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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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Live inhibition of Cdc42 in HCC1954 cells transiently expressing Raichu-Cdc42 biosensor.Fluorescence intensity (A, left) and acceptor fluorescence anisotropy maps (A, right) of live HCC1954 cells transiently expressing Raichu-Cdc42 biosensor before (A. top) and after (A. bottom) addition of Cdc42 inhibitor. The scale bar represents 5 µm. (B) Corresponding representative histograms of the fluorescence acceptor anisotropy before (A top right) and after inhibition (A bottom right) are shown. (C) Time lapse of acceptor fluorescence anisotropy (±SD) after 30 µM addition of Cdc42 inhibitor for cell expressing respectively Raichu-Cdc42 biosensor (pink), Cdc42 (blue) or after addition of 30 µM DMSO for cell expressing Raichu-Cdc42 biosensor (green). Images were taken every 5 minutes (200 ms exposure for EMCCD) for 50 minutes. (D) Ensemble steady state acceptor fluorescence anisotropy values obtained on HCC1954 cells expressing Raichu-Cdc42biosensor before (pre) and after (post) addition of Cdc42 inhibitor, or before and after addition of DMSO, or for cells expressing Cdc42 before and after addition of Cdc42 inhibitor. Cells were imaged live at 37°C, excited in TIRF excitation.
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pone-0110695-g007: Live inhibition of Cdc42 in HCC1954 cells transiently expressing Raichu-Cdc42 biosensor.Fluorescence intensity (A, left) and acceptor fluorescence anisotropy maps (A, right) of live HCC1954 cells transiently expressing Raichu-Cdc42 biosensor before (A. top) and after (A. bottom) addition of Cdc42 inhibitor. The scale bar represents 5 µm. (B) Corresponding representative histograms of the fluorescence acceptor anisotropy before (A top right) and after inhibition (A bottom right) are shown. (C) Time lapse of acceptor fluorescence anisotropy (±SD) after 30 µM addition of Cdc42 inhibitor for cell expressing respectively Raichu-Cdc42 biosensor (pink), Cdc42 (blue) or after addition of 30 µM DMSO for cell expressing Raichu-Cdc42 biosensor (green). Images were taken every 5 minutes (200 ms exposure for EMCCD) for 50 minutes. (D) Ensemble steady state acceptor fluorescence anisotropy values obtained on HCC1954 cells expressing Raichu-Cdc42biosensor before (pre) and after (post) addition of Cdc42 inhibitor, or before and after addition of DMSO, or for cells expressing Cdc42 before and after addition of Cdc42 inhibitor. Cells were imaged live at 37°C, excited in TIRF excitation.

Mentions: Cdc42 inhibition was achieved on Raichu-Cdc42 biosensor expressed in HCC1954 cells and imaged live at 37°C, before and after Cdc42 GTPase inhibitor addition (Fig.7.A&B). As can be seen on the fluorescence anisotropy maps pre- and post-addition of the inhibitor (Fig. 7.A) and the corresponding histograms (Fig.7.B), a clear increase of the acceptor fluorescence anisotropy of the Raichu-Cdc42 biosensor was measured 50 minutes after addition of the inhibitor. Furthermore, time-lapse experiments acquiring images every 5 minutes to dynamically monitor Cdc42 inhibition at the cell plasma membrane were undertaken. From these time lapse data, both total intensity and the corresponding acceptor fluorescence anisotropy maps were extracted (Movies S1&S2). The average fluorescence anisotropy extracted from this time lapse highlights a clear increase of acceptor fluorescence anisotropy after Cdc42 inhibition (Fig.7.C). These measurements were repeated in 10 cells and the anisotropy measured before and after inhibition showed a significant increase of the fluorescence anisotropy after inhibition (Table 4 and Fig.7.D, p = 0.004, **). In order to confirm that the increase in fluorescence anisotropy was directly related to Cdc42 inhibition, control measurements were undertaken by imaging Raichu-Cdc42 biosensor in 30 µM DMSO in order to ensure that the effect on the fluorescence anisotropy was linked to the inhibitor and not to the carrier. Measurements of the fluorescence anisotropy before and after addition of the inhibitor for several cells (Fig.7.D), and time-lapse experiments (Fig. 7.C) indicate a stable and high activity of the biosensor over the imaging period, without phototoxicity/photodamage induced by the illumination or effect of the carrier. The reference value for the open conformation (i.e. non interacting form) was given by imaging cells expressing Cdc42 –mRFP1 in presence of the inhibitor. In that case too, no effect of the inhibitor was observed either on time lapse (Fig.7.C) or on ensemble measurements (Table 4 and Fig.7.D). As seen in Figure 7.C, after 50 minutes of treatment with the inhibitor, the fluorescence anisotropy of the Raichu-Cdc42 biosensor went from low anisotropy values (comparable to when imaged in DMSO) to reach the fluorescence anisotropy of cells expressing Cdc42-mRFP1 (i.e. baseline value for absence of FRET). Thus, the increase in the acceptor fluorescence anisotropy is directly related to a decrease in the FRET efficiency and consequently to a change of conformation of the Raichu-Cdc42 biosensor from an active (GTP-bound) conformation to an inactive (GDP-bound) conformation. We noticed that the response to inhibition was cell specific in terms of efficiency and time to response after inhibitor addition. On average, the fluorescence anisotropy was increased by 25% after 50 minutes treatment (Table 4). This imaging time window seemed to be optimal in order to have an effective inhibition of Cdc42 activity without inducing phototoxicity/photodamage. In addition to a clear decrease of the intramolecular FRET of the Raichu-Cdc42 biosensor (Fig.7.C), intensity images also showed morphological changes of the cell shape, which might be linked to the induced inhibition of Cdc42 related filopodia formation and cell migration (Movie S1).


