Curated Optogenetic Publication Database

Abstract: Light-controlled transcriptional activation is a commonly used optogenetic strategy that allows researchers to regulate gene expression with high spatiotemporal precision. The vast majority of existing tools are, however, limited to light-triggered induction of gene expression. Here, we inverted this mode of action and created optogenetic systems capable of efficiently terminating transcriptional activation in response to blue light. First, we designed highly compact regulators by photo-controlling the VP16 (pcVP16) transactivation peptide. Then, applying a two-hybrid strategy, we engineered LOOMINA (light off-operated modular inductor of transcriptional activation), a versatile transcriptional control platform for mammalian cells that is compatible with various effector proteins. Leveraging the flexibility of CRISPR systems, we combined LOOMINA with dCas9 to control transcription with blue light from endogenous promoters with exceptionally high dynamic ranges in multiple cell lines. Functionally and mechanistically, the versatile LOOMINA platform and the exceptionally compact pcVP16 transactivator represent valuable additions to the optogenetic repertoire for transcriptional regulation.

Abstract: Research for biopharmaceutical production processes with mammalian cells steadily aims to enhance the cell-specific productivity as a means for optimizing total productivities of bioreactors. Whereas current technologies such as pH, temperature, and osmolality shift require modifications of the cultivation medium, the use of optogenetic switches in recombinant producer cells might be a promising contact-free alternative. However, the proper application of optogenetically engineered cells requires a detailed understanding of basic cellular responses of cells that do not yet contain the optogenetic switches. The knowhow of ideal light exposure to enable the optimum use of related approaches is missing so far. Consequently, the current study set out to find optimum conditions for IgG1 producing Chinese hamster ovary (CHO) cells which were exposed to blue LED light. Growth characteristics, cell-specific productivity using enzyme-linked immunosorbent assay, as well as cell cycle distribution using flow cytometry were analyzed. Whereas too harsh light exposure causes detrimental growth effects that could be compensated with antioxidants, a surprising boost of cell-specific productivity by 57% occurred at optimum high light doses. The increase coincided with an increased number of cells in the G1 phase of the cell cycle after 72 h of illumination. The results present a promising new approach to boost biopharmaceutical productivity of mammalian cells simply by proper light exposure without any further optogenetic engineering. KEY POINTS: • Blue LED light hinders growth in CHO DP-12 cells • Antioxidants protect to a certain degree from blue light effects • Illumination with blue LED light raises cell-specific productivity.

Abstract: Light-Oxygen-Voltage (LOV) domains are responsible for detecting blue light (BL) and regulating the activities of effector domains in various organisms. Photozipper (PZ), an N-terminally truncated aureochrome-1 protein, contains a LOV domain and a basic leucin zipper (bZIP) domain and plays a role as a light-activatable transcription factor. PZ is monomeric in the dark state and undergoes non-covalent dimerization upon illumination with BL, subsequently increasing its affinity for the target DNA. To clarify the molecular mechanism of aureochromes, we prepared site-directed mutants of PZ and performed quantitative analyses in the dark and light states. Although the amino acid substitutions in the hinge region between the LOV core and A'α helix had minor effects on the dimerization and DNA-binding properties of PZ, the substitutions in the β-sheet region of the LOV core and in the A'α helix significantly affected these properties. We found that light signals are transmitted from the LOV core to the effector bZIP domain via the hydrophobic residues on the β-sheet. The light-induced conformational change possibly deforms the hydrophobic regions of the LOV core and induces the detachment of the A'α helix to expose the dimerization surface, likely activating the bZIP domain in a light-dependent manner.

Abstract: Herein, a set of optogenetic tools (designated LiPOP) that enable photoswitchable necroptosis and pyroptosis in live cells with varying kinetics, is introduced. The LiPOP tools allow reconstruction of the key molecular steps involved in these two non-apoptotic cell death pathways by harnessing the power of light. Further, the use of LiPOPs coupled with upconversion nanoparticles or bioluminescence is demonstrated to achieve wireless optogenetic or chemo-optogenetic killing of cancer cells in multiple mouse tumor models. LiPOPs can trigger necroptotic and pyroptotic cell death in cultured prokaryotic or eukaryotic cells and in living animals, and set the stage for studying the role of non-apoptotic cell death pathways during microbial infection and anti-tumor immunity.

Abstract: Plant-based photosensors, such as the light-oxygen-voltage sensing domain 2 (LOV2) from oat phototropin 1, can be modularly wired into cell signaling networks to remotely control protein activity and physiological processes. However, the applicability of LOV2 is hampered by the limited choice of available caging surfaces and its preference to accommodate the effector domains downstream of the C-terminal Jα helix. Here, we engineered a set of LOV2 circular permutants (cpLOV2) with additional caging capabilities, thereby expanding the repertoire of genetically encoded photoswitches to accelerate the design of optogenetic devices. We demonstrate the use of cpLOV2-based optogenetic tools to reversibly gate ion channels, antagonize CRISPR-Cas9-mediated genome engineering, control protein subcellular localization, reprogram transcriptional outputs, elicit cell suicide and generate photoactivatable chimeric antigen receptor T cells for inducible tumor cell killing. Our approach is widely applicable for engineering other photoreceptors to meet the growing need of optogenetic tools tailored for biomedical and biotechnological applications.