Simultaneous readout of multiple FRET pairs using photochromism.

Area of Science:

• Molecular Biology
• Biophysics
• Cellular Imaging

Background:

• Förster resonant energy transfer (FRET) is a key technique for studying molecular interactions in living cells.
• Simultaneous FRET measurements are often limited by spectral overlap of donor and acceptor fluorophores.
• Distinguishing multiple FRET signals within the same cell requires advanced spectral unmixing or orthogonal readouts.

Purpose of the Study:

• To develop a novel FRET-based approach for distinguishing spectrally overlapping FRET pairs.
• To enable simultaneous multiplexed measurements of intracellular signaling pathways.
• To overcome limitations in current FRET methodologies for complex cellular analysis.

Main Methods:

• Utilized photochromism of donor fluorophores to differentiate FRET pairs with identical spectral properties.
• Developed theoretical framework and validated with numerical simulations.
• Engineered photochromic biosensors (rsAKARev for PKA, EKARev for ERK) and integrated a third spectrally-shifted sensor for multiplexed measurements.

Main Results:

• Demonstrated successful discrimination of overlapping FRET pairs by exploiting donor photochromism.
• Achieved simultaneous readout of cAMP-dependent protein kinase (PKA) and extracellular signal-regulated kinase (ERK) activities in the same cell.
• Successfully performed multiplexed measurements of PKA, ERK, and calcium signaling pathways.

Conclusions:

• Exploiting donor photochromism in FRET offers a powerful strategy for multiplexed molecular interaction analysis.
• This method significantly enhances the capability of FRET to probe complex cellular signaling networks.
• The developed photochromic biosensors provide a versatile platform for simultaneous monitoring of multiple cellular events.