cell fate is largely determined by cellular signaling networks and is heavily dependent on the supplementation of exogenous recombinant proteins into culture media; however, uneven distribution and inconsistent stability of recombinant proteins are closely associated with the spontaneous differentiation of pluripotent stem cells (PSCs) and result in significant costs in large-scale manufacturing. Here, we report a novel PSC culture system via wirelessly controllable optical activation of the fibroblast growth factor (FGF) signaling pathway without the need for supplementation of recombinant FGF2 protein, a key molecule for maintaining pluripotency of PSCs. Using a fusion protein between the cytoplasmic region of the FGF receptor-1 and a light-oxygen-voltage domain, we achieved tunable, blue light-dependent activation of FGF signaling in human and porcine PSCs. Our data demonstrate that a highly controllable optical stimulation of the FGF signaling pathway is sufficient for long-term maintenance of PSCs, without the loss of differentiation potential into three germ layers. This culture system will be a cost-effective platform for a large-scale stem cell culture.

Abstract: Optogenetic approaches facilitate the study of signaling and metabolic pathways in animal cell systems. In the past 10 years, a plethora of light-regulated switches for the targeted control over the induction of gene expression, subcellular localization of proteins, membrane receptor activity, and other cellular processes have been developed and successfully implemented. However, only a few tools have been engineered toward the quantitative and spatiotemporally resolved downregulation of proteins. Here we present a protocol for reversible and rapid blue light-induced reduction of protein levels in mammalian cells. By implementing a dual-regulated optogenetic switch (Blue-OFF), both repression of gene expression and degradation of the target protein are triggered simultaneously. We apply this system for the blue light-mediated control of programmed cell death. HEK293T cells are transfected with the proapoptotic proteins PUMA and BID integrated into the Blue-OFF system. Overexpression of these proteins leads to programmed cell death, which can be prevented by irradiation with blue light. This experimental approach is very straightforward, requires just simple hardware, and therefore can be easily implemented in state-of-the-art equipped mammalian cell culture labs. The system can be used for targeted cell signaling studies and biotechnological applications.

Abstract: Optogenetic tools allow for use of light as an external input to control cellular processes. When applied to regulate the function of transcription factors, optogenetic approaches provide a tunable, reversible, and bidirectional method to control gene expression. Herein, we present a detailed method to induce gene expression in mammalian cells using the light dependent dimerization of cryptochrome 2 (CRY2) and CIB1 to complement a split transcription factor. We also describe a protocol to disrupt gene expression with light by fusing a dimeric transcription factor to CRY2. When combined with a light-induced degron attached to the gene product, this method allows for rapid modulation of target protein abundance.

Abstract: cAMP is a crucial mediator of multiple cell signaling pathways. This cyclic nucleotide requires strict spatiotemporal control for effective function. Light-activated proteins have become a powerful tool to study signaling kinetics due to having quick on/off rates and minimal off-target effects. The photoactivated adenylyl cyclase from Beggiatoa (bPAC) produces cAMP rapidly upon stimulation with blue light. However, light delivery is not always feasible, especially in vivo. Hence, we created a luminescence-activated cyclase by fusing bPAC with nanoluciferase (nLuc) to allow chemical activation of cAMP activity. This dual-activated adenylyl cyclase can be stimulated using short bursts of light or long-term chemical activation with furimazine and other related luciferins. Together these can be used to mimic transient, chronic, and oscillating patterns of cAMP signaling. Moreover, when coupled to compartment-specific targeting domains, these reagents provide a new powerful tool for cAMP spatiotemporal dynamic studies. Here, we describe detailed methods for working with bPAC-nLuc in mammalian cells, stimulating cAMP production with light and luciferins, and measuring total cAMP accumulation.

Abstract: Synthetic extracellular matrices with reversibly adjustable mechanical properties are essential for the investigation of how cells respond to dynamic mechanical cues as occurring in living organisms. One interesting approach to engineer dynamic biomaterials is the incorporation of photoreceptors from cyanobacteria or plants into polymer materials. Here, we give an overview of existing photoreceptor-based biomaterials and describe a detailed protocol for the synthesis of a phytochrome-based extracellular matrix (CyPhyGel). Using cell-compatible light in the red and far-red spectrum, the mechanical properties of this matrix can be adjusted in a fully reversible, wavelength-specific, and dose-dependent manner with high spatiotemporal control.

