Final Report Summary - TOPAS (Towards the Quantal Nature of Receptor/cAMP Signals)
Signals inside cells are often transmitted via small chemical signals termed second messengers. It was generally assumed that these second messengers diffuse freely in the cells and that their concentrations change gradually. The project TOPAS aimed to assess activation of receptors and determination of the intracellular second messenger cyclic AMP (cAMP) with utmost sensitivity and to establish their discrete character. This aim was achieved by the creation of a number of fluorescent biosensors that can measure receptor activation and determine the levels of cAMP, and by the building of microscope capable of detecting these sensors at single molecule sensitivity. The project achieved to measure the speed of receptor activation and signaling and to demonstrate that such signals are not uniform. It further succeeded in imaging the movements of individual receptors at the cell surface and their interactions with individual components of the signaling machinery. Imaging of cAMP was achieved not only in isolated cells but also in vivo, notably in Drosophila fruitflies. The suitability of these techniques for practical applications (e.g. high throughput screening) is currently studied in a proof-of-concept project.