Curated Optogenetic Publication Database

Abstract: Regulation of receptor tyrosine kinase (RTK) activity is necessary for studying cell signaling pathways in health and disease. We developed a generalized approach for engineering RTKs optically controlled with far-red light. We targeted the bacterial phytochrome DrBphP to the cell surface and allowed its light-induced conformational changes to be transmitted across the plasma membrane via transmembrane helices to intracellular RTK domains. Systematic optimization of these constructs has resulted in optically regulated epidermal growth factor receptor, HER2, TrkA, TrkB, FGFR1, IR1, cKIT and cMet, named eDrRTKs. eDrRTKs induced downstream signaling in mammalian cells in tens of seconds. The ability to activate eDrRTKs with far-red light enabled spectral multiplexing with fluorescent probes operating in a shorter spectral range, allowing for all-optical assays. We validated eDrTrkB performance in mice and found that minimally invasive stimulation in the neocortex with penetrating via skull far-red light-induced neural activity, early immediate gene expression and affected sleep patterns.

Abstract: Optically controlled receptor tyrosine kinases (opto-RTKs) allow regulation of RTK signaling using light. Until recently, the majority of opto-RTKs were activated with blue-green light. Fusing a photosensory core module of Deinococcus radiodurans bacterial phytochrome (DrBphP-PCM) to the kinase domains of neurotrophin receptors resulted in opto-RTKs controlled with light above 650 nm. To expand this engineering approach to RTKs of other families, here we combined the DrBpP-PCM with the cytoplasmic domains of EGFR and FGFR1. The resultant Dr-EGFR and Dr-FGFR1 opto-RTKs are rapidly activated with near-infrared and inactivated with far-red light. The opto-RTKs efficiently trigger ERK1/2, PI3K/Akt, and PLCγ signaling. Absence of spectral crosstalk between the opto-RTKs and green fluorescent protein-based biosensors enables simultaneous Dr-FGFR1 activation and detection of calcium transients. Action mechanism of the DrBphP-PCM-based opto-RTKs is considered using the available RTK structures. DrBphP-PCM represents a versatile scaffold for engineering of opto-RTKs that are reversibly regulated with far-red and near-infrared light.

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.