Learn more about…
…our general research interest
…ongoing research projects
…methods that we established
…our funding sources
…recent and upcoming talks of Harald

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 physiology in health and disease. 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 cancer cells and nerve cells. 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 disease models. Our interdisciplinary research builds on the development of new molecular tools and their applications in cell and animal models (mouse and Drosophila).

You can read more about our projects below.

Or click here for a list of recent and upcoming presentations of Harald.

ONGOING RESEARCH PROJECTS

Cell proliferation and survival in several disorders
Cell proliferation and survival 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, whereas many disparate diseases, including Type I and Parkinson’s disease, are characterized by excessive cell death. Growth factors are a driver of pro-survival and proliferative pathways both in cancer and in cells affected by excessive disease-related death. Many growth factor receptors have widespread tissue distribution and thus systemic administration results in undesired signaling in cells other than those targeted. For this 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.

Further reading:
– Our paper on in vivo light-activated proliferation control in PLoS Genetics 2021
– Our collaborative mouse study with Sabine Werner’s group from ETH Zurich on challenges in optical growth factor control in vivo in LifeScienceAlliance 2021
– Our Current Opinion 2022 on the design of optogenetic cell proliferation/survival receptors
– Our perspective with Sonja Kleinlogel from Uni Berne on optogenetic therapies in NRR 2022

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 drives non-small cell lung cancer and that we activated using light in the absence of the unknown ligand. We have also submitted a patent application on the action of AV-951 on Crizotinib-resistant variants of ROS1.

Our all-optical method can be easily adapted to many drug targets and cellular processes because of the many optical control tools available and we are excited to do this in the future also with new partners.

Further reading:
– Our paper in Nature Chemical Biology 2015
– 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
– Our Current Opinion 2017 with Viviana Agus from Axxam S.p.A.
– Our Methods 2020 paper with with Viviana, specifically in the context of ion channels

Function and physiology of orphan receptors / New OptoXR designs
Many GPCRs 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 animal models. Our primary approach utilized the OptoXR methodology where the known stimulus is a light signal activating rhodopsin. 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).

Within the OptoXR approach, we have established a new structure-based design methodology. It is to the best of our knowledge the first study describing a rational receptor engineering approach and one of very few studies in which the coupling profile of a GPCR was rationally reengineered.

Further reading:
– 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
– Our Current Opinion 2019 on the design of GPCRs with new functions
– Our paper on structure-based light-activated GPCR design in Cell Structure 2022

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 collaborative work with Christian Henneberger’s (University of Bonn) and Colin Jackson’s (Australian National University) lab.

Further reading:
– Our collaborative paper in Nature Chemical Biology 2018
– 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.

Further reading:
– 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.

Further reading:
– Our JMB 2019 paper describing a useful vector collection
– 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.

Further reading:
– Our paper in Electrophoresis 2015
– Wikipedia.org reference to our paper here

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 RESEARCH IS CURRENTLY FUNDED BY…
Flinders University
College of Medicine and Public Health
Australian Research Council (ARC)
National Health and Medical Research Council (NHMRC)
Industry partners

IN THE PAST, OUR RESEARCH WAS FUNDED BY…
Monash University
The Australian Regenerative Medicine Institute (ARMI)
EMBL Australia
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 (PAST 5 YEARS) AND UPCOMING INVITED PRESENTATIONS
2023, March 30, Synthetic Biology Australia – Adelaide Node Seminar Series, Adelaide, SA
2022, December 6, 40th Australasian Neuroscience Society Annual Meeting, Melbourne, VIC
2022, September 12, South Australian Neuroscience Alliance Seminar Series, Adelaide, SA
2021, December 1, Photopharmacology III, New York, NY
2021, October 28, Optogenetics in complex systems, Lausanne, Switzerland
2021, October 8, The University of Melbourne, Melbourne, VIC
2021, September 14, Synthetic Biology Australasia (SBA) Conference 2021, global virtual meeting
2019, November 29, Neuroplasticity – Systems, Synapses and Tools Symposium, Hobart, TAS
2019, October 3, Charles Perkins Centre, University of Sydney, NSW
2019, September 20, University of New South Wales, Sydney, NSW
2019, September 8-13, 13th Australian Peptide Conference, Port Douglas, QLD
2019, May 3, Baker Heart and Diabetes Institute, Melbourne, VIC
2019, April 30, Department of Diabetes, Monash University, Melbourne, VIC
2019, April 16, St Vincent’s Institute of Medical Research, Melbourne, VIC
2018, November 30, 11th Australian Islet Study Group Meeting, Canberra, ACT
2018, October 25&27, International Symposium on Growth Factors in Disease and Therapy, Hangzhou and Wenzhou, China
2018, October 2, Genetic Manipulation of Neuronal Activity V, Janelia Farms, VA
2018, September 20, Science without borders symposium, EMBL Heidelberg, Heidelberg, Germany
2018, July 15, Chinese University of Hong Kong, Hong Kong SAR
2018, June 20, Baker Heart and Diabetes Institute, Melbourne, VIC
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
2018, February 5, The 43rd Lorne Conference on Protein Structure and Function 2018, Lorne, VIC