Curated Optogenetic Publication Database

Abstract: Optogenetics and photonic technologies are changing the future of medicine. To implement light‐based therapies in the clinic, patient‐friendly devices that can deliver light inside the body while offering tunable properties and compatibility with soft tissues are needed. Here extrusion printing of degradable, hydrogel‐based optical waveguides with optical losses as low as 0.1 dB cm−1 at visible wavelengths is described. Core‐only and core‐cladding fibers are printed at room temperature from polyethylene glycol (PEG)‐based and PEG/Pluronic precursors, and cured by in situ photopolymerization. The obtained waveguides are flexible, with mechanical properties tunable within a tissue‐compatible range. Degradation times are also tunable by adjusting the molar mass of the diacrylate gel precursors, which are synthesized by linking PEG diacrylate (PEGDA) with varying proportions of DL‐dithiothreitol (DTT). The printed waveguides are used to activate photochemical and optogenetic processes in close‐to‐physiological environments. Light‐triggered migration of cells in a photoresponsive 3D hydrogel and drug release from an optogenetically‐engineered living material by delivering light across >5 cm of muscle tissue are demonstrated. These results quantify the in vitro performance, and reflect the potential of the printed degradable fibers for in vivo and clinical applications.

Abstract: The expression of a gene to a protein is one of the most vital biological processes. The use of light to control biology offers unparalleled spatiotemporal resolution from an external, orthogonal signal. A variety of methods have been developed that use light to control the steps of transcription and translation of specific genes into proteins, for cell-free to in vivo biotechnology applications. These methods employ techniques ranging from the modification of small molecules, nucleic acids and proteins with photocages, to the engineering of proteins involved in gene expression using naturally light-sensitive proteins. Although the majority of currently available technologies employ ultraviolet light, there has been a recent increase in the use of functionalities that work at longer wavelengths of light, to minimise cellular damage and increase tissue penetration. Here, we discuss the different chemical and biological methods employed to control gene expression, while also highlighting the central themes and the most exciting applications within this diverse field.

Abstract: Protein degradation technology, which is one of the most direct and effective ways to regulate the life activities of cells, is expected to be applied to the treatment of various diseases. However, current protein degradation technologies such as some small-molecule degraders which are unable to achieve spatiotemporal regulation, making them difficult to transform into clinical applications. In this article, an upconversion optogenetic nanosystem was designed to attain accurate regulation of protein degradation. This system worked via two interconnected parts: 1) the host cell expressed light-sensitive protein that could trigger the ubiquitinproteasome pathway upon blue-light exposure; 2) the light regulated light-sensitive protein by changing light conditions to achieve regulation of protein degradation. Experimental results based on model protein (Green Fluorescent Protein, GFP) validated that this system could fulfill protein degradation both in vitro (both Hela and 293T cells) and in vivo (by upconversion optogenetic nanosystem), and further demonstrated that we could reach spatiotemporal regulation by changing the illumination time (0–25 h) and the illumination frequency (the illuminating frequency of 0–30 s every 1 min). We further took another functional protein (The Nonstructural Protein 9, NSP9) into experiment. Results confirmed that the proliferation of porcine reproductive and respiratory syndrome virus (PRRSV) was inhibited by degrading the NSP9 in this light-induced system, and PRRSV proliferation was affected by different light conditions (illumination time varies from 0–24 h). We expected this system could provide new perspectives into spatiotemporal regulation of protein degradation and help realize the clinical application transformation for treating diseases of protein degradation technology.

Abstract: Many cell- and tissue-level functions are coordinated by intracellular signaling pathways that trigger the expression of context-specific target genes. Yet the input-output relationships that link pathways to the genes they activate are incompletely understood. Mapping the pathway-decoding logic of natural target genes could also provide a basis for engineering novel signal-decoding circuits. Here we report the construction of synthetic immediate-early genes (SynIEGs), target genes of Erk signaling that implement complex, user-defined regulation and can be monitored by using live-cell biosensors to track their transcription and translation. We demonstrate the power of this approach by confirming Erk duration-sensing by FOS, elucidating how the BTG2 gene is differentially regulated by external stimuli, and designing a synthetic immediate-early gene that selectively responds to the combination of growth factor and DNA damage stimuli. SynIEGs pave the way toward engineering molecular circuits that decode signaling dynamics and combinations across a broad range of cellular contexts.

Abstract: Monobodies are synthetic non-immunoglobulin customizable protein binders invaluable to basic and applied research, and of considerable potential as future therapeutics and diagnostic tools. The ability to reversibly control their binding activity to their targets on demand would significantly expand their applications in biotechnology, medicine, and research. Here we present, as proof-of-principle, the development of a light-controlled monobody (OptoMB) that works in vitro and in cells and whose affinity for its SH2-domain target exhibits a 330-fold shift in binding affinity upon illumination. We demonstrate that our αSH2-OptoMB can be used to purify SH2-tagged proteins directly from crude E. coli extract, achieving 99.8% purity and over 40% yield in a single purification step. By virtue of their ability to be designed to bind any protein of interest, OptoMBs have the potential to find new powerful applications as light-switchable binders of untagged proteins with the temporal and spatial precision afforded by light.

