Curated Optogenetic Publication Database

Abstract: General methods for spatiotemporal control of specific endogenous proteins would be broadly useful for probing protein function in living cells. Synthetic protein binders that bind and inhibit endogenous protein targets can be obtained from nanobodies, designed ankyrin repeat proteins (DARPins), and other small protein scaffolds, but generalizable methods to control their binding activity are lacking. Here, we report robust single-chain photoswitchable DARPins (psDARPins) for bidirectional optical control of endogenous proteins. We created topological variants of the DARPin scaffold by computer-aided design so fusion of photodissociable dimeric Dronpa (pdDronpa) results in occlusion of target binding at baseline. Cyan light induces pdDronpa dissociation to expose the binding surface (paratope), while violet light restores pdDronpa dimerization and paratope caging. Since the DARPin redesign leaves the paratope intact, the approach was easily applied to existing DARPins for GFP, ERK, and Ras, as demonstrated by relocalizing GFP-family proteins and inhibiting endogenous ERK and Ras with optical control. Finally, a Ras-targeted psDARPin was used to determine that, following EGF-activation of EGFR, Ras is required for sustained EGFR to ERK signaling. In summary, psDARPins provide a generalizable strategy for precise spatiotemporal dissection of endogenous protein function.

Abstract: Hyperactivation of phosphatidylinositol 3-kinase (PI3K) signaling is a prominent feature in cancer cells. However, the mechanism underlying malignant behaviors in the state remains unknown. Here, we describe a mechanism of cancer drug resistance through the protein synthesis pathway, downstream of PI3K signaling. An optogenetic tool (named PPAP2) controlling PI3K signaling was developed. Melanoma cells stably expressing PPAP2 (A375-PPAP2) acquired resistance to a cancer drug in the hyperactivation state. Proteome analyses revealed that expression of the antiapoptotic factor tumor necrosis factor alpha-induced protein 8 (TNFAIP8) was upregulated. TNFAIP8 upregulation was mediated by protein translation from preexisting mRNA. These results suggest that cancer cells escape death via upregulation of TNFAIP8 expression from preexisting mRNA even though alkylating cancer drugs damage DNA.

Abstract: How protein signaling networks respond to different input strengths is an important but poorly understood problem in cell biology. For example, the small GTPase RhoA regulates both focal adhesion (FA) growth or disassembly, but whether RhoA serves as a switch selecting between cellular outcomes, or if outcomes are simply modulated by additional factors in the cell, is not clear. Here, we develop a photoswitchable RhoA guanine exchange factor, psRhoGEF, to precisely control endogenous RhoA activity. We also develop a FRET-based biosensor to allow visualization of RhoA activity together with psRhoGEF control. Using these new optical tools, we discover that low levels of RhoA activation preferentially induce FA disassembly in a Src-dependent manner, while high levels induce both FA growth and disassembly in a ROCK-dependent manner. Thus, rheostatic control of RhoA activation with photoswitchable RhoGEF reveals that cells can use signal amplitude to produce multiple responses to a single biochemical signal.

Abstract: Pulsing cellular dynamics in genetic circuits have been shown to provide critical capabilities to cells in stress response, signaling and development. Despite the fascinating discoveries made in the past few years, the mechanisms and functional capabilities of most pulsing systems remain unclear, and one of the critical challenges is the lack of a technology that allows pulsatile regulation of transgene expression both in vitro and in vivo. Here, we describe the development of a synthetic BRET-based transgene expression (LuminON) system based on a luminescent transcription factor, termed luminGAVPO, by fusing NanoLuc luciferase to the light-switchable transcription factor GAVPO. luminGAVPO allows pulsatile and quantitative activation of transgene expression via both chemogenetic and optogenetic approaches in mammalian cells and mice. Both the pulse amplitude and duration of transgene expression are highly tunable via adjustment of the amount of furimazine. We further demonstrated LuminON-mediated blood-glucose homeostasis in type 1 diabetic mice. We believe that the BRET-based LuminON system with the pulsatile dynamics of transgene expression provides a highly sensitive tool for precise manipulation in biological systems that has strong potential for application in diverse basic biological studies and gene- and cell-based precision therapies in the future.

Abstract: Focusing light deep by engineering wavefronts toward guide stars inside scattering media has potential biomedical applications in imaging, manipulation, stimulation, and therapy. However, the lack of endogenous guide stars in biological tissue hinders its translations to in vivo applications. Here, we use a reversibly switchable bacterial phytochrome protein as a genetically encoded photochromic guide star (GePGS) in living tissue to tag photons at targeted locations, achieving light focusing inside the tissue by wavefront shaping. As bacterial phytochrome-based GePGS absorbs light differently upon far-red and near-infrared illumination, a large dynamic absorption contrast can be created to tag photons inside tissue. By modulating the GePGS at a distinctive frequency, we suppressed the competition between GePGS and tissue motions and formed tight foci inside mouse tumors in vivo and acute mouse brain tissue, thus improving light delivery efficiency and specificity. Spectral multiplexing of GePGS proteins with different colors is an attractive possibility.

Abstract: Optical control over the activity of receptor tyrosine kinases (RTKs) provides an efficient way to reversibly and non-invasively map their functions. We combined catalytic domains of Trk (tropomyosin receptor kinase) family of RTKs, naturally activated by neurotrophins, with photosensory core module of DrBphP bacterial phytochrome to develop opto-kinases, termed Dr-TrkA and Dr-TrkB, reversibly switchable on and off with near-infrared and far-red light. We validated Dr-Trk ability to reversibly light-control several RTK pathways, calcium level, and demonstrated that their activation triggers canonical Trk signaling. Dr-TrkA induced apoptosis in neuroblastoma and glioblastoma, but not in other cell types. Absence of spectral crosstalk between Dr-Trks and blue-light-activatable LOV-domain-based translocation system enabled intracellular targeting of Dr-TrkA independently of its activation, additionally modulating Trk signaling. Dr-Trks have several superior characteristics that make them the opto-kinases of choice for regulation of RTK signaling: high activation range, fast and reversible photoswitching, and multiplexing with visible-light-controllable optogenetic tools.

Abstract: Several light-regulated genetic circuits have been applied to spatiotemporally control transgene expression in mammalian cells. However, simultaneous regulation of multiple genes using one genetic device by light has not yet been reported. In this study, we engineered a bidirectional expression module based on LightOn system. Our data showed that both reporter genes could be regulated at defined and quantitative levels. Simultaneous regulation of four genes was further achieved in cultured cells and mice. Additionally, we successfully utilized the bidirectional expression module to monitor the expression of a suicide gene, showing potential for photodynamic gene therapy. Collectively, we provide a robust and useful tool to simultaneously control multiple genes expression by light, which will be widely used in biomedical research and biotechnology.