@article{8674, abstract = {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.}, author = {Henneberger, Christian and Bard, Lucie and Panatier, Aude and Reynolds, James P. and Kopach, Olga and Medvedev, Nikolay I. and Minge, Daniel and Herde, Michel K. and Anders, Stefanie and Kraev, Igor and Heller, Janosch P. and Rama, Sylvain and Zheng, Kaiyu and Jensen, Thomas P. and Sanchez-Romero, Inmaculada and Jackson, Colin J. and Janovjak, Harald L and Ottersen, Ole Petter and Nagelhus, Erlend Arnulf and Oliet, Stephane H.R. and Stewart, Michael G. and Nägerl, U. VAlentin and Rusakov, Dmitri A. }, issn = {10974199}, journal = {Neuron}, number = {5}, pages = {P919--936.E11}, publisher = {Elsevier}, title = {{LTP induction boosts glutamate spillover by driving withdrawal of perisynaptic astroglia}}, doi = {10.1016/j.neuron.2020.08.030}, volume = {108}, year = {2020}, } @article{6025, abstract = {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.}, author = {Capek, Daniel and Smutny, Michael and Tichy, Alexandra Madelaine and Morri, Maurizio and Janovjak, Harald L and Heisenberg, Carl-Philipp J}, journal = {eLife}, publisher = {eLife Sciences Publications}, title = {{Light-activated Frizzled7 reveals a permissive role of non-canonical wnt signaling in mesendoderm cell migration}}, doi = {10.7554/eLife.42093}, volume = {8}, year = {2019}, } @article{6564, abstract = {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.}, author = {Tichy, Alexandra-Madelaine and Gerrard, Elliot J. and Legrand, Julien M.D. and Hobbs, Robin M. and Janovjak, Harald L}, issn = {10898638}, journal = {Journal of Molecular Biology}, number = {17}, pages = {3046--3055}, publisher = {Elsevier}, title = {{Engineering strategy and vector library for the rapid generation of modular light-controlled protein–protein interactions}}, doi = {10.1016/j.jmb.2019.05.033}, volume = {431}, year = {2019}, } @article{7406, abstract = {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.}, author = {Mckenzie, Catherine and Spanova, Miroslava and Johnson, Alexander J and Kainrath, Stephanie and Zheden, Vanessa and Sitte, Harald H. and Janovjak, Harald L}, issn = {0165-0270}, journal = {Journal of Neuroscience Methods}, pages = {114--121}, publisher = {Elsevier}, title = {{Isolation of synaptic vesicles from genetically engineered cultured neurons}}, doi = {10.1016/j.jneumeth.2018.11.018}, volume = {312}, year = {2019}, } @phdthesis{7132, abstract = {A major challenge in neuroscience research is to dissect the circuits that orchestrate behavior in health and disease. Proteins from a wide range of non-mammalian species, such as microbial opsins, have been successfully transplanted to specific neuronal targets to override their natural communication patterns. The goal of our work is to manipulate synaptic communication in a manner that closely incorporates the functional intricacies of synapses by preserving temporal encoding (i.e. the firing pattern of the presynaptic neuron) and connectivity (i.e. target specific synapses rather than specific neurons). Our strategy to achieve this goal builds on the use of non-mammalian transplants to create a synthetic synapse. The mode of modulation comes from pre-synaptic uptake of a synthetic neurotransmitter (SN) into synaptic vesicles by means of a genetically targeted transporter selective for the SN. Upon natural vesicular release, exposure of the SN to the synaptic cleft will modify the post-synaptic potential through an orthogonal ligand gated ion channel. To achieve this goal we have functionally characterized a mixed cationic methionine-gated ion channel from Arabidopsis thaliana, designed a method to functionally characterize a synthetic transporter in isolated synaptic vesicles without the need for transgenic animals, identified and extracted multiple prokaryotic uptake systems that are substrate specific for methionine (Met), and established a primary/cell line co-culture system that would allow future combinatorial testing of this orthogonal transmitter-transporter-channel trifecta. Synthetic synapses will provide a unique opportunity to manipulate synaptic communication while maintaining the electrophysiological integrity of the pre-synaptic cell. In this way, information may be preserved that was generated in upstream circuits and that could be essential for concerted function and information processing.}, author = {Mckenzie, Catherine}, issn = {2663-337X}, pages = {95}, publisher = {Institute of Science and Technology Austria}, title = {{Design and characterization of methods and biological components to realize synthetic neurotransmission}}, doi = {10.15479/at:ista:7132}, year = {2019}, } @article{137, abstract = {Fluorescent sensors are an essential part of the experimental toolbox of the life sciences, where they are used ubiquitously to visualize intra- and extracellular signaling. In the brain, optical neurotransmitter sensors can shed light on temporal and spatial aspects of signal transmission by directly observing, for instance, neurotransmitter release and spread. Here we report the development and application of the first optical sensor for the amino acid glycine, which is both an inhibitory neurotransmitter and a co-agonist of the N-methyl-d-aspartate receptors (NMDARs) involved in synaptic plasticity. Computational design of a glycine-specific binding protein allowed us to produce the optical glycine FRET sensor (GlyFS), which can be used with single and two-photon excitation fluorescence microscopy. We took advantage of this newly developed sensor to test predictions about the uneven spatial distribution of glycine in extracellular space and to demonstrate that extracellular glycine levels are controlled by plasticity-inducing stimuli.