Abstract: Understanding how the activity of membrane receptors and cellular signaling pathways shapes cell behavior is of fundamental interest in basic and applied research. Reengineering receptors to react to light instead of their cognate ligands allows for generating defined signaling inputs with high spatial and temporal precision and facilitates the dissection of complex signaling networks. Here, we describe fundamental considerations in the design of light-regulated receptor tyrosine kinases (Opto-RTKs) and appropriate control experiments. We also introduce methods for transient receptor expression in HEK293 cells, quantitative assessment of signaling activity in reporter gene assays, semiquantitative assessment of (in)activation time courses through Western blot (WB) analysis, and easy to implement light stimulation hardware.

Abstract: 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.

Abstract: The transport of proteins between the nucleus and the cytosol is a vital process regulating cellular activity. The ability to spatiotemporally control the nucleocytoplasmic transport of a protein of interest allows for elucidating its function taking into account the dynamic and heterogeneous nature of biological processes contrary to conventional knockin, knockout, and chemically induced overexpression strategies. We recently developed two optogenetic tools, called LINuS and LEXY, for reversibly controlling with blue light the nuclear import and export of proteins of interest, respectively. Here we describe how to use them to control the localization of a protein of interest in cultured mammalian cells using a fluorescence microscope.

Abstract: G protein-coupled receptors (GPCRs) form the largest class of membrane receptors in the mammalian genome with nearly 800 human genes encoding for unique subtypes. Accordingly, GPCR signaling is implicated in nearly all physiological processes. However, GPCRs have been difficult to study due in part to the complexity of their function which can lead to a plethora of converging or diverging downstream effects over different time and length scales. Classic techniques such as pharmacological control, genetic knockout and biochemical assays often lack the precision required to probe the functions of specific GPCR subtypes. Here we describe the rapidly growing set of optogenetic tools, ranging from methods for optical control of the receptor itself to optical sensing and manipulation of downstream effectors. These tools permit the quantitative measurements of GPCRs and their downstream signaling with high specificity and spatiotemporal precision.

Abstract: CRISPR labeling is a powerful technique to study the chromatin architecture in live cells. In CRISPR labeling, a catalytically dead CRISPR-Cas9 mutant is employed as programmable DNA-binding domain to recruit fluorescent proteins to selected genomic loci. The fluorescently labeled loci can then be identified as fluorescent spots and tracked over time by microscopy. A limitation of this approach is the lack of temporal control of the labeling process itself: Cas9 binds to the g(uide)RNA-complementary target loci as soon as it is expressed. The decoration of the genome with Cas9 molecules will, however, interfere with gene regulation and-possibly-affect the genome architecture itself. The ability to switch on and off Cas9 DNA binding in CRISPR labeling experiments would thus be important to enable more precise interrogations of the chromatin spatial organization and dynamics and could further be used to study Cas9 DNA binding kinetics directly in living human cells.Here, we describe a detailed protocol for light-inducible CRISPR labeling. Our method employs CASANOVA, an engineered, optogenetic anti-CRISPR protein, which efficiently traps the Streptococcus pyogenes (Spy)Cas9 in the dark, but permits Cas9 DNA targeting upon illumination with blue light. Using telomeres as exemplary target loci, we detail the experimental steps required for inducible CRISPR labeling with CASANOVA. We also provide instructions on how to analyze the resulting microscopy data in a fully automated fashion.

Abstract: Since the breakthrough discoveries that CRISPR-Cas9 nucleases can be easily programmed and employed to induce targeted double-strand breaks in mammalian cells, the gene editing field has grown exponentially. Today, CRISPR technologies based on engineered class II CRISPR effectors facilitate targeted modification of genes and RNA transcripts. Moreover, catalytically impaired CRISPR-Cas variants can be employed as programmable DNA binding domains and used to recruit effector proteins, such as transcriptional regulators, epigenetic modifiers or base-modifying enzymes, to selected genomic loci. The juxtaposition of CRISPR and optogenetics enables spatiotemporally confined and highly dynamic genome perturbations in living cells and animals and holds unprecedented potential for biology and biomedicine.Here, we provide an overview of the state-of-the-art methods for light-control of CRISPR effectors. We will detail the plethora of exciting applications enabled by these systems, including spatially confined genome editing, timed activation of endogenous genes, as well as remote control of chromatin-chromatin interactions. Finally, we will discuss limitations of current optogenetic CRISPR tools and point out routes for future innovation in this emerging field.

