Realising the optogenetic potential of JellyOp: an opsin photopigment from the box jellyfish

The development of drugs that can influence physiology has played a crucial role in the huge advances in biological understanding that have occurred over the last century. However, this pharmaceutical approach to manipulating animal physiology has a number of well-known limitations. Drugs are commonly not very selective, having unintended effects on parts of the body, or physiological systems other than those of interest (so-called 'side effects'). Moreover, in comparison with the natural changes in physiology that they hope to regulate, drug effects build up rather slowly and persist for a long time. For these reasons, there would be a huge advantage in developing new ways of manipulating physiological systems that go beyond the achievements of pharmacy. We propose developing such a technology. Our approach will be to use a light sensitive version of a protein called a GPCR. GPCRs appear in practically every cell of our body. Their job is to fine tune the cell's activity and physiology according to signals released from neighbouring cells and other parts of the body. Because they are so influential they have long been recognised as a good way of treating the symptoms of disease. Indeed more than half of currently prescribed drugs are designed to alter their activity. By making photosensitive GPCRs we will be able to use light rather than drugs to tweak their activity. Unlike drugs, light can be switched on and off very rapidly. Light can also be applied at high doses to a single group of cells without influencing the rest of the body. These features mean that we will be able to use the new GPCRs to fine tune physiology with a resolution way beyond what is currently possible using drugs. This breakthrough will allow researchers to experimentally reproduce 'naturalistic' modulations in cell activity and examine their consequences. This is a great approach to understanding how the body works.

We have recently shown that an opsin photopigment from the box jellyfish (JellyOp) expresses efficiently in mammalian cells to allow light dependent regulation of Gas signalling and subsequent increases in the second messenger cAMP. JellyOp drives high amplitude responses and, unlike currently available alternatives, is resistant to bleach, allowing light to be used to achieve repeated or sustained activation of G-protein signalling. JellyOp thus has excellent potential as an optogenetic tool. Here we propose exploiting that potential by producing a number of structural variants of JellyOp that differ in sensory and/or signalling characteristics. Our goal is to produce versions of JellyOp that can couple to two other major classes of G-protein (Gi and q); are sensitive to different wavelengths; have different temporal resolution; and differ in their ability to interact with G-protein vs non G-protein dependent signalling cascades. Structural variants will be generated using standard methods of site directed mutagenesis and their sensory characteristics screened using established real-time reporter assays in cell culture. The most promising will then be expressed in Drosophila to confirm in vivo function. The endpoint for this project will thus be production of a toolbox of photopigments capable of regulating GPCR activity with unprecedented sophistication. These will be made available to the research community.

As this grant is primarily concerned with tool development, the immediate beneficiaries of our efforts will be fellow researchers who will be able to apply our methods to their research programmes. Importantly, this benefit will accrue to researchers from industry as well as academia and working in almost any aspect of animal biology. In order to facilitate this application of our work, we need to ensure both that relevant communities are aware of our findings and that the tools we develop are readily available. In terms of the former we know of no better approach than the accepted methods of academic dissemination; publishing high quality data in readily accessible journals; giving presentations at national/international conferences; and writing review articles. We have, of course, requested funds to support all of those activities. We propose a number of approaches to making the tools developed as part of this proposal widely available. Plasmids containing the JellyOp structural variants will be deposited with Addgene and transgenic flies with the Bloomington Drosophila stock center at Indiana University, US.

While our project will have little immediate application for the general public, optogenetics is a great vehicle for public understanding of science. The ability to modify animal behaviour and physiology using light sounds sufficiently 'science fiction' to capture the public imagination. Optogenetic experiments are thereafter conceptually straightforward, providing a simple illustration of the scientific method. They also provide an easy way to explain how cellular events underpin complex aspects of our biology. With this in mind, we additionally propose to develop a teaching tool for schools (key stage 3/4) on optogenetics as an exciting new field of science. This will involve creating a website as an aid for both teachers and students, together with a workshop that we will deliver to a number of local schools. The workshop will involve looking at a range of transgenic flies and stimulating muscle contractions with optogenetic tools and lights, as well as examining cells under the microscope. This will be done in conjunction with the Manchester Museum of Science and Industry, who are the STEM program co-ordinators for the region and with whom Helena Bailes has helped with several school science events.