Abstract: Phototropins are photoreceptor proteins, which regulate blue light dependent biological processes for efficient photosynthesis in plants and algae. The proteins consist of a photosensory domain that responds to the ambient light and an output module that triggers cellular responses. The photosensory domain of phototropin from Chlamydomonas reinhardtii contains two conserved LOV (Light-Oxygen-Voltage) domains with flavin chromophores. Blue light triggers the formation of a covalent cysteine-flavin adduct and upregulates the phototropin kinase activity. Little is known about the structural mechanism which leads to kinase activation and how the two LOV domains contribute to this. Here, we investigate the role of the LOV1 domain from Chlamydomonas reinhardtii phototropin by characterizing the structural changes occurring after blue light illumination with nano- millisecond time-resolved X-ray solution scattering. By structurally fitting the data with atomic models generated by molecular dynamics simulations, we find that the adduct formation induces a rearrangement of the hydrogen bond network from the buried chromophore to the protein surface. Particularly, the change in conformation and associated hydrogen bonding of the conserved glutamine 120 induce a global movement of the β-sheet, ultimately driving a change in electrostatic potential on the protein surface. Based on the change of electrostatics, we propose a structural model of how LOV1 and LOV2 domains interact and regulate the full-length phototropin from Chlamydomonas reinhardtii. This provides a rationale for how LOV photosensor proteins function and contributes to the optimal design of optogenetic tools based on LOV domains.

Abstract: A growing number of optogenetic tools have been developed to reversibly control binding between two engineered protein domains. In contrast, relatively few tools confer light-switchable binding to a generic target protein of interest. Such a capability would offer substantial advantages, enabling photoswitchable binding to endogenous target proteins in cells or light-based protein purification in vitro. Here, we report the development of opto-nanobodies (OptoNBs), a versatile class of chimeric photoswitchable proteins whose binding to proteins of interest can be enhanced or inhibited upon blue light illumination. We find that OptoNBs are suitable for a range of applications including reversibly binding to endogenous intracellular targets, modulating signaling pathway activity, and controlling binding to purified protein targets in vitro. This work represents a step towards programmable photoswitchable regulation of a wide variety of target proteins.

Abstract: Flavoproteins are important blue light sensors in photobiology and play a key role in optogenetics. The characterization of their excited state structure and dynamics is thus an important objective. Here, we present a detailed study of excited state vibrational spectra of flavin mononucleotide (FMN), in solution and bound to the LOV-2 (Light-Oxygen-Voltage) domain of Avena sativa phototropin. Vibrational frequencies are determined for the optically excited singlet state and the reactive triplet state, through resonant ultrafast femtosecond stimulated Raman spectroscopy (FSRS). To assign the observed spectra, vibrational frequencies of the excited states are calculated using density functional theory, and both measurement and theory are applied to four different isotopologues of FMN. Excited state mode assignments are refined in both states, and their sensitivity to deuteration and protein environment are investigated. We show that resonant FSRS provides a useful tool for characterizing photoactive flavoproteins and is able to highlight chromophore localized modes and to record hydrogen/deuterium exchange.

Abstract: Cells have the ability to sense different environmental signals and position themselves accordingly in order to support their survival. Introducing analogous capabilities to the bottom-up assembled minimal synthetic cells is an important step for their autonomy. Here, a minimal synthetic cell which combines a multistimuli sensitive adhesion unit with an energy conversion module is reported, such that it can adhere to places that have the right environmental parameters for ATP production. The multistimuli sensitive adhesion unit senses light, pH, oxidative stress, and the presence of metal ions and can regulate the adhesion of synthetic cells to substrates in response to these stimuli following a chemically coded logic. The adhesion unit is composed of the light and redox responsive protein interaction of iLID and Nano and the pH sensitive and metal ion mediated binding of protein His-tags to Ni2+ -NTA complexes. Integration of the adhesion unit with a light to ATP conversion module into one synthetic cell allows it to adhere to places under blue light illumination, non-oxidative conditions, at neutral pH and in the presence of metal ions, which are the right conditions to synthesize ATP. Thus, the multistimuli responsive adhesion unit allows synthetic cells to self-position and execute their functions.

