Visualization of the Activity of Rac1 Small GTPase in a Cell.

Abstract

Rho family G proteins including Rac regulate a variety of cellular functions, such as morphology, motility, and gene expression. Here we developed a fluorescence resonance energy transfer-based analysis in which we could monitor the activity of Rac1. To detect fluorescence resonance energy transfer, yellow fluorescent protein fused Rac1 and cyan fluorescent protein fused Cdc42-Rac1-interaction-binding domain of Pak1 protein were used as intermolecular probes of FRET. The fluorophores were separated with linear unmixing method. The fluorescence resonance energy transfer efficiency was measured by acceptor photobleaching assisted assay. With these methods, the Rac1 activity was visualized in a cell. The present findings indicate that this approach is sensitive enough to achieve results similar to those from ratiometric fluorescence resonance energy transfer analysis.

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Acta Histochem. Cytochem. 43 (6): 163–168, 2010
doi:10.1267/ahc.10025
© 2010 The Japan Society of Histochemistry and Cytochemistry
Advance Publication
AHC Acta Histochemica et Cytochemica0044-5991 1347-5800Japan Society of Histochemistry and CytochemistryTokyo, JapanAHC10025 10.1267/ahc.10025Regular Article
Visualization of the Activity of Rac1 Small GTPase in a Cell
Morihiro Higashi*1,2, Jianyong Yu*2, Hiroshi Tsuchiya2, Teruyoshi Saito2,
Toshinao Oyama2, Hidetada Kawana3, Motoo Kitagawa2, Jun-ichi Tamaru1 and
Kenichi Harigaya2
1Department of Pathology, Saitama Medical Center, Saitama Medical University, 1981 Kamoda, Kawagoe, Saitama 350–8550,
Japan, 2Molecular and Tumor Pathology, Chiba University Graduate School of Medicine, 1–8–1 Inohana, Chuo-ku, Chiba
260–8670, Japan and 3Division of Pathology, Chiba Cancer Center, 666–2 Nitona, Chuo-ku, Chiba 260–8717
Correspondence to: Morihiro Higashi, Department of Pathology,
Saitama Medical Center, Saitama Medical University, 1981 Kamoda,
Kawagoe, Saitama 350–8550, Japan.
E-mail: mhigashi@saitama-med.ac.jp
*All of these authors contributed equally to this work
30
Received September 1, 2010; accepted November 17, 2010; published online December 23, 2010
© 2010 The Japan Society of Histochemistry and Cy-
Rho family G proteins including Rac regulate a variety of cellular functions, such as mor-
phology, motility, and gene expression. Here we developed a fluorescence resonance
energy transfer-based analysis in which we could monitor the activity of Rac1. To detect
fluorescence resonance energy transfer, yellow fluorescent protein fused Rac1 and cyan
fluorescent protein fused Cdc42-Rac1-interaction-binding domain of Pak1 protein were used
as intermolecular probes of FRET. The fluorophores were separated with linear unmixing
method. The fluorescence resonance energy transfer efficiency was measured by acceptor
photobleaching assisted assay. With these methods, the Rac1 activity was visualized in a
cell. The present findings indicate that this approach is sensitive enough to achieve results
similar to those from ratiometric fluorescence resonance energy transfer analysis.
Key words: Rac1, FRET, cell imaging
I.Introduction
Rac protein is a member of Rho family GTPases and
plays a central role in cell migration by inducing the exten-
sion of lamellipodia [5, 22]. The Rho family GTPases are
thought to act as morphological switches by cycling between
a GTP-bound (active) and GDP-bound (inactive) state. The
activity of these proteins is regulated by the interaction of
Rho family GTPases with guanine-nucleotide exchange
factors (GEFs) and GTPase-activating proteins (GAPs). The
activated small GTPases bind to their effectors. For exam-
ple, the GTP-bound Rac1 bind to p21 protein (Cdc42/Rac)-
activated kinase 1 (PAK) protein through its Cdc42-Rac-
interactive-binding (CRIB) domain [10].