}, author = {Zhang, William and Herde, Michel and Mitchell, Joshua and Whitfield, Jason and Wulff, Andreas and Vongsouthi, Vanessa and Sanchez Romero, Inmaculada and Gulakova, Polina and Minge, Daniel and Breithausen, Björn and Schoch, Susanne and Janovjak, Harald L and Jackson, Colin and Henneberger, Christian}, journal = {Nature Chemical Biology}, number = {9}, pages = {861 -- 869}, publisher = {Nature Publishing Group}, title = {{Monitoring hippocampal glycine with the computationally designed optical sensor GlyFS}}, doi = {10.1038/s41589-018-0108-2}, volume = {14}, year = {2018}, } @article{5984, abstract = {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.}, author = {Morri, Maurizio and Sanchez-Romero, Inmaculada and Tichy, Alexandra-Madelaine and Kainrath, Stephanie and Gerrard, Elliot J. and Hirschfeld, Priscila and Schwarz, Jan and Janovjak, Harald L}, issn = {2041-1723}, journal = {Nature Communications}, number = {1}, publisher = {Springer Nature}, title = {{Optical functionalization of human class A orphan G-protein-coupled receptors}}, doi = {10.1038/s41467-018-04342-1}, volume = {9}, year = {2018}, } @phdthesis{418, abstract = {The aim of this thesis was the development of new strategies for optical and optogenetic control of proliferative and pro-survival signaling, and characterizing them from the molecular mechanism up to cellular effects. These new light-based methods have unique features, such as red light as an activator, or the avoidance of gene delivery, which enable to overcome current limitations, such as light delivery to target tissues and feasibility as therapeutic approach. A special focus was placed on implementing these new light-based approaches in pancreatic β-cells, as β-cells are the key players in diabetes and especially their loss in number negatively affects disease progression. Currently no treatment options are available to compensate the lack of functional β-cells in diabetic patients. In a first approach, red-light-activated growth factor receptors, in particular receptor tyrosine kinases were engineered and characterized. Receptor activation with light allows spatio-temporal control compared to ligand-based activation, and especially red light exhibits deeper tissue penetration than other wavelengths of the visible spectrum. Red-light-activated receptor tyrosine kinases robustly activated major growth factor related signaling pathways with a high temporal resolution. Moreover, the remote activation of the proliferative MAPK/Erk pathway by red-light-activated receptor tyrosine kinases in a pancreatic β-cell line was also achieved, through one centimeter thick mouse tissue. Although red-light-activated receptor tyrosine kinases are particularly attractive for applications in animal models due to the deep tissue penetration of red light, a drawback, especially with regard to translation into humans, is the requirement of gene therapy. In a second approach an endogenous light-sensitive mechanism was identified and its potential to promote proliferative and pro-survival signals was explored, towards light-based tissue regeneration without the need for gene transfer. Blue-green light illumination was found to be sufficient for the activation of proliferation and survival promoting signaling pathways in primary pancreatic murine and human islets. Blue-green light also led to an increase in proliferation of primary islet cells, an effect which was shown to be mostly β-cell specific in human islets. Moreover, it was demonstrated that this approach of pancreatic β-cell expansion did not have any negative effect on the β-cell function, in particular on their insulin secretion capacity. In contrast, a trend for enhanced insulin secretion under high glucose conditions after illumination was detected. In order to unravel the detailed characteristics of this endogenous light-sensitive mechanism, the precise light requirements were determined. In addition, the expression of light sensing proteins, OPN3 and rhodopsin, was detected. The observed effects were found to be independent of handling effects such as temperature differences and cytochrome c oxidase dependent ATP increase, but they were found to be enhanced through the knockout of OPN3. The exact mechanism of how islets cells sense light and the identity of the photoreceptor remains unknown. Summarized two new light-based systems with unique features were established that enable the activation of proliferative and pro-survival signaling pathways. While red-light-activated receptor tyrosine kinases open a new avenue for optogenetics research, by allowing non-invasive control of signaling in vivo, the identified endogenous light-sensitive mechanism has the potential to be the basis of a gene therapy-free therapeutical approach for light-based β-cell expansion.