embo practical course on quantitative fret, frap and fcs live-cell fret

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