Detecting Protein–Protein Interactions with
CFP-YFP FRET by Acceptor Photobleaching
UNIT 12.7
This unit presents a protocol for detecting molecular interactions between two proteins
(Protein1 and Protein2) at a specific location within a cell (Karpova et al., 2003). The
assay is based on FRET (Förster resonance energy transfer) by acceptor photobleaching.
Stable or transfected cell lines containing the following constructs are required: (1)
Protein1-YFP (yellow fluorescent protein) and Protein2-CFP (cyan fluorescent protein);
(2) CFP fused to YFP (positive control); (3) unconjugated CFP (negative control no.
1); (4) unconjugated YFP (imaging calibration); and (5) unconjugated CFP mixed with
unconjugated YFP (negative control no. 2). Live or fixed cells are imaged on a Zeiss
LSM510 or comparable confocal microscope equipped with an argon laser and configured
to detect emission from CFP (excited at 458 nm) and YFP (excited at 514 nm). After
bleaching of the acceptor (Protein1-YFP) at a specific location within the cell using
the 514-nm laser line, the change in donor fluorescence (Protein2-CFP) is quantified by
comparing prebleach and post-bleach images. FRET is detected if donor fluorescence
increases by significantly more than what is detected in the two negative controls. This
indicates molecular interactions between Protein1-YFP and Protein2-CFP at the location
of the photobleach. The positive control using the CFP-YFP fusion protein establishes
the maximum detectable level of FRET under the imaging conditions in use.
FRET FOR FIXED CELLS
Materials
BASIC
PROTOCOL
Plasmids (Ausubel et al., 2005) for Protein1-YFP and Protein2-CFP (to test for
FRET between Protein1 and Protein2) and for CFP-YFP fusion, unconjugated
CFP, and unconjugated YFP (as controls)
Cell line of interest
2% (v/v) formaldehyde in PBS
Phosphate-buffered saline (PBS; APPENDIX 2A)
Mounting medium without antifade
No. 1.5 coverslips
6-well tissue-culture plate
Laser scanning confocal microscope: e.g., Zeiss LSM510 or similar system with:
Argon laser tuned to 458-nm and 514-nm laser lines
470- to 500-nm band-pass emission filter (BP470-500) for CFP
530-nm long-pass emission filter (LP530) for YFP
Chroma q500Ip (505 nm) beam splitter
LSM510 physiology software (Zeiss) or comparable software enabling
quantification of intensities
Additional reagents and equipment for transfection (Seidman et al., 1997) and
culture (APPENDIX 3B) of mammalian cells
Prepare plasmids and cell lines
1. To test for FRET between Protein1 and Protein2, construct or obtain plasmids for
Protein1-YFP and Protein2-CFP.
Ausubel (2005) contains detailed protocols for construction, manipulation, and purification of plasmids.
Consider using brighter forms of CFP and YFP molecules (Nagai et al., 2002; Rizzo
et al., 2004), and monomeric mutations (Zacharias et al., 2002).
Contributed by Tatiana Karpova and James G. McNally
Current Protocols in Cytometry (2006) 12.7.1-12.7.11
C 2006 by John Wiley & Sons, Inc.
Copyright Cellular and
Molecular
Imaging
12.7.1
Supplement 35
2. Perform appropriate experiments to evaluate functionality of the fusion proteins.
These might include in vitro assays using isolated fusion proteins, rescue of a gene
knockout strain with the fusion protein, or localization of the fusion protein in cells.
3. As FRET controls, construct or obtain plasmids for CFP-YFP fusion, unconjugated
CFP, and unconjugated YFP.
4. Perform single and double transfections of the cells (Seidman et al., 1997) with the
constructs obtained in steps 1 and 3 using standard transfection procedures to obtain
five different cell lines containing:
a.
b.
c.
d.
e.
Protein1-YFP and Protein2-CFP.
CFP fused to YFP.
Unconjugated CFP.
Unconjugated YFP.
Unconjugated CFP mixed with unconjugated YFP.
