Fig. 1: Three-colour FRET-Cascade construct. | Communications Chemistry

Fig. 1: Three-colour FRET-Cascade construct.

From: Development of three-colour FRET cascade for force sensing of the putative RIAM-vinculin interaction in fibroblasts

Fig. 1

A A graphical representation of the fluorophores used in the three-colour FRET cascade module with specific energy transfers (\(\Gamma\)) labelled. The purple lightning bolt represents excitation at 435 nm. Solid arrows show the direction of energy cascade through mVenus, dashed arrow shows mTurq2 to mScarlet-I direct energy transfer. B Excitation spectra for mTurq2 and the emission spectra for mTurq2 (blue), mTurq2-mVenusG68A-mScarlet-I (yellow), and mTurq2-mVenus-mScarlet-I (red), where mTurq2 alone is excited at 435 nm (excitation spectrum indicated by black dashed line). C An AlphaFold3 model of the three-colour protein complex, regions with a high predicted local distance difference test (pLDDT) score >90 are dark blue, 70 > 90 light blue, 50 > 70, yellow and those with a very low pLDDT score ( <  50) are indicated in orange. D Average FRET efficiencies for the various FRET pairs measured using our custom TCSPC-FLIM system, n = 9 individual cells measured per condition across N = 3 technical repeats. Bars are the mean FRET efficiency ± SEM (error bars); comparisons of means are calculated by one-way ANOVA using Dunnett T3 correction for multiple comparisons: : P ≥ 0.123 ns (ns), P ≤ 0.0332 (*), P ≤ 0.0021 (**), P ≤ 0.0002 (***), P ≤ 0.0001 (****), p = 0.0614 (mVenus-mScarlet vs. mTurq2-mVenus-mScarlet-I).

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