FastTrack GECI: Development of novel fast calcium indicators for intracellular, extracellular and in vivo imaging

To understand and specifically target processes that occur in living organisms, microscopy techniques are useful. Using light emission as indicator of nerve impulses or showing the movements of components such as molecules or ions is an attractive approach with minimum invasion. To be informative, indicator molecules need to be designed that are specific and report biological processes with maximum fidelity. An important mediator between stimulus and effect is the calcium ion. It acts as a messenger for and controls a wide range of functions from muscle contraction and heart beat to learning and forming memories. Nerve conduction and muscle contraction are rapid processes, switched on in 1/1000 of a second. Appropriate molecular indicators should be just as fast. Our proposal addresses the issue that the indicators that have been developed are much slower than the processes. By understanding the molecular attributes that determine how fast the indicator's response is, we applied a number of modifications to the molecular structure and demonstrated that we can increase the response times. We will next apply the same strategy to a newer, brighter but still slow family of indicators. The application of these improved probes will allow more faithful visualisation of nerve and heart activity. However, even the fast probes have a property which limits their use, they require a certain level of calcium to detect it and furthermore will show the same brightness regardless of any higher calcium levels. To counter this problem, we propose a set of probes whose signal will increase with the level of calcium proportionately. Calcium is a messenger not only within the cell but also in the space between cells. Here the calcium levels are over 10000-fold higher than in the cell and monitoring such levels requires that the indicator has low affinity for calcium. Our third set of probes addresses this issues by the rational design referred to above. Finally, with the advanced microscopy techniques that are available, we can monitor the actions of calcium not only in cells but also in specific parts of the cell. The ability to do so has great potential for more detailed understanding of how the cell works and also to identify abnormal calcium levels and time courses in disease conditions. Calcium concentrations are precisely timed and locally changed and there is much evidence that in many diseases the ability of the cell to precisely adjust calcium levels is damaged. This is relevant in aging, epilepsy, dementia and memory disorders and also causes problems in the heart.
Our proposed probes will enable the better understanding of the underlying condition and in the long-term point to treatment targets.

Genetically encoded calcium indicators are useful probes but their rise and decay kinetics are slow in response to calcium concentration changes, which, in the case of action potentials, occur on the sub ms to ms time scale. The relatively slow kinetics is not surprising given the fact that formation of the RS20-calmodulin complex involves steric constraints by the interjecting cpGFP and that the RS20-calmodulin complex has a sub nM Kd. Thus to address the slow calcium rise and decay kinetics of GCaMPs the hypothesis was successfully tested that reducing the binding affinity between the peptide and calmodulin by selectively disabling its calcium binding sites would result in faster kinetics. Using the mutagenesis strategy successfully applied to GCaMP3 will be used to improve the kinetics of the new generation GCaMP6 probes.
Novel fast probe designs based on the interaction of IQ peptides with calmodulin and of secretagog EF-hand pairs with SNAP-25 (Kd ~ 1- 100 microM) will be tested. GCaMP3 and our new mutants are highly cooperative with respect to calcium concentration. However for quantification of calcium flux, linear indicator response is required. As a homolog of the calmodulin - MLCK peptide complex fused with cpGFP, we propose the troponin C - troponin I (TnC - TnI) complex as a novel and fast probe with the potential of giving a linear response to calcium concentration. Our G-GECO EF-2:3 probe has a Kd of 1.8 mM. This will be tagged for membrane anchoring and export to the extracellular face of the plasma membrane and will be tested as an extracellular calcium indicator in hippocampal tissue slices. The same mutations and others which caused similarly high Kd will be performed on GCaMP6 and any of the novel probes e.g Tn-based. Probes will be targeted to synaptic membranes for investigation of pre- and post-synaptic calcium signalling in various stimulation paradigms including those that evoke long-term potentiation and excitotoxicity in brain ce

Impact summary
The proposed research will make both immediate and long-term academic and societal impact in a number of ways.
Contribution to academic advances, across and within disciplines
One of the ways the proposed research represents value for public money is through informing the teaching of students. Final year specialisation, BSc and Master level research projects are based on on-going research and thus scientific research in the PI's laboratory will be beneficial by offering both a wider perspective on the direction of scientific research and the ability to learn and use state-of-the-art methodology.
An important contribution is made by the training of the post-doctoral researcher. Specifically in the case of Dr Helassa, his training at this stage is aimed at facilitating his transition to PI, to develop an independent research programme and to be able to conduct world-class research in his area of expertise. His successful transition to PI will be the best possible demonstration of value for public money as his training will be repaid by his becoming a future research leader and by his transferring his knowledge and skills to future students.
Training at the more technical level for the requested Technician contributes to his/her employment prospects and enriches the skills base that is essential for the economy to function.
The aims of our proposed research are the development of research tools for interdisciplinary research in bioscience and the understanding of the biophysical mechanisms of the probes. This understanding enhances the outcome of the subsequent physiological research by our collaborators and it also benefits the wider scientific community. Thus, our aim is to publish our work in high impact journals that serve the wider scientific community. Thereby increasing the competitiveness of UK research, further academic and societal benefits will justify the expenditure of public funds.
Enhancing quality of life, health and creative output
Beyond the advances in methodology and theory that our proposed research impacts directly and immediately, the tools that we propose to develop will enable advances in scientific disciplines e.g. neuro- and cardiac physiology as well as in aging research which have the potential for more direct future economic and societal impact thereby contributing to enhancing quality of life, health and creative output.
Specifically, for neuroscience, advances in understanding network activities in the brain in animal models will first of all increase our insight into our highest level of functioning. Secondly, through disease models, the potential for understanding, diagnosing and ultimately treating diseases such as dementia, Alzheimer's disease, epilepsy will be enhanced. The economic benefits that could derive from that will be significant.
For cardiac physiology, our tools that more faithfully report activity in cardiac myocytes will be used for detailed investigation of subcellular functions. This has the potential for the development of precisely and specifically targeted treatment of disorders of myocyte function.
Our research tools will enhance the research capabilities in the priority area of healthy aging. Calcium homeostasis is a proven vulnerability in aging. Probes of calcium signalling directed at monitoring changes in calcium are applicable ubiquitously to any cell type thus enabling investigation of characteristics of the aging process in a number of model systems. The understanding of processes obtained in cell systems will help identify targets for intervention beyond the completion of our project.