}, author = {Gschaider-Reichhart, Eva}, issn = {2663-337X}, pages = {107}, publisher = {Institute of Science and Technology Austria}, title = {{Optical and optogenetic control of proliferation and survival }}, doi = {10.15479/AT:ISTA:th_913}, year = {2018}, } @phdthesis{6266, abstract = {A major challenge in neuroscience research is to dissect the circuits that orchestrate behavior in health and disease. Proteins from a wide range of non-mammalian species, such as microbial opsins, have been successfully transplanted to specific neuronal targets to override their natural communication patterns. The goal of our work is to manipulate synaptic communication in a manner that closely incorporates the functional intricacies of synapses by preserving temporal encoding (i.e. the firing pattern of the presynaptic neuron) and connectivity (i.e. target specific synapses rather than specific neurons). Our strategy to achieve this goal builds on the use of non-mammalian transplants to create a synthetic synapse. The mode of modulation comes from pre-synaptic uptake of a synthetic neurotransmitter (SN) into synaptic vesicles by means of a genetically targeted transporter selective for the SN. Upon natural vesicular release, exposure of the SN to the synaptic cleft will modify the post-synaptic potential through an orthogonal ligand gated ion channel. To achieve this goal we have functionally characterized a mixed cationic methionine-gated ion channel from Arabidopsis thaliana, designed a method to functionally characterize a synthetic transporter in isolated synaptic vesicles without the need for transgenic animals, identified and extracted multiple prokaryotic uptake systems that are substrate specific for methionine (Met), and established a primary/cell line co-culture system that would allow future combinatorial testing of this orthogonal transmitter-transporter-channel trifecta. Synthetic synapses will provide a unique opportunity to manipulate synaptic communication while maintaining the electrophysiological integrity of the pre-synaptic cell. In this way, information may be preserved that was generated in upstream circuits and that could be essential for concerted function and information processing. }, author = {Mckenzie, Catherine}, issn = {2663-337X}, pages = {95}, publisher = {Institute of Science and Technology Austria}, title = {{Design and characterization of methods and biological components to realize synthetic neurotransmission }}, doi = {10.15479/at:ista:th_1055}, year = {2018}, } @article{538, abstract = {Optogenetik und Photopharmakologie ermöglichen präzise räumliche und zeitliche Kontrolle von Proteinwechselwirkung und -funktion in Zellen und Tieren. Optogenetische Methoden, die auf grünes Licht ansprechen und zum Trennen von Proteinkomplexen geeignet sind, sind nichtweitläufig verfügbar, würden jedoch mehrfarbige Experimente zur Beantwortung von biologischen Fragestellungen ermöglichen. Hier demonstrieren wir die Verwendung von Cobalamin(Vitamin B12)-bindenden Domänen von bakteriellen CarH-Transkriptionsfaktoren zur Grünlicht-induzierten Dissoziation von Rezeptoren. Fusioniert mit dem Fibroblasten-W achstumsfaktor-Rezeptor 1 führten diese im Dunkeln in kultivierten Zellen zu Signalaktivität durch Oligomerisierung, welche durch Beleuchten umgehend aufgehoben wurde. In Zebrafischembryonen, die einen derartigen Rezeptor exprimieren, ermöglichte grünes Licht die Kontrolle über abnormale Signalaktivität während der Embryonalentwicklung. }, author = {Kainrath, Stephanie and Stadler, Manuela and Gschaider-Reichhart, Eva and Distel, Martin and Janovjak, Harald L}, journal = {Angewandte Chemie}, number = {16}, pages = {4679 -- 4682}, publisher = {Wiley}, title = {{Grünlicht-induzierte Rezeptorinaktivierung durch Cobalamin-bindende Domänen}}, doi = {10.1002/ange.201611998}, volume = {129}, year = {2017}, } @inbook{957, abstract = {Small molecule biosensors based on Forster resonance energy transfer (FRET) enable small molecule signaling to be monitored with high spatial and temporal resolution in complex cellular environments. FRET sensors can be constructed by fusing a pair of fluorescent proteins to a suitable recognition domain, such as a member of the solute-binding protein (SBP) superfamily. However, naturally occurring SBPs may be unsuitable for incorporation into FRET sensors due to their low thermostability, which may preclude imaging under physiological conditions, or because the positions of their N- and C-termini may be suboptimal for fusion of fluorescent proteins, which may limit the dynamic range of the resulting sensors. Here, we show how these problems can be overcome using ancestral protein reconstruction and circular permutation. Ancestral protein reconstruction, used as a protein engineering strategy, leverages phylogenetic information to improve the thermostability of proteins, while circular permutation enables the termini of an SBP to be repositioned to maximize the dynamic range of the resulting FRET sensor. We also provide a protocol for cloning the engineered SBPs into FRET sensor constructs using Golden Gate assembly and discuss considerations for in situ characterization of the FRET sensors.}, author = {Clifton, Ben and Whitfield, Jason and Sanchez Romero, Inmaculada and Herde, Michel and Henneberger, Christian and Janovjak, Harald L and Jackson, Colin}, booktitle = {Synthetic Protein Switches}, editor = {Stein, Viktor}, issn = {10643745}, pages = {71 -- 87}, publisher = {Springer}, title = {{Ancestral protein reconstruction and circular permutation for improving the stability and dynamic range of FRET sensors}}, doi = {10.1007/978-1-4939-6940-1_5}, volume = {1596}, year = {2017}, } @inbook{958, abstract = {Biosensors that exploit Forster resonance energy transfer (FRET) can be used to visualize biological and physiological processes and are capable of providing detailed information in both spatial and temporal dimensions. In a FRET-based biosensor, substrate binding is associated with a change in the relative positions of two fluorophores, leading to a change in FRET efficiency that may be observed in the fluorescence spectrum. As a