5. Let the transfected cells grow on no. 1.5 coverslips placed in wells of a 6-well plate
until full expression of the transfected proteins is attained (usually 24 hr).
APPENDIX 3B
contains detailed protocols for growth of mammalian cells.
Grow the cells under conditions where they will attach to the coverglass if possible, or
immobilize the cells in some other way, such that cells will not drift out of focus during
the FRET procedure. The simplest way to accomplish this is to grow the cells on no. 1.5
coverslips deposited in wells of a 6-well plate.
6. Fix the transfected cells by incubating ≥20 min in 2% formaldehyde.
7. Wash the cells in PBS, then mount the coverslips with the cells on a microscope slide
coverslip. Do not use antifade reagents in the mounting medium.
Antifade reagents will significantly reduce the efficiency of the intentional photobleach
required for FRET by acceptor photobleaching.
Configure the confocal microscope for CFP/YFP imaging
8. Prepare the LSM510 confocal microscope (or comparable system) with a 40-mW
argon laser set at 75% efficiency and tuned to lines at 458, 488, and 514 nm.
A weaker argon laser (25 mW) can also be used, but at 100% efficiency. In general, laser
power should be set such that a rapid (∼1 sec) photobleach can be achieved, but not so
high that it still causes bleaching when the beam is attenuated during normal imaging
of the cells (see step 18 below).
If not already installed in the confocal light path, the two filters and beam splitter must
be purchased and then installed by a service engineer. For initial focusing and centering
of labeled cells, a GFP filter set should be available on the microscope itself.
9. Choose objective magnification and zoom settings such that the region for FRET
measurement occupies ∼ 10% of the image.
10. Select “Multi track” mode in the Configuration control window (sequential imaging
of the two channels).
11a. If default CFP/YFP setting is available: Select this setting, then proceed to step 18.
Detecting
Protein–Protein
Interactions with
CFP-YFP FRET
by Acceptor
Photobleaching
11b. If default CFP/YFP setting is not available: Implement this setting by following
steps 12 to 17.
12. Select the Line option (line switching between the channels) in the Configuration
Control window. Select 12-bit imaging in the Scan Control window.
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Current Protocols in Cytometry
13. Select the 458/514-nm double dichroic mirror for insertion in the excitation path to
permit excitation of CFP with the 458-nm argon laser line and YFP with the 514-nm
argon laser line.
14. Select the q500Ip (505-nm) beam splitter for insertion in the light path to direct YFP
emission to photomultiplier tube 1 (PMT1) and CFP emission to photomultiplier
tube 3.
PMT2 could also be used to detect CFP emission, but PMT3 is recommended on the Zeiss
510 because the light path is more direct.
15. Select the 530-nm long-pass filter (LP530) for insertion in the light path in front of
PMT1 to improve specific detection of YFP emission.
16. Select the 470- to 500-nm band-pass filter (BP470-500) for insertion in the light path
in front of PMT3 to improve specific detection of CFP emission.
17. Save the CFP/YFP configuration for future use.
IMPORTANT NOTE: From this step on, the term “CFP channel” will be used to refer to
the combination of the 458-nm laser-line excitation with the BP470-500 emission filter
and PMT3, and the term “YFP channel” will be used to refer to the combination of the
514-nm laser-line excitation with the LP530 emission filter and PMT1.
Adjust the confocal microscope for dual imaging of CFP and YFP
Steps 18 to 25 are to be performed first with the unconjugated CFP slide, then with
the unconjugated YFP slide, and finally repeated with the unconjugated CFP slide. This
ensures that the microscope gain and offset are adjusted to eliminate bleed-through
between the two channels.
18. Find the cells and focus on them using the microscope oculars, a GFP filter set, and
the microscope’s mercury arc lamp. Start with cells containing unconjugated CFP
as prepared in step 7. Find some representative fluorescent cells, and quickly center
them in the middle of the field.
Do not use the mercury arc lamp for fine focusing, as this will induce bleaching of the
specimen.
19. Switch to confocal mode, then adjust the laser transmission to 0.15% using the
acousto-optical tunable filter (AOTF).
