1. Gert-Jan Kremers FRET and Live Cell Imaging Wednesday, May 21, QFM 2014

2. FRET microscopy Outline 1.What is FRET and what is it used for? 2.Requirements for FRET? 3.How is FRET measured? 4.FRET in the real world

3. Fluorescence microscopy can be used to determine if two proteins are co-localized. This means that they are found within the same ~250 nm x ~500 nm region of the specimen. However, proteins are much smaller than the resolution limit of fluorescence. Is there any way we can use fluorescence microscopy to detect where specific protein-protein interactions occur?

4. Yes, using FRET!

5. What is FRET? –Förster (fluorescence) resonance energy transfer, or FRET, is a distance-dependent, photophysical process between a donor and acceptor fluorophore –FRET results in a loss of donor fluorescence and an increase in donor fluorescence –FRET occurs between fluorophores separated by distances of less than 10nm –FRET can easily be measured by fluorescence microscopy –FRET can thus be used to report on the proximity of molecules over much smaller distances than can be resolved by standard fluorescence or even super-resolution approaches

6. What is FRET used for? Protein-protein interaction (intermolecular FRET) Conformational change (intramolecular FRET)

7. What is FRET used for? http://zeiss.magnet.fsu.edu/ Ca 2 + Genetically encoded “biosensors”

8. Example: Androgen receptor Ligand-dependent transcription factor t = 45min Martin van Royen, Erasmus MC

9. Androgen receptor DBDNTDYFPCFPLBD DBDNTDYFP CFP LBD DBDNTDYFPLBD DBDNTDYFPLBD DBDNTDCFPLBD DBDNTD CFP LBD Both intra- and intermolecular N/C interaction Intermolecular N/C interaction only Questions: 1.Do androgen receptors dimerize? (Intermolecular N-C interactions) 2.Does ligand binding induce conformational change? (Intramolecular N-C interaction) 3.In what order do these events occur? DBDNTDLBD DBDNTDLBD DBDNTDLBD DBDNTDLBD NTD DBDLBD DBDLBD

10. Schaufele, et al. (2005) PNAS Van Royen et al. (2007) JCB

11. What are the requirements for FRET to occur? 1.Spectral overlap between donor emission and acceptor absorbance spectra 2.Favorable orientation of fluorophores 3.Close proximity (less than 10nm) 4.Förster radius (R 0 )

12. 1.Spectral overlap between donor emission and acceptor absorbance spectra Add legend to graph

13. 2. Favorable orientation Vogel et al 2006

14. 3. Close proximity of donor and acceptor Vogel et al 2006

15. 3. Close proximity of donor and acceptor E = 1 / [1 + (r/R o ) 6 ]

16. 4. Förster radius (R 0 ) R 0 = [2.8x10 -11 ∙ κ 2 ∙ QY D ∙ ε A ∙ J(λ)] 1/6 nm Κ 2 = Orientation factor, range 0 to 4, but 2/3 for randomly oriented D and A. QY D = donor quantum yield ε A = acceptor extinction coefficient J(λ)= spectral overlap integral = ∫ F D (λ) ∙ E A (λ) ∙ λ 4 dλ

17. R 0 for common FRET pairs DonorAcceptorR 0 (nm) FITCTMR5.5 Cy3Cy55.0 ECFPEYFP4.7 EGFPmCherry5.3 mTurq2mVenus5.8 mClovermRuby26.3

18. FRET microscopy Outline 1.What is FRET and what is it used for? 2.Requirements for FRET? 3.How is FRET measured? 4.FRET in the real world

19. How is FRET measured?

20. 1.Sensitized acceptor fluorescence 2.Spectral imaging 3.Fluorescence lifetime imaging (FLIM) 4.Polarization anisotropy 5.Acceptor photobleaching

21. Sensitized acceptor fluorescence Excite donor, measure acceptor fluorescence +True readout of FRET +Easy qualitative measurements –Quantitation requires many correction for bleedthrough, etc. –Sensitive to photobleaching Vogel et al 2006

22. FRET with CFP and YFP –Excitation of CFP leads to some YFP excitation because YFP is brighter than CFP. –CFP emission also bleeds into the YFP channel (i.e. there will always be some apparent “FRET” signal).