Fluorescence resonance energy transfer (FRET) is a
nonradiative transfer of energy between two fluorophores
that are placed in close proximity and in a proper relative
angular orientation [6]. Variants of green fluorescent protein
(GFP) provide genetically encoded fluorophores that serve
as the donor and the acceptor in FRET. Some difficulties
exist to measure FRET. One of them is spectral bleed
through, i.e. the contribution of donor and acceptor fluores-
cence emission to the FRET channel [2]. Several researcher
groups have developed single-molecule probes to detect
the activity of Rac1, in which the monitor peptides are
sandwiched with the two GFP variants [7, 13–15, 19]. To
visualize the FRET efficiency, the ratio of the intensity of
donors and the acceptors are mapped in an image of a
cell. However, it has been restricted to qualitative or relative
assessments owing to the spectral bleed-through contamina-
tion resulting from fluorescence overlap between the donor
and the acceptor.
We develop a new method to visualize the Rac1 activi-
ty in a cell using a pair of intermolecular probes and spectral
unmixing and acceptor photobleaching assisted FRET assay.
II.Materials and Methods
Cell culture
Human glioblastoma cell line U251MG was obtained

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from American Type Culture Collection (ATCC, Rockville,
MD, USA) and was maintained in Iscove’s modified
Dulbecco’s medium (Invitrogen, Carlsbad, CA, USA), sup-
plemented with 10% fetal bovine serum. The following anti-
bodies were used: anti-Rac1) (BD Biosciences Pharmingen,
San Diego, CA, USA) and, and anti-β-tubulin (Santa Cruz
Biotechnology, Santa Cruz, CA). PDBF-BB was purchased
from Sigma-Aldrich (St Louis, MO, USA)
Plasmids
Rac1 cDNA was cloned in frame with Venus, a mono-
meric yellow fluorescent protein (YFP) as a C-terminal
fusion. Venus was a gift from Dr. A Miyawaki, Riken Brain
Science Institute, and cDNAs for Rac1 (wild type, dominant
negative form and constitutive active form) were gifts from
Dr. Y. Takai, Osaka University. CRIB domain of PAK1
fused to cyan fluorescent protein (CFP) as a C-terminal
fusion. Transfections were performed with Lipofectamine
2000 (Invitrogen) as directed by the manufacturer.
Live cell imaging
Cells transfected with YFP or CFP constructs were
plated on 35 mm-diameter glass-based Petri dishes
(Matsunami Glass Industries Ltd., Tokyo, Japan). Cells were
imaged on a Zeiss LSM 510 META confocal microscope
(Carl Zeiss, Jena, Germany) equipped with temperature and
CO2 controls, an argon laser, a helium/neon and Plan
Apochromat 40× or 63× oil Iris lenses.
Spectral linear unmixing
To separate signals from fluorescent proteins in a cell,
we used a linear unmixing method, as described previously
[25, 26]. Briefly, images were acquired from five spectral
channels simultaneously with a Zeiss LSM 510 META
confocal microscope (63× NA 1.4 oil-immersion objective),
covering important parts of the emission from both fluoro-
phores (477.9–584.9 nm) with a 21.4 nm spectral resolution
at an excitation wavelength of 458 nm. The acquired 4D
image sequence (x, y, time and spectrum) was first back-
ground subtracted and separated to two fluorophores (YFP
and CFP) using the linear unmixing method implemented
in Mathematica software (Wolfram Research, Inc., Cham-
paign, IL, USA). The fluorescence emission spectrum of a
mixed specimen is an addition of the abundance-weighted
spectral response of all constructs. In the two element case
it is expressed by,
Fm(x, y, λ)=a(x, y)Fa(λ)+b(x, y)Fb(λ)
where a(x, y) and b(x, y) are unknown abundance factors of
the two fluorophores at pixel location (x, y), whose spectral
responses are expressed as Fa(λ) and Fb(λ), respectively.
Fa(λ) and Fb(λ) are measured using single fluorophore
specimens. a(x, y) and b(x, y) are estimated using a least-
square fitting. One of the pair of fluorophores was used
for a reference spectra of a fluorophore. For example, in
the u-adFRET assay of YFP-wtRac1 and CFP-Pak-CRIB
pair, we used YFP-wtRac1 for reference of YFP, and CFP-
Pak-CRIB for reference of CFP.
