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

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

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?

Yes, using FRET!

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

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

What is FRET used for? Ca 2 + Genetically encoded “biosensors”

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

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

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

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 )

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

2. Favorable orientation Vogel et al 2006

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

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

4. Förster radius (R 0 ) R 0 = [2.8x ∙ 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

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

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

How is FRET measured?

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

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

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).

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

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

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.

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

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

Polarization anisotropy imaging +Fast +Nondestructive +Single sample +Can also measure homoFRET –Difficult with high NA lenses

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

Quantifying FRET using acceptor photobleaching Post bleach Pre bleach E = (I D - I DA )/I D E = ( )/ 63 = % FRET E = (I D - I DA )/I D E = ( )/ 94 = 0.35

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)

Piston and Kremers (2007) TIBS 32:407

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

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

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

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

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

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

Resources scence/fret/fretintro.html etintro.html campus.magnet.fsu.edu/referencelibrary/fret.html with-flim/ nce/fret/fretintro.html