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

Lead Research Organisation: King's College London
Department Name: Physics

Abstract

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.

Technical Summary

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.

Publications

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George R (2009) A complex of Shc and Ran-GTPase localises to the cell nucleus. in Cellular and molecular life sciences : CMLS

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Levitt JA (2009) Fluorescence lifetime and polarization-resolved imaging in cell biology. in Current opinion in biotechnology

 
Description The GFP refractive index sensing distance is 100s of nanometers, thus it is difficult to probe the local environment of GFP-tagged proteins using the fluorescence decay of GFP (J Biomed Opt, SPIE Proc). However, FRET studies allow nanometer proximity information to be obtained to study protein-protein interaction (Biochem J, Cell Mol Life Sci, Cell Signal)



The fluorescence lifetimes of molecular rotors can be used to directly map viscosity in living cells (JACS, J Phys Chem C). Commercially available rotors such as DCVJ have very short lifetimes (a few picoseconds), bodipy-based lipophilic rotors have many 100ps or nanoseconds which can easily be measured (JACS, J Phys Chem C). Porphyrin-based molecular rotors (Org Biomol Chem) can act as photosensitisers to kill cancer cells and viscosity probes at the same time (Nat Chem). The main conclusion is that environment of these molecular rotors is much more viscous than water with the concomitant effect on diffusion (JACS, J Phys Chem C, Nat Chem).



FRAP and time-resolved fluorescence anisotropy measurements of GFP-tagged proteins in cell membranes and the immune synapse also yields diffusion coefficients much higher than in an aqueous environment. (SPIE Proc and papers in preparation.)
Exploitation Route KCL business
Sectors Pharmaceuticals and Medical Biotechnology

 
Description outreach - talk at Science Museum lates event (Crick, the future of biomedical discovery)
First Year Of Impact 2014
Sector Culture, Heritage, Museums and Collections
Impact Types Cultural,Societal

 
Description MRC Course in Advanced Optical Microscopy 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact "MRC Course in Advanced Optical Microscopy" in the Marine Biology Laboratory, Plymouth. The lecturers are invited, while the number of attendees is restricted to 20 who are selected by the organisers. I have been regularly speaking at this workshop since 2004, except 2013.
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,2009,2010,2011,2012,2014,2015,2016,2017,2018
URL https://www.mba.ac.uk/practical-course-advanced-optical-microscopy-cell-biology
 
Description Optical Imaging and Electrophysiological Recording in Neuroscience 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Optical Imaging and Electrophysiological Recording in Neuroscience, Paris Neuroscience School, Federation of Neurosciences, Université Paris Descartes, IBENS, École Normale Supérieure Paris, 14-23 May 2018. The lecturers are invited, while the number of attendees is restricted to 20 who are selected by the organisers. I have been regularly speaking at this workshop since it was founded in 2009.
Year(s) Of Engagement Activity 2009,2011,2012,2013,2014,2015,2016,2017,2018
URL https://parisneuro.ovh/