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

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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.