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EMBO Practical course on Quantitative FRET, FRAP and FCS Live-cell FRET

EMBO Practical course on Quantitative FRET, FRAP and FCS Live-cell FRET. ZMBH. Victor Sourjik ZMBH, University of Heidelberg. Measuring FRET in vivo. Define the goal. Choose fluorescent labels. Choose your method. Get data !. I. Goals of in vivo FRET measurements.

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EMBO Practical course on Quantitative FRET, FRAP and FCS Live-cell FRET

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  1. EMBO Practicalcourse on Quantitative FRET, FRAP and FCSLive-cell FRET ZMBH Victor Sourjik ZMBH, University of Heidelberg

  2. Measuring FRET in vivo Definethegoal Choosefluorescentlabels Chooseyourmethod Getdata!

  3. I. Goals of in vivo FRET measurements • Measuringmoleculardistances • Detectingconformationalchanges • Detectinginteractions • Localizinginteractions • Followinginteractiondynamics • Reporting enzymaticactivities and intracellularconditions

  4. Measuringmoleculardistancesusing FRET High efficiency • FRET efficiencyisvery sensitive to the distance betweenfluorophores  potential of FRET as a molecularruler 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

  5. Measuringmoleculardistancesusing FRET • FRET efficiencyisvery sensitive to the distance betweenfluorophores  potential of FRET as a molecularruler • Problems of in vivo FRET • Fluorophoresareusually large (fluorescentproteins) and coupledwith flexible linkers • Limitedattachmentsitesforfluorophores • Weakspecificfluorescence (duelow to moderate proteinlevels) • Highautofluorescencebackground • Non-opimalratio of donor to acceptor

  6. Measuringmoleculardistancesusing FRET • FRET efficiencyisvery sensitive to the distance betweenfluorophores  potential of FRET as a molecularruler • Problems of in vivo FRET • Fluorophoresareusually large (fluorescentproteins) and coupledwith flexible linkers • Limitedattachmentsitesforfluorophores • Weakspecificfluorescence (duelow to moderate proteinlevels) • Highautofluorescencebackground • Non-opimalratio of donor to acceptor Possible (althoughnot ideal) solution: Fix thecells and usefluorescently-labeledmonoclonalantibodies

  7. Measuringmoleculardistancesusing FRET • FRET efficiencyisvery sensitive to the distance betweenfluorophores  potential of FRET as a molecularruler • Problems of in vivo FRET • Fluorophoresareusually large (fluorescentproteins) and coupledwith flexible linkers • Limitedattachmentsitesforfluorophores • Weakspecificfluorescence (duelow to moderate proteinlevels) • Highautofluorescencebackground • Non-opimalratio of donor to acceptor Ideal solution: Labelingwithsmalldyes

  8. Detectingconformationalchangesusing FRET P Low efficiency High efficiency

  9. Detectingconformationalchangesusing FRET • Advantages • Ratio of donor to acceptorisfixed P • Problems • Precisionisfrequentlynot high enough (generalformeasuringdistances) • Limitedattachmentsitesforfluorophores

  10. Detectingconformationalchangesusing FRET • Advantages • Ratio of donor to acceptorisfixed P • Problems • Precisionisfrequentlynot high enough (generalformeasuringdistances) • Limitedattachmentsitesforfluorophores • Most common current uses: • Conformational changes in complexes • Reporter of intracellular conditions

  11. Detectingconformationalchanges in complexes • Advantages • Conformationalchangesaretypically larger • Problems • Ratio of donor to acceptorisnotfixed P P

  12. Detectingconformationalchanges in complexes • Advantages • Conformationalchangesaretypically larger • Problems • Ratio of donor to acceptorisnotfixed Possiblesolution: Useonlyonefluorophore (homo-FRET) P P

  13. FRET as reporter of intracellularconditions • Advantages • Sensors areengineered to exhibit large conformationalchangesuponligandbindingormodification CaM • Problems • Only a limitednumber of sensorsisavailable: Ca2+, cAMP, severalkinases... Ca2+ CaM Based on conformational chenge, e.g. Cameleon (calcium sensor)

