embo practical course on quantitative fret, frap and fcs live-cell fret
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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. - PowerPoint PPT PresentationTRANSCRIPT
Victor Sourjik ZMBH, University of Heidelberg
EMBO Practical course on Quantitative FRET, FRAP and FCS
Live-cell FRET
ZMBH
Measuring FRET in vivoDefine the goal
Choose fluorescent
labels
Choose your method
Get data!
I. Goals of in vivo FRET measurements
Measuring molecular distancesDetecting conformational
changesDetecting interactionsLocalizing interactionsFollowing interaction dynamicsReporting enzymatic activities
and intracellular conditions
Measuring molecular distances using FRET
FRET efficiency is very sensitive to the distance between fluorophores
potential of FRET as a molecular ruler
R0 R06
J*QD*n-4*2
FRET Efficiency:E = R0
6/(R06+R6) 1/R6
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
Measuring molecular distances using FRET
FRET efficiency is very sensitive to the distance between fluorophores
potential of FRET as a molecular rulerProblems 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 rulerProblems 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 rulerProblems 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
High efficiency Low efficiency
P
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
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 complexesReporter of intracellular conditions
Detecting conformational changes in complexes
P
Problems Ratio of donor to acceptor is not
fixed
Advantages Conformational changes
are typically larger
P
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)
FRET as reporter of intracellular conditions
Problems Only a limited number of
sensors is available: Ca2+, cAMP, several kinases...
Advantages Sensors are engineered
to exhibit large conformational changes upon ligand binding or modification
Ca2+
CaM
CaM
Based on conformational chenge, e.g. Cameleon (calcium sensor)
FRET as reporter of intracellular conditions
Problems Only a limited number of
sensors is available: Ca2+, 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
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
PromisesFRET as a generalized
interaction-mapping technique
Interacting proteins (or, more exactly, proteins in one complex)
Non-interacting proteins
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
II. Fluorescent labels for in vivo FRET measurements
Fluorescent proteinsIn-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
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
http://zeiss-campus.magnet.fsu.edu
CFP = cyan fluorescent protein (donor)YFP = yellow fluorescent protein (acceptor)
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
Caution: FRET efficiency with FPs as FRET pair is always far below 100%
http://zeiss-campus.magnet.fsu.edu
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
Combining proteins and dyes for in vivo FRET measurements
Roger Y. Tsien’s web site
III. Methods to measure FRET in vivo
Spectral measurementsTwo-channel FRET (sensitized
emission)One-channel FRET (acceptor
photobleaching)One-channel FRET (donor
photobleaching)Polarization imagingLife-time imaging
Spectral measurement of FRET
Advantages Complete spectral informationDrawbacks Requires a specialized system (e.g.,
Zeiss LSM 710) Requires carefull image analysis
http://zeiss-campus.magnet.fsu.edu
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
Advantages Can be performed on a simple wide-
field microscopeDrawbacks Limited spectral information Requires carefull image analysis
http://zeiss-campus.magnet.fsu.edu
Two-channel measurement of FRET
Sensitized emission
Leica Microsystems
http://zeiss-campus.magnet.fsu.edu
Linear spectral unmixing
A B C
One-channel measurement of FRET
Acceptor photobleachinghttp://zeiss-campus.magnet.fsu.edu
Procedure:Acquire signal of donor
fluorescenceBleach acceptorAcquire signal of donor
fluorescence again
510 nm
One-channel measurement of FRET
Acceptor photobleachinghttp://zeiss-campus.magnet.fsu.edu
Advantages Is very simple and reliableDrawbacks One-time experiment
510 nm
One-channel measurement of FRET
Acceptor photobleaching
http://zeiss-campus.magnet.fsu.edu
Can be done either in imaging or whole-field acquisition mode
ImagingWhole-field acquisition
CFP
YFP
510 nm
One-channel measurement of FRET
Donor photobleaching
Time (sec)
Dono
r (CF
P) fl
uore
scen
ce
- FRET+ FRET
Procedure:Follow kinetics of donor
bleaching
Advantages Is comparatively simpleDrawbacks One-time experiment Can be affected by other
intracellular factors
Polarization (anisotropy) measurement of FRET
Procedure:Excite with polarized lightMeasure emission in two
orthogonal directions of polarization
Advantages Allows measuring homo-FRET Is comparatively simpleDrawbacks Requires specialized
equipment Can be affected by other
intracellular factors
Weak (no) FRET = high anisotropyStrong FRET = low anisotropy
Homo-FRET
Life-time measurement of FRET
Time (sec)
Phizicky et al., Nature. 2003 422:208-15
http://micro.magnet.fsu.edu/primer/index.html
fs
ps
ns
Life-time measurement of FRET
Advantages Reports both FRET efficiency and
fraction of interacting proteins Not sensitive to acceptor concentrationDrawbacks Limited speed Limited spatial resolution
Time (sec)
Phizicky et al., Nature. 2003 422:208-15
http://micro.magnet.fsu.edu/primer/index.html
Our own work (just one slide!)FRET as a network mapping technique
Bacterial chemotaxis network
A
B