Signal transduction pathways inside cells involve the coupling of ligand-receptor interactions to many intracellular events. These events include phosphorylation by tyrosine kinases and/or serine/threonine kinases. Protein phosphorylation change enzyme activities and protein conformations. The eventual outcome is an alteration in cellular activity and changes in the program of genes expressed within the responding cells. Phosphorylation dynamics can be imaged by FRET, by labelling two proteins-, domains-, or phospho-epitopes that come in close proximity during a phosphorylation event.
Epidermal-Growth Factor Receptor (EGFR) phosphorylation with the eYFP-(acceptor)-labelled phosphotyrosine-binding domain and eCFP (donor)-tagged EGFR.Beta-secretase (BACE) phosphorylation with BACE-GFP (donor) transfected cells fixed and stained with phosphoserine-Cy3 (acceptor).
When the enzyme is labelled by one fluorophore of a FRET pair, and the substrate by the other, FRET is expected when the enzyme cleaves the substrate.
Presenilin 1 (PS1) is a critical component of the gamma-secretase complex. This complex is involved in the cleavage of several substrates, including the amyloid precursor protein (APP). By FLIM-FRET is shown that the low-density receptor-related protein (LRP) is a PS1 interactor and can compete with APP for gamma-secretase enzymatic activity.
Endosome fusion can also be imaged by FRET:
Fusion between primary endocytic vesicles and/ or sorting endosomes.
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When the N-terminus is tagged with the donor fluorophore and the C-terminus with the acceptor fluorophore (or vice versa), the conformational change of the macromolecule can be visualised by the occurrence of FRET. In the 'open' conformation no FRET will occur, while the 'closed' conformation will cause FRET. Different dyes bind to different regions in DNA and so FRET occurrence can give information on the condensation of DNA:
An example is the staining of nuclei with Hoechst, that binds to AT-rich regions and with 7-AAD (7-aminoactinomycin D) that binds to GC-rich regions. These stained nuclei give a non-homogenous FRET signal in total nuclei, hence an increased FRET efficiency is shown when the cell progresses from G1 to G2/M (condensed DNA formation) phase.
Oligomerization kinetics is used to reveal the composition of macromolecules, and can be observed by FRET:
Conformation of leptin receptors expressed in the cell membrane.
Interactions between lipids and proteins can be visualised by FRET by incorporation of fluorescent lipids in the membrane and fluorescence-tagged peripheral membrane proteins.