Chapter 16
Multicolor FRET-FLIM Microscopy to Analyze Multiprotein
Interactions in Live Cells
Abdullah Ahmed, Jennifer Schoberer, Emily Cooke,
and Stanley W. Botchway
Abstract
The need to describe and understand signaling pathways in live cell is seen as a primary route to identifying
and developing targeted medicines. Signaling cascade is also seen as a complex communication and involves
interactions between multiple interconnecting proteins. Where subcellularly and how different proteins
interact need to be preserved during investigation. Furthermore, these complex events occurring simultaneously may lead to a single or multiple end point or cell function such as protein synthesis, cell cytoskeleton formation, DNA damage repair, or autophagy. There is therefore a need of real-time noninvasive
methods for protein assays to enable direct visualization of the interactions in their natural environment and
hence overcome the limitations of methods that rely on invasive cell disruption techniques. Fo ¨rster
resonance energy transfer (FRET) coupled with fluorescence lifetime imaging microscopy (FLIM) is an
advanced imaging method to observe protein–protein interactions at nanometer scale inside single living
cells in real-time. Here we describe the development and use of two-channel pulsed interleave excitation
(PIE) for multiple protein interactions in the mTORC1 pathway. The proteins were first tagged with
multiple color fluorescent protein derivatives. The FRET-FLIM combination means that the information
gained from using standard steady-state FRET between interacting proteins is considerably improved by
monitoring changes in the excited-state lifetime of the donor fluorophore where its quenching in the
presence of the acceptor is evidence for a direct physical interaction.
Key words mTOR, Enzymes, Protein complex, Fluorescence imaging, Confocal microscopy,
Excited-state lifetime, TCSPC, FRET, FLIM GFP-tag, DNA, Three-color
1 Introduction
All cellular processes are driven by the action of proteins, from ion
channels across phospholipid membranes to DNA synthesis and cell
growth. For example, the mechanistic or mammalian target of
rapamycin (mTOR) pathway functions in the coordination of
energy, nutrients, and growth factor availability to regulate key
biological processes including cellular growth, metabolism, and
protein synthesis through the phosphorylation of downstream substrates, ribosomal protein, S6 Kinase 1 (S6K1), and 4E-binding
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_16, © Springer Science+Business Media, LLC, part of Springer Nature 2021
287
Multicolor FRET-FLIM Microscopy to Analyze Multiprotein
Interactions in Live Cells
Abdullah Ahmed, Jennifer Schoberer, Emily Cooke,
and Stanley W. Botchway
Abstract
The need to describe and understand signaling pathways in live cell is seen as a primary route to identifying
and developing targeted medicines. Signaling cascade is also seen as a complex communication and involves
interactions between multiple interconnecting proteins. Where subcellularly and how different proteins
interact need to be preserved during investigation. Furthermore, these complex events occurring simultaneously may lead to a single or multiple end point or cell function such as protein synthesis, cell cytoskeleton formation, DNA damage repair, or autophagy. There is therefore a need of real-time noninvasive
methods for protein assays to enable direct visualization of the interactions in their natural environment and
hence overcome the limitations of methods that rely on invasive cell disruption techniques. Fo ¨rster
resonance energy transfer (FRET) coupled with fluorescence lifetime imaging microscopy (FLIM) is an
advanced imaging method to observe protein–protein interactions at nanometer scale inside single living
cells in real-time. Here we describe the development and use of two-channel pulsed interleave excitation
(PIE) for multiple protein interactions in the mTORC1 pathway. The proteins were first tagged with
multiple color fluorescent protein derivatives. The FRET-FLIM combination means that the information
gained from using standard steady-state FRET between interacting proteins is considerably improved by
monitoring changes in the excited-state lifetime of the donor fluorophore where its quenching in the
presence of the acceptor is evidence for a direct physical interaction.
Key words mTOR, Enzymes, Protein complex, Fluorescence imaging, Confocal microscopy,
Excited-state lifetime, TCSPC, FRET, FLIM GFP-tag, DNA, Three-color
1 Introduction
All cellular processes are driven by the action of proteins, from ion
channels across phospholipid membranes to DNA synthesis and cell
growth. For example, the mechanistic or mammalian target of
rapamycin (mTOR) pathway functions in the coordination of
energy, nutrients, and growth factor availability to regulate key
biological processes including cellular growth, metabolism, and
protein synthesis through the phosphorylation of downstream substrates, ribosomal protein, S6 Kinase 1 (S6K1), and 4E-binding
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_16, © Springer Science+Business Media, LLC, part of Springer Nature 2021
287