Optimized design and in vivo application of optogenetically functionalized Drosophila dopamine receptors.

Neuromodulatory signaling via G protein-coupled receptors (GPCRs) plays a pivotal role in regulating neural network function and animal behavior. The recent development of optogenetic tools to induce G protein-mediated signaling provides 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 signaling in vivo remains challenging. Here we optimize the design of optoXRs by considering evolutionary conserved GPCR-G protein interactions and demonstrate the feasibility of this approach using two Drosophila Dopamine receptors (optoDopRs). These optoDopRs exhibit high signaling specificity and light sensitivity in vitro. In vivo, we show receptor and cell type-specific effects of dopaminergic signaling in various behaviors, including the ability of optoDopRs to rescue the loss of the endogenous receptors. This work demonstrates that optoXRs can enable optical control of neuromodulatory receptor-specific signaling in functional and behavioral studies.

CDK6 activity in a recurring convergent kinase network motif.

In humans, more than 500 kinases phosphorylate ~15% of all proteins in an emerging phosphorylation network. Convergent local interaction motifs, in which ≥two kinases phosphorylate the same substrate, underlie feedback loops and signal amplification events but have not been systematically analyzed. Here, we first report a network-wide computational analysis of convergent kinase-substrate relationships (cKSRs). In experimentally validated phosphorylation sites, we find that cKSRs are common and involve >80% of all human kinases and >24% of all substrates. We show that cKSRs occur over a wide range of stoichiometries, in many instances harnessing co-expressed kinases from family subgroups. We then experimentally demonstrate for the prototypical convergent CDK4/6 kinase pair how multiple inputs phosphorylate the tumor suppressor retinoblastoma protein (RB) and thereby hamper in situ analysis of the individual kinases. We hypothesize that overexpression of one kinase combined with a CDK4/6 inhibitor can dissect convergence. In breast cancer cells expressing high levels of CDK4, we confirm this hypothesis and develop a high-throughput compatible assay that quantifies genetically modified CDK6 variants and inhibitors. Collectively, our work reveals the occurrence, topology, and experimental dissection of convergent interactions toward a deeper understanding of kinase networks and functions.

Structure-guided optimization of light-activated chimeric G-protein-coupled receptors.

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 ß2AR that including Gα contacts increased signaling 7- to 20-fold compared with other designs. In turn, contact elimination diminished function. Finally, this platform allowed optimization of a further OptoXR and spectral tuning. Our work exemplifies structure-based OptoXR development for targeted cell and network manipulation.

Light-activated receptor tyrosine kinases: Designs and applications.

Receptor tyrosine kinases (RTKs) are a large and essential membrane receptor family. The molecular mechanisms and physiological consequences of RTK activation depend on, for example, ligand identity, subcellular localization, and developmental or disease stage. In the past few years, genetically-encoded light-activated RTKs (Opto-RTKs) have been developed to dissect these complexities by providing reversible and spatio-temporal control over cell signaling. These methods have very recently matured to include highly-sensitive multi-color actuators. The new ability to regulate RTK activity with high precision has been recently harnessed to gain mechanistic insights in subcellular, tissue, and animal models. Because of their sophisticated engineering, Opto-RTKs may only mirror some aspects of natural activation mechanisms but nevertheless offer unique opportunities to study RTK signaling and physiology.

A Rationally and Computationally Designed Fluorescent Biosensor for d-Serine.

Solute-binding proteins (SBPs) have evolved to balance the demands of ligand affinity, thermostability, and conformational change to accomplish diverse functions in small molecule transport, sensing, and chemotaxis. Although the ligand-induced conformational changes that occur in SBPs make them useful components in biosensors, they are challenging targets for protein engineering and design. Here, we have engineered a d-alanine-specific SBP into a fluorescence biosensor with specificity for the signaling molecule d-serine (D-serFS). This was achieved through binding site and remote mutations that improved affinity (KD = 6.7 ± 0.5 µM), specificity (40-fold increase vs glycine), thermostability (Tm = 79 °C), and dynamic range (∼14%). This sensor allowed measurement of physiologically relevant changes in d-serine concentration using two-photon excitation fluorescence microscopy in rat brain hippocampal slices. This work illustrates the functional trade-offs between protein dynamics, ligand affinity, and thermostability and how these must be balanced to achieve desirable activities in the engineering of complex, dynamic proteins.

Acute and chronic effects of a light-activated FGF receptor in keratinocytes in vitro and in mice.

FGFs and their high-affinity receptors (FGFRs) play key roles in development, tissue repair, and disease. Because FGFRs bind overlapping sets of ligands, their individual functions cannot be determined using ligand stimulation. Here, we generated a light-activated FGFR2 variant (OptoR2) to selectively activate signaling by the major FGFR in keratinocytes. Illumination of OptoR2-expressing HEK 293T cells activated FGFR signaling with remarkable temporal precision and promoted cell migration and proliferation. In murine and human keratinocytes, OptoR2 activation rapidly induced the classical FGFR signaling pathways and expression of FGF target genes. Surprisingly, multi-level counter-regulation occurred in keratinocytes in vitro and in transgenic mice in vivo, including OptoR2 down-regulation and loss of responsiveness to light activation. These results demonstrate unexpected cell type-specific limitations of optogenetic FGFRs in long-term in vitro and in vivo settings and highlight the complex consequences of transferring optogenetic cell signaling tools into their relevant cellular contexts.