result, their design requires a ligand-binding protein that exhibits a conformational change upon binding. However, not all ligand-binding proteins produce responsive sensors upon conjugation to fluorescent proteins or dyes, and identifying the optimum locations for the fluorophores often involves labor-intensive iterative design or high-throughput screening. Combining the genetic fusion of a fluorescent protein to the ligand-binding protein with site-specific covalent attachment of a fluorescent dye can allow fine control over the positions of the two fluorophores, allowing the construction of very sensitive sensors. This relies upon the accurate prediction of the locations of the two fluorophores in bound and unbound states. In this chapter, we describe a method for computational identification of dye-attachment sites that allows the use of cysteine modification to attach synthetic dyes that can be paired with a fluorescent protein for the purposes of creating FRET sensors.}, author = {Mitchell, Joshua and Zhang, William and Herde, Michel and Henneberger, Christian and Janovjak, Harald L and O'Mara, Megan and Jackson, Colin}, booktitle = {Synthetic Protein Switches}, editor = {Stein, Viktor}, issn = {10643745}, pages = {89 -- 99}, publisher = {Springer}, title = {{Method for developing optical sensors using a synthetic dye fluorescent protein FRET pair and computational modeling and assessment}}, doi = {10.1007/978-1-4939-6940-1_6}, volume = {1596}, year = {2017}, } @article{1026, abstract = {The optogenetic revolution enabled spatially-precise and temporally-precise control over protein function, signaling pathway activation, and animal behavior with tremendous success in the dissection of signaling networks and neural circuits. Very recently, optogenetic methods have been paired with optical reporters in novel drug screening platforms. In these all-optical platforms, light remotely activated ion channels and kinases thereby obviating the use of electrophysiology or reagents. Consequences were remarkable operational simplicity, throughput, and cost-effectiveness that culminated in the identification of new drug candidates. These blueprints for all-optical assays also revealed potential pitfalls and inspire all-optical variants of other screens, such as those that aim at better understanding dynamic drug action or orphan protein function.}, author = {Agus, Viviana and Janovjak, Harald L}, issn = {09581669}, journal = {Current Opinion in Biotechnology}, pages = {8 -- 14}, publisher = {Elsevier}, title = {{Optogenetic methods in drug screening: Technologies and applications}}, doi = {10.1016/j.copbio.2017.02.006}, volume = {48}, year = {2017}, } @article{1028, abstract = {Optogenetics and photopharmacology provide spatiotemporally precise control over protein interactions and protein function in cells and animals. Optogenetic methods that are sensitive to green light and can be used to break protein complexes are not broadly available but would enable multichromatic experiments with previously inaccessible biological targets. Herein, we repurposed cobalamin (vitamin B12) binding domains of bacterial CarH transcription factors for green-light-induced receptor dissociation. In cultured cells, we observed oligomerization-induced cell signaling for the fibroblast growth factor receptor 1 fused to cobalamin-binding domains in the dark that was rapidly eliminated upon illumination. In zebrafish embryos expressing fusion receptors, green light endowed control over aberrant fibroblast growth factor signaling during development. Green-light-induced domain dissociation and light-inactivated receptors will critically expand the optogenetic toolbox for control of biological processes.}, author = {Kainrath, Stephanie and Stadler, Manuela and Gschaider-Reichhart, Eva and Distel, Martin and Janovjak, Harald L}, issn = {14337851}, journal = {Angewandte Chemie - International Edition}, number = {16}, pages = {4608--4611}, publisher = {Wiley-Blackwell}, title = {{Green-light-induced inactivation of receptor signaling using cobalamin-binding domains}}, doi = {10.1002/anie.201611998}, volume = {56}, year = {2017}, } @article{1440, author = {Janovjak, Harald L}, journal = {Structure}, number = {2}, pages = {213 -- 215}, publisher = {Cell Press}, title = {{Light at the end of the protein: Crystal structure of a C-terminal light-sensing domain}}, doi = {10.1016/j.str.2016.01.002}, volume = {24}, year = {2016}, } @article{1101, abstract = {Optical sensors based on the phenomenon of Förster resonance energy transfer (FRET) are powerful tools that have advanced the study of small molecules in biological systems. However, sensor construction is not trivial and often requires multiple rounds of engineering or an ability to screen large numbers of variants. A method that would allow the accurate rational design of FRET sensors would expedite the production of biologically useful sensors. Here, we present Rangefinder, a computational algorithm that allows rapid in silico screening of dye attachment sites in a ligand-binding protein for the conjugation of a dye molecule to act as a Förster acceptor for a fused fluorescent protein. We present three ratiometric fluorescent sensors designed with Rangefinder, including a maltose sensor with a dynamic range of >300% and the first sensors for the most abundant sialic acid in human cells, N-acetylneuraminic acid. Provided a ligand-binding protein exists, it is our expectation that this model will facilitate the design of an optical sensor for any small molecule of interest.