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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 live MDA-MB 231 cells transiently expressing Raichu-Cdc42 or its different mutants.Fluorescence intensity (A. top) and acceptor fluorescence anisotropy maps (A, bottom) of live MDA-MB 231 cells transiently expressing Raichu-Cdc42 biosensor, T17N mutant, Y40C, mutant or Cdc42 respectively imaged at 20°C. Representative histograms of the fluorescence acceptor fluorescence anisotropy of the corresponding cells (B). The scale bar represents 5 µm.
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pone-0110695-g005: Acceptor fluorescence anisotropy of live MDA-MB 231 cells transiently expressing Raichu-Cdc42 or its different mutants.Fluorescence intensity (A. top) and acceptor fluorescence anisotropy maps (A, bottom) of live MDA-MB 231 cells transiently expressing Raichu-Cdc42 biosensor, T17N mutant, Y40C, mutant or Cdc42 respectively imaged at 20°C. Representative histograms of the fluorescence acceptor fluorescence anisotropy of the corresponding cells (B). The scale bar represents 5 µm.

Mentions: Having observed significant differences of activity of the Raichu-Cdc42 biosensor depending on excitation condition, we imaged T17N and Y40C mutant biosensors, which constitute negative controls [48]. Cells expressing Cdc42-mRFP1 provided a baseline value for the acceptor fluorescence anisotropy in the absence of donor (i.e. no FRET could occur). Determination of the acceptor fluorescence anisotropy of several cells under epifluorescence illumination for the different constructs and statistical analysis resulted in only a statistically significant difference between Raichu-Cdc42 biosensor and its Y40C mutant (p = 0.015,*, Fig. 4.C). In contrast, under TIRF excitation (Fig.4.D), the four different constructs were clearly distinguished with high significance in the case of Raichu-Cdc42 and its Y40C mutant (p = 0.0004, ***) and good significance for Raichu-Cdc42 and its T17N mutant (p = 0.008,**) as well as Cdc42-mRFP1 and the Y40C mutant (p = 0.03,*). Due to the additional spatial information provided by TIRF excitation combined with aaFRET, we were able to clearly distinguish Raichu-Cdc42 and its different mutants and thus to show differences in activity for the different constructs (Table 3). Spatial differences of the fluorescence anisotropy of the different constructs can be seen in the anisotropy maps (Fig.5.A). This result is further supported by the respective distinct histograms of the acceptor anisotropy (Fig.5.B) which showed a significant increase of the acceptor fluorescence anisotropy in the case of theY40C mutant compared to Raichu-Cdc42 biosensor. This confirmed that both Y40C and T17N mutants are negative controls [48], which could not be resolved in epifluorescence excitation linked to both low signal-to-noise ratio (resulting predominantly from fluorescence contamination from out of focus light) and lower observed activity of Raichu-Cdc42 biosensor in the cellular milieu in comparison to at the plasma membrane.


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 live MDA-MB 231 cells transiently expressing Raichu-Cdc42 or its different mutants.Fluorescence intensity (A. top) and acceptor fluorescence anisotropy maps (A, bottom) of live MDA-MB 231 cells transiently expressing Raichu-Cdc42 biosensor, T17N mutant, Y40C, mutant or Cdc42 respectively imaged at 20°C. Representative histograms of the fluorescence acceptor fluorescence anisotropy of the corresponding cells (B). The scale bar represents 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110695-g005: Acceptor fluorescence anisotropy of live MDA-MB 231 cells transiently expressing Raichu-Cdc42 or its different mutants.Fluorescence intensity (A. top) and acceptor fluorescence anisotropy maps (A, bottom) of live MDA-MB 231 cells transiently expressing Raichu-Cdc42 biosensor, T17N mutant, Y40C, mutant or Cdc42 respectively imaged at 20°C. Representative histograms of the fluorescence acceptor fluorescence anisotropy of the corresponding cells (B). The scale bar represents 5 µm.
Mentions: Having observed significant differences of activity of the Raichu-Cdc42 biosensor depending on excitation condition, we imaged T17N and Y40C mutant biosensors, which constitute negative controls [48]. Cells expressing Cdc42-mRFP1 provided a baseline value for the acceptor fluorescence anisotropy in the absence of donor (i.e. no FRET could occur). Determination of the acceptor fluorescence anisotropy of several cells under epifluorescence illumination for the different constructs and statistical analysis resulted in only a statistically significant difference between Raichu-Cdc42 biosensor and its Y40C mutant (p = 0.015,*, Fig. 4.C). In contrast, under TIRF excitation (Fig.4.D), the four different constructs were clearly distinguished with high significance in the case of Raichu-Cdc42 and its Y40C mutant (p = 0.0004, ***) and good significance for Raichu-Cdc42 and its T17N mutant (p = 0.008,**) as well as Cdc42-mRFP1 and the Y40C mutant (p = 0.03,*). Due to the additional spatial information provided by TIRF excitation combined with aaFRET, we were able to clearly distinguish Raichu-Cdc42 and its different mutants and thus to show differences in activity for the different constructs (Table 3). Spatial differences of the fluorescence anisotropy of the different constructs can be seen in the anisotropy maps (Fig.5.A). This result is further supported by the respective distinct histograms of the acceptor anisotropy (Fig.5.B) which showed a significant increase of the acceptor fluorescence anisotropy in the case of theY40C mutant compared to Raichu-Cdc42 biosensor. This confirmed that both Y40C and T17N mutants are negative controls [48], which could not be resolved in epifluorescence excitation linked to both low signal-to-noise ratio (resulting predominantly from fluorescence contamination from out of focus light) and lower observed activity of Raichu-Cdc42 biosensor in the cellular milieu in comparison to at the plasma membrane.

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