Curated Optogenetic Publication Database

Abstract: Although the tools based on split proteins have found broad applications, ranging from controlled biological signaling to advanced molecular architectures, many of them suffer from drawbacks such as background reassembly, low thermodynamic stability, and static structural features. Here, we present a chemically inducible protein assembly method enabled by the dissection of the carboxyl-terminal domain of a B12-dependent photoreceptor, CarHC. The resulting segments reassemble efficiently upon addition of cobalamin (AdoB12, MeB12, or CNB12). Photolysis of the cofactors such as AdoB12 and MeB12 further leads to stable protein adducts harboring a bis-His-ligated B12. Split CarHC enables the creation of a series of protein hydrogels, of which the mechanics can be either photostrengthened or photoweakened, depending on the type of B12. These materials are also well suited for three dimensional cell culturing. Together, this new protein chemistry, featuring negligible background autoassembly, stable conjugation, and phototunability, has opened up opportunities for designing smart materials.

Abstract: Wearable smart electronic devices, such as smart watches, are generally equipped with green-light-emitting diodes, which are used for photoplethysmography to monitor a panoply of physical health parameters. Here, we present a traceless, green-light-operated, smart-watch-controlled mammalian gene switch (Glow Control), composed of an engineered membrane-tethered green-light-sensitive cobalamin-binding domain of Thermus thermophilus (TtCBD) CarH protein in combination with a synthetic cytosolic TtCBD-transactivator fusion protein, which manage translocation of TtCBD-transactivator into the nucleus to trigger expression of transgenes upon illumination. We show that Apple-Watch-programmed percutaneous remote control of implanted Glow-controlled engineered human cells can effectively treat experimental type-2 diabetes by producing and releasing human glucagon-like peptide-1 on demand. Directly interfacing wearable smart electronic devices with therapeutic gene expression will advance next-generation personalized therapies by linking biopharmaceutical interventions to the internet of things.

Abstract: The regulation of cell–cell adhesions in space and time plays a crucial role in cell biology, especially in the coordination of multicellular behavior. Therefore, tools that allow for the modulation of cell–cell interactions with high precision are of great interest to a better understanding of their roles and building tissue‐like structures. Herein, the green light‐responsive protein CarH is expressed at the plasma membrane of cells as an artificial cell adhesion receptor, so that upon addition of its cofactor vitamin B12 specific cell–cell interactions form and lead to cell clustering in a concentration‐dependent manner. Upon green light illumination, the CarH based cell–cell interactions disassemble and allow for their reversion with high spatiotemporal control. Moreover, these artificial cell–cell interactions impact cell migration, as observed in a wound‐healing assay. When the cells interact with each other in the presence of vitamin B12 in the dark, the cells form on a solid front and migrate collectively; however, under green light illumination, individual cells migrate randomly out of the monolayer. Overall, the possibility of precisely controlling cell–cell interactions and regulating multicellular behavior is a potential pathway to gaining more insight into cell–cell interactions in biological processes.

Abstract: The quest to engineer increasingly complex synthetic gene networks in mammalian and plant cells requires an ever-growing portfolio of orthogonal gene expression systems. To control gene expression, light is of particular interest due to high spatial and temporal resolution, ease of dosage and simplicity of administration, enabling increasingly sophisticated man-machine interfaces. However, the majority of applied optogenetic switches are crowded in the UVB, blue and red/far-red light parts of the optical spectrum, limiting the number of simultaneously applicable stimuli. This problem is even more pertinent in plant cells, in which UV-A/B, blue, and red light-responsive photoreceptors are already expressed endogenously. To alleviate these challenges, we developed a green light responsive gene switch, based on the light-sensitive bacterial transcription factor CarH from Thermus thermophilus and its cognate DNA operator sequence CarO. The switch is characterized by high reversibility, high transgene expression levels, and low leakiness, leading to up to 350-fold induction ratios in mammalian cells. In this chapter, we describe the essential steps to build functional components of the green light-regulated gene switch, followed by detailed protocols to quantify transgene expression over time in mammalian cells. In addition, we expand this protocol with a description of how the optogenetic switch can be implemented in protoplasts of A. thaliana.

Abstract: Dynamically tunable biomaterials are of particular interest in the field of biomedical engineering because of the potential utility for shape-change materials, drug and cell delivery and tissue regeneration. Stimuli-responsive proteins formed into hydrogels are potential candidates for such systems, due to the genetic tailorability and control over structure-function relationships. Here we report the synthesis of genetically engineered Silk-Elastin-Like Protein (SELP) photoresponsive hydrogels. Polymerization of the SELPs and monomeric adenosylcobalamin (AdoB12)-dependent photoreceptor C-terminal adenosylcobalamin binding domain (CarHC) was achieved using genetically encoded SpyTag-SpyCatcher peptide-protein pairs under mild physiological conditions. The hydrogels exhibited a partial collapse of the crosslinked molecular network with both decreased loss and storage moduli upon exposure to visible light. The materials were also evaluated for cytotoxicity and the encapsulation and release of L929 murine fibroblasts from 3D cultures. The design of these photo-responsible proteins provides new stimuli-responsive SELP-CarHC hydrogels for dynamically tunable protein-based materials.