20. Select a pinhole of 1 Airy disk unit in the CFP channel, then select a pinhole in the
YFP channel such that the optical section thickness is identical to that in the CFP
channel.
21. Set the offset in the CFP and YFP channels such that the average background does
not exceed 150.
22. Set the amplifier gain to 1.
23. Set the detector gains such that the maximum values in the image do not exceed
3000 in the CFP channel and 3900 in the YFP channel.
24. If the intensity values of images in either the CFP or YFP channel do not exceed
1200, then increase the size of the pinhole to 1.25 in step 19 and repeat steps 21 to
22. Continue to increment the size of the pinhole until intensity values exceed 1200.
25. Save the adjusted imaging parameters (Steps 19 to 24) and do not change them in
subsequent imaging.
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Perform the acceptor photobleaching
Steps 26 to 33 are to be performed with slides a, b, c, and e prepared in step 7 (see step 4
for the contents of each slide).
26. Place the slide on the microscope stage, and focus and center as described in step 18.
27. Open the Edit Bleach window on the Zeiss LSM510 to define the bleaching conditions.
28. Set up bleaching with a 514-nm laser line with the AOTF at 100%.
If bleaching of the donor is also detected (see Critical Parameters and Troubleshooting),
the strength of the photobleach should be reduced by decreasing the AOTF transmission.
29. Define the area to be bleached by drawing a region of interest (ROI) on top of the
cell image.
The ROI should be large enough to enable reliable intensity measurements, preferably
containing at least 900 pixels ( e.g., a rectangle 30 × 30 pixels). Smaller regions encompassing particular structures of interest can also be examined, but the data will be
noisier.
More than one ROI may be selected per cell, although this generally increases the
time required to perform the photobleach. If FRET is expected to occur in a certain
compartment of the cell, an ROI can be selected there, and another ROI can be selected
somewhere else in the same cell to serve as a negative control.
30. Choose 20 iterations for the bleach to enable sufficient bleaching of the YFP-tagged
protein.
31. Using the Time Series window of the LSM510, configure image acquisition for five
prebleach and five post-bleach images.
This time series enables assessment of the rate of photobleaching resulting from image
acquisition, which, if large, can interfere with FRET detection.
32. Acquire the time series with photobleach.
33. Repeat steps 29 to 32 with a total of at least 30 independent ROIs from at least 10
different cells.
Perform the FRET calculations
Steps 34 to 40 are to be performed with the data obtained from each of the four slides
(a, b, c, and e, prepared in step 7) in steps 26 to 33.
34. Using LSM510 software (or another quantification program) measure average intensity in the bleached ROIs.
35. Measure the average intensity of the background (area outside the cell) and subtract
this value from average intensities in the bleached ROIs.
36. Calculate the FRET energy transfer efficiency (EF ) using the formula EF = (I6 –
I5 )/I6 , where In is the CFP average intensity at the nth time point after background
subtraction.
As the bleach occurs between time points 5 and 6, this formula yields the increase in
CFP fluorescence following the bleach of YFP, normalized by CFP fluorescence after the
bleach.
Detecting
Protein–Protein
Interactions with
CFP-YFP FRET
by Acceptor
Photobleaching
37. Average the measurements from the different ROIs to get a mean EF value.
38. As a control, use an identical ROI (the control ROI) and position it over a nonbleached
region of the specimen to calculate CF = (I6 – I5 )/I6 .
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39. Subtract the average background intensity from the average intensities in the control
ROI.
40. Average the measurements from the different control ROIs to get a mean CF value.
41. Use the values for EF and CF for each of the four different slides to assess whether
FRET occurs between Protein1 and Protein2, as described under Anticipated Results.
FRET FOR LIVE CELLS
The Basic Protocol may be applied without modification to live cells if the proteins under
study do not exchange rapidly. This can be tested by photobleaching each protein in live
cells and then collecting time-lapse images to see if there is recovery of fluorescence
within the bleach spot. If there is any recovery, then the FRET procedure outlined above
should be modified as follows.