Abstract: Centrosomes, the main microtubule organizing centers (MTOCs) of metazoan cells, contain an older "mother" and a younger "daughter" centriole. Stem cells either inherit the mother or daughter-centriole-containing centrosome, providing a possible mechanism for biased delivery of cell fate determinants. However, the mechanisms regulating centrosome asymmetry and biased centrosome segregation are unclear. Using 3D-structured illumination microscopy (3D-SIM) and live-cell imaging, we show in fly neural stem cells (neuroblasts) that the mitotic kinase Polo and its centriolar protein substrate Centrobin (Cnb) accumulate on the daughter centriole during mitosis, thereby generating molecularly distinct mother and daughter centrioles before interphase. Cnb's asymmetric localization, potentially involving a direct relocalization mechanism, is regulated by Polo-mediated phosphorylation, whereas Polo's daughter centriole enrichment requires both Wdr62 and Cnb. Based on optogenetic protein mislocalization experiments, we propose that the establishment of centriole asymmetry in mitosis primes biased interphase MTOC activity, necessary for correct spindle orientation.

Abstract: The cellular architecture of the ventral tegmental area (VTA), the main hub of the brain reward system, remains only partially characterized. To extend the characterization to inhibitory neurons, we have identified three distinct subtypes of somatostatin (Sst)-expressing neurons in the mouse VTA. These neurons differ in their electrophysiological and morphological properties, anatomical localization, as well as mRNA expression profiles. Importantly, similar to cortical Sst-containing interneurons, most VTA Sst neurons express GABAergic inhibitory markers, but some of them also express glutamatergic excitatory markers and a subpopulation even express dopaminergic markers. Furthermore, only some of the proposed marker genes for cortical Sst neurons were expressed in the VTA Sst neurons. Physiologically, one of the VTA Sst neuron subtypes locally inhibited neighboring dopamine neurons. Overall, our results demonstrate the remarkable complexity and heterogeneity of VTA Sst neurons and suggest that these cells are multifunctional players in the midbrain reward circuitry.

Abstract: The transcriptional inducer anhydrotetracycline (aTc) and the bacteriostatic antibiotic tetracycline (Tc) are commonly used in all fields of biology for control of transcription or translation. A drawback of these and other small molecule inducers is the difficulty of their removal from cell cultures, limiting their application for dynamic control. Here, we describe a simple method to overcome this limitation, and show that the natural photosensitivity of aTc/Tc can be exploited to turn them into highly predictable optogenetic transcriptional- and growth-regulators. This new optogenetic class uniquely features both dynamic and setpoint control which act via population-memory adjustable through opto-chemical modulation. We demonstrate this method by applying it for dynamic gene expression control and for enhancing the performance of an existing optogenetic system. We then expand the utility of the aTc system by constructing a new chemical bandpass filter that increases its aTc response range. The simplicity of our method enables scientists and biotechnologists to use their existing systems employing aTc/Tc for dynamic optogenetic experiments without genetic modification.

Abstract: Lipid asymmetry in biological membranes is essential for various cell functions, such as cell polarity, cytokinesis, and apoptosis. P4-ATPases (flippases) are involved in the generation of such asymmetry. In Saccharomyces cerevisiae, the protein kinases Fpk1p/Fpk2p activate the P4-ATPases Dnf1p/Dnf2p by phosphorylation. Previously, we have shown that a blue-light-dependent protein kinase, phototropin from Chlamydomonas reinhardtii (CrPHOT), complements defects in an fpk1Δ fpk2Δ mutant. Herein, we investigated whether CrPHOT optically regulates P4-ATPase activity. First, we demonstrated that the translocation of NBD-labelled phospholipids to the cytoplasmic leaflet via P4-ATPases was promoted by blue-light irradiation in fpk1Δ fpk2Δ cells with CrPHOT. In addition, blue light completely suppressed the defects in membrane functions (such as endocytic recycling, actin depolarization, and apical-isotropic growth switching) caused by fpk1Δ fpk2Δ mutations. All responses required the kinase activity of CrPHOT. Hence, these results indicate the utility of CrPHOT as a powerful and first tool for optogenetic manipulation of P4-ATPase activity.

Abstract: Since the neurobiological inception of optogenetics, light-controlled molecular perturbations have been applied in many scientific disciplines to both manipulate and observe cellular function. Proteins exhibiting light-sensitive conformational changes provide researchers with avenues for spatiotemporal control over the cellular environment and serve as valuable alternatives to chemically inducible systems. Optogenetic approaches have been developed to target proteins to specific subcellular compartments, allowing for the manipulation of nuclear translocation and plasma membrane morphology. Additionally, these tools have been harnessed for molecular interrogation of organelle function, location, and dynamics. Optogenetic approaches offer novel ways to answer fundamental biological questions and to improve the efficiency of bioengineered cell factories by controlling the assembly of synthetic organelles. This review first provides a summary of available optogenetic systems with an emphasis on their organelle-specific utility. It then explores the strategies employed for organelle targeting and concludes by discussing our perspective on the future of optogenetics to control subcellular structure and organization. This article is categorized under: Laboratory Methods and Technologies > Genetic/Genomic Methods Physiology > Physiology of Model Organisms Biological Mechanisms > Regulatory Biology Models of Systems Properties and Processes > Cellular Models.

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: 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: 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: 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: 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: 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.