Abstract: Axon regeneration constitutes a fundamental challenge for regenerative neurobiology, which necessitates the use of tailor-made biomaterials for controllable delivery of cells and biomolecules. An increasingly popular approach for creating these materials is to directly assemble engineered proteins into high-order structures, a process that often relies on sophisticated protein chemistry. Here, we present a simple approach for creating injectable, photoresponsive hydrogels via metal-directed assembly of His6-tagged proteins. The B12-dependent photoreceptor protein CarHC can complex with transition metal ions through an amino-terminal His6-tag, which can further undergo a sol-gel transition upon addition of AdoB12, leading to the formation of hydrogels with marked injectability and photodegradability. The inducible phase transitions further enabled facile encapsulation and release of cells and proteins. Injecting the Zn2+-coordinated gels decorated with leukemia inhibitory factor into injured mouse optic nerves led to prolonged cellular signaling and enhanced axon regeneration. This study illustrates a powerful strategy for designing injectable biomaterials.

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: Spatiotemporal control of integrin-mediated cell adhesions to extracellular matrix regulates cell behavior with has numerous implications for biotechnological applications. In this work, two approaches for regulating cell adhesions in space and time with high precision are reported, both of which utilize green light. In the first design, CarH, which is a tetramer in the dark, is used to mask cRGD adhesion-peptides on a surface. Upon green light illumination, the CarH tetramer dissociates into its monomers, revealing the adhesion peptide so that cells can adhere. In the second design, the RGD motif is incorporated into the CarH protein tetramer such that cells can adhere to surfaces functionalized with this protein. The cell adhesions can be disrupted with green light, due to the disassembly of the CarH-RGD protein. Both designs allow for photoregulation with noninvasive visible light and open new possibilities to investigate the dynamical regulation of cell adhesions in cell biology.

Abstract: The ever-increasing complexity of synthetic gene networks and applications of synthetic biology requires precise and orthogonal gene expression systems. Of particular interest are systems responsive to light as they enable the control of gene expression dynamics with unprecedented resolution in space and time. While broadly used in mammalian backgrounds, however, optogenetic approaches in plant cells are still limited due to interference of the activating light with endogenous photoreceptors. Here, we describe the development of the first synthetic light-responsive system for the targeted control of gene expression in mammalian and plant cells that responds to the green range of the light spectrum in which plant photoreceptors have minimal activity. We first engineered a system based on the light-sensitive bacterial transcription factor CarH6 and its cognate DNA operator sequence CarO from Thermus thermophilus to control gene expression in mammalian cells. The system was functional in various mammalian cell lines, showing high induction (up to 350-fold) along with low leakiness, as well as high reversibility. We quantitatively described the systems characteristics by the development and experimental validation of a mathematical model. Finally, we transferred the system into A. thaliana protoplasts and demonstrated gene expression in response to green light. We expect that this system will provide new opportunities in applications based on synthetic gene networks and will open up perspectives for optogenetic studies in mammalian and plant cells.

Abstract: Thanks to the precise control over their structural and functional properties, genetically engineered protein-based hydrogels have emerged as a promising candidate for biomedical applications. Given the growing demand for creating stimuli-responsive "smart" hydrogels, here we show the synthesis of entirely protein-based photoresponsive hydrogels by covalently polymerizing the adenosylcobalamin (AdoB12)-dependent photoreceptor C-terminal adenosylcobalamin binding domain (CarHC) proteins using genetically encoded SpyTag-SpyCatcher chemistry under mild physiological conditions. The resulting hydrogel composed of physically self-assembled CarHC polymers exhibited a rapid gel-sol transition on light exposure, which enabled the facile release/recovery of 3T3 fibroblasts and human mesenchymal stem cells (hMSCs) from 3D cultures while maintaining their viability. A covalently cross-linked CarHC hydrogel was also designed to encapsulate and release bulky globular proteins, such as mCherry, in a light-dependent manner. The direct assembly of stimuli-responsive proteins into hydrogels represents a versatile strategy for designing dynamically tunable materials.

Abstract: Optogenetics and photopharmacology provide spatiotemporally precise control over protein interactions and protein function in cells and animals. Optogenetic methods that are sensitive to green light and can be used to break protein complexes are not broadly available but would enable multichromatic experiments with previously inaccessible biological targets. Herein, we repurposed cobalamin (vitamin B12) binding domains of bacterial CarH transcription factors for green-light-induced receptor dissociation. In cultured cells, we observed oligomerization-induced cell signaling for the fibroblast growth factor receptor 1 fused to cobalamin-binding domains in the dark that was rapidly eliminated upon illumination. In zebrafish embryos expressing fusion receptors, green light endowed control over aberrant fibroblast growth factor signaling during development. Green-light-induced domain dissociation and light-inactivated receptors will critically expand the optogenetic toolbox for control of biological processes.