ALTERNATE
PROTOCOL
Additional Materials (also see Basic Protocol)
Environmental chamber or a stage heater, such as a Nevtek ASI400
Follow steps 1 to 5 of the Basic Protocol, then omit steps 6 and 7 (i.e., the fixation steps).
Cells should be grown in a chamber suitable for live cell imaging, e.g., a chambered
coverglass (Lab-Tek II; Nalgene Nunc). Follow steps 8 to 28 of the Basic Protocol, as
described above, then proceed with the remainder of the protocol, modified as follows at
the indicated steps.
29. Define the region to be bleached as the entire cell, such that all acceptor fluorescence
will be destroyed after bleaching.
This draconian measure is required to prevent the recovery of fluorescent acceptor
molecules in the bleach spot, which would negate some or all of the effect of the photobleach on donor fluorescence.
33. Acquire the bleach sequence for at least 10 cells. Repeat this procedure for at least
10 more cells, but now with the AOTF transmission for the photobleach set at 0%.
As the whole cell is bleached in this procedure, nonbleached controls must be acquired
separately.
When bleaching the whole cell, there are several choices for ROIs to measure changes
in intensity. The whole cell can be selected, but it is also advisable to measure smaller
ROIs in case FRET is compartmentalized. The same-sized ROIs can then be used to make
control (CF ) measurements in cells that were imaged without the intentional photobleach.
COMMENTARY
Background Information
FRET basics
Protein-protein interactions are critical for
many cellular processes. Such interactions include the stable association of proteins within
large complexes and the transient association
of regulatory proteins. For many years, these
types of protein-protein interactions have been
analyzed in vitro. However, in vitro methods
have limitations with regard to determining the
timing and spatial localization of protein interactions within the cell, and can also be subject
to artifacts resulting from the extraction and
isolation procedures employed.
Some of these limitations are potentially
addressed by light microscopy, which has the
capacity to examine either intact fixed or living
cells. As a start, intact cells can be examined by
immunofluorescence microscopy to determine
if two proteins colocalize at a particular cellular site, but colocalization does not prove interaction. Rather, owing to the resolution limits
of light microscopy, colocalization indicates
only that the two proteins are located within
the same 200 × 200 × 500–nm volume element (voxel). Since typical globular proteins
are only 5 to 10 nm in diameter, many such
molecules can reside within the same optically
resolvable voxel and still not directly interact.
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Molecular
Imaging
12.7.5
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Supplement 35
This limitation of colocalization can be overcome by FRET (Herman, 1989), which can
demonstrate that two proteins are less than 10
nm apart and therefore must form a complex.
FRET is based on the ability of a fluorophore to transfer some energy from its
excited state to an adjacent fluorophore, instead of converting this energy into fluorescence. For FRET to occur, the first fluorophore,
the “donor,” must have an emission spectrum
overlapping the excitation spectrum of the second fluorophore, the “acceptor.” In addition,
FRET requires that the two fluorophores be
very close. The amount of FRET, or its efficiency E, is given by E = R0 6 /(R0 6 + r6 ),
where r is the distance between the two fluorophores and R0 is the distance at which 50%
energy transfer takes place. The R0 value is
fluorophore dependent, and is determined by
the amount of overlap between the excitation
and emission spectra of the donor and acceptor, the relative orientation of the donor
and acceptor, and the “brightness” or quantum yield of the donor. The R0 value for the
donor-acceptor CFP/YFP FRET pair is 4.92
nm (Patterson et al., 2000). Other fluorophore
pairs yield R0 values in the range of 2 to 6
nm. Owing to the sixth-power dependence on
the spatial separation, r, the FRET efficiency
drops dramatically with increase in r beyond
R0 , so FRET can detect only nanometer-scale
apposition of fluorophores.
