FRET Basics and Applications an EAMNET teaching module

Transcription

1 FRET Basics and Applications an EAMNET teaching module Timo Zimmermann + Stefan Terjung Advanced Light Microscopy Facility European Molecular Biology Laboratory, Heidelberg

2 Overview 1) Fluorescence Resonance Energy Transfer Basics 2) Confocal FRET detection techniques 3) FRET and fluorescent proteins 4) A new GFP FRET pair with increased efficiency

3 The resolving power of light microscopes is limited to distances of hundreds of nanometers (<organelles). Fluorescence Resonance Energy Transfer (FRET) allows the detection of molecule-molecule interactions in the nanometer range with light microscopy. FRET is sometimes also called Förster Resonance Energy Transfer, as Förster was the first who published quantitative theory of molecular resonance energy transfer (Förster 1946, Förster 1948). Organelles Cells Worm Housefly Human FRET 1 Å m 1 nm 10-9 m 1 µm 10-6 m 1 mm 10-3 m 1 cm 10-2 m 1 m light microscopy resolution limit

4 Fluorescence Resonance Energy transfer (FRET) FRET is a non-radiative transfer of energy from an excited donor molecule to a suitable acceptor molecule in close proximity. Fluorescence Resonance Energy Transfer Wouters et al. (2001), TICB 11/5 In the case of FRET, excitation of the donor fluorophore results not only in donor emission, but partially also in emission characteristic for the acceptor fluorophore.

5 Dependence on distance and spectral overlap The efficiency of energy transfer strongly depends on the distance between the donor and acceptor molecules and on overlap of the donor molecule emission and acceptor molecule excitation spectra high specificity. FRET efficiency is depends on molecule distance The FRET efficiency depends on the distance between the two interacting molecules. At the distance of the Förster radius R 0 between the molecules, the FRET efficiency is 50%. The typical R 0 is around 3 nm.

6 Donor/Acceptor Pairs Examples for common FRET Donor/Acceptor pairs: Donor (Em.) Acceptor (Exc.) FITC (520 nm) Cy3 (566 nm) EGFP(508 nm) CFP (477 nm) EGFP (508 nm) TRITC (550 nm) Cy5 (649 nm) Cy3 (554 nm) YFP (514 nm) YFP (514 nm)

7 FRET detection methods A variety of FRET detection methods exist for light microscopy Acceptor photobleaching Donor photobleaching Ratio imaging Sensitized emission Fluorescence lifetime measurements

8 FRET detection methods The detection methods have different properties and are suited to different samples Detection of changes: Acceptor photobleaching Donor photobleaching Information self-contained: Ratio imaging Sensitized emission => fixed samples => in vivo Fluorescence lifetime measurements

9 Acceptor Photobleaching Experimental steps of acceptor photobleaching measurements In acceptor photobleaching, the acceptor molecule of the FRET pair is bleached, resulting in a brightening (unquenching) of the donor fluorescence. Prebleach Image Zoom 4x Bleaching Original Zoom Postbleach Image Median Filtering Subtraction: Postbleach Prebleach Division: Subtraction/ Postbleach GFP GFP 543 Cy Cy3 An apparent FRET efficiency (product of the efficiency of the FRET pair and the amount of interacting donor) can be calculated Acquisition Processing

10 Acceptor photobleaching Shift Correction by Cross-Correlation helps avoiding edge artifacts in the comparison of pre- and postbleach images. Edge artifacts Without correction With correction

11 Donor photobleaching The bleaching rate of the donor fluorophore is affected by FRET. Measuring the bleaching of the donor in the presence/absence of acceptor is a possibility to detect FRET. Fluorescence lifetime FRET decreases donor fluorescence lifetime => decreased likeliness of bleaching => decreased bleaching rate An apparent FRET efficiency (product of the efficiency of the FRET pair and the amount of interacting donor) can be calculated. However: Quantitation is problematic due to direct and indirect bleaching of acceptor

12 Overview 1) Fluorescence Resonance Energy Transfer Basics 2) Confocal FRET detection techniques 3) FRET and fluorescent proteins 4) A new GFP FRET pair with increased efficiency

13 FRET and Fluorescent Proteins (FPs) Protein-Protein Interactions: - FRET between an FP and a dye -FRET betweenfps Cameleons: In vivo measurements of physiological changes (ratio imaging)

14 Measurement of Protein Phosphorylation by FRET Application example: An acceptor-labelled antibody against a phosphorylated residue can be used to detect the phosphorylation status of a GFP-fusion protein by FRET Cy3 anti-p-thyr d>ro GFP-Protein Not Phosphorylated P d<ro GFP-Protein Phosphorylated Verveer, et al. 2000

15 Acceptor photobleaching Receptor phosphorylation after EGF-Stimulation 0 min 2 min 5 min ErbB1-GFP/Cy3 FRET (receptor phosphorylation), Verveer, et al. 2000

