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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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Ensemble steady state acceptor fluorescence anisotropy values of MDA-MB 231 cells expressing different Cdc42 constructs.MDA-MB 231 cells were expressing different Cdc42 constructs and imaged live at 37°C, excited respectively in epifluorescence and in TIRF excitation. Measurements were compared using either two-tailed paired t-test (A) or two-tailed unpaired t-test with 95% confidence intervals (B, C) (***p<0.001,**p<0.01, *p<0.05, ns non significant).
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pone-0110695-g006: Ensemble steady state acceptor fluorescence anisotropy values of MDA-MB 231 cells expressing different Cdc42 constructs.MDA-MB 231 cells were expressing different Cdc42 constructs and imaged live at 37°C, excited respectively in epifluorescence and in TIRF excitation. Measurements were compared using either two-tailed paired t-test (A) or two-tailed unpaired t-test with 95% confidence intervals (B, C) (***p<0.001,**p<0.01, *p<0.05, ns non significant).

Mentions: To monitor Cdc42 activity with changes in temperature, we reproduced the previous experiments at 37°C for the different Cdc42 constructs. Figure 6.A gathers all the data obtained on the different mutants at 37°C and compared the effect of the illumination. It shows that there was a significant decrease of the acceptor fluorescence anisotropy for both Raichu-Cdc42 and its T17N mutant in TIRF excitation compared to epifluorescence excitation which was not the case for the Y40C mutant or the Cdc42-mRFP1 constructs. This is perhaps surprising since T17N mutant is inactive given its GDP-bound state. The Cdc42 component of the sensor has, therefore, a reduced affinity to the PAK1-CRIB binding domain, but not abrogation of binding. As such we must consider the potential energy landscape is sufficiently modified at 37°C to allow significant FRET to occur. Comparing results obtained respectively in epifluorescence illumination (Fig.6.B) and in TIRF (Fig.6.C), we observed higher activity of both Raichu-Cdc42 and T17N at the plasma membrane of the cells compared to intracellularly. Furthermore, in epifluorescence excitation, at 37°C, all constructs were more active since the average acceptor fluorescence anisotropy values were lower compared to those obtained at 20°C (Table 3). Given that no effect of temperature was seen on Cdc42 for both illumination modes (p = 0.46 insignificant in epifluorescence, p = 0.33 insignificant for TIRF excitation), variations in activity of Cdc42, which must occur due to imaging at physiological temperature, are the most probable cause, although possible phase changes at the plasma membrane [98] and increased probe/effector mobility could also contribute to this effect. This increased activity of the biosensors at 37°C already enabled us to distinguish the different constructs in epifluorescence excitation (although the two negative mutants T17N and Y40C could not be differentiated) (Fig.6.B) and in TIRF excitation, all constructs could be clearly differentiated (Fig. 6.A & C). Use of TIRF excitation for Raichu-Cdc42 constructs and T17N mutant resulted in anisotropy values with a narrow distribution, which is predominantly due to the increase in signal-to-noise ratio in TIRF excitation compared to epifluorescence illumination. The effect of temperature was noticeable on Raichu-Cdc42 and on Y40C in epifluorescence excitation, whereas on the other constructs, no effect of the temperature could be detected for both excitation types. These results reflect the dynamic nature of protein orientation in the biosensor. For the Y40C construct, the binding of Cdc42 to the Cdc42 -interactive binding motif (CRIB) of PAK1 is modified whereas the T17N mutant is trapped in a GDP-bound state (inactive) and the affinity of binding of Cdc42 to PAK1 in presence of guanine nucleotide is reduced but binding of the sensor domain is unaffected. In contrast for Y40C, the binding domain of Cdc42 to PAK1 has been modified preventing binding of Cdc42 and thus the biosensor will always be in an open conformation so FRET is unlikely. Thus, the true negative control upon which we determine the dynamic range of the biosensor must be the Y40C.