Detecting
Protein–Protein
Interactions with
CFP-YFP FRET
by Acceptor
Photobleaching
FRET applications
The advent of GFP derivatives suitable for
FRET has stimulated a resurgence of interest
in this technique. Before the GFP era, FRET
required either direct labeling of proteins with
donor or acceptor or indirect labeling using
antibodies conjugated to the donor and acceptor. Direct labeling is a laborious procedure
that requires protein isolation, dye conjugation, purification, and incorporation into the
cell. Indirect antibody labeling is considerably
simpler, but the size of the antibody molecules
can place the donor and acceptor at distances
that preclude FRET. The advantages of GFP
fusion technology for FRET are (1) the direct labeling of the proteins of interest; (2) the
relative simplicity of constructing the GFP fusions and incorporating them into cells; and (3)
the ability to perform FRET measurements in
live cells and thereby examine the dynamics
of protein-protein interactions.
GFP labeling technology and FRET have
now been applied to a number of important
problems. These include investigations of
where and when different proteins interact
within cells, such as the dimerization of receptors on plasma membranes (Cornea and Conn,
2002) and the interactions of nuclear transport
factors and nuclear pore proteins (Damelin and
Silver, 2000). Special FRET-based indicator
molecules have also been developed to detect specific protein modifications or changes
in molecular conformation (reviewed in Pertz
and Hahn, 2004, and Zaccolo, 2004).
Despite these and other successes, FRET
is far from routine. Success hinges on optimal choice of the fluorophore pair and optimal orientation of the two fluorophores within
the protein complex. The second condition depends in part on chance, and therefore can fail
even when the proteins in question are interacting. Thus, a negative result in FRET does
not mean that the proteins do not interact. The
one exception to this rule of thumb is if FRET
is present but then disappears following some
cellular perturbation. In this special case, the
absence of FRET is meaningful.
Techniques for FRET measurement
FRET can be detected by three different
light microscopy approaches: (1) FLIM (fluorescence lifetime imaging microscopy); (2)
sensitized emission; and (3) photobleaching.
FRET by FLIM (Gadella and Jovin, 1995)
provides perhaps the most sensitive approach
for FRET detection, but also requires the most
complicated instrumentation, which is still not
widely available. This approach is based on
measurement of the fluorescence lifetime of
the donor. The lifetime is the time that a
molecule remains in the excited state before
returning to the ground state. When FRET
occurs, the donor fluorescence lifetime decreases. This can be detected with special instrumentation.
In FRET by sensitized emission, the donor
is excited and the increased fluorescence of
the acceptor is queried. This method can
be performed on conventional wide-field or
confocal microscopes, but requires somewhat
complicated correction and normalization procedures. These arise from the fact that three
independent processes contribute to fluorescence in the acceptor emission channel: (1) fluorescence of the acceptor as a result of FRET;
(2) fluorescence of the acceptor induced inadvertently by light intended to excite the donor;
and (3) bleeding of donor emission into the acceptor emission channel. To filter out the second and third effects, correction procedures are
applied. Several competing algorithms for this
purpose are available, and debate continues
about which is the best (Youvan et al., 1997;
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Current Protocols in Cytometry
Gordon et al., 1998; Xia and Liu, 2001; Elangovan et al., 2003).
In the third approach, bleaching of either
the donor or the acceptor is used to detect
FRET (Jovin and Arndt-Jovin, 1989; Kenworthy, 2001; Karpova et al., 2003; Agresti
et al., 2005). Like sensitized emission, these
approaches also have the advantage that they
can be performed on conventional wide-field
or confocal microscopes. Of these two photobleaching approaches to FRET, the easier to
interpret is acceptor photobleaching, in which
the acceptor is totally bleached and the donor
fluorescence before the bleach is compared to
that after the bleach (reviewed in Kenworthy,
2001). If FRET was present before the photobleach, then the donor fluorescence should
increase after the photobleach, since there is
no longer a flow of excited-state energy from
the donor to the acceptor. In principle, this
procedure requires no corrections, since only
the donor channel fluorescence is measured;
bleed-through from the acceptor is negligible
under normal conditions and virtually nonexistent after the photobleach. In practice, the authors have found that a correction for “pseudo
FRET” is required owing to an increase in
donor fluorescence that sometimes occurs for
unknown reasons after a photobleach, even
when no acceptor is present (Karpova et al.,
2003). A second disadvantage of this photobleaching approach is that it typically can be
applied only once in a live cell. The reason is
that most proteins exhibit fairly rapid turnover,
and so photobleached acceptor molecules will
be replaced by nonbleached acceptors from
other parts of the cell. In this situation, FRET
by acceptor photobleaching requires that acceptors throughout the entire cell be bleached
to ensure no fluorescence recovery; as a result, time-lapse FRET measurements become
impossible.