A Light-Oxygen-Voltage Receptor Integrates Light and Temperature.

Sensory photoreceptors enable organisms to adjust their physiology, behavior, and development in response to light, generally with spatiotemporal acuity and reversibility. These traits underlie the use of photoreceptors as genetically encoded actuators to alter by light the state and properties of heterologous organisms. Subsumed as optogenetics, pertinent approaches enable regulating diverse cellular processes, not least gene expression. Here, we controlled the widely used Tet repressor by coupling to light-oxygen-voltage (LOV) modules that either homodimerize or dissociate under blue light. Repression could thus be elevated or relieved, and consequently protein expression was modulated by light. Strikingly, the homodimeric RsLOV module from Rhodobacter sphaeroides not only dissociated under light but intrinsically reacted to temperature. The limited light responses of wild-type RsLOV at 37 °C were enhanced in two variants that exhibited closely similar photochemistry and structure. One variant improved the weak homodimerization affinity of 40 µM by two-fold and thus also bestowed light sensitivity on a receptor tyrosine kinase. Certain photoreceptors, exemplified by RsLOV, can evidently moonlight as temperature sensors which immediately bears on their application in optogenetics and biotechnology. Properly accounted for, the temperature sensitivity can be leveraged for the construction of signal-responsive cellular circuits.

Optogenetic delivery of trophic signals in a genetic model of Parkinson's disease.

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.

Microbial methionine transporters and biotechnological applications.

Methionine (Met) is an essential amino acid with commercial value in animal feed, human nutrition, and as a chemical precursor. Microbial production of Met has seen intensive investigation towards a more sustainable alternative to the chemical synthesis that currently meets the global Met demand. Indeed, efficient Met biosynthesis has been achieved in genetically modified bacteria that harbor engineered enzymes and streamlined metabolic pathways. Very recently, the export of Met as the final step during its fermentative production has been studied and optimized, primarily through identification and expression of microbial Met efflux transporters. In this mini-review, we summarize the current knowledge on four families of Met export and import transporters that have been harnessed for the production of Met and other valuable biomolecules. These families are discussed with respect to their function, gene regulation, and biotechnological applications. We cover methods for identification and characterization of Met transporters as the basis for the further engineering of these proteins and for exploration of other solute carrier families. The available arsenal of Met transporters from different species and protein families provides blueprints not only for fermentative production but also synthetic biology systems, such as molecular sensors and cell-cell communication systems. KEY POINTS: • Sustainable production of methionine (Met) using microbes is actively explored. • Met transporters of four families increase production yield and specificity. • Further applications include other biosynthetic pathways and synthetic biology.

Formation of Kiss1R/GPER Heterocomplexes Negatively Regulates Kiss1R-mediated Signalling through Limiting Receptor Cell Surface Expression.

Kisspeptin receptor (Kiss1R) is an important receptor that plays central regulatory roles in reproduction by regulating hormone release in the hypothalamus. We hypothesize that the formation of heterocomplexes between Kiss1R and other hypothalamus G protein-coupled receptors (GPCRs) affects their cellular signaling. Through screening of potential interactions between Kiss1R and hypothalamus GPCRs, we identified G protein-coupled estrogen receptor (GPER) as one interaction partner of Kiss1R. Based on the recognised function of kisspeptin and estrogen in regulating the reproductive system, we investigated the Kiss1R/GPER heterocomplex in more detail and revealed that complex formation significantly reduced Kiss1R-mediated signaling. GPER did not directly antagonize Kiss1R conformational changes upon ligand binding, but it rather reduced the cell surface expression of Kiss1R. These results therefore demonstrate a regulatory mechanism of hypothalamic hormone receptors via receptor cooperation in the reproductive system and modulation of receptor sensitivity.

LTP Induction Boosts Glutamate Spillover by Driving Withdrawal of Perisynaptic Astroglia.

Extrasynaptic actions of glutamate are limited by high-affinity transporters expressed by perisynaptic astroglial processes (PAPs): this helps maintain point-to-point transmission in excitatory circuits. Memory formation in the brain is associated with synaptic remodeling, but how this affects PAPs and therefore extrasynaptic glutamate actions is poorly understood. Here, we used advanced imaging methods, in situ and in vivo, to find that a classical synaptic memory mechanism, long-term potentiation (LTP), triggers withdrawal of PAPs from potentiated synapses. Optical glutamate sensors combined with patch-clamp and 3D molecular localization reveal that LTP induction thus prompts spatial retreat of astroglial glutamate transporters, boosting glutamate spillover and NMDA-receptor-mediated inter-synaptic cross-talk. The LTP-triggered PAP withdrawal involves NKCC1 transporters and the actin-controlling protein cofilin but does not depend on major Ca2+-dependent cascades in astrocytes. We have therefore uncovered a mechanism by which a memory trace at one synapse could alter signal handling by multiple neighboring connections.

