Short talk:
To unlock sensitive molecular switches in live cells using high throughput FRET

Nicolaas van der Voort1, Nina M Bartels2, Claus AM Seidel1, Cornelia Monzel2

1Molecular Physical Chemistry, HHU, Düsseldorf, Germany,
2Experimental Medical Physics, HHU, Düsseldorf, Germany

high throughput FRET, single-cell, time-resolved FRET, CD95, CTLA4

The field of molecular biology is rapidly evolving from studying binary yes/no relationships to probing complex signal transduction mechanisms. To study a system where the outcome depends quantitively on the concentration of input signaling molecules, a method is required that can measure interactions with high sensitivity, cover large concentration ranges and gather sufficient statistics to study the natural variability in live cells. Here we present high throughput FRET (htFRET), which uses EGFP-mCherry lifetime information from cellular data to perform FRET sensitized donor decay (ε(t)) fits1 to detect FRET fractions as low as 1%. Using our highly automated acquisition and data processing pipeline we can measure > 3000 cells in one experiment while obtaining phenotype information such as cell fate and protein expression level.  

For the CD95 protein in presence of its ligand, we convert FRET fractions to oligomer fraction using Accessible Volume Simulations to reveal that 10 % oligomer formation in median is sufficient to trigger apoptosis. Furthermore, we study dimerization of the CTLA4 membrane receptor protein and find that the dimerization fraction depends on concentration, enabling us to obtain the dimerization constant Kd. An upper limit in the concentration accessible by FRET is posed by unspecific interaction due to proximity FRET. We quantify and discuss strategies to overcome this limit. Our method is easily transferable to measure molecular interaction < 10 nm in any cellular organelle.

1Greife, A., Peulen, T.O., et al. Functional FRET imaging in living cells with sub-nanometer resolution, in preparation

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