The protocol presented in this unit for
FRET by acceptor photobleaching is adaptable to scanning confocal microscopes with
an argon laser. These microscopes are the most
convenient systems to perform this technique
because a laser enables fast (a few seconds)
and targeted (ROI) bleaching. Bleaching with
a mercury or xenon arc lamp (Kenworthy et al.,
2000; Llopis et al., 2000; Day et al., 2001)
often requires several minutes, during which
time the cells could move out of focus. The
confocal system also enables bleaching in a
series of different-shaped ROIs, enabling more
straightforward FRET analysis in different cellular compartments and/or direct comparison
of experimental and control regions in the
same cell.
Critical Parameters and
Troubleshooting
Dual imaging of CFP and YFP
Although CFP and YFP are convenient labels, their spectra overlap considerably more
than is optimal for FRET, and this can lead
to artifacts in FRET experiments. Therefore,
it is essential to eliminate bleed-through between the CFP and YFP channels. This can
be accomplished first by judicious choice of
the emission filters and beam splitter (steps 8
to 17) and second by tuning the detector gain
and offset (steps 19 to 25).
Once the gain and offset parameters are set,
they should not be changed midway through
the experiment, otherwise it will be impossible
to make comparisons among the experimental
data and the positive and negative controls.
To avoid this, it is often necessary to initially
examine all five slides (a, b, c, d and e) and establish the range of intensities that are shared
by at least a subset of cells on each slide. Imaging cells in this common intensity range then
makes it possible to maintain the same gain
and offset throughout the experiment.
Laser power and AOTF settings
A compromise must be struck between two
competing factors that influence on the choice
of laser power and the transmission throughput
of the AOTF. These competing factors are the
ability to rapidly bleach the acceptor without
bleaching the donor and the ability to collect
images after the bleach with minimal additional photobleaching.
There are two types of undesirable photobleaching, each detectable by straightforward
procedures. First, unintentional bleaching of
the donor during the YFP acceptor photobleach can be recognized if EF < CF for the
unconjugated CFP slide (also see Anticipated
Results). Second, acquisition photobleaching
can be recognized by plotting the average intensities for the CFP and YFP channels as a
function of time (timepoints, n = 1 to 10). This
plot will always show some decrease in intensities, but if more than a 25% drop in intensities
occurs over a span of ten images, then there is
significant photobleaching during acquisition.
If both of these unintentional photobleaching effects are observed, the laser power can
be reduced to a level where the acceptor
is still rapidly bleached but both inadvertent
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Molecular
Imaging
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Current Protocols in Cytometry
Supplement 35
bleaching of the donor and acquisition photobleaching become minimal. If only donor photobleaching is detected, then the AOTF setting
during the photobleach can be reduced. If, on
the other hand, only acquisition photobleaching is detected, then the AOTF transmission
during imaging can be reduced.
Detecting
Protein–Protein
Interactions with
CFP-YFP FRET
by Acceptor
Photobleaching
Pseudo FRET
Once unintentional bleaching of the donor
has been significantly reduced (see preceding
section), then the observation of EF < CF in
any of the four slides (a, b, c, and e) indicates that an unbleached region of the specimen shows an increase in donor fluorescence
resulting from a bleaching at some other location in the same specimen. This is evidence
for “pseudo FRET” (Karpova et al., 2003), a
fairly common occurrence in the authors’ experience whose underlying cause remains unknown. The most telling diagnostic for pseudo
FRET comes when EF < CF for the positive
control (the CFP-YFP fusion), since, in this
case, FRET is expected and therefore must
yield a larger value for EF . However, this
pseudo FRET behavior can be sporadic and
emerge in only a subset of slides. Although
in the negative controls EF and CF should be
similar, there is again no reason for CF to be
significantly larger than EF . Therefore, when
this is detected on any of the slides, caution
must be exercised in interpreting the data.