}, author = {Mitchell, Joshua and Whitfield, Jason and Zhang, William and Henneberger, Christian and Janovjak, Harald L and O'Mara, Megan and Jackson, Colin}, journal = {ACS SENSORS}, number = {11}, pages = {1286 -- 1290}, publisher = {American Chemical Society}, title = {{Rangefinder: A semisynthetic FRET sensor design algorithm}}, doi = {10.1021/acssensors.6b00576}, volume = {1}, year = {2016}, } @phdthesis{1124, author = {Morri, Maurizio}, issn = {2663-337X}, pages = {129}, publisher = {Institute of Science and Technology Austria}, title = {{Optical functionalization of human class A orphan G-protein coupled receptors}}, year = {2016}, } @article{1441, abstract = {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.}, author = {Gschaider-Reichhart, Eva and Inglés Prieto, Álvaro and Tichy, Alexandra-Madelaine and Mckenzie, Catherine and Janovjak, Harald L}, journal = {Angewandte Chemie - International Edition}, number = {21}, pages = {6339 -- 6342}, publisher = {Wiley}, title = {{A phytochrome sensory domain permits receptor activation by red light}}, doi = {10.1002/anie.201601736}, volume = {55}, year = {2016}, } @article{1100, abstract = {During metazoan development, the temporal pattern of morphogen signaling is critical for organizing cell fates in space and time. Yet, tools for temporally controlling morphogen signaling within the embryo are still scarce. Here, we developed a photoactivatable Nodal receptor to determine how the temporal pattern of Nodal signaling affects cell fate specification during zebrafish gastrulation. By using this receptor to manipulate the duration of Nodal signaling in vivo by light, we show that extended Nodal signaling within the organizer promotes prechordal plate specification and suppresses endoderm differentiation. Endoderm differentiation is suppressed by extended Nodal signaling inducing expression of the transcriptional repressor goosecoid (gsc) in prechordal plate progenitors, which in turn restrains Nodal signaling from upregulating the endoderm differentiation gene sox17 within these cells. Thus, optogenetic manipulation of Nodal signaling identifies a critical role of Nodal signaling duration for organizer cell fate specification during gastrulation.}, author = {Sako, Keisuke and Pradhan, Saurabh and Barone, Vanessa and Inglés Prieto, Álvaro and Mueller, Patrick and Ruprecht, Verena and Capek, Daniel and Galande, Sanjeev and Janovjak, Harald L and Heisenberg, Carl-Philipp J}, journal = {Cell Reports}, number = {3}, pages = {866 -- 877}, publisher = {Cell Press}, title = {{Optogenetic control of nodal signaling reveals a temporal pattern of nodal signaling regulating cell fate specification during gastrulation}}, doi = {10.1016/j.celrep.2016.06.036}, volume = {16}, year = {2016}, } @inbook{1549, abstract = {Nature has incorporated small photochromic molecules, colloquially termed 'photoswitches', in photoreceptor proteins to sense optical cues in photo-taxis and vision. While Nature's ability to employ light-responsive functionalities has long been recognized, it was not until recently that scientists designed, synthesized and applied synthetic photochromes to manipulate many of which open rapidly and locally in their native cell types, biological processes with the temporal and spatial resolution of light. Ion channels in particular have come to the forefront of proteins that can be put under the designer control of synthetic photochromes. Photochromic ion channel controllers are comprised of three classes, photochromic soluble ligands (PCLs), photochromic tethered ligands (PTLs) and photochromic crosslinkers (PXs), and in each class ion channel functionality is controlled through reversible changes in photochrome structure. By acting as light-dependent ion channel agonists, antagonist or modulators, photochromic controllers effectively converted a wide range of ion channels, including voltage-gated ion channels, 'leak channels', tri-, tetra- and pentameric ligand-gated ion channels, and temperaturesensitive ion channels, into man-made photoreceptors. Control by photochromes can be reversible, unlike in the case of 'caged' compounds, and non-invasive with high spatial precision, unlike pharmacology and electrical manipulation. Here, we introduce design principles of emerging photochromic molecules that act on ion channels and discuss the impact that these molecules are beginning to have on ion channel biophysics and neuronal physiology.}, author = {Mckenzie, Catherine and Sanchez Romero, Inmaculada and Janovjak, Harald L}, booktitle = {Novel chemical tools to study ion channel biology}, isbn = {978-1-4939-2844-6}, pages = {101 -- 117}, publisher = {Springer}, title = {{Flipping the photoswitch: Ion channels under light control}}, doi = {10.1007/978-1-4939-2845-3_6}, volume = {869}, year = {2015}, } @article{1611, abstract = {Biosensors for signaling molecules allow the study of physiological processes by bringing together the fields of protein engineering, fluorescence imaging, and cell biology. Construction of genetically encoded biosensors generally relies on the availability of a binding "core" that is both specific and stable, which can then be combined with fluorescent molecules to create a sensor. However, binding proteins with the desired properties are often not available in nature and substantial improvement to sensors can be required, particularly with regard to their durability. Ancestral protein reconstruction is a powerful protein-engineering tool able to generate highly stable and functional proteins. In this work, we sought to establish the utility of ancestral protein reconstruction to biosensor development, beginning with the construction of an l-arginine biosensor. l-arginine, as the immediate precursor to nitric oxide, is an important molecule in many physiological contexts including brain function. Using a combination of ancestral reconstruction and circular permutation, we constructed a Förster resonance energy transfer (FRET) biosensor for l-arginine (cpFLIPR). cpFLIPR displays high sensitivity and specificity, with a Kd of ∼14 μM and a maximal dynamic range of 35%. Importantly, cpFLIPR was highly robust, enabling accurate l-arginine measurement at physiological temperatures. We established that cpFLIPR is compatible with two-photon excitation fluorescence microscopy and report l-arginine concentrations in brain tissue.