  14. FRET as reporter of intracellularconditions • Advantages • Sensors areengineered to exhibit large conformationalchangesuponligandbindingormodification Phosphorylation domain Binding domain • Problems • Only a limitednumber of sensorsisavailable: Ca2+, cAMP, severalkinases... P Based on intramolecular binding, e.g. kinase reporters

  15. Detectingproteininteractionsusing 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

  16. Detectingproteininteractionsusing 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

  17. II. Fluorescentlabelsfor in vivo FRET measurements • Fluorescent proteins • In-vivo labeling with fluorescent dyes

  18. Proteins vsdyes in fluorescencemicroscopy • Fluorescentproteins • Canbegeneticallyencoded (high specificity) • Proteins arebulky (5 nm) • Spectraarebroad (strongcross-talk) • Not verybright and photostable • In-vivolabelingwithfluorescentdyes • Small size • Bright and relativelyphotostable • Narrowspectra and large spectralchoice • Specificin-vivolabelingisdifficult

  19. Spectralrequirementsfor 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

  20. Fluorescentproteinsfor 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)

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

  22. Fluorescentdyesfor 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

  23. Combiningproteins and dyesfor in vivo FRET measurements Roger Y. Tsien’s web site

  24. III. Methods to measure FRET in vivo • Spectralmeasurements • Two-channel FRET (sensitizedemission) • One-channel FRET (acceptorphotobleaching) • One-channel FRET (donorphotobleaching) • Polarizationimaging • Life-timeimaging

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

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

  27. Spectral measurement of FRET In a generalcase (so-called linear spectralunmixing): • Acquirespectra at donor and acceptorexcitationwavelength • Acquirespectraforcontrolsampleswithonlydonor and onlyacceptor • Subtractdonor and acceptorcross-talk (bleed-through) to gettrue FRET signal http://zeiss-campus.magnet.fsu.edu

  28. Two-channelmeasurement 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

  29. Two-channelmeasurement of FRETSensitizedemission http://zeiss-campus.magnet.fsu.edu A B C Linear spectral unmixing Leica Microsystems

  30. One-channelmeasurement of FRETAcceptorphotobleaching http://zeiss-campus.magnet.fsu.edu Procedure: • Acquiresignal of donorfluorescence • Bleachacceptor • Acquiresignal of donorfluorescenceagain 510 nm

  31. One-channelmeasurement of FRETAcceptorphotobleaching http://zeiss-campus.magnet.fsu.edu Advantages • Is very simple and reliable Drawbacks • One-time experiment 510 nm

  32. One-channelmeasurement of FRETAcceptorphotobleaching Imaging Can be done either in imaging or whole-field acquisition mode Whole-field acquisition YFP CFP http://zeiss-campus.magnet.fsu.edu 510 nm

  33. One-channelmeasurement of FRETDonorphotobleaching Advantages • Iscomparatively simple Drawbacks • One-timeexperiment • Canbeaffectedbyotherintracellularfactors Donor (CFP) fluorescence + FRET - FRET Time (sec) Procedure: • Follow kinetics of donor bleaching

  34. Polarization (anisotropy) measurement of FRET Advantages • Allowsmeasuringhomo-FRET • Iscomparatively simple Drawbacks • Requiresspecializedequipment • Canbeaffectedbyotherintracellularfactors Weak (no) FRET = high anisotropy Strong FRET = low anisotropy Homo-FRET Procedure: • Excitewithpolarized light • Measureemission in two orthogonal directions of polarization

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

  36. Life-timemeasurement of FRET Advantages • Reports both FRET efficiency and fraction of interactingproteins • Not sensitive to acceptorconcentration Drawbacks • Limitedspeed • Limitedspatialresolution http://micro.magnet.fsu.edu/primer/index.html Time (sec) Phizicky et al., Nature. 2003 422:208-15

  37. Ourownwork (just oneslide!)FRET as a networkmappingtechnique Bacterial chemotaxis network A B

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