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Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane.

Devauges V, Matthews DR, Aluko J, Nedbal J, Levitt JA, Poland SP, Coban O, Weitsman G, Monypenny J, Ng T, Ameer-Beg SM - PLoS ONE (2014)

Bottom Line: Higher activity of the probe was found at the cell plasma membrane compared to intracellularly.Imaging fluorescence anisotropy in TIRF allowed clear differentiation of the Raichu-Cdc42 biosensor from negative control mutants.Finally, inhibition of Cdc42 was imaged dynamically in live cells, where we show temporal changes of the activity of the Raichu-Cdc42 biosensor.

View Article: PubMed Central - PubMed

Affiliation: Richard Dimbleby Cancer Research Laboratory, Division of Cancer Studies and Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom.

ABSTRACT
We present a novel imaging system combining total internal reflection fluorescence (TIRF) microscopy with measurement of steady-state acceptor fluorescence anisotropy in order to perform live cell Förster Resonance Energy Transfer (FRET) imaging at the plasma membrane. We compare directly the imaging performance of fluorescence anisotropy resolved TIRF with epifluorescence illumination. The use of high numerical aperture objective for TIRF required correction for induced depolarization factors. This arrangement enabled visualisation of conformational changes of a Raichu-Cdc42 FRET biosensor by measurement of intramolecular FRET between eGFP and mRFP1. Higher activity of the probe was found at the cell plasma membrane compared to intracellularly. Imaging fluorescence anisotropy in TIRF allowed clear differentiation of the Raichu-Cdc42 biosensor from negative control mutants. Finally, inhibition of Cdc42 was imaged dynamically in live cells, where we show temporal changes of the activity of the Raichu-Cdc42 biosensor.

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Acceptor fluorescence anisotropy of fixed MCF7 cells transiently expressing eGFP/mRFP1 FRET rulers.Fluorescence intensity (A) and acceptor fluorescence anisotropy maps (B) of fixed MCF7 cells transiently expressing Cdc42-mRFP1 (left side) as a reference, eGFP-32aa-mRFP1 (middle) and eGFP-7aa-mRFP1 (right). Representative histograms of the acceptor fluorescence anisotropy of the corresponding cells (C). Mean values obtained from these histograms for the different cells imaged are then compared using unpaired t-test with Welch's correction with 95% confidence intervals (***p<0.001) (D). The scale bar represents 5 µm.
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pone-0110695-g003: Acceptor fluorescence anisotropy of fixed MCF7 cells transiently expressing eGFP/mRFP1 FRET rulers.Fluorescence intensity (A) and acceptor fluorescence anisotropy maps (B) of fixed MCF7 cells transiently expressing Cdc42-mRFP1 (left side) as a reference, eGFP-32aa-mRFP1 (middle) and eGFP-7aa-mRFP1 (right). Representative histograms of the acceptor fluorescence anisotropy of the corresponding cells (C). Mean values obtained from these histograms for the different cells imaged are then compared using unpaired t-test with Welch's correction with 95% confidence intervals (***p<0.001) (D). The scale bar represents 5 µm.

Mentions: Fixed MCF7 cells expressing eGFP-32aa-mRFP1 and eGFP-7aa-mRFP1 for the FRET rulers and Cdc42-mRFP1 as a reference value for acceptor anisotropy were used to calibrate the aaFRET measurements under epifluorescence fluorescence excitation. The steady-state acceptor fluorescence anisotropy was calculated using equations 3 with equation 4 a&b and total intensity and anisotropy maps were deduced as shown in Figure 3. A & B.


Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane.

Devauges V, Matthews DR, Aluko J, Nedbal J, Levitt JA, Poland SP, Coban O, Weitsman G, Monypenny J, Ng T, Ameer-Beg SM - PLoS ONE (2014)

Acceptor fluorescence anisotropy of fixed MCF7 cells transiently expressing eGFP/mRFP1 FRET rulers.Fluorescence intensity (A) and acceptor fluorescence anisotropy maps (B) of fixed MCF7 cells transiently expressing Cdc42-mRFP1 (left side) as a reference, eGFP-32aa-mRFP1 (middle) and eGFP-7aa-mRFP1 (right). Representative histograms of the acceptor fluorescence anisotropy of the corresponding cells (C). Mean values obtained from these histograms for the different cells imaged are then compared using unpaired t-test with Welch's correction with 95% confidence intervals (***p<0.001) (D). The scale bar represents 5 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215982&req=5

pone-0110695-g003: Acceptor fluorescence anisotropy of fixed MCF7 cells transiently expressing eGFP/mRFP1 FRET rulers.Fluorescence intensity (A) and acceptor fluorescence anisotropy maps (B) of fixed MCF7 cells transiently expressing Cdc42-mRFP1 (left side) as a reference, eGFP-32aa-mRFP1 (middle) and eGFP-7aa-mRFP1 (right). Representative histograms of the acceptor fluorescence anisotropy of the corresponding cells (C). Mean values obtained from these histograms for the different cells imaged are then compared using unpaired t-test with Welch's correction with 95% confidence intervals (***p<0.001) (D). The scale bar represents 5 µm.
Mentions: Fixed MCF7 cells expressing eGFP-32aa-mRFP1 and eGFP-7aa-mRFP1 for the FRET rulers and Cdc42-mRFP1 as a reference value for acceptor anisotropy were used to calibrate the aaFRET measurements under epifluorescence fluorescence excitation. The steady-state acceptor fluorescence anisotropy was calculated using equations 3 with equation 4 a&b and total intensity and anisotropy maps were deduced as shown in Figure 3. A & B.

Bottom Line: Higher activity of the probe was found at the cell plasma membrane compared to intracellularly.Imaging fluorescence anisotropy in TIRF allowed clear differentiation of the Raichu-Cdc42 biosensor from negative control mutants.Finally, inhibition of Cdc42 was imaged dynamically in live cells, where we show temporal changes of the activity of the Raichu-Cdc42 biosensor.

View Article: PubMed Central - PubMed

Affiliation: Richard Dimbleby Cancer Research Laboratory, Division of Cancer Studies and Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom.

ABSTRACT
We present a novel imaging system combining total internal reflection fluorescence (TIRF) microscopy with measurement of steady-state acceptor fluorescence anisotropy in order to perform live cell Förster Resonance Energy Transfer (FRET) imaging at the plasma membrane. We compare directly the imaging performance of fluorescence anisotropy resolved TIRF with epifluorescence illumination. The use of high numerical aperture objective for TIRF required correction for induced depolarization factors. This arrangement enabled visualisation of conformational changes of a Raichu-Cdc42 FRET biosensor by measurement of intramolecular FRET between eGFP and mRFP1. Higher activity of the probe was found at the cell plasma membrane compared to intracellularly. Imaging fluorescence anisotropy in TIRF allowed clear differentiation of the Raichu-Cdc42 biosensor from negative control mutants. Finally, inhibition of Cdc42 was imaged dynamically in live cells, where we show temporal changes of the activity of the Raichu-Cdc42 biosensor.

Show MeSH
Related in: MedlinePlus