DYNACA-DA · Dynamic Calcium Clamp: Design and Applications

Periodic Report Summary - DYNACA-DA (Dynamic Calcium Clamp: Design and Applications)

Project context and objectives The project aims at creating a tool to control the intracellular concentration of calcium ions ([Ca2+]i). This tool will be particularly important for most of the fields in cellular biology since [Ca2+]i is one of the most important second messengers in virtually all cell types: from life onset to cell death through almost every physiological process (muscle contraction and heart physiology, as well as coagulation and immune system control, and even cognitive fields such as autism or sleep). The resting [Ca2+]i is usually low and its sensors have a fairly high affinity, therefore even slight changes can lead to dramatic effects. In neurons, the importance of Ca2+ is even more obvious, for it takes part in the following: - the computation of the information received by the neuron, e.g. via the modifications of the membrane potential induced by the activation of specific Ca2+ selective voltage-dependent channels and calcium-dependent potassium channels; - the synaptic transmission via the calcium-dependent release of synaptic vesicles; - the plasticity of the synaptic efficacy. There are several ways to image the variations of [Ca2+]i ; however, no tool was available to set the [Ca2+]i to a given value. Using the neuron as a model cell type to demonstrate the tool’s effectiveness, the objectives of the project are: 1. to develop the theoretical framework to realise the new tool, called a dynamic calcium clamp; 2. to convert this theoretical work into the actual tool; 3. to prove the effectiveness of the tool; 4. to apply it to two scientific questions (4a and 4b). Work performed Goals 1 to 4a were supposed to be carried out during the first period (outgoing phase) in Boston, (USA), while the return phase was meant to transfer the technology back to Europe, and apply it to the scientific question in goal 4b. The outgoing phase was successful and almost all the objectives were reached. The data recorded during the outgoing phase was analysed during the return phase and it led to the publication of the results in 'PLoS ONE'. Briefly, we designed an electro-optical technique to quantitatively set [Ca2+]i, in real time and with sub-cellular resolution, using two-photon Ca2+ uncaging and dynamic-clamp and called it a Dynamic Two-photon Calcium controller (DTC). For a number of non-scientific reasons (funding and administrative issues), the technology transfer could not occur because a key component necessary to rebuild the setup was not available at the return host. This component was a tunable Titanium:Sapphire laser, the pulsed nature of which is the very foundation of 2-photon excitation. To work around this issue, the researcher laid out all the theory behind a conversion of this technology from 2 to 1-photon. In the meantime, he performed all the control experiments, which did not require the uncaging system to work. These experiments are being analysed at the time of writing. The publication in 'PLoS ONE' has been seen by more than 650 people so far, which denotes a vast interest from the scientific community about this work.

Data: CORDIS, © European Union