23. Sensitized acceptor fluorescence CFP-YFP Donor channel Acceptor channel “FRET” channel CFP YFP

24. Wavelength excitation FluorophoreDonor emission images Acceptor emission images D Dab Ac D + Aef A Ad g Need 3 filter sets and 3 cell preparations D, donor; A, acceptor D, donor excitation wavelength; A, acceptor excitation wavelength Calculating Bleedthrough Factors Precision FRET (PFRET) Algorithm

25. PFRET = UFRET – DSBT – ASBT UFRET (image f) is uncorrected FRET ASBT is the acceptor spectral bleedthrough ASBT = ([c]/[d]) x [g]. DSBT is the donor spectral bleedthrough DSBT = ([b]/[a]) x [e]. !Assumes the images of single and double-labeled samples are collected under identical conditions. !Assumes bleedthrough is proportional to the amount of donor and acceptor present in a given cell.

26. Spectral imaging Excite donor, measure emission spectra +Spectral information +Easy qualitative measurement –Sensitive to photobleaching –Low S/N ratio FRET No FRET

27. Fluorescence lifetime imaging +Concentration independent +Acceptor does not need to be fluorescent +Quantify fraction of interacting molecules –Special (expensive) instrumentation –Low number of photons

28. Polarization anisotropy imaging http://www.microscopyu.com/articles/fluorescence/fret/fretintro.html +Fast +Nondestructive +Single sample +Can also measure homoFRET –Difficult with high NA lenses

29. Acceptor photobleaching / donor dequenching Bleach acceptor Donor quenched due to FRET Release of donor quenching +Straightforward and quantitative: E = (I D - I DA )/I D +Performed on single sample –Destructive –Beware of artifacts due to acceptor photoconversion upon bleaching

30. Quantifying FRET using acceptor photobleaching Post bleach Pre bleach E = (I D - I DA )/I D E = (63 - 42)/ 63 = 0.34 34% FRET E = (I D - I DA )/I D E = (94 - 61)/ 94 = 0.35

31. FRET microscopy Outline 1.What is FRET and what is it used for? 2.Requirements for FRET? 3.How is FRET measured? 4.FRET in the real world (the good, the bad and the ugly)

32. Piston and Kremers (2007) TIBS 32:407

33. If FRET is so great, why isn’t everyone doing it? FRET analysis requires rigorous controls and careful quantification –Acceptor is often excited directly –Spectral overlap Not all molecules within FRET proximity undergo FRET (orientation effects) Not all interacting proteins will show FRET FRET is not immune to experimental artifacts

34. Special considerations for FRET studies using fluorescent proteins Remember that FRET measures the distance between the fluorophores, which in this case are buried inside the beta barrel of the FPs Many fluorescent proteins themselves can form dimers or higher-level oligomers when present at high concentrations, and this can give rise to a false positive –See Zacharias et al (2002) Science 296:913 –Thus is important to use monomeric forms for FRET studies

35. Biophys J. 2006 December 15; 91(12): L99–L101 A good way to get started: FRET standards as a positive control

36. Another type of positive control: known interacting proteins LC3 and Atg4B C74A have been reported to directly interact Kraft and Kenworthy 2012 J Biomed Optics

39. Nature Methods 2, 801 (2005) doi:10.1038/nmeth1105-801 Valentin et al. Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments Example of a FRET artifact

40. Examples of ways to demonstrate FRET is “specific” Include negative controls matched for expression level and localization Demonstrate FRET is eliminated when interacting domains are mutated Show physiological regulation of FRET Unlabeled proteins should compete with labeled proteins

41. Resources http://www.olympusmicro.com/primer/techniques/fluore scence/fret/fretintro.html http://www.microscopyu.com/articles/fluorescence/fret/fr etintro.html http://zeiss- campus.magnet.fsu.edu/referencelibrary/fret.html http://www.leica-microsystems.com/science-lab/fret- with-flim/ http://micro.magnet.fsu.edu/primer/techniques/fluoresce nce/fret/fretintro.html