Protein-protein interactions at the cellular interface: Biophotonics approaches to live cell FRET measurements

The control of cellular function and intracellular signaling is clearly complex and highly regulated. Many signaling cascades have been extensively investigated and the relationships between proteins have been partially delineated using biochemical techniques. Microscopical techniques coupled with immunocytochemical methods have allowed researchers to image the relative localization of multiple signaling molecules. However, both types of measurement assume (different degrees of) cell homogeneity, which means that important variations in behaviour across the cell, and localisation of signaling events to specific parts of the cell, will be missed. Many intracellular structures are heterogeneous below the 200 nm length scale resolvable by widefield microscopy. Measurement of the near-field localization of protein complexes may be achieved by the detection of Förster resonant energy transfer (FRET) between protein-conjugated fluorophores1. FRET is a non radiative, dipole-dipole coupling process whereby energy from an excited donor fluorophore is transferred to an acceptor fluorophore in close proximity2,3. The dependence of the coupling efficiency varies with the inverse sixth power of the distance between acceptor and donor and is typically described in terms of the Förster radius (distance at which the efficiency of energy transfer is 50%), typically of the order 1-10 nm. Since the process depletes the excited state population of the donor, FRET will both reduce the fluorescence intensity and fluorescence lifetime of the donor. The advantage of using donor fluorescence lifetime to detect FRET is that the method is independent of fluorophore concentration, donor-acceptor stoichiometry and optical path length and is therefore well suited to studies in intact cells4,5. Combined with confocal or multiphoton techniques to examine the localization of effects in cellular compartments, FLIM/FRET allows us to determine populations of interacting protein species on a point-by-point basis at each resolved voxel in the cell6-8. The use of ‘ensemble’ FRET/FLIM techniques to probe protein-protein interactions in intact cells is now an established technique9. We and others have adapted the FLIM-based protein-protein interaction assays to directly monitor post-translational modifications (PTMs) of proteins (such as phosphorylation by PKC10, ubiquitination11 and sumoylation12) within live and fixed cells.
In this seminar, I will discuss recent developments in high-speed, multiphoton FLIM that allows us to image fluorescence lifetime changes due to FRET in a fraction of a second using multifocal multiphoton microscopy. Examples of protein-protein interactions in the ErbB network will be shown along with future developments. Furthermore, I will present a recently developed FRET technique13, which allows quantification of protein interaction by acceptor fluorescence anisotropy (aaFRET) and shows significant promise in simplicity and speed. aaFRET provides a direct read-out of interaction with a good dynamic range and also permits elimination of false positives linked to direct excitation of the acceptor14. I will show that this analysis method is comparable to FLIM for quantification of protein-protein interactions by FRET – and offers significant advantages in terms of speed and dynamic range at typically observed FRET efficiencies.

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