OUR RESEARCH INTEREST
Our work lies at the interface of synthetic biology and mammalian physiology in the new research field synthetic physiology. The past decades have seen the emergence of synthetic biology, and with its paradigm ‘build to understand’, synthetic biology has proven to tackle the most complex biological problems. We are exploring synthetic principles for understanding and manipulating animal physiology. In past few years, our focus was on the ‘synthetic’ of synthetic physiology and we established new methods to control the signaling and behavior of nerve cells, cancer cells and key cell populations involved in metabolism. Our methods offer spatial and temporal precision and included but were not limited to optogenetics. We are currently transitioning to the ‘physiology’ of synthetic physiology with a focus on understanding/manipulating cell signaling and cell behavior in tissues affected by degeneration. Our interdisciplinary research builds on the development of new molecular tools and their applications in animal models (mouse and Drosophila) of disease.
Click here for a list of recent and upcoming presentations of Harald.
ONGOING RESEARCH PROJECTS
Cell proliferation and survival in degenerative disorders
Cell survival and proliferation are stringently regulated in all organisms and dysregulation towards too much cell death or too much cell growth can be equally fatal. Cancer is the most prominent disease characterized by uncontrolled cell growth, while many disparate diseases, including Type I and Parkinson’s disease, are characterized by excessive cell death. Growth factors are capable of activating pro-survival and proliferative pathways in cells affected by excessive disease-related death (e.g. pancreatic beta-cells, dopaminergic neurons and spinal cord neurons), but difficulties and concerns in using growth factors as research tools and in translation remain. Many growth factor receptors have widespread tissue distribution and thus systemic administration results in undesired signaling and toxicity in cells other than those targeted. In addition, many growth factors exhibit limited half-life in the circulation and cannot be delivered to some critical tissues (e.g. the brain). Finally, growth signals delivered by gene therapy are stationary and permanent. For these and other reasons, it is currently not possible to selectively and reversibly regulate the survival and proliferation of specific cell populations in a tissue or organism. We tackle this challenge by exploring new synthetic strategies for site-targeted administration of pro-survival and proliferative signals. In light of the known efficacy of growth factors but confronted with the above challenges, the new strategies may in the future be applied to control survival and proliferation of cells ex vivo or in translation.
Function and physiology of orphan receptors
Many membrane receptors are classified as ‘orphans’ because limited information is available on their ligands, coupling to the cellular signaling machinery and physiological functions. Many orphan receptors are conserved across species, strongly suggesting the existence of physiological functions, and orphans have been implicated in a variety of dysfunctions, including cancer, metabolic disorders and brain disorders. Currently, it is not possible to activate orphan receptors in situ and in real time and consequently at least two major questions remain: (i) Do orphan receptors couple to canonical cellular signaling pathways? (ii) What are the physiological roles of orphan receptors in health and disease? We tackle these questions based on the hypothesis that ‘functionalized’ orphan receptors, in which activation by an unknown ligand is replaced with that of a known stimulus, enable probing their function in cells and animals models. This work shines light on one of the longest-standing problems in physiology by revealing new biological insight on the signaling function and physiology of orphan receptors (this information will be valuable in basic research and drug discovery) and by providing chemical-genetic and optogenetic approaches to activate receptors in the absence of ligands (conventionally, ligands are considered essential for experiments with receptors). In our all-optical drug screen (see below), we already identified a drug candidate targeting an orphan receptor tyrosine kinase that is implicated in non-small cell lung cancer.
– Our paper on light-activated GPCR signaling in Nature Communications 2018
– Identifiers and sequences of light-activated variants of 63 understudied and orphan GPCRs can be found in Nature Communications 2018 Supplementary Table 3 and Supplementary Data 1 to 4
– Our modified Golden Gate cloning procedure and the corresponding expression vectors can be found in Nature Communications 2018 Supplementary Figures 1 and 2 and Supplementary Tables 4 and 5
– Available genes and construct information can be found here, including light-activated variants of nine prototypical GPCRs that served as controls
All-optical drug discovery
Already some years ago we and others have proposed that the non-invasive nature of optogenetics may be exploited to create new experimental paradigms in systems biology and small molecule screening. In both cases the use of optogenetics was suggested for creating more powerful, more reproducible and more economic high-throughput cellular assays. We recently demonstrated the first of these optogenetics-assisted small molecule screening methods. In our ‘all-optical’ screen, light acted both as activator and read-out to obviate the use of assay chemicals (e.g. peptide ligands or reagents for read-out), to limit the number of required operational steps (e.g. solution exchange or other invasive actions using robotics) and to improve specificity. In our first screen, we identified AV-951 (Tivozanib) as a potent and selective inhibitor of ROS1, an orphan kinase that we activated using light in the absence of the unknown ligand. Our method can be easily adapted to many drug targets and cellular processes because of the many optical control tools available (see below for a link to a review of Mueller and Weber) and we are excited to do this in the future also with new partners.
