ShadowG and ShadowY are efficient dark acceptors for mTurquoise2 in the AURKA biosensor. A. Model illustrating the mode of action of the ShadowG-AURKA-mTurquoise2 or the ShadowY-AURKA-mTurquoise2 biosensors. These biosensors switch from an open-to-close conformation upon autophosphorylation of AURKA on Thr288, bringing the donor and the acceptor in vicinity and allowing FRET detection. B. and C. (Upper panels) Representative fluorescence (mTurquoise2 channel) and Lifetime (donor onlybiosensor) images of U2OS cells expressing the indicated constructs and synchronised at mitosis. (Lower panel) Corresponding Lifetime quantification at the mitotic spindle. ShG: ShadowG; ShY: ShadowY; mTurq2: mTurquoise2. Lifetime values for individual cells are represented as black dots in each boxplot. The bar in boxplots represents the median; whiskers extend from the 10th to the 90th percentiles. n=10 cells per condition of one representative experiment (of three). Scale bar: 10 nm. ***P<0.001 against the 'AURKA-mTurquoise2' condition; a P<0.001 compared to the 'ShadowGAURKA-mTurquoise2' condition in B. or a P<0.05 compared to the 'ShadowY-AURKA-mTurquoise2' condition in C.

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... and of 800 psec when ShadowY was used ( Supplementary Fig. 1A), demonstrating that both fluorophores are excellent acceptors for mTurquoise2. We then replaced the GFP-mCherry donor-acceptor pair of the original AURKA biosensor with mTurquoise2 and ShadowG or ShadowY, thereby creating ShadowG-AURKA-mTurquoise2 or ShadowY- AURKA-mTurquoise2 (Fig. 2B). We then tested the conformational changes of these two biosensors by FRET/FLIM in U2OS cells synchronised at mitosis, by calculating the net difference in the lifetime (Lifetime) between the donor-only construct (AURKA-mTurquoise2) and ShadowG-AURKA- mTurquoise2 or ShadowY-AURKA-mTurquoise2 at the mitotic spindle. We measured a mean ...

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... changes of these two biosensors by FRET/FLIM in U2OS cells synchronised at mitosis, by calculating the net difference in the lifetime (Lifetime) between the donor-only construct (AURKA-mTurquoise2) and ShadowG-AURKA- mTurquoise2 or ShadowY-AURKA-mTurquoise2 at the mitotic spindle. We measured a mean Lifetime of 150 psec for both biosensors ( Fig. 2B-C), which was similar to the decrease observed for the original GFP-AURKA-mCherry one (Bertolin et al., 2016). With the same approach, we also tested the impact of kinase-dead AURKA on FRET efficiencies. Lifetime values for both biosensors carrying the Lys162Met mutation were half than their wild-type counterparts, indicating lower FRET ...

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... by superYFP, a novel dimeric yellow fluorescent protein with high brightness (118 for the monomer); the full characterisation of the physicochemical properties of superYFP will be described elsewhere. We coupled superYFP to mTurquoise2, as this appeared to be an efficient donor fluorophore in the dark versions of the AURKA biosensor (Fig. 2). We then tested the capacity of mTurquoise2 to act as a donor of energy for superYFP in a tandem construct. mTurquoise2 displayed a Lifetime of 600 psec when superYFP was present in U2OS cells synchronised at mitosis ( Supplementary Fig. 1B), indicating that this donor-acceptor couple is suitable for FRET/FLIM ...

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... ANOVA and the Dunnet's method were used to compare the effects of the transfected AURKA plasmids on GFP fluorescence anisotropy (Fig. 1C). One-way ANOVA and the Tukey's method were used to compare the effect of the transfected vectors on fluorescence lifetime ( Fig. 2B; Supplementary Fig. 1A). One-way ANOVA on ranks and the Kruskal-Wallis method were used to compare the effect of the transfected vectors on fluorescence lifetime (Fig. 2C, 4B).The Student's t-test was used to compare the effect of a GFP monomer and a tandem dimer on fluorescence anisotropy (Fig. 1B) or of a fluorescence tandem on Lifetime (Supplementary ...

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... plasmids on GFP fluorescence anisotropy (Fig. 1C). One-way ANOVA and the Tukey's method were used to compare the effect of the transfected vectors on fluorescence lifetime ( Fig. 2B; Supplementary Fig. 1A). One-way ANOVA on ranks and the Kruskal-Wallis method were used to compare the effect of the transfected vectors on fluorescence lifetime (Fig. 2C, 4B).The Student's t-test was used to compare the effect of a GFP monomer and a tandem dimer on fluorescence anisotropy (Fig. 1B) or of a fluorescence tandem on Lifetime (Supplementary Fig. 1 B); the Mann-Whitney test was used to compare the cross-correlation/auto-correlation ratio between normal and Lys162Met AURKA (Fig. 3 and ...