Abstract: It is widely understood that CRISPR-Cas9 technology is revolutionary, with well-recognized issues including the potential for off-target edits and the attendant need for spatiotemporal control of editing. Here, we describe a far-red light (FRL)–activated split-Cas9 (FAST) system that can robustly induce gene editing in both mammalian cells and mice. Through light-emitting diode–based FRL illumination, the FAST system can efficiently edit genes, including nonhomologous end joining and homology-directed repair, for multiple loci in human cells. Further, we show that FAST readily achieves FRL-induced editing of internal organs in tdTomato reporter mice. Finally, FAST was demonstrated to achieve FRL-triggered editing of the PLK1 oncogene in a mouse xenograft tumor model. Beyond extending the spectrum of light energies in optogenetic toolbox for CRISPR-Cas9 technologies, this study demonstrates how FAST system can be deployed for programmable deep tissue gene editing in both biological and biomedical contexts toward high precision and spatial specificity.

Abstract: Animal embryos are patterned by a handful of highly conserved inductive signals. Yet, in most cases, it is unknown which pattern features (i.e., spatial gradients or temporal dynamics) are required to support normal development. An ideal experiment to address this question would be to "paint" arbitrary synthetic signaling patterns on "blank canvas" embryos to dissect their requirements. Here, we demonstrate exactly this capability by combining optogenetic control of Ras/extracellular signal-related kinase (ERK) signaling with the genetic loss of the receptor tyrosine-kinase-driven terminal signaling patterning in early Drosophila embryos. Blue-light illumination at the embryonic termini for 90 min was sufficient to rescue normal development, generating viable larvae and fertile adults from an otherwise lethal terminal signaling mutant. Optogenetic rescue was possible even using a simple, all-or-none light input that reduced the gradient of Erk activity and eliminated spatiotemporal differences in terminal gap gene expression. Systematically varying illumination parameters further revealed that at least three distinct developmental programs are triggered at different signaling thresholds and that the morphogenetic movements of gastrulation are robust to a 3-fold variation in the posterior pattern width. These results open the door to controlling tissue organization with simple optical stimuli, providing new tools to probe natural developmental processes, create synthetic tissues with defined organization, or directly correct the patterning errors that underlie developmental defects.

Abstract: We present a new concept for whole-cell biosensors that couples the response to Hg2+ with bioluminescence and bacterial aggregation. This allows us to use the bacterial aggregation to preconcentrate the bioluminescent bacteria at the substrate surface and increase the sensitivity of Hg2+ detection. This whole-cell biosensor combines a Hg2+-sensitive bioluminescence reporter and light-responsive bacterial cell-cell adhesions. We demonstrate that the blue luminescence in response to Hg2+ is able to photoactivate bacterial aggregation, which provides a second readout for Hg2+ detection. In return, the Hg2+-triggered bacterial aggregation leads to faster sedimentation and more efficient formation of biofilms. At low Hg2+ concentrations, the enrichment of the bacteria in biofilms leads to an up to 10-fold increase in the signal. The activation of photoswitchable proteins with biological light is a new concept in optogenetics, and the presented bacterial biosensor design is transferable to other bioluminescent reporters with particular interest for environmental monitoring.

Abstract: Amyloids are protein polymers that were initially linked to human diseases. Across the whole Tree of Life, many disease-unrelated proteins are now emerging for which amyloids represent distinct functional states. Most bacterial amyloids described are extracellular, contributing to biofilm formation. However, only a few have been found in the bacterial cytosol. This paper reviews from the perspective of synthetic biology (SynBio) our understanding of the subtle line that separates functional from pathogenic and transmissible amyloids (prions). In particular, it is focused on RepA-WH1, a functional albeit unconventional natural amyloidogenic protein domain that participates in controlling DNA replication of bacterial plasmids. SynBio approaches, including protein engineering and the design of allosteric effectors such as diverse ligands and an optogenetic module, have enabled the generation in RepA-WH1 of an intracellular cytotoxic prion-like agent in bacteria. The synthetic RepA-WH1 prion has the potential to develop into novel antimicrobials.

Abstract: Photo-induced structural rearrangements of chromophore-containing proteins are essential for various light-dependent signaling pathways and optogenetic applications. Ultrafast structural and spectroscopic methods have offered insights into these structural rearrangements across many timescales. However, questions still remain about exact mechanistic details, especially regarding photoisomerization of the chromophore within these proteins femtoseconds to picoseconds after photoexcitation. Instrumentation advancements for time-resolved crystallography and ultrafast electron diffraction provide a promising opportunity to study these reactions, but achieving enough signal-to-noise is a constant challenge. Here we present four new photoactive yellow protein constructs and one new fluorescent protein construct that contain heavy atoms either within or around the chromophore and can be expressed with high yields. Structural characterization of these constructs, most at atomic resolution, show minimal perturbation caused by the heavy atoms compared to wild-type structures. Spectroscopic studies report the effects of the heavy atom identity and location on the chromophore's photophysical properties. None of the substitutions prevent photoisomerization, although certain rates within the photocycle may be affected. Overall, these new proteins containing heavy atoms are ideal samples for state-of-theart time-resolved crystallography and electron diffraction experiments to elucidate crucial mechanistic information of photoisomerization.