At present, the cause of pseudo FRET is
unknown, so it is difficult to eliminate it. However, it might arise from some feature of the
mounting medium or the fixation procedure,
so one option is to alter these variables and
see if that eliminates the behavior. Another
possibility is that the phenomenon arises from
to local heating effects (Sylvain Costes, pers.
comm.), so an option here is to try a chamber
with a larger aqueous volume that might dissipate local heating effects more quickly. It must
be noted, however, that this pseudo FRET effect is distinct from what has been termed false
FRET. The latter arises from the photoconversion of YPF to a CFP-like molecule following
excitation with 514-nm light (Valentin et al.,
2005). False FRET only can occur at the site
of photobleaching, whereas FRET occurs both
at the site of bleaching and beyond it. Under
the authors’ imaging conditions and with the
specimens tested in the authors’ laboratory, the
false FRET effect is negligible, as assayed by
measuring the change in fluorescence in the
CFP channel for cells containing only YFP.
No FRET
If EF is not significantly larger than CF for
the experimental slide (see Anticipated Results), then FRET was not detected between
Protein1 and Protein2. This could indicate that
these proteins do not interact, but, as noted
above, it could also mean that the FRET procedure was not optimized. Two general tactics
for optimizing FRET are manipulating the fluorophores or attempting an alternative FRET
measurement.
Perhaps the simplest approach to increasing
FRET sensitivity is to increase the levels of the
fluorophores. Since the increase in donor fluorescence after acceptor photobleaching is generally a relatively small fraction (often 10% or
less) of the initial donor fluorescence, brighter
cells will give rise to a larger absolute increase relative to the noise background. Thus,
transfection levels can be increased to obtain
brighter cells. In this case, however, it is important to check whether the transfected proteins are still properly localized. High levels of
expressed proteins can produce artifactual interactions of the proteins or can even alter normal cellular processes, so these caveats must
be kept in mind when increasing the protein
expression levels.
A failure to detect FRET might also indicate
that the CFP and YFP molecules are too widely
separated, even though the proteins to which
they attach do interact. Thus, different fusion
sites for the CFP and YFP moieties should be
tried, namely C- and N-terminal fusions for
each of the proteins. Sometimes only one of
these four possible combinations will produce
detectable FRET. FRET efficiencies may also
be increased by adding a flexible linker between the proteins and the fused CFP or YFP
moieties. Even though linkers may increase the
distance between the fluorophores, the added
rotational freedom of the fluorophores could
enhance the chances for FRET.
In addition to the CFP-YFP FRET pair,
other fluorophores can also be tested. One possibility is to use GFP and a monomeric DsRed
derivative—one of the mRFP variants such as
mCherry (Shaner et al., 2004)). Like CFP and
YFP, there is considerable overlap in the GFP
and mRFP spectra, so these fluorophores will
also require careful implementation of the dual
imaging conditions. Evidence for FRET between these partners exists as detected by both
acceptor photobleaching and sensitized emission (Yang et al., 2005).
The acceptor photobleaching approach has
also been demonstrated for another pair of
12.7.8
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FRET partners, Cy3 and Cy5 (Kenworthy,
2001). This pair is well suited to FRET
measurements, but complications arise in introducing these labels into cells. The most
straightforward approach is to examine fixed
cells in which antibodies to the proteins of interest are available. These primary antibodies
can be labeled with Cy3 and Cy5, and this may
yield a positive FRET result. Direct labeling
of the proteins under study would be even better, but this involves significantly more labor
to isolate, tag, purify, and then reintroduce the
labeled proteins into cells.
The second general tactic to consider if
no FRET is detected is to employ an altogether different FRET detection procedure.
