Investigation of the molecular dynamics of intercellular communication with advanced multi-dimensional fluorescence spectroscopy and imaging

All living organisms are made of cells, and there are many of them. You have more cells in your body than there are stars in our galaxy! Understanding how cells function and interact is understanding life itself. Cells are made of atoms and molecules, and not only is it important that they are in the right proportions, but also how these molecules are organized and what patterns they form. For example, there is a constant struggle between your white blood cells and invading disease organisms like bacteria and viruses which is happening right now, inside you, on a microscopic scale. We know that the white blood cells scan the molecules on other cell surfaces and somehow recognise the invaders, but if we could understand more about how it works, how cells communicate, we could help the white cells exterminate the invaders and protect us from disease. So we need to look at living cells bumping into each other, and the best way is to do this is to use a microscope. We cannot see the molecules directly because they are so small, but we can label the type of molecule we want to watch by attaching a particular fluorescent 'tag'. The revolution sparked by the development of the green fluorescent protein means that we can genetically label a particular molecule and watch where it goes. It's a bit like following a firefly around, it's easy to see where it goes because it glows. It's very similar to what we can do with a fluorescence microscope and fluorescent molecules in cells. But we want to do more than just locate fluorescent molecules. We want to understand more about what they are doing and experiencing. The solution is to use the properties of the fluorescent light to tell us about the environment of the fluorescent molecules themselves. If we use the fluorescence lifetime and the fluorescence polarization, and some clever reasoning, we can get information about the local environment on the cell surface, and how molecules interact when cells bump into each other. For example, which types of molecules are close together, and how close. And we can also see whether they move to the part of the cell surface that's in contact with another cell and what happens to them there. This will help to answer the question: How do cells communicate? How does this work? That's what we want to solve using new high-tech fluorescence imaging technology.

The proposed interdisciplinary collaboration brings together expertise in cell biology and advanced multi-dimensional fluorescence spectroscopy and imaging to provide important insight and understanding of intercellular communication between Natural Killer (NK) cells and target cells. Intercellular communication is of fundamental importance to many biological processes, for example for the workings of the immune system. The formation of an immune or immunological synapse (IS), at which cell surface proteins form large supramolecular clusters at an intercellular contact, is a general property of immune cells and is critical in bringing relevant receptors and signalling molecules together and excluding irrelevant ones. Recognition mechanisms employed by Natural Killer (NK) cells depend on a large number of activating and inhibitory receptors, the balance of which regulates NK cell activation. We propose to apply advanced multidimensional fluorescence spectroscopy and imaging (fluorescence correlation spectroscopy (FCS), fluorescence lifetime imaging (FLIM), time-resolved fluorescence anisotropy imaging (TR-FAIM) of fluorescent proteins and membrane probes to understanding intercellular communication between NK cells and target cells by studying the interaction and biophysical environment of specific proteins.