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)

Live inhibition of Cdc42 in HCC1954 cells transiently expressing Raichu-Cdc42 biosensor.Fluorescence intensity (A, left) and acceptor fluorescence anisotropy maps (A, right) of live HCC1954 cells transiently expressing Raichu-Cdc42 biosensor before (A. top) and after (A. bottom) addition of Cdc42 inhibitor. The scale bar represents 5 µm. (B) Corresponding representative histograms of the fluorescence acceptor anisotropy before (A top right) and after inhibition (A bottom right) are shown. (C) Time lapse of acceptor fluorescence anisotropy (±SD) after 30 µM addition of Cdc42 inhibitor for cell expressing respectively Raichu-Cdc42 biosensor (pink), Cdc42 (blue) or after addition of 30 µM DMSO for cell expressing Raichu-Cdc42 biosensor (green). Images were taken every 5 minutes (200 ms exposure for EMCCD) for 50 minutes. (D) Ensemble steady state acceptor fluorescence anisotropy values obtained on HCC1954 cells expressing Raichu-Cdc42biosensor before (pre) and after (post) addition of Cdc42 inhibitor, or before and after addition of DMSO, or for cells expressing Cdc42 before and after addition of Cdc42 inhibitor. Cells were imaged live at 37°C, excited in TIRF excitation.
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pone-0110695-g007: Live inhibition of Cdc42 in HCC1954 cells transiently expressing Raichu-Cdc42 biosensor.Fluorescence intensity (A, left) and acceptor fluorescence anisotropy maps (A, right) of live HCC1954 cells transiently expressing Raichu-Cdc42 biosensor before (A. top) and after (A. bottom) addition of Cdc42 inhibitor. The scale bar represents 5 µm. (B) Corresponding representative histograms of the fluorescence acceptor anisotropy before (A top right) and after inhibition (A bottom right) are shown. (C) Time lapse of acceptor fluorescence anisotropy (±SD) after 30 µM addition of Cdc42 inhibitor for cell expressing respectively Raichu-Cdc42 biosensor (pink), Cdc42 (blue) or after addition of 30 µM DMSO for cell expressing Raichu-Cdc42 biosensor (green). Images were taken every 5 minutes (200 ms exposure for EMCCD) for 50 minutes. (D) Ensemble steady state acceptor fluorescence anisotropy values obtained on HCC1954 cells expressing Raichu-Cdc42biosensor before (pre) and after (post) addition of Cdc42 inhibitor, or before and after addition of DMSO, or for cells expressing Cdc42 before and after addition of Cdc42 inhibitor. Cells were imaged live at 37°C, excited in TIRF excitation.
Mentions: Cdc42 inhibition was achieved on Raichu-Cdc42 biosensor expressed in HCC1954 cells and imaged live at 37°C, before and after Cdc42 GTPase inhibitor addition (Fig.7.A&B). As can be seen on the fluorescence anisotropy maps pre- and post-addition of the inhibitor (Fig. 7.A) and the corresponding histograms (Fig.7.B), a clear increase of the acceptor fluorescence anisotropy of the Raichu-Cdc42 biosensor was measured 50 minutes after addition of the inhibitor. Furthermore, time-lapse experiments acquiring images every 5 minutes to dynamically monitor Cdc42 inhibition at the cell plasma membrane were undertaken. From these time lapse data, both total intensity and the corresponding acceptor fluorescence anisotropy maps were extracted (Movies S1&S2). The average fluorescence anisotropy extracted from this time lapse highlights a clear increase of acceptor fluorescence anisotropy after Cdc42 inhibition (Fig.7.C). These measurements were repeated in 10 cells and the anisotropy measured before and after inhibition showed a significant increase of the fluorescence anisotropy after inhibition (Table 4 and Fig.7.D, p = 0.004, **). In order to confirm that the increase in fluorescence anisotropy was directly related to Cdc42 inhibition, control measurements were undertaken by imaging Raichu-Cdc42 biosensor in 30 µM DMSO in order to ensure that the effect on the fluorescence anisotropy was linked to the inhibitor and not to the carrier. Measurements of the fluorescence anisotropy before and after addition of the inhibitor for several cells (Fig.7.D), and time-lapse experiments (Fig. 7.C) indicate a stable and high activity of the biosensor over the imaging period, without phototoxicity/photodamage induced by the illumination or effect of the carrier. The reference value for the open conformation (i.e. non interacting form) was given by imaging cells expressing Cdc42 –mRFP1 in presence of the inhibitor. In that case too, no effect of the inhibitor was observed either on time lapse (Fig.7.C) or on ensemble measurements (Table 4 and Fig.7.D). As seen in Figure 7.C, after 50 minutes of treatment with the inhibitor, the fluorescence anisotropy of the Raichu-Cdc42 biosensor went from low anisotropy values (comparable to when imaged in DMSO) to reach the fluorescence anisotropy of cells expressing Cdc42-mRFP1 (i.e. baseline value for absence of FRET). Thus, the increase in the acceptor fluorescence anisotropy is directly related to a decrease in the FRET efficiency and consequently to a change of conformation of the Raichu-Cdc42 biosensor from an active (GTP-bound) conformation to an inactive (GDP-bound) conformation. We noticed that the response to inhibition was cell specific in terms of efficiency and time to response after inhibitor addition. On average, the fluorescence anisotropy was increased by 25% after 50 minutes treatment (Table 4). This imaging time window seemed to be optimal in order to have an effective inhibition of Cdc42 activity without inducing phototoxicity/photodamage. In addition to a clear decrease of the intramolecular FRET of the Raichu-Cdc42 biosensor (Fig.7.C), intensity images also showed morphological changes of the cell shape, which might be linked to the induced inhibition of Cdc42 related filopodia formation and cell migration (Movie S1).

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