Victor Sourjik ZMBH, University of Heidelberg EMBO Practical course on Quantitative FRET, FRAP and FCS Live-cell FRET Victor Sourjik ZMBH, University of Heidelberg

Choose fluorescent labels Measuring FRET in vivo Define the goal Choose fluorescent labels Choose your method Get data!

I. Goals of in vivo FRET measurements Measuring molecular distances Detecting conformational changes Detecting interactions Localizing interactions Following interaction dynamics Reporting enzymatic activities and intracellular conditions

Measuring molecular distances using FRET High efficiency FRET efficiency is very sensitive to the distance between fluorophores  potential of FRET as a molecular ruler FRET efficiency for CFP/YFP FRET pair FRET Efficiency: E = R06/(R06+R6)  1/R6 No FRET at R > 11 nm (100 Å) GFP size ~ 5 nm (50 Å) R R0 R06  J*QD*n-4*2 Low efficiency

Measuring molecular distances using FRET FRET efficiency is very sensitive to the distance between fluorophores  potential of FRET as a molecular ruler Problems of in vivo FRET Fluorophores are usually large (fluorescent proteins) and coupled with flexible linkers Limited attachment sites for fluorophores Weak specific fluorescence (due low to moderate protein levels) High autofluorescence background Non-opimal ratio of donor to acceptor

Measuring molecular distances using FRET FRET efficiency is very sensitive to the distance between fluorophores  potential of FRET as a molecular ruler Problems of in vivo FRET Fluorophores are usually large (fluorescent proteins) and coupled with flexible linkers Limited attachment sites for fluorophores Weak specific fluorescence (due low to moderate protein levels) High autofluorescence background Non-opimal ratio of donor to acceptor Possible (although not ideal) solution: Fix the cells and use fluorescently-labeled monoclonal antibodies

Measuring molecular distances using FRET FRET efficiency is very sensitive to the distance between fluorophores  potential of FRET as a molecular ruler Problems of in vivo FRET Fluorophores are usually large (fluorescent proteins) and coupled with flexible linkers Limited attachment sites for fluorophores Weak specific fluorescence (due low to moderate protein levels) High autofluorescence background Non-opimal ratio of donor to acceptor Ideal solution: Labeling with small dyes

Detecting conformational changes using FRET P High efficiency Low efficiency

Detecting conformational changes using FRET Advantages Ratio of donor to acceptor is fixed P Problems Precision is frequently not high enough (general for measuring distances) Limited attachment sites for fluorophores

Detecting conformational changes using FRET Advantages Ratio of donor to acceptor is fixed P Problems Precision is frequently not high enough (general for measuring distances) Limited attachment sites for fluorophores Most common current uses: Conformational changes in complexes Reporter of intracellular conditions

Detecting conformational changes in complexes Advantages Conformational changes are typically larger Problems Ratio of donor to acceptor is not fixed P P

Detecting conformational changes in complexes Advantages Conformational changes are typically larger Problems Ratio of donor to acceptor is not fixed Possible solution: Use only one fluorophore (homo-FRET) P P

FRET as reporter of intracellular conditions Advantages Sensors are engineered to exhibit large conformational changes upon ligand binding or modification CaM Problems Only a limited number of sensors is available: Ca2+, cAMP, several kinases... Ca2+ CaM Based on conformational chenge, e.g. Cameleon (calcium sensor)

FRET as reporter of intracellular conditions Advantages Sensors are engineered to exhibit large conformational changes upon ligand binding or modification Binding domain Phosphorylation domain Problems Only a limited number of sensors is available: Ca2+, cAMP, several kinases... P Based on intramolecular binding, e.g. kinase reporters

Detecting protein interactions using FRET Interacting proteins (or, more exactly, proteins in one complex) Promises FRET as a generalized interaction- mapping technique Problems Strong spectral cross-talk between typical fluorophores (fluorescent proteins) Typically low FRET efficiency Limited attachment sites for fluorophores Weak specific fluorescence Non-opimal ratio of donor to acceptor Bulky fluorophores  Detection of absolute strength of physiological interactions is non-trivial Non-interacting proteins

Detecting protein interactions using FRET + Stimulus Possible solution: Detecting changes in protein interactions Relative concentrations of donor and acceptor do not change upon stimulation (i.e., internal control)  Changes in FRET are more reliably detected than absolute values P - Stimulus

