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Photoreceptors added to optogenetic toolbox

After light-gated ion channels in nerve cells had successfully paved the way for optogenetics, numerous tools have been added to the optogenetic toolbox. Photoreceptors are novel optogenetic tools, which, when coupled to enzymes and kinases, can trigger certain cell functions upon illumination with light. Prof. Dr. Wilfried Weber, synthetic biologist at BIOSS (Centre for Biological Signalling Studies) at the University of Freiburg, is one of the scientists who have been the major drivers in the use of photoreceptors as optogenetic tools. The trend is moving towards being able to operate any controllable cellular process with light.

Light is essential for life on Earth; all organisms, whether they are mice, trees or bacteria, are able to perceive light with specifically adapted structures known as photoreceptors. Only specialised cells are able to absorb and interpret different wavelengths. “The majority of cells know nothing about optogenetics,” said Prof. Dr. Wilfried Weber from the University of Freiburg. “We need to teach them. And we do this by introducing relevant optogenetic components.” What the researchers are in fact doing is making light-insensitive cells optically responsive, i.e. making them respond to light. “This can be done with genetic engineering,” said Weber.

Plants help to expand the optogenetic toolbox

Weber and his team of researchers transfer photoreceptors from plants into mammalian cells and couple them with suitable effector molecules with the goal of establishing an optogenetic system with which they can achieve specific effects. “A broad range of components are nowadays known and can be combined in such a way that the most diverse processes can be controlled with light,” Weber explained.

The ability to control protein activity with photoreceptors has hugely broadened the applications for optogenetics over the last two years. In addition to being used for manipulating ion channels and neurons, photoreceptors can now also be used for the manipulation of virtually all cells. Weber is sure that this ability has triggered the creativity of scientists around the world enormously. “This is a perfect field for applying synthetic biology,” said Weber delighted with recent developments. These developments have also attracted the attention of numerous researchers who are focussed on cellular regulatory mechanisms. They are now able to control processes of interest with light and gain completely new insights.

Different building blocks can be combined

Remote-controlled mammalian cells: targets of light-controlled manipulation (circled green: extracellular matrix, signalling chain, nuclear transport and promoter activation). © Prof. Dr. Wilfried Weber and Hannes Beyer, University of Freiburg
Plants use the protein phytochrome B (Phy B), a photoreceptor that detects red (660 mm) and far-red light (740 mm), and the phytochrome interacting factor (PIF) to detect light. The detection of light is a reversible mechanism: Illumination with red light leads to the binding of PIF and Phy B, which triggers a specific signalling cascade. The interaction between PIF and PhyB can be abolished by illumination with far-red light. The genetic information for Phy B and PIF can be obtained from plant cells and introduced into human or animal cell cultures. In the optimal case, these cells then produce the coveted receptor. These tools can in principle be used to control the activity of any protein. Many cellular kinases are inactive as monomers and only become active upon dimerisation. Protein kinase activity can be controlled by coupling one kinase molecule to Phy B and the other to PIF. Illumination with red light leads to the binding of PIF and Phy B, and hence to the dimerisation and activation of the kinase molecules. These optogenetic tools can also be used to turn on and off the effectiveness of other enzymes, protein transport and gene activity processes within seconds. “There are examples that show that the entire signalling chain from the extracellular matrix to receptors, phosphorylation cascades, nuclear transport, promoter activation and to the stability of the final product can be excellently controlled with light. We can thus excellently control these cellular processes,” Weber said. “The nice thing about light is that it provides us with a high temporal and spatial resolution. This means that we can precisely determine the cell in which we want to turn on genes and the point in time they are activated.”

Revolution in tool development

The promoters of the genes shown are activated in relation to illumination with red, blue or UV light. © Prof. Dr. Wilfried Weber and Hannes Beyer, University of Freiburg

Weber has been exploring the regulation of genes on the promoter level for ten years now. In early 2013, Weber published a study describing the first red/far-red light-triggered gene switch for achieving gene expression control within seconds and has since received many requests from all areas of research. Synthetic biologists, developmental biologists, immunobiologists and cell biologists have shown great interest in Weber’s plasmids that encode the Phy B and PIF system.

Weber is specifically focussed on promoters that are switched on by an activation domain. This works as follows: A bacterial protein that binds to the promoter region is coupled with a PIF molecule and a Phy B molecule is coupled to the activation domain. “Illumination with red light causes the two to come together. The activation domain switches on the promoter and the gene is transcribed,” Weber explained. Illumination with far-red light leads to the dissociation of the molecules; the activation domain is absent, and the promoter is not switched on. Weber highlighted that this development is mainly the result of work done by researchers from the University of Freiburg’s Department of Plant Physiology, Prof. Dr. Eberhard Schäfer and Prof. Dr. Andreas Hiltbrunner, who have made a great contribution to the characterisation of Phy B function.

Potential for application in all disciplines

Cell cultures can be illuminated with a mobile phone light pattern… © Hannes Beyer

The applications will not be long in coming. Weber and his colleagues have created a kind of universal switch that can be used by researchers from different disciplines and adapted to their specific research interests. Weber and his team are working with a group of researchers from Zurich University Hospital with the aim of using the switch for inducing angiogenic growth factors. The initial results are quite promising. Kinases also appear to play a major role in tumour signalling pathways and Weber’s switch now enables the effective optogenetic control of kinase activity.

... which in turn leads to the activation of the gene promoter. © Hannes Beyer

Wilfried Weber is already working on putting a second and third promoter under the control of blue-light and UV-light dependent proteins with the goal of being able to differentially induce up to three genes in a single cell culture in response to light of different wavelengths. Illumination with a photomask, i.e. a plate with holes that allow light to shine through in a defined pattern, leads to the induction of genes that are exposed to light. Genes that remain in the dark are not expressed.

Weber’s latest idea is to illuminate cell cultures with a mobile phone screen with a certain light pattern and induce the activation of gene promoters. “The pixels on the screen are the same size as cells, i.e. 20 micrometres,” said Weber. “So we are using the same level of resolution as cells, which facilitates the activation of target promoters. And this works amazingly well.” Welcome to the future!

Further information:

Prof. Dr. Wilfried Weber
Institute of Biology II and BIOSS
University of Freiburg
Schänzlestr. 18
79104 Freiburg
Tel.: +49 761 203 97654
E-mail: wilfried.weber(at)bioss.uni-freiburg.de
Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/photoreceptors-added-to-optogenetic-toolbox