}, author = {Whitfield, Jason and Zhang, William and Herde, Michel and Clifton, Ben and Radziejewski, Johanna and Janovjak, Harald L and Henneberger, Christian and Jackson, Colin}, journal = {Protein Science}, number = {9}, pages = {1412 -- 1422}, publisher = {Wiley}, title = {{Construction of a robust and sensitive arginine biosensor through ancestral protein reconstruction}}, doi = {10.1002/pro.2721}, volume = {24}, year = {2015}, } @article{1867, abstract = {Cultured mammalian cells essential are model systems in basic biology research, production platforms of proteins for medical use, and testbeds in synthetic biology. Flavin cofactors, in particular flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are critical for cellular redox reactions and sense light in naturally occurring photoreceptors and optogenetic tools. Here, we quantified flavin contents of commonly used mammalian cell lines. We first compared three procedures for extraction of free and noncovalently protein-bound flavins and verified extraction using fluorescence spectroscopy. For separation, two CE methods with different BGEs were established, and detection was performed by LED-induced fluorescence with limit of detections (LODs 0.5-3.8 nM). We found that riboflavin (RF), FMN, and FAD contents varied significantly between cell lines. RF (3.1-14 amol/cell) and FAD (2.2-17.0 amol/cell) were the predominant flavins, while FMN (0.46-3.4 amol/cell) was found at markedly lower levels. Observed flavin contents agree with those previously extracted from mammalian tissues, yet reduced forms of RF were detected that were not described previously. Quantification of flavins in mammalian cell lines will allow a better understanding of cellular redox reactions and optogenetic tools.}, author = {Hühner, Jens and Inglés Prieto, Álvaro and Neusüß, Christian and Lämmerhofer, Michael and Janovjak, Harald L}, journal = {Electrophoresis}, number = {4}, pages = {518 -- 525}, publisher = {Wiley}, title = {{Quantification of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in mammalian model cells by CE with LED-induced fluorescence detection}}, doi = {10.1002/elps.201400451}, volume = {36}, year = {2015}, } @article{1678, abstract = {High-throughput live-cell screens are intricate elements of systems biology studies and drug discovery pipelines. Here, we demonstrate an optogenetics-assisted method that avoids the need for chemical activators and reporters, reduces the number of operational steps and increases information content in a cell-based small-molecule screen against human protein kinases, including an orphan receptor tyrosine kinase. This blueprint for all-optical screening can be adapted to many drug targets and cellular processes.}, author = {Inglés Prieto, Álvaro and Gschaider-Reichhart, Eva and Muellner, Markus and Nowak, Matthias and Nijman, Sebastian and Grusch, Michael and Janovjak, Harald L}, journal = {Nature Chemical Biology}, number = {12}, pages = {952 -- 954}, publisher = {Nature Publishing Group}, title = {{Light-assisted small-molecule screening against protein kinases}}, doi = {10.1038/nchembio.1933}, volume = {11}, year = {2015}, } @article{1844, abstract = {Local protein interactions ("molecular context" effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and β-lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations.}, author = {Risso, Valeria and Manssour Triedo, Fadia and Delgado Delgado, Asuncion and Arco, Rocio and Barroso Deljesús, Alicia and Inglés Prieto, Álvaro and Godoy Ruiz, Raquel and Gavira, Josè and Gaucher, Eric and Ibarra Molero, Beatriz and Sánchez Ruiz, Jose}, journal = {Molecular Biology and Evolution}, number = {2}, pages = {440 -- 455}, publisher = {Oxford University Press}, title = {{Mutational studies on resurrected ancestral proteins reveal conservation of site-specific amino acid preferences throughout evolutionary history}}, doi = {10.1093/molbev/msu312}, volume = {32}, year = {2014}, } @article{2032, abstract = {As light-based control of fundamental signaling pathways is becoming a reality, the field of optogenetics is rapidly moving beyond neuroscience. We have recently developed receptor tyrosine kinases that are activated by light and control cell proliferation, epithelial–mesenchymal transition, and angiogenic sprouting—cell behaviors central to cancer progression.