Single-molecule imaging provides a powerful way to study biochemical processes in live cells, yet it remains challenging to track single molecules while simultaneously detecting their interactions. Here, we describe a novel property of rhodamine dyes, proximity-assisted photoactivation (PAPA), in which one fluorophore (the 'sender') can reactivate a second fluorophore (the 'receiver') from a dark state. PAPA requires proximity between the two fluorophores, yet it operates at a longer average intermolecular distance than Förster resonance energy transfer (FRET). We show that PAPA can be used in live cells both to detect protein-protein interactions and to highlight a subpopulation of labeled protein complexes in which two different labels are in proximity. In proof-of-concept experiments, PAPA detected the expected correlation between androgen receptor self-association and chromatin binding at the single-cell level. These results establish a new way in which a photophysical property of fluorophores can be harnessed to study molecular interactions in single-molecule imaging of live cells.
Keywords: cell biology; fluorophore photoactivation; human; live-cell imaging; molecular biophysics; protein–protein interactions; single-molecule fluorescence; single-particle tracking (SPT); structural biology.
A human body is made up of trillions of cells, each containing millions of proteins working to keep our bodies going. Since the invention of the microscope four hundred years ago, scientists have made large strides in visualizing cells and even single protein molecules within cells. To do this, proteins of interest are labeled with fluorescent dyes that absorb – or are ‘excited’ by – light of one color, and then give off light of a different color. The labeled proteins are excited by a powerful laser, and a sensitive camera detects the light emitted by single molecules of dye. This technique is called single-particle tracking (SPT), and it can reveal how proteins move around inside a cell. Because most proteins work together in teams or complexes, it would be useful to track the movement of proteins while at the same time observing their interactions. Unfortunately, SPT does not typically allow scientists to watch how proteins interact with each other. Graham et al. accidentally discovered how to do precisely this. First, they labeled proteins with two different colored dyes. Then, the dyes were excited using alternating red and green lasers. Repeated excitation destroys the fluorescent dye molecules, and sure enough, red-excited dye molecules went dark over time. Unexpectedly, however, molecules of the dye that had been excited with red light reappeared after exciting the second dye with green light. The fluorescent molecules were not dead, just sleeping. ‘Resuscitating’ one dye with the other required that they be close together, and therefore this process was called proximity-assisted photoactivation (PAPA for short). PAPA was able to detect interactions between proteins labeled with different dyes in live human cells, and combining PAPA with SPT allowed Graham et al. to distinguish protein molecules labeled with two different dyes from those labeled with a single dye. Finally, Graham et al. labeled molecules of the androgen receptor protein with two different dyes to monitor how they responded to testosterone. Combining PAPA and SPT measurements successfully detected the pairing of androgen receptor molecules, as well as increased binding of these paired androgen receptor molecules to DNA. This new way of observing how proteins interact will be useful for studying where and how fast these interactions happen in living cells. Understanding how teams of proteins work together under normal conditions will also shed light on how they misbehave in diseases.
© 2022, Graham et al.