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Neuromodulatory signalingviaG protein-coupled receptor (GPCRs) plays a pivotal role in regulating neural network function and animal behavior. Recent efforts have led to the development of optogenetic tools to induce G protein-mediated signaling, with the promise of acute and cell type-specific manipulation of neuromodulatory signals. However, designing and deploying optogenetically functionalized GPCRs (optoXRs) with accurate specificity and activity to mimic endogenous signalingin vivoremains challenging. Here we optimized the design of optoXRs by considering evolutionary conserved GPCR-G protein interactions and demonstrate the feasibility of this approach using twoDrosophilaDopamine receptors (optoDopRs). We validated these optoDopRs showing that they exhibit high signaling specificity and light sensitivityin vitro.In vivowe detected receptor and cell type-specific effects of dopaminergic signaling in various behaviors including the ability of optoDopRs to rescue loss of the endogenous receptors. This work demonstrates that OptoXRs can enable optical control of neuromodulatory receptor specific signaling in functional and behavioral studies.

G-protein-coupled receptors (GPCRs) are the largest human receptor family and involved in virtually every physiological process. One hallmark of their function is specific coupling to selected signaling pathways. The ability to tune this coupling would make development of receptors with new capabilities possible. Complexes of GPCRs and G-proteins have recently been resolved at high resolution, but this information was in only few cases harnessed for rational receptor engineering. Here, we demonstrate structure-guided optimization of light-activated OptoXRs. Our hypothesis was that incorporation of GPCR-Gα contacts would lead to improved coupling. We first evaluated structure-based alignments for chimeric receptor fusion. We then show in a light-activated β

G-protein coupled receptors (GPCRs) are the largest human receptor family and involved in virtually every physiological process. One hallmark of GPCR function is the specific coupling of activated receptors to selected downstream signaling pathways. The ability to tune this coupling would permit the development of receptors with new capabilities. GPCRs and G-proteins have been recently resolved structurally at high resolution, but this information was in only very few cases harnessed for a rational engineering of these protein complexes. Here, we demonstrate the structure-guided optimization of coupling in chimeric light-activated GPCRs (OptoXRs). Our hypothesis was that the incorporation of structural GPCR-Gα contacts will lead to improved receptor activity. We first evaluated structure-based alignments as complements to existing sequence-based methods for generation of chimeric receptors. We then show in a prototypical light-activated β2AR that inclusion of α-helical residues forming structural contacts to Gα resulted in receptors with 7- to 20-fold increased function compared to other design strategies. In turn, elimination of GPCR-Gα contacts diminished function. Finally, the efficient receptor design served as a platform for the optimization of a further light-activated receptor and spectral tuning of the photoreceptor core domain. Our work exemplifies how increased OptoXR potency and new functionalities can be achieved through structure-based design towards targeted inputs into cells and cellular networks.

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.

Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson’s disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair., Author summary The death of physiologically important cells and tissues underlies of a wide range of diseases, including the neurodegenerative disorder Parkinson’s disease. Currently, the two major strategies to counter cell degeneration are the injection of soluble growth factor peptides and growth factor gene therapy. Importantly, both strategies can lead to the undesired activation of healthy bystander cells or the non-natural permanent modification of cells and their internal signals. Here, we developed a light-based method to overcome these limitations. The use of optogenetics allowed delivering cell type-specific pro-survival signals in a genetic model of Parkinson’s disease. In Drosophila and human cells that exhibit loss of the PINK1 gene, akin to autosomal recessive Parkinson’s disease, we efficiently suppressed disease phenotypes using a light-activated tyrosine kinase receptor. This work demonstrates a ‘remote controlled’ and thus spatio-temporally precise strategy to interfere with degeneration and may open new avenues towards tissue repair in a variety of disease models, including but not limited to diseases of the brain.

Light-activated chimeric GPCRs, termed OptoXRs, can elicit cell signalling responses with the high spatial and temporal precision of light. In recent years, an expanding OptoXR toolkit has been applied to, for example, dissect neural circuits in awake rodents, guide cell migration during vertebrate development and even restore visual responses in a rodent model of blindness. OptoXRs have been further developed through incorporation of highly sensitive photoreceptor domains and a plethora of signalling modules. The availability of new high-resolution structures of GPCRs and a deeper understanding of GPCR function allows critically revisitation of the design of OptoXRs. Next-generation OptoXRs will build on advances in structural biology, receptor function and photoreceptor diversity to manipulate GPCR signalling with unprecedented accuracy and precision.

10.7554/eLife.42093.001Non-canonical Wnt signaling plays a central role for coordinated cell polarization and directed migration in metazoan development. While spatiotemporally restricted activation of non-canonical Wnt-signaling drives cell polarization in epithelial tissues, it remains unclear whether such instructive activity is also critical for directed mesenchymal cell migration. Here, we developed a light-activated version of the non-canonical Wnt receptor Frizzled 7 (Fz7) to analyze how restricted activation of non-canonical Wnt signaling affects directed anterior axial mesendoderm (prechordal plate, ppl) cell migration within the zebrafish gastrula. We found that Fz7 signaling is required for ppl cell protrusion formation and migration and that spatiotemporally restricted ectopic activation is capable of redirecting their migration. Finally, we show that uniform activation of Fz7 signaling in ppl cells fully rescues defective directed cell migration in fz7 mutant embryos. Together, our findings reveal that in contrast to the situation in epithelial cells, non-canonical Wnt signaling functions permissively rather than instructively in directed mesenchymal cell migration during gastrulation.

Introduction Xenogeneic extracellular matrix (ECM) hydrogels have shown promise in remediating cardiac ischemia damage in animal models, yet analogous human ECM hydrogels have not been well development. An original human placenta-derived hydrogel (hpECM) preparation was thus generated for assessment in cardiomyocyte cell culture and therapeutic cardiac injection applications. Methods and results Hybrid orbitrap-quadrupole mass spectrometry and ELISAs showed hpECM to be rich in collagens, basement membrane proteins, and regenerative growth factors (e.g. VEGF-B, HGF). Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes synchronized and electrically coupled on hpECM faster than on conventional cell culture environments, as validated by intracellular calcium measurements. In vivo, injections using biotin-labeled hpECM confirmed its spatially discrete localization to the myocardium proximal to the injection site. hpECM was injected into rat myocardium following an acute myocardium infarction induced by left anterior descending artery ligation. Compared to sham treated animals, which exhibited aberrant electrical activity and larger myocardial scars, hpECM injected rat hearts showed a significant reduction in scar volume along with normal electrical activity of the surviving tissue, as determined by optical mapping. Conclusion Placental matrix and growth factors can be extracted as a hydrogel that effectively supports cardiomyocytes in vitro, and in vivo reduces scar formation while maintaining electrophysiological activity when injected into ischemic myocardium. Statement of Significance This is the first report of an original extracellular matrix hydrogel preparation isolated from human placentas (hpECM). hpECM is rich in collagens, laminin, fibronectin, glycoproteins, and growth factors, including known pro-regenerative, pro-angiogenic, anti-scarring, anti-inflammatory, and stem cell-recruiting factors. hpECM supports the culture of cardiomyocytes, stem cells and blood vessels assembly from endothelial cells. In a rat model of myocardial infarction, hpECM injections were safely deliverable to the ischemic myocardium. hpECM injections repaired the myocardium, resulting in a significant reduction in infarct size, more viable myocardium, and a normal electrophysiological contraction profile. hpECM thus has potential in therapeutic cardiovascular applications, in cellular therapies (as a delivery vehicle), and is a promising biomaterial for advancing basic cell-based research and regenerative medicine applications.

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.