}, author = {Inglés Prieto, Álvaro and Gschaider-Reichhart, Eva and Schelch, Karin and Janovjak, Harald L and Grusch, Michael}, journal = {Molecular and Cellular Oncology}, number = {4}, publisher = {Taylor & Francis}, title = {{The optogenetic promise for oncology: Episode I}}, doi = {10.4161/23723548.2014.964045}, volume = {1}, year = {2014}, } @article{2084, abstract = {Receptor tyrosine kinases (RTKs) are a large family of cell surface receptors that sense growth factors and hormones and regulate a variety of cell behaviours in health and disease. Contactless activation of RTKs with spatial and temporal precision is currently not feasible. Here, we generated RTKs that are insensitive to endogenous ligands but can be selectively activated by low-intensity blue light. We screened light-oxygen-voltage (LOV)-sensing domains for their ability to activate RTKs by light-activated dimerization. Incorporation of LOV domains found in aureochrome photoreceptors of stramenopiles resulted in robust activation of the fibroblast growth factor receptor 1 (FGFR1), epidermal growth factor receptor (EGFR) and rearranged during transfection (RET). In human cancer and endothelial cells, light induced cellular signalling with spatial and temporal precision. Furthermore, light faithfully mimicked complex mitogenic and morphogenic cell behaviour induced by growth factors. RTKs under optical control (Opto-RTKs) provide a powerful optogenetic approach to actuate cellular signals and manipulate cell behaviour.}, author = {Grusch, Michael and Schelch, Karin and Riedler, Robert and Gschaider-Reichhart, Eva and Differ, Christopher and Berger, Walter and Inglés Prieto, Álvaro and Janovjak, Harald L}, journal = {EMBO Journal}, number = {15}, pages = {1713 -- 1726}, publisher = {Wiley-Blackwell}, title = {{Spatio-temporally precise activation of engineered receptor tyrosine kinases by light}}, doi = {10.15252/embj.201387695}, volume = {33}, year = {2014}, } @article{2471, abstract = {The impact of disulfide bonds on protein stability goes beyond simple equilibrium thermodynamics effects associated with the conformational entropy of the unfolded state. Indeed, disulfide crosslinks may play a role in the prevention of dysfunctional association and strongly affect the rates of irreversible enzyme inactivation, highly relevant in biotechnological applications. While these kinetic-stability effects remain poorly understood, by analogy with proposed mechanisms for processes of protein aggregation and fibrillogenesis, we propose that they may be determined by the properties of sparsely-populated, partially-unfolded intermediates. Here we report the successful design, on the basis of high temperature molecular-dynamics simulations, of six thermodynamically and kinetically stabilized variants of phytase from Citrobacter braakii (a biotechnologically important enzyme) with one, two or three engineered disulfides. Activity measurements and 3D crystal structure determination demonstrate that the engineered crosslinks do not cause dramatic alterations in the native structure. The inactivation kinetics for all the variants displays a strongly non-Arrhenius temperature dependence, with the time-scale for the irreversible denaturation process reaching a minimum at a given temperature within the range of the denaturation transition. We show this striking feature to be a signature of a key role played by a partially unfolded, intermediate state/ensemble. Energetic and mutational analyses confirm that the intermediate is highly unfolded (akin to a proposed critical intermediate in the misfolding of the prion protein), a result that explains the observed kinetic stabilization. Our results provide a rationale for the kinetic-stability consequences of disulfide-crosslink engineering and an experimental methodology to arrive at energetic/structural descriptions of the sparsely populated and elusive intermediates that play key roles in irreversible protein denaturation.}, author = {Sanchez Romero, Inmaculada and Ariza, Antonio and Wilson, Keith and Skjøt, Michael and Vind, Jesper and De Maria, Leonardo and Skov, Lars and Sánchez Ruiz, Jose}, journal = {PLoS One}, number = {7}, publisher = {Public Library of Science}, title = {{Mechanism of protein kinetic stabilization by engineered disulfide crosslinks}}, doi = {10.1371/journal.pone.0070013}, volume = {8}, year = {2013}, } @article{2857, abstract = {In the vibrant field of optogenetics, optics and genetic targeting are combined to commandeer cellular functions, such as the neuronal action potential, by optically stimulating light-sensitive ion channels expressed in the cell membrane. One broadly applicable manifestation of this approach are covalently attached photochromic tethered ligands (PTLs) that allow activating ligand-gated ion channels with outstanding spatial and temporal resolution. Here, we describe all steps towards the successful development and application of PTL-gated ion channels in cell lines and primary cells. The basis for these experiments forms a combination of molecular modeling, genetic engineering, cell culture, and electrophysiology. The light-gated glutamate receptor (LiGluR), which consists of the PTL-functionalized GluK2 receptor, serves as a model.