Design and Application of Light-Regulated Receptor Tyrosine Kinases.

Understanding how the activity of membrane receptors and cellular signaling pathways shapes cell behavior is of fundamental interest in basic and applied research. Reengineering receptors to react to light instead of their cognate ligands allows for generating defined signaling inputs with high spatial and temporal precision and facilitates the dissection of complex signaling networks. Here, we describe fundamental considerations in the design of light-regulated receptor tyrosine kinases (Opto-RTKs) and appropriate control experiments. We also introduce methods for transient receptor expression in HEK293 cells, quantitative assessment of signaling activity in reporter gene assays, semiquantitative assessment of (in)activation time courses through Western blot (WB) analysis, and easy to implement light stimulation hardware.

All-Optical Miniaturized Co-culture Assay of Voltage-Gated Ca2+ Channels.

Light-activated proteins enable the reversible and spatiotemporal control of cellular events in optogenetics. Optogenetics is also rapidly expanding into the field of drug discovery where it provides cost-effective and noninvasive approaches for cell manipulation in high-throughput screens. Here, we present a prototypical cell-based assay that applies Channelrhodopsin2 (ChR2) to recapitulate physiological membrane potential changes and test for voltage-gated ion channel (VGIC) blockade. ChR2 and the voltage-gated Ca2+ channel 1.2 (CaV1.2) are expressed in individual HEK293 cell lines that are then co-cultured for formation of gap junctions and an electrical syncytium. This co-culture allows identification of blockers using parallel fluorescence plate readers in the 384-well plate format in an all-optical mode of operation. The assay is transferable to other VGICs by modularly combining new and existing cell lines and potentially also to other drug targets.

Optogenetic control of excitatory post-synaptic differentiation through neuroligin-1 tyrosine phosphorylation.

Neuroligins (Nlgns) are adhesion proteins mediating trans-synaptic contacts in neurons. However, conflicting results around their role in synaptic differentiation arise from the various techniques used to manipulate Nlgn expression level. Orthogonally to these approaches, we triggered here the phosphorylation of endogenous Nlgn1 in CA1 mouse hippocampal neurons using a photoactivatable tyrosine kinase receptor (optoFGFR1). Light stimulation for 24 hr selectively increased dendritic spine density and AMPA-receptor-mediated EPSCs in wild-type neurons, but not in Nlgn1 knock-out neurons or when endogenous Nlgn1 was replaced by a non-phosphorylatable mutant (Y782F). Moreover, light stimulation of optoFGFR1 partially occluded LTP in a Nlgn1-dependent manner. Combined with computer simulations, our data support a model by which Nlgn1 tyrosine phosphorylation promotes the assembly of an excitatory post-synaptic scaffold that captures surface AMPA receptors. This optogenetic strategy highlights the impact of Nlgn1 intracellular signaling in synaptic differentiation and potentiation, while enabling an acute control of these mechanisms.

Light-activated chimeric GPCRs: limitations and opportunities.

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.

Engineering Strategy and Vector Library for the Rapid Generation of Modular Light-Controlled Protein-Protein Interactions.

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.

Light-activated Frizzled7 reveals a permissive role of non-canonical wnt signaling in mesendoderm cell migration.

Non-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.

Isolation of synaptic vesicles from genetically engineered cultured neurons.

BACKGROUND: Synaptic vesicles (SVs) are an integral part of the neurotransmission machinery, and isolation of SVs from their host neuron is necessary to reveal their most fundamental biochemical and functional properties in in vitro assays. Isolated SVs from neurons that have been genetically engineered, e.g. to introduce genetically encoded indicators, are not readily available but would permit new insights into SV structure and function. Furthermore, it is unclear if cultured neurons can provide sufficient starting material for SV isolation procedures. NEW METHOD: Here, we demonstrate an efficient ex vivo procedure to obtain functional SVs from cultured rat cortical neurons after genetic engineering with a lentivirus. RESULTS: We show that ∼108 plated cortical neurons allow isolation of suitable SV amounts for functional analysis and imaging. We found that SVs isolated from cultured neurons have neurotransmitter uptake comparable to that of SVs isolated from intact cortex. Using total internal reflection fluorescence (TIRF) microscopy, we visualized an exogenous SV-targeted marker protein and demonstrated the high efficiency of SV modification. COMPARISON WITH EXISTING METHODS: Obtaining SVs from genetically engineered neurons currently generally requires the availability of transgenic animals, which is constrained by technical (e.g. cost and time) and biological (e.g. developmental defects and lethality) limitations. CONCLUSIONS: These results demonstrate the modification and isolation of functional SVs using cultured neurons and viral transduction. The ability to readily obtain SVs from genetically engineered neurons will permit linking in situ studies to in vitro experiments in a variety of genetic contexts.