– Our paper in Nature Chemical Biology 2015
– Our Current Opinion 2017 with Viviana Agus from Axxam S.p.A.
– Our bioinformatics procedure to identify orphan (receptor) tyrosine kinases can be found in the Nature Chemical Biology 2015 Supplementary Figure 7 and Supplementary Table 2
– Press coverage and articles about this work can be found here
Sensors for neurotransmitters and synthetic neurotransmission
Our work in the context of our HFSP Young Investigator grant focused on understanding and manipulating neurotransmitter signaling. Historically, acetylcholine was the first chemical neurotransmitter to be synthesized, and it was shown to activate cells about sixty years before the genetic identity of acetylcholine receptors was revealed. While the components of chemical neurotransmission have been at least in part demystified in the past, an on-going challenge is to more sensitively detect neurotransmitter signaling, e.g. using optical sensors, and to create orthogonal neurotransmitter systems that will allow manipulating neural circuits in a manner that strictly relies on synaptic connectivity and activity. We address these two challenges in ongoing work.
– Our series of articles in the Synthetic Protein Switches 2017 issue of Methods in Molecular Biology and in ACS Sensors 2017
– Our first joint paper in Protein Science 2015
– The webpage of our collaborator Colin Jackson (Australian National University)
– The webpage of our collaborator Christian Henneberger (University of Bonn)
ESTABLISHED METHODOLOGICAL FOUNDATION
Light-controlled receptor tyrosine kinases (Opto-RTKs)
Optogenetics has revolutionized the field of neuroscience and is starting to have a big impact on cell and developmental biology as well. Advantages of optogenetics include the ease with which signals can be applied and withdrawn and the ability of rapid and local signal activation. Complementary to light-sensing ion channels and ion pumps, we developed light-activated receptor tyrosine kinases, such as Opto-FGFR1 or Opto-EGFR, the light-activated fibroblast growth factor receptor 1 or the light-activated epidermal growth factor receptor, along with light-activated neurotrophic factor receptors, as well as a light-INactivated fibroblast growth factor receptor 1. In cells expressing these receptors, light controls signaling with spatial and temporal precision and faithfully mimics complex mitogenic and morphogenic cell behavior. The receptors are available here. We are currently continuing this project in many directions by both further engineering as well as applying the receptors to selected questions in physiology.
– Our papers in EMBO Journal 2014 (blue light), Angewandte Chemie Int. Ed 2016 (red light) and Angewandte Chemie Int. Ed 2017 (green light INactivated)
– Our perspective in Molecular and Cellular Oncology 2015
– Press coverage and articles about this work can be found here (blue light), here (red light) and here (green light)
– Interview with Harald in LabTimes 2014
– Available genes and construct information can be found here
Light-induced homodimerization of receptors and other proteins
While working on the Opto-RTKs project, we have identified through database and literature searches LOV domains that change oligomerization state in response to blue light stimulation. The proteins allow for in vivo spatio-temporally precise regulation of many dimerization reactions in response to blue light with different lifetimes and dissociation constant. The domains are available here, along with the red-light induced homodimerizing phytochrome sensory domain.
– EMBO Journal 2014 supplement Table S1, S2 and S4
– A modified Table S1 for the blue light-activated proteins with estimated, in vitro dissociation constants and additional references can be found here
– Available genes and construct information can be found here
Optogenetics meets analytical chemistry
All optogenetic tools require chemical co-factors (e.g. flavins in LOV domains, retinal in opsins or tetrapyrroles in phytochromes) for light sensing, but surprisingly the quantity and distribution of these molecules in cells and tissues are poorly understood, even for those molecules that are present in all cells. To address this, we started a new line of research that combines analytical chemistry with optogenetics. In our first study, we quantified flavin contents in different cell types using a modified, LED-based capillary electrophoresis technique. We found that free flavin contents were limited and are potentially limiting in optogenetic experiments and other synthetic biology studies that express flavin-binding proteins. We are now expanding this line of research to the other co-factors and we explore how co-factor household is regulated.