II. Fluorescent labels for in vivo FRET measurements Fluorescent proteins In-vivo labeling with fluorescent dyes

Proteins vs dyes in fluorescence microscopy Fluorescent proteins Can be genetically encoded (high specificity) Proteins are bulky (5 nm) Spectra are broad (strong cross-talk) Not very bright and photostable In-vivo labeling with fluorescent dyes Small size Bright and relatively photostable Narrow spectra and large spectral choice Specific in-vivo labeling is difficult

Spectral requirements for FRET labels CFP = cyan fluorescent protein (donor) YFP = yellow fluorescent protein (acceptor) http://zeiss-campus.magnet.fsu.edu Requirements for the FRET pair: excitation spectra of donor and acceptor are separated emission spectrum of donor overlaps with excitation spectrum of acceptor emission spectra of donor and acceptor are separated

Fluorescent proteins for in vivo FRET measurements Nathan C. Shaner, Paul A. Steinbach, & Roger Y. Tsien. 2005 Nature Methods, Vol. 2: 905 – 909 Any two proteins with overlapping emission spectrum of donor and excitation spectrum of acceptor can be used a FRET pair (including the same protein as donor and acceptor)

Fluorescent proteins for in vivo FRET measurements http://zeiss-campus.magnet.fsu.edu Caution: FRET efficiency with FPs as FRET pair is always far below 100%

Fluorescent dyes for in vivo FRET measurements Fluorescent dyes with relatively specific binding to short peptide sequences (e.g., FlAsH or ReAsH) Miyawaki et al., supplement to Nature Cell Biol., 5 Fluorescent dyes specifically binding to protein tags (e.g., SNAP-tag or HaloTag) HaloTag, Promega Corporation

Combining proteins and dyes for in vivo FRET measurements Roger Y. Tsien’s web site

III. Methods to measure FRET in vivo Spectral measurements Two-channel FRET (sensitized emission) One-channel FRET (acceptor photobleaching) One-channel FRET (donor photobleaching) Polarization imaging Life-time imaging

Spectral measurement of FRET http://zeiss-campus.magnet.fsu.edu Advantages Complete spectral information Drawbacks Requires a specialized system (e.g., Zeiss LSM 710) Requires carefull image analysis

Spectral measurement of FRET http://zeiss-campus.magnet.fsu.edu

Spectral measurement of FRET http://zeiss-campus.magnet.fsu.edu In a general case (so-called linear spectral unmixing): Acquire spectra at donor and acceptor excitation wavelength Acquire spectra for control samples with only donor and only acceptor Subtract donor and acceptor cross-talk (bleed-through) to get true FRET signal

Two-channel measurement of FRET http://zeiss-campus.magnet.fsu.edu Advantages Can be performed on a simple wide-field microscope Drawbacks Limited spectral information Requires carefull image analysis

Two-channel measurement of FRET Sensitized emission http://zeiss-campus.magnet.fsu.edu A B C Linear spectral unmixing Leica Microsystems

One-channel measurement of FRET Acceptor photobleaching http://zeiss-campus.magnet.fsu.edu Procedure: Acquire signal of donor fluorescence Bleach acceptor Acquire signal of donor fluorescence again 510 nm

One-channel measurement of FRET Acceptor photobleaching http://zeiss-campus.magnet.fsu.edu Advantages Is very simple and reliable Drawbacks One-time experiment 510 nm

One-channel measurement of FRET Acceptor photobleaching Imaging Whole-field acquisition YFP CFP http://zeiss-campus.magnet.fsu.edu 510 nm Can be done either in imaging or whole-field acquisition mode

One-channel measurement of FRET Donor photobleaching Donor (CFP) fluorescence + FRET - FRET Time (sec) Advantages Is comparatively simple Drawbacks One-time experiment Can be affected by other intracellular factors Procedure: Follow kinetics of donor bleaching

Polarization (anisotropy) measurement of FRET Weak (no) FRET = high anisotropy Strong FRET = low anisotropy Homo-FRET Advantages Allows measuring homo-FRET Is comparatively simple Drawbacks Requires specialized equipment Can be affected by other intracellular factors Procedure: Excite with polarized light Measure emission in two orthogonal directions of polarization

Life-time measurement of FRET Time (sec) http://micro.magnet.fsu.edu/primer/index.html ps fs ns Phizicky et al., Nature. 2003 422:208-15

Life-time measurement of FRET Time (sec) http://micro.magnet.fsu.edu/primer/index.html Advantages Reports both FRET efficiency and fraction of interacting proteins Not sensitive to acceptor concentration Drawbacks Limited speed Limited spatial resolution Phizicky et al., Nature. 2003 422:208-15

Our own work (just one slide!) FRET as a network mapping technique Bacterial chemotaxis network A B