Abstract: Light oxygen voltage-sensing (LOV) domains are widely found in photoreceptor proteins of plants, algae, fungi, and bacteria. Structural studies of LOV domains suggest that Phe and Gln residues located in the proximity of the chromophore undergo conformational changes upon illumination; however, the molecular mechanism associated with activation of the effector domain remains to be elucidated. Photozipper (PZ) protein is an N-terminally truncated aureochrome-1 comprising a LOV domain and a basic leucine zipper domain. Blue light (BL) induces PZ dimerization and subsequently increases its affinity for target DNA. In this study, we prepared PZ mutants with substitutions of F298 and Q317 and performed quantitative analyses in dark and light states. Substitutions of Q317 significantly reduced the light-induced changes in PZ affinity for the target DNA, especially in the case of the high affinities observed in the dark state. Upon illumination, all PZ mutants showed increased affinity for the target sequence, which demonstrated a clear correlation with the dimer fraction of each PZ mutant. These results suggest the existence of a conformational equilibrium and that its shift by a synergistic interaction between the chromophore and protein moiety probably enables BL-regulated switching of aureochrome-1.

Abstract: Optogenetics is the genetic approach for controlling cellular processes with light. It provides spatiotemporal, quantitative and reversible control over biological signaling and metabolic processes, overcoming limitations of chemically inducible systems. However, optogenetics lags in plant research because ambient light required for growth leads to undesired system activation. We solved this issue by developing plant usable light-switch elements (PULSE), an optogenetic tool for reversibly controlling gene expression in plants under ambient light. PULSE combines a blue-light-regulated repressor with a red-light-inducible switch. Gene expression is only activated under red light and remains inactive under white light or in darkness. Supported by a quantitative mathematical model, we characterized PULSE in protoplasts and achieved high induction rates, and we combined it with CRISPR-Cas9-based technologies to target synthetic signaling and developmental pathways. We applied PULSE to control immune responses in plant leaves and generated Arabidopsis transgenic plants. PULSE opens broad experimental avenues in plant research and biotechnology.

Abstract: Bacterial phytochrome photoreceptors usually belong to two-component signaling systems which transmit environmental stimuli to a response regulator through a histidine kinase domain. Phytochromes switch between red light-absorbing and far-red light-absorbing states. Despite exhibiting extensive structural responses during this transition, the model bacteriophytochrome from Deinococcus radiodurans (DrBphP) lacks detectable kinase activity. Here, we resolve this long-standing conundrum by comparatively analyzing the interactions and output activities of DrBphP and a bacteriophytochrome from Agrobacterium fabrum (AgP1). Whereas AgP1 acts as a conventional histidine kinase, we identify DrBphP as a light-sensitive phosphatase. While AgP1 binds its cognate response regulator only transiently, DrBphP does so strongly, which is rationalized at the structural level. Our data pinpoint two key residues affecting the balance between kinase and phosphatase activities, which immediately bears on photoreception and two-component signaling. The opposing output activities in two highly similar bacteriophytochromes inform the use of light-controllable histidine kinases and phosphatases for optogenetics.

Abstract: Actomyosin-mediated apical constriction drives a wide range of morphogenetic processes. Activation of myosin-II initiates pulsatile cycles of apical constrictions followed by either relaxation or stabilization (ratcheting) of the apical surface. While relaxation leads to dissipation of contractile forces, ratcheting is critical for the generation of tissue-level tension and changes in tissue shape. How ratcheting is controlled at the molecular level is unknown. Here, we show that the actin crosslinker βH-spectrin is upregulated at the apical surface of invaginating mesodermal cells during Drosophila gastrulation. βH-spectrin forms a network of filaments which co-localize with medio-apical actomyosin fibers, in a process that depends on the mesoderm-transcription factor Twist and activation of Rho signaling. βH-spectrin knockdown results in non-ratcheted apical constrictions and inhibition of mesoderm invagination, recapitulating twist mutant embryos. βH-spectrin is thus a key regulator of apical ratcheting during tissue invagination, suggesting that actin cross-linking plays a critical role in this process.