Förster Resonance Energy Transfer (FRET)-based techniques are gaining an increasing importance in cell biology and cell-matrix adhesion studies because they allow both the detection of conformational changes of target proteins and their localisation in cells. Frequency Domain-Fluorescence Lifetime Microscopy (FD-FLIM) is currently considered one of the most reliable methods to measure FRET in live cells. However, due to its dependence on many technical prerequisites, its use is not yet widespread. The purpose of this work was to first establish FD-FLIM measurements of FRET on a new FD-FLIM microscope module. Then we aimed to apply FD-FLIM-FRET measurements to the study of conformational changes of the cell matrix-adhesion proteins vinculin and integrin and of the growth factor receptor Tie-2. In the first part of the work, published FRET probes including distance-sensors and two sets of vinculin-based probes were extensively tested with FD-FLIM, sensitised emission and ratiometric FRET. FD-FLIM was shown to be the most accurate method in approximating molecular distances between fluorophores. Moreover this study unveiled specific caveats associated with both existing vinculin FRET probes. FD-FLIM was then used to study conformational changes of the extracellular matrix receptor alphavβ3 integrin and of the angiopoietin receptor Tie-2 using specific FRET probes designed by us. While data showed that the alphav-integrin-FRET probe localised to adhesion sites, more experiments will be required to evaluate its full functionality. The Tie-2-FRET probe was fully functional and, upon ligand binding, allowed the detection of a bending movement of the extracellular domain towards the cell membrane. Finally, a combination of FRET, immunofluorescence and tension release experiments were used to show that intracellular tension is not required to maintain integrins in their activated conformation. However, intracellular tension is required to recruit other key proteins such as vinculin, talin and tensin to adhesions sites. Overall this work demonstrates the importance of FD-FLIM-FRET as a tool to investigate conformational changes of adhesion proteins and transmembrane receptors within the cell environment.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:756796 |
| Date | January 2015 |
| Creators | Balloi, Eleonora |
| Contributors | Humphries, Martin ; Ballestrem, Christoph |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/investigating-conformational-changes-of-proteins-using-frster-resonance-energy-transfer(fd5f53e7-9464-40bf-9c87-6fbd0a3d8804).html |
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