REAGENTS AND COLLABORATIONS
We believe in sharing of published but also unpublished materials without strings attached. Have a look at our genes and reagents here and drop us a line if you think we might have something that is useful for your research (even if it is not listed on that page).
OUR MISSION STATEMENT
“Lights will guide you home.” – Coldplay
Our common research goal is to shed light on biological signals with the help of synthetic molecular torches. Each of us contributes a unique background ranging from biology to neuroscience, physics and chemistry. This enables us to design novel and multidisciplinary ways to approach classic problems in cellular signaling in mammalian systems. As an international group we work together following principles of collegiality by supporting each other to achieve our goals. Our dedication to research builds on the desire for innovation and intellectual curiosity.
OUR RESEARCH WAS/IS FUNDED BY…
The Juvenile Diabetes Research Foundation (JDRF)
European Union Seventh Framework Programme (FP7)
Human Frontier Science Program (HFSP)
Austrian Science Fund (FWF)
Austrian Research Promotion Agency (FFG)
Austrian Academy of Sciences (OEAW)
Dan David Foundation
Ramon Areces Foundation
RECENT AND UPCOMING INVITED PRESENTATIONS
2018, March 25, Gordon Research Conference on Fibroblast Growth Factors in Development & Disease, Ventura, CA
2018, March 22, School of Medicine, University of California Davis, CA
2018, March 20, Department of Chemistry, Stanford University, CA
2018, March 5, Gordon Research Conference on Photosensory Receptors and Signal Transduction, Il Ciocco, Italy
2017, December 5, The VIP-PACAP Congress 2017, Hong Kong SAR
2017, November 30, Molecular Health Sciences, ETH Zurich, Zurich, Switzerland
2017, September 14, With new molecular probes and modulators towards novel pharmacological concepts, Bonn, Germany
2017, September 1, Design and Light Control, Frankfurt, Germany
2017, August 27, Paul Ehrlich Symposium, Vienna, Austria
2017, August 24, International Biophysics Summer School, Kleinwalsertal, Austria
2017, August 22, 26th ISN-ESN Biennial Meeting, International Society for Neurochemistry, Paris, France
2017, April 6, 14th Life Science meeting, IMC University of Applied Sciences, Krems, Austria
2017, April 13, Neuronal Patterning and Connectivity, Center for Molecular Neurobiology, Hamburg, Germany
2016, December 15, BioMedX, Heidelberg, Germany
2016, December 8, iBV CNRS INSERM, Université de Nice Sophia Antipolis, France
2016, October 19, Genetic Manipulation of Neuronal Activity IV, Janelia Farms, VA
2016, October 5, 17th International Conference on Retinal Proteins, Potsdam, Germany
2016, September 21, Pharmacology of 7TM-receptors and downstream signaling pathways, Bonn, Germany
2016, July 16, A Passion for Proteins: From Structure and Folding to Understanding and Design, Bayreuth, Germany
2016, June 8, From Solid State to BioPhysics VIII, Dubrovnik, Croatia
2016, March 24, Faculty of Health Sciences, University of Macao, Macao SAR
2016, March 21, School of Aerospace, Mechanical and Mechatronic Engineering, University of Sydney, Australia
2016, March 14, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, Melbourne, Australia
2016, January 28, Modern Concepts in Structural Biology, Vienna Biocenter, Austria
2015, December 1, bioss – Centre for Biological Signalling Studies, Freiburg, Germany
2015, September 24, Ludwig Institute for Cancer Research, Oxford, UK
2015, July 1, Institute of Chemical Biology, University of Vienna, Austria
2015, June 9, Signal Processing in Neurons (SPIN), Innsbruck, Austria
2015, March 20, Bordeaux Neurocampus, France
2015, March 3, Medical University of Graz, Austria
2014, October 15, University Hospital Heidelberg, Germany
2014, October 14, European Molecular Biology Laboratory, Heidelberg, Germany
2014, September 26, Beilstein Conference on Molecular Switches, Chiemsee, Germany
2014, September 12, 16th International Congress on Photobiology, Cordoba, Argentina
2014, June 9, Salk Institute for Biological Studies, CA
2014, June 6, University of California Irvine, CA
2014, May 7, Casimir Spring School, Arnemuiden, Netherlands
2014, February 4, CIM IMPRS, Münster, Germany