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

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Presentation on theme: "Victor Sourjik ZMBH, University of Heidelberg EMBO Practical course on Quantitative FRET, FRAP and FCS Live-cell FRET ZMBH."— Presentation transcript:

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

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

3 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

4 Measuring molecular distances using FRET FRET efficiency is very sensitive to the distance between fluorophores potential of FRET as a molecular ruler R0R0 R 0 6 J*Q D *n -4 * 2 FRET Efficiency: E = R 0 6 /(R 0 6 +R 6 ) 1/R 6 No FRET at R > 11 nm (100 Å) GFP size ~ 5 nm (50 Å) FRET efficiency for CFP/YFP FRET pair R High efficiency Low efficiency

5 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

6 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

7 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

8 Detecting conformational changes using FRET High efficiency Low efficiency P

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

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

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

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

13 FRET as reporter of intracellular conditions Problems Only a limited number of sensors is available: Ca 2+, cAMP, several kinases... Advantages Sensors are engineered to exhibit large conformational changes upon ligand binding or modification Ca 2+ CaM Based on conformational chenge, e.g. Cameleon (calcium sensor)

14 FRET as reporter of intracellular conditions Problems Only a limited number of sensors is available: Ca 2+, cAMP, several kinases... Advantages Sensors are engineered to exhibit large conformational changes upon ligand binding or modification Based on intramolecular binding, e.g. kinase reporters Binding domain P Phosphorylation domain

15 Detecting protein interactions using FRET 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 Promises FRET as a generalized interaction- mapping technique Interacting proteins (or, more exactly, proteins in one complex) Non-interacting proteins

16 Detecting protein interactions using FRET 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 - Stimulus

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

18 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

19 Spectral requirements for FRET labels 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 CFP = cyan fluorescent protein (donor) YFP = yellow fluorescent protein (acceptor)

20 Fluorescent proteins for in vivo FRET measurements Nathan C. Shaner, Paul A. Steinbach, & Roger Y. Tsien 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)

21 Fluorescent proteins for in vivo FRET measurements Caution: FRET efficiency with FPs as FRET pair is always far below 100%

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

23 Combining proteins and dyes for in vivo FRET measurements Roger Y. Tsiens web site

24 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

25 Spectral measurement of FRET Advantages Complete spectral information Drawbacks Requires a specialized system (e.g., Zeiss LSM 710) Requires carefull image analysis

26 Spectral measurement of FRET

27 Spectral measurement of FRET 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

28 Two-channel measurement of FRET Advantages Can be performed on a simple wide-field microscope Drawbacks Limited spectral information Requires carefull image analysis

29 Two-channel measurement of FRET Sensitized emission Leica Microsystems Linear spectral unmixing ABC

30 One-channel measurement of FRET Acceptor photobleaching Procedure: Acquire signal of donor fluorescence Bleach acceptor Acquire signal of donor fluorescence again 510 nm

31 One-channel measurement of FRET Acceptor photobleaching Advantages Is very simple and reliable Drawbacks One-time experiment 510 nm

32 One-channel measurement of FRET Acceptor photobleaching Can be done either in imaging or whole-field acquisition mode Imaging Whole-field acquisition CFP YFP 510 nm

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

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

35 Life-time measurement of FRET Time (sec) Phizicky et al., Nature : fs ps ns

36 Life-time measurement of FRET Advantages Reports both FRET efficiency and fraction of interacting proteins Not sensitive to acceptor concentration Drawbacks Limited speed Limited spatial resolution Time (sec) Phizicky et al., Nature :

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

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