Journal
ELIFE
Volume 11, Issue -, Pages -Publisher
eLIFE SCIENCES PUBL LTD
DOI: 10.7554/eLife.76870
Keywords
single-molecule fluorescence; single-particle tracking (SPT); protein-protein interactions; live-cell imaging; fluorophore photoactivation; Human
Categories
Funding
- Howard Hughes Medical Institute
- Jane Coffin Childs Memorial Fund for Medical Research
- Life Sciences Research Foundation
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Single-molecule imaging is a powerful tool for studying biochemical processes in live cells, but tracking single molecules and detecting their interactions simultaneously remains challenging. This study describes a novel property of rhodamine dyes, proximity-assisted photoactivation (PAPA), which allows the detection of protein-protein interactions in live cells using two different labeled protein complexes in proximity.
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 Forster 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.
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