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)

Ensemble steady state acceptor fluorescence anisotropy values of MDA-MB 231 cells expressing different Cdc42 constructs.MDA-MB 231 cells were expressing different Cdc42 constructs and imaged live at 37°C, excited respectively in epifluorescence and in TIRF excitation. Measurements were compared using either two-tailed paired t-test (A) or two-tailed unpaired t-test with 95% confidence intervals (B, C) (***p<0.001,**p<0.01, *p<0.05, ns non significant).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110695-g006: Ensemble steady state acceptor fluorescence anisotropy values of MDA-MB 231 cells expressing different Cdc42 constructs.MDA-MB 231 cells were expressing different Cdc42 constructs and imaged live at 37°C, excited respectively in epifluorescence and in TIRF excitation. Measurements were compared using either two-tailed paired t-test (A) or two-tailed unpaired t-test with 95% confidence intervals (B, C) (***p<0.001,**p<0.01, *p<0.05, ns non significant).
Mentions: To monitor Cdc42 activity with changes in temperature, we reproduced the previous experiments at 37°C for the different Cdc42 constructs. Figure 6.A gathers all the data obtained on the different mutants at 37°C and compared the effect of the illumination. It shows that there was a significant decrease of the acceptor fluorescence anisotropy for both Raichu-Cdc42 and its T17N mutant in TIRF excitation compared to epifluorescence excitation which was not the case for the Y40C mutant or the Cdc42-mRFP1 constructs. This is perhaps surprising since T17N mutant is inactive given its GDP-bound state. The Cdc42 component of the sensor has, therefore, a reduced affinity to the PAK1-CRIB binding domain, but not abrogation of binding. As such we must consider the potential energy landscape is sufficiently modified at 37°C to allow significant FRET to occur. Comparing results obtained respectively in epifluorescence illumination (Fig.6.B) and in TIRF (Fig.6.C), we observed higher activity of both Raichu-Cdc42 and T17N at the plasma membrane of the cells compared to intracellularly. Furthermore, in epifluorescence excitation, at 37°C, all constructs were more active since the average acceptor fluorescence anisotropy values were lower compared to those obtained at 20°C (Table 3). Given that no effect of temperature was seen on Cdc42 for both illumination modes (p = 0.46 insignificant in epifluorescence, p = 0.33 insignificant for TIRF excitation), variations in activity of Cdc42, which must occur due to imaging at physiological temperature, are the most probable cause, although possible phase changes at the plasma membrane [98] and increased probe/effector mobility could also contribute to this effect. This increased activity of the biosensors at 37°C already enabled us to distinguish the different constructs in epifluorescence excitation (although the two negative mutants T17N and Y40C could not be differentiated) (Fig.6.B) and in TIRF excitation, all constructs could be clearly differentiated (Fig. 6.A & C). Use of TIRF excitation for Raichu-Cdc42 constructs and T17N mutant resulted in anisotropy values with a narrow distribution, which is predominantly due to the increase in signal-to-noise ratio in TIRF excitation compared to epifluorescence illumination. The effect of temperature was noticeable on Raichu-Cdc42 and on Y40C in epifluorescence excitation, whereas on the other constructs, no effect of the temperature could be detected for both excitation types. These results reflect the dynamic nature of protein orientation in the biosensor. For the Y40C construct, the binding of Cdc42 to the Cdc42 -interactive binding motif (CRIB) of PAK1 is modified whereas the T17N mutant is trapped in a GDP-bound state (inactive) and the affinity of binding of Cdc42 to PAK1 in presence of guanine nucleotide is reduced but binding of the sensor domain is unaffected. In contrast for Y40C, the binding domain of Cdc42 to PAK1 has been modified preventing binding of Cdc42 and thus the biosensor will always be in an open conformation so FRET is unlikely. Thus, the true negative control upon which we determine the dynamic range of the biosensor must be the Y40C.

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