The virtue of the acceptor photobleaching approach is its relative simplicity, but a drawback of this approach is that the occurrence
of pseudo FRET can reduce the sensitivity of
the technique. An alternative microscopy approach to FRET, fluorescence lifetime imaging, is likely to be more sensitive than acceptor photobleaching, and also more suitable
for examining temporal changes in live cells
(Wallrabe and Periasamy, 2005). The instrumentation for lifetime imaging is still not
commonly available, but a number of such setups do exist. Sensitized emission is another
alternative that has the advantage that it can
be accomplished with more conventional instrumentation such as a confocal microscope.
Indeed, both Zeiss and Leica confocal systems now offer software for performing the
normalization and bleed-through corrections
required for sensitized-emission FRET. To the
authors’ knowledge, careful comparisons of
acceptor photobleaching and sensitized emission have not been performed, so it is not clear
whether any improvement in sensitivity is expected with the sensitized-emission approach.
Anticipated Results
The authors recommend a four-step procedure to evaluate whether or not FRET has been
detected.
The first step is to examine the results with
the positive control, CFP fused to YFP. This
slide should yield EF significantly greater than
CF , as determined by Student’s t test. If EF is
less than CF , this indicates inadvertent donor
photobleaching by the 514-nm laser line (also
see Critical Parameters and Troubleshooting).
If EF is approximately equal to CF , this suggests a problem with either the microscope
configuration or the FRET analysis procedure;
when properly implemented, the protocols in
this unit normally yield a positive FRET signal
for this fusion protein.
The second step is to examine the results
with the negative control for “random” FRET
owing to the mix of CFP and YFP. EF for
this mix should be significantly less than EF
for the positive control (the CFP-YFP fusion).
This indicates that, as expected, there is significantly more FRET when the two molecules
are fused than when the two molecules are separate. EF for this CFP and YFP mixture should
also be compared to CF for the same mixture. EF for this mixture, although less than
EF for the CFP-YFP fusion, is expected either to be equal to or somewhat greater than
CF for the mixture. A somewhat larger value of
EF compared to CF is expected here, since random interactions of CFP and YFP and/or weak
dimerization between them can arise and lead
to a small amount of FRET.
The third step is to examine the measurements for the unconjugated CFP slide. Here,
the expected result is that EF will be about
the same as or somewhat less than EF for the
mixture of unconjugated CFP and YFP. The
reason is that no FRET is expected in the unconjugated CFP slide, and only weak FRET,
at best, is expected in the mixture of unconjugated CFP and YFP. In addition to this comparison between slides, it is also useful to compare
EF from the unconjugated CFP slide with CF
from the same slide. These two values should
be similar because FRET should be impossible in either case (i.e., with or without a photobleach). However, sometimes it is found that
EF (measured from the photobleached region)
is less than CF (measured from the nonphotobleached region). This will arise if the photobleach of the acceptor was too strong, leading to inadvertent photobleaching of the donor
and therefore to artifactual reduction of EF (see
Critical Parameters and Troubleshooting).
The fourth and final step is to examine the
experimental slide (Protein1-YFP, Protein2CFP). If the two proteins are interacting, then
FRET should occur, and EF from the experimental slide should be significantly larger than
EF for the mixture of CFP and YFP. At the
same time, EF for the experimental slide is in
general expected to be less than EF for the
positive control (CFP-YFP fusion), since this
positive control yields rather strong FRET. Finally, EF for the experimental slide should also
be significantly larger than EF for the unconjugated CFP slide, since this slide is a second
negative control for the absence of FRET. If
all these conditions are met, then there is good
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evidence that the proteins in question do indeed interact.
Time Considerations
FRET by photobleaching tests may be performed in 4 hr, spending 1 hr on each cell
line. It is advisable to conduct three separate
4-hr sessions to obtain three independent repetitions of the same experiment.
Acknowledgments
This research was supported by the intramural program of the NIH, National Cancer
Institute, Center for Cancer Research. The authors thank Amrie Grammer, LiuSheng He and
Peter Lipsky (NIH, NIAMS) for their suggestions in the development of this protocol.
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Contributed by Tatiana Karpova and
James G. McNally
National Institutes of Health
Bethesda, Maryland
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