}, author = {Szobota, Stephanie and Mckenzie, Catherine and Janovjak, Harald L}, journal = {Methods in Molecular Biology}, pages = {417 -- 435}, publisher = {Springer}, title = {{Optical control of ligand-gated ion channels}}, doi = {10.1007/978-1-62703-351-0_32}, volume = {998}, year = {2013}, } @article{2856, abstract = {G protein–coupled receptors (GPCRs), the largest family of membrane signaling proteins, respond to neurotransmitters, hormones and small environmental molecules. The neuronal function of many GPCRs has been difficult to resolve because of an inability to gate them with subtype specificity, spatial precision, speed and reversibility. To address this, we developed an approach for opto-chemical engineering of native GPCRs. We applied this to the metabotropic glutamate receptors (mGluRs) to generate light-agonized and light-antagonized mGluRs (LimGluRs). The light-agonized LimGluR2, on which we focused, was fast, bistable and supported multiple rounds of on/off switching. Light gated two of the primary neuronal functions of mGluR2: suppression of excitability and inhibition of neurotransmitter release. We found that the light-antagonized tool LimGluR2-block was able to manipulate negative feedback of synaptically released glutamate on transmitter release. We generalized the optical control to two additional family members: mGluR3 and mGluR6. This system worked in rodent brain slices and in zebrafish in vivo, where we found that mGluR2 modulated the threshold for escape behavior. These light-gated mGluRs pave the way for determining the roles of mGluRs in synaptic plasticity, memory and disease.}, author = {Levitz, Joshua and Pantoja, Carlos and Gaub, Benjamin and Janovjak, Harald L and Reiner, Andreas and Hoagland, Adam and Schoppik, David and Kane, Brian and Stawski, Philipp and Schier, Alexander and Trauner, Dirk and Isacoff, Ehud}, journal = {Nature Neuroscience}, pages = {507 -- 516}, publisher = {Nature Publishing Group}, title = {{Optical control of metabotropic glutamate receptors}}, doi = {10.1038/nn.3346}, volume = {16}, year = {2013}, } @article{505, abstract = {Alkyd resins are polyesters containing unsaturated fatty acids that are used as binding agents in paints and coatings. Chemical drying of these polyesters is based on heavy metal catalyzed cross-linking of the unsaturated fatty acid moieties. Among the heavy-metal catalysts, cobalt complexes are the most effective, yet they have been proven to be carcinogenic. Therefore, strategies to replace the cobalt-based catalyst by environmentally friendlier and less toxic alternatives are under development. Here, we demonstrate for the first time that a laccase-mediator system can effectively replace the heavy-metal catalyst and cross-link alkyd resins. Interestingly, the biocatalytic reaction does not only work in aqueous media, but also in a solid film, where enzyme diffusion is limited. Within the catalytic cycle, the mediator oxidizes the alkyd resin and is regenerated by the laccase, which is uniformly distributed within the drying film as evidenced by confocal laser scanning microscopy. During gradual build-up of molecular weight, there is a concomitant decrease of the oxygen content in the film. A new optical sensor to follow oxygen consumption during the cross-linking reaction was developed and validated with state of the art techniques. A remarkable feature is the low sample amount required, which allows faster screening of new catalysts.}, author = {Greimel, Katrin and Perz, Veronika and Koren, Klaus and Feola, Roland and Temel, Armin and Sohar, Christian and Herrero Acero, Enrique and Klimant, Ingo and Guebitz, Georg}, journal = {Green Chemistry}, number = {2}, pages = {381 -- 388}, publisher = {Royal Society of Chemistry}, title = {{Banning toxic heavy-metal catalysts from paints: Enzymatic cross-linking of alkyd resins}}, doi = {10.1039/c2gc36666e}, volume = {15}, year = {2013}, } @inbook{10896, abstract = {Under physiological conditions the brain, via the purine salvage pathway, reuses the preformed purine bases hypoxanthine, derived from ATP degradation, and adenine (Ade), derived from polyamine synthesis, to restore its ATP pool. However, the massive degradation of ATP during ischemia, although providing valuable neuroprotective adenosine, results in the accumulation and loss of diffusible purine metabolites and thereby leads to a protracted reduction in the post-ischemic ATP pool size. In vivo, this may both limit the ability to deploy ATP-dependent reparative mechanisms and reduce the subsequent availability of adenosine, whilst in brain slices results in tissue with substantially lower levels of ATP than in vivo. In the present review, we describe the mechanisms by which brain tissue replenishes its ATP, how this can be improved with the clinically tolerated chemicals D-ribose and adenine, and the functional, and potential therapeutic, implications of doing so.}, author = {zur Nedden, Stephanie and Doney, Alexander S. and Frenguelli, Bruno G.}, booktitle = {Adenosine}, editor = {Masino, Susan and Boison, Detlev}, isbn = {9781461439028}, pages = {109--129}, publisher = {Springer}, title = {{The double-edged sword: Gaining Adenosine at the expense of ATP. How to balance the books}}, doi = {10.1007/978-1-4614-3903-5_6}, year = {2012}, } @article{3405, abstract = {Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system and gates non-selective cation channels. The origins of glutamate receptors are not well understood as they differ structurally and functionally from simple bacterial ligand-gated ion channels. Here we report the discovery of an ionotropic glutamate receptor that combines the typical eukaryotic domain architecture with the 'TXVGYG' signature sequence of the selectivity filter found in K+ channels. This receptor exhibits functional properties intermediate between bacterial and eukaryotic glutamate-gated ion channels, suggesting a link in the evolution of ionotropic glutamate receptors.}, author = {Janovjak, Harald L and Sandoz, Guillaume and Isacoff, Ehud}, journal = {Nature Communications}, number = {232}, pages = {1 -- 6}, publisher = {Nature Publishing Group}, title = {{Modern ionotropic glutamate receptor with a K+ selectivity signature sequence}}, doi = {10.1038/ncomms1231}, volume = {2}, year = {2011}, }