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

Viviane Devauges,Tony Ng,James A Levitt,Simon P Poland,James Monypenny,Daniel R Matthews,Justin Aluko,Oana Coban,Gregory Weitsman,Simon M Ameer-Beg,Jakub Nedbal

doi:10.1371/journal.pone.0110695

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.

Highlights

Many biological processes occur at the cell plasma membrane which constitutes a signalling platform for the cell
Total internal reflection fluorescence (TIRF) microscopy is the method of choice to image events occurring at the plasma membrane since this technique enables excitation of only those fluorophores that are located very close to the glass coverslip [1], by generating an evanescent wave at an interface between substrates with refractive index mismatch
TIRF microscopy has already been applied to measurement of protein-protein interactions using Forster resonance energy transfer (FRET) [13,14], by fluorescence lifetime imaging microscopy (FLIM) [12,15,16,17] and by acceptor photobleaching [18,19,20,21]

Summary

Introduction

Many biological processes occur at the cell plasma membrane which constitutes a signalling platform for the cell. We perform steady-state acceptor fluorescence anisotropy under evanescent excitation to monitor the activity of Cdc42 at the cell plasma membrane using a Raichu-Cdc42 FRET biosensor probe.

Results

Conclusion