16 CFP/YFP The combination of cyan and yellow fluorescent protein is the most commonly used fluorescent protein FRET pair

17 Fluorescence Resonance Energy Transfer Cameleon Tandem constructs CFP YFP Pollock and Heim TiCB 1999, Miyawaki et al. Nature 1997

18 In vivo CFP/YFP cameleon measurements Measurements caried out on the Leica SP2 AOBS at 405 nm excitation: 2 µm Ionomycin + 20mM CaCl 2 Histamine EGTA

19 Cross-talk and cross-excitation of two fluorophores is an intrinsic problem of multichannel measurements and is also present in FRET measurements Channel 1: nm Channel 2: nm RGB Overlay CFP only YFP only CFP+ YFP

20 Sensitized emission detection D D A D A D A A Ratiometric imaging can only be done in samples with a fixed stochiometry of donor and acceptor (e.g. Cameleons) A A A A D D D A A D D A In samples with variable stochiometries, the detected acceptor fluorescence has to be corrected for emission cross-talk and for cross-excitation A A

21 Sensitized emission detection Predetermined factors with pure samples of donor and acceptor: Donor cross-talk : R D Acceptor cross-excitation: R E Required images: Donor channel Donor excitation F D Acceptor channel Donor excitation F DA Acceptor channel Acceptor excitation F A F DA corr/f A => Donor cross-talk correction Acceptor cross-excitation correction corr

22 Overview 1) Fluorescence Resonance Energy Transfer Basics 2) Confocal FRET detection techniques 3) FRET and fluorescent proteins 4) A new GFP FRET pair with increased efficiency

23 CFP/YFP Cyan and yellow fluorescent protein is the most commonly used fluorescent protein FRET pair

24 Requirements for a good FRET pair -Maximal overlap of donor emission and acceptor excitation -Minimal direct excitation of the acceptor at the excitation maximum of the donor

25 Spectral overlap of FRET Pairs The spectral overlap of donor emission and acceptor excitation is only partial for CFP/YFP and much better for GFP/YFP pairs

26 Requirements for a good FRET pair -Maximal overlap of donor emission and acceptor excitation -Minimal direct excitation of the acceptor at the excitation maximum of the donor

27 Different Cross-Excitation of FRET Pairs Using a suitable laser excitation for CFP, YFP is directly excited significantly (=> high background signal) GFP2 is excitable around 400 nm, where YFP is almost not excitable (=> low background signal)

28 Comparison of CFP/YFP and GFP2/YFP FRET pairs CFP YFP glycine linker exc. 405/458 nm GFP2 YFP glycine linker exc. 405 nm

29 Acceptor photobleaching Comparison of CFP and GFP2 in the same construct Before After CFP-YFP: FRET efficiency 20% GFP2-YFP: FRET efficiency 30% => 50% increase

30 Improved FRET Efficiency significantly improves Detection Whereas the differences between FRET pairs are not significant at high transfer efficiencies, a more efficient FRET pair significantly improves the detectable FRET interaction in cases of low FRET efficiency.

31 Sensitized emission of GFP2-YFP FRET pairs GFP2 excitation GFP2 emission GFP2 excitation YFP emission YFP excitation YFP emission YFP (sensitized emission) YFP (direct excitation) GFP2+YFP Coexpression GFP2-YFP linked Data are shown after linear unmixing of the GFP2 and YFP emission signals.

32 Comparison of CFP/YFP and GFP2/YFP FRET pairs - 32% increased overlap of donor emission and acceptor excitation - Higher absorbance and quantum efficiency of the donor - Higher Foerster Radius (approx. 5.5 nm) - Increased FRET efficiency, especially at longer distances - Suitable for donor photobleaching - However: Linear unmixing of the strongly overlapping emission signals required

33 ALMF: Rainer Pepperkok Jens Rietdorf Stefan Terjung GFP2/YFP project: Andreas Girod Virginie Georget Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2 -YFP FRET pair T. Zimmermann, J. Rietdorf, A. Girod, V. Georget, R. Pepperkok, FEBS Letters 531 (2002)

34 Literature T. Förster (1946): Naturwissenschaften 6, 166 T. Förster (1948): Ann. Phys. (Leipzig) 2, 55 A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura and R. Y. Tsien (1997): Nature 388, B.A. Pollok and R. Heim (1999): Trends in Cell Biology 9, P.J. Verveer, F.S. Wouters, A.R. Reynolds, P.I. Bastiaens (2000): Science 290, F.S. Wouters, P. J. Verveer and P. I. H. Bastiaens (2001): Trends Cell Biol 11, T. Zimmermann, J. Rietdorf, A. Girod, V. Georget, R. Pepperkok (2002): FEBS Letters 531,