Transcript EMBO Practical course on Quantitative FRET, FRAP and FCS

ZMBH
EMBO Practical course on Quantitative FRET, FRAP and FCS
Live-cell FRET
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
 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
Low efficiency
R06  J*QD*n-4*2
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
Possible (although not ideal) solution:
Fix the cells and use fluorescently-labeled
monoclonal antibodies
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
Ideal solution:
Labeling with small dyes
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
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
Most common current uses:
Conformational changes in complexes
Reporter of intracellular conditions
Problems
 Precision is frequently not high enough
(general for measuring distances)
 Limited attachment sites for fluorophores
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
CaM
Advantages
 Sensors are engineered to exhibit
large conformational changes upon
ligand binding or modification
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
Phosphorylation domain
Binding domain
Advantages
 Sensors are engineered to exhibit
large conformational changes upon
ligand binding or modification
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 interactionmapping technique
Non-interacting proteins
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
Detecting protein interactions using
FRET
+ Stimulus
Possible solution:
Detecting changes in protein
interactions
P
 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
- 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, & RogerY.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
RogerY. 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
A
B
C
http://zeiss-campus.magnet.fsu.edu
Linear spectral unmixing
Leica Microsystems
One-channel measurement of FRET
Acceptor photobleaching
http://zeiss-campus.magnet.fsu.edu
510 nm
Procedure:
 Acquire signal of donor fluorescence
 Bleach acceptor
 Acquire signal of donor fluorescence again
One-channel measurement of FRET
Acceptor photobleaching
http://zeiss-campus.magnet.fsu.edu
510 nm
Advantages
 Is very simple and reliable
Drawbacks
 One-time experiment
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 (CFP) fluorescence
Donor photobleaching
+ FRET
- FRET
Time (sec)
Procedure:
 Follow kinetics of donor bleaching
Advantages
 Is comparatively simple
Drawbacks
 One-time experiment
 Can be affected by other intracellular
factors
Polarization (anisotropy) measurement
of FRET
Weak (no) FRET = high anisotropy
Strong FRET
= low anisotropy
Homo-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
Life-time measurement of FRET
http://micro.magnet.fsu.edu/primer/index.html
ps
Time (sec)
Phizicky et al., Nature. 2003 422:208-15
fs
ns
Life-time measurement of FRET
http://micro.magnet.fsu.edu/primer/index.html
Time (sec)
Phizicky et al., Nature. 2003 422:208-15
Advantages
 Reports both FRET efficiency and fraction of
interacting proteins
 Not sensitive to acceptor concentration
Drawbacks
 Limited speed
 Limited spatial resolution
Our own work (just one slide!)
FRET as a network mapping technique
Bacterial chemotaxis network
A
B