TY - JOUR AB - Optogenetics enables the spatio-temporally precise control of cell and animal behavior. Many optogenetic tools are driven by light-controlled protein–protein interactions (PPIs) that are repurposed from natural light-sensitive domains (LSDs). Applying light-controlled PPIs to new target proteins is challenging because it is difficult to predict which of the many available LSDs, if any, will yield robust light regulation. As a consequence, fusion protein libraries need to be prepared and tested, but methods and platforms to facilitate this process are currently not available. Here, we developed a genetic engineering strategy and vector library for the rapid generation of light-controlled PPIs. The strategy permits fusing a target protein to multiple LSDs efficiently and in two orientations. The public and expandable library contains 29 vectors with blue, green or red light-responsive LSDs, many of which have been previously applied ex vivo and in vivo. We demonstrate the versatility of the approach and the necessity for sampling LSDs by generating light-activated caspase-9 (casp9) enzymes. Collectively, this work provides a new resource for optical regulation of a broad range of target proteins in cell and developmental biology. AU - Tichy, Alexandra-Madelaine AU - Gerrard, Elliot J. AU - Legrand, Julien M.D. AU - Hobbs, Robin M. AU - Janovjak, Harald L ID - 6564 IS - 17 JF - Journal of Molecular Biology SN - 00222836 TI - Engineering strategy and vector library for the rapid generation of modular light-controlled protein–protein interactions VL - 431 ER - TY - JOUR AB - G-protein-coupled receptors (GPCRs) form the largest receptor family, relay environmental stimuli to changes in cell behavior and represent prime drug targets. Many GPCRs are classified as orphan receptors because of the limited knowledge on their ligands and coupling to cellular signaling machineries. Here, we engineer a library of 63 chimeric receptors that contain the signaling domains of human orphan and understudied GPCRs functionally linked to the light-sensing domain of rhodopsin. Upon stimulation with visible light, we identify activation of canonical cell signaling pathways, including cAMP-, Ca2+-, MAPK/ERK-, and Rho-dependent pathways, downstream of the engineered receptors. For the human pseudogene GPR33, we resurrect a signaling function that supports its hypothesized role as a pathogen entry site. These results demonstrate that substituting unknown chemical activators with a light switch can reveal information about protein function and provide an optically controlled protein library for exploring the physiology and therapeutic potential of understudied GPCRs. AU - Morri, Maurizio AU - Sanchez-Romero, Inmaculada AU - Tichy, Alexandra-Madelaine AU - Kainrath, Stephanie AU - Gerrard, Elliot J. AU - Hirschfeld, Priscila AU - Schwarz, Jan AU - Janovjak, Harald L ID - 5984 IS - 1 JF - Nature Communications SN - 2041-1723 TI - Optical functionalization of human class A orphan G-protein-coupled receptors VL - 9 ER - TY - JOUR AB - Optogenetics and photopharmacology enable the spatio-temporal control of cell and animal behavior by light. Although red light offers deep-tissue penetration and minimal phototoxicity, very few red-light-sensitive optogenetic methods are currently available. We have now developed a red-light-induced homodimerization domain. We first showed that an optimized sensory domain of the cyanobacterial phytochrome 1 can be expressed robustly and without cytotoxicity in human cells. We then applied this domain to induce the dimerization of two receptor tyrosine kinases—the fibroblast growth factor receptor 1 and the neurotrophin receptor trkB. This new optogenetic method was then used to activate the MAPK/ERK pathway non-invasively in mammalian tissue and in multicolor cell-signaling experiments. The light-controlled dimerizer and red-light-activated receptor tyrosine kinases will prove useful to regulate a variety of cellular processes with light. Go deep with red: The sensory domain (S) of the cyanobacterial phytochrome 1 (CPH1) was repurposed to induce the homodimerization of proteins in living cells by red light. By using this domain, light-activated protein kinases were engineered that can be activated orthogonally from many fluorescent proteins and through mammalian tissue. Pr/Pfr=red-/far-red-absorbing state of CPH1. AU - Gschaider-Reichhart, Eva AU - Inglés Prieto, Álvaro AU - Tichy, Alexandra-Madelaine AU - Mckenzie, Catherine AU - Janovjak, Harald L ID - 1441 IS - 21 JF - Angewandte Chemie - International Edition TI - A phytochrome sensory domain permits receptor activation by red light VL - 55 ER -