Colocalization analysis
To verify colocalization, time-lapse images were
analyzed with the “colocalization” plugin of ImageJ. The
random or codependent nature were tested using intensity
correlation analysis (ICA) [18] in which the distribution of
the intensity value for each pixel of a channel is plotted
against the product of the difference of the mean (PDM)
of the two channels. The PDM value is expressed as
PDM = (red intensity–mean red intensity) ×
(green intensity–mean green intensity)
The PDM image where each pixel is equal to the PDM
value at that location is pseudocoloured in yellow and the
areas in blue represent the areas of positive and negative
PDM values, corresponding to the presence and absence of
colocalization, respectively.
Rac activation assay (pulldown assay)
pGEX-PAK-CRIB [1] was introduced into the
Rosetta2 (DE3) strain of E. coli, and GST fusion protein
was expressed and purified. Cells were washed with
ice-cold PBS and harvested in lysis buffer (20 mM Hepes-
NaOH, pH 7.9, 300 mM NaCl, 1 mM EDTA, 10 mM
NaF, 15% Glycerol, 0.5% Nonidet P-40, and protease
inhibitor mixture). After lysis for 15 min at 4°C, the samples
were centrifuged at 14,000×g at 4°C. Five hundred μg of
the lysate was mixed with 30 μg of the PAK-CRIB as a GST
fusion protein for 2 hr at 4°C. Then the samples were washed
four times. Finally, the pelleted beads were resuspended in
15 μL of Laemmli’s sample buffer and subjected to SDS-
polyacrylamide gel electrophoresis (15%). Bound Rac1
were detected by Western blotting using the antibodies
against Rac1.
FRET analysis (u-adFRET)
For unmixing-acceptor depletion FRET (u-ad FRET),
cells were fixed in 4% paraformaldehyde in PBS for 20 min
at room temperature, washed with PBS, and mounted in
Mowiol reagent containing 10% Mowiol 4–88 (Calbiochem,
Beeston, UK), 25% glycerol, and 2.5% 1.4-diazabicyclo
[2, 2, 2] octane (Sigma, Poole, UK) in 50 mM Tris/HCl,
pH 8.5. One or two ROIs within a field were acceptor-
photobleached. The 458 nm laser line was used for imaging
as it can excite both CFP and YFP. The 514 nm laser line
was used for the acceptor-photobleaching. The acquired
image series were subjected to the linear unmixing method
and separated images were processed using Mathematica
software according to the algorithm described in the pre-
vious report [11] and below.
FRET efficiency is calculated from the unmixed
images by the following equation,
E = (1 – fd(x, y)/fdph(x, y))100%

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Visualization of Rac1 Activity in a Cell
165
where fd(x, y) and fdph(x, y) are the fluorescence of the
donor before and after photobleaching. Maps of E were
depicted using MetaMorph software (Molecular Devices,
Sunnyvale, CA, USA).
III. Results
Localization of YFP-Rac1 and CFP-Pak-CRIB
We constructed intermolecular probes for Rac1 activity
that consisted of full-length Rac1 fused to YFP (YFP-Rac1)
and CRIB domain of the Pak1 fused to CFP (CFP-Pak-
CRIB). In these probes, we expected that binding CFP-
Pak-CRIB to YFP-Rac1 would bring CFP close to YFP,
increasing FRET from CFP to YFP (Fig. 1A).
First, we used fluorescent microscopy to detect the
subcellular localization of YFP-Rac1 and CFP-Pak-CRIB in
human glioblastoma cell line, U251MG cells. In U251MG
cells, lamellipodial protrusion was clearly observable.
YFP-Rac1 and CFP-Pak-CRIB were localized in the plasma
membrane corresponding to lamellipodia and diffused
throughout the cytosol (Fig. 1B and C). The subcellular
localizations of YFP-Rac1 and CFP-Pak-CRIB were identi-
cal to the localization of endogenous Rac1 or full length
PAK1, respectively, in previous reports [8, 23]. These
results validated the use of YFP-Rac1 and CFP-Pak-CRIB
for monitoring Rac activity in a cell.
Intensity correlation analysis
We next examined a time-lapse colocalization analysis
between YFP-Rac1 and CFP-Pak-CRIB in U251MG cells
in order to detect the interaction in vivo. Images were ob-
tained every thirty seconds for five minutes with a confocal
laser microscope. After acquisition of the spectral images,
YFP and CFP were unmixed using a linear spectral unmix-
ing method to exclude the fluorescence cross-talk of the
fluorophores (Fig. 2A and B). The merged images indicated
that YFP-Rac1 and CFP-Pak-CRIB colocalized at the
lamellipodia (Fig. 2C). To quantify the co-localization of
the proteins, we used intensity correlation analysis (ICA)
[18]. A pseudocoloured image, where each pixel is equal to
the PDM (product from the differences from the means;
Fig. 1.
Distribution of YFP-Rac1 and CFP-Pak-CRIB in U251MG
cells. (A) Schematic representations of YFP-Rac1 and CFP-Pak-
CRIB bound to GDP or GTP. When Rac1 is bound to GDP,
fluorescence of 475 nm emanates from CFP with excitation of 433
nm. When Rac1 is bound to GTP, Pak-CRIB brings CFP into close
proximity to CFP, which causes FRET and fluorescence of 527 nm
from YFP. (B) Distribution of YFP-wild type Rac1. YFP-wtRac1
localizes in the lamellipodia. (C) Distribution of CFP-Pak-CRIB.
CFP-Pak-CRIB localizes in the lamellipodia.
Fig. 2.
Intensity correlation analysis of YFP-Rac1 and CFP-Pak-
CRIB. (A–C) Time-lapse imaging of YFP-Rac1 (A), CFP-Pak-
CRIB (B), merged images (C) upon stimulation of PDGF. (D)
Intensity correlation analysis. PDM plot showed a high codepen-
dency of Rac1 and Pak-CRIB distribution in lamellipodia. (E) Time
sequence of PDM value. After stimuli of PDGF, the PDM value
was increased in about 200 seconds.

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Higashi et al.
166
see Materials and Methods) value at that location (Fig. 2D),
showed a high codependency of YFP-Rac1 and CFP-Pak-
CRIB in the lamellipodia. Upon the stimulation of platelet
derived growth factor (PDGF), the values of PDM within the
membrane were increased at two to five minutes (Fig. 2E).
These results indicate that the colocalization of YFP-Rac1
and CFP-Pak-CRIB was increased in the lamellipodia and
suggest that the CFP-Pak-CRIB binds to the YFP-Pak-CRIB
according to the upregulation of the activity of Rac1.
Stimulation of PDGF enhances Rac1 activity
To confirm that the stimulation of PDGF actually
elicits the activation of Rac biochemically, we performed a
Rac pull-down assay. A GST fusion of Rac/Cdc42 binding
(CRIB) motif of PAK was used to affinity precipitate the
activated form of Rac [1]. U251MG cells were treated
with 20 ng/ml of PDGF and total cell lysates were obtained
at the indicated time. Obtained cell lysates were incubated
with GST-PAK-CRIB and bound Rac1 were detected with
Western blotting using a Rac1 antibody (Fig. 3 upper panel).
Amount of total Rac1 was also detected with Western
blotting (Fig 3. lower panel). As shown in Figure 3, upon
stimulation of PDGF, cells showed increased Rac activity
in two to three minutes after the stimulation (Fig. 3). The
results were comparable to that of ICA analysis, and showed
the activation of endogenous Rac1 by PDGF stimuli.
Imaging of Rac1 activity in U251MG cells
We next detected the protein interaction between
YFP-Rac1 and CFP-Pak-CRIB in vivo by a FRET-based
assay. U251MG cells co-transfected with YFP-Rac1 and
CFP-Pak-CRIB were fixed and used for a quantitative
acceptor-depletion-FRET approach combining linear spec-
tral unmixing (u-adFRET) [9, 11]. In this approach, the
cross-talk of the fluorophores can be excluded. FRET effi-
ciency (E) is calculated from the unmixed donor and ac-
ceptor emission before and after acceptor photobleaching.
Figure 4A shows the example of the time sequence of the
Fig. 3.
Rac activation assay. Active Rac1 was pulled down with a
GST-Pak-CRIB after stimuli of PDGF. U251MG cells were treated
with 20 ng/ml of PDGF. Total cell lysates obtained at the indicated
time were incubated with GST-PAK-CRIB and bound Rac1 were
detected with Western blotting using a Rac1 antibody (upper
panel). Amount of total Rac1 was also detected with Western
blotting (lower panel).
Fig. 4.
Imaging of Rac activity in U251MG cells using a u-adFRET
assay. U251MG cells co-transfected with YFP-Rac1 and CFP-
Pak-CRIB were replated onto glass-bottom dishes. YFP and CFP
images were obtained from spectral images using the linear unmix-
ing method. (A) Example profiles of fluorescence of donor (blue)
and acceptor (yellow) before and after acceptor-photobleaching.
(B) Visualization of the Rac1 activities in cells co-transfected with
the combination of CFP-Pak-CRIB and YFP-fused dominant nega-
tive Rac1 (upper left panel, as negative control) or wild type Rac1
(lower panels) or constitutive Rac1 (upper right panel, as positive
control). (C) Mean FRET. In cells co-transfected with Pak-CRIB
and wtRac1, the FRET efficiency is increased with the stimulation
of PDGF.

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Visualization of Rac1 Activity in a Cell
167
mean fluorescence of the donors and acceptors in the region
of interest (ROI). After acceptor photobleaching (Fig. 4A,
yellow line), donor emission within ROI was increased
(Fig. 4A, blue line), indicating that FRET had occurred.
Maps of FRET efficiency indicated that Rac1 was activated
in the lamellipodia upon the stimulation of PDGF (Fig. 4B).
Statistical analysis showed that FRET efficiency of the cell
stimulated with PDGF was higher than that of control cell
(Fig. 4C).
IV. Discussion
We describe a FRET-based visualization of Rac1 activ-
ity using intermolecular probes as a novel technical assay.
FRET is one of the most useful and widely applied tools in
use today to measure distances on the molecular scale in
cells [16]. A number of trials were done to detect the activity
of Rac1 with FRET-based assay. Most of these used intra-
molecular probes in which FRET pairs consisted of a
single molecule [21]. Intramolecular probes have the advan-
tage to detect the activity in a living cell as the probes
have equal molar amount of YFP and CFP [16]. However,
it requires intensive effort to construct the probes so as to
obtain adequate FRET efficiency as small amounts of FRET
will occur due to the short distance between the fluorophore
pairs in the intramolecular probes. In our system using inter-
molecular probes, by contrast, the probes can be constructed
conveniently and the distance between the fluorophores is
enough to suppress the basement FRET. It is also important
to place the probes at the proper intracellular localization.
In the system using a native Rac1, the subcellular localiza-
tion of the Rac1 is thought to be identical to the endogenous
Rac1 as the CAAX box and the poly-basic region in its C-
terminus, which are important to target membrane localiza-
tion, are intact [14, 24].
To detect FRET rigorously, it is important to increase
signal/noise ratio. The major factor to repress signal/noise
ratio is the spectral bleed through [2]. Using the emission
spectra of specified pure fluorochromes as a reference, the
fluorescence intensity of the corresponding fluorochorme
within the mixture can therefore be precisely determined
from its composite spectrum [26].
Although the acceptor photobleaching method is re-
stricted to the fixed specimen, the assay is applicable to the
detection of the FRET in a tissue section if the appropriate
fluorescent dyes are selected. FRET analysis using a pair of
fluorescent dyes such as FITC and Cy3 [4, 12] or Cy3 and
Cy5 have been reported [17]. Recently, Blanco et al. report-
ed a way to visualize the RNA splicing variants in cells by
a combined method of in situ hybridization and FRET
analysis using Cy3 and Cy5 labeled probes [3]. The activity
of many signaling molecules, such as small GTPases and
tyrosine kinases, can be detected as protein-protein inter-
action [20]. Applications of the assay in the tissue section
might improve the ability of immunohistochemistry methods
to detect the activity of signaling molecules.
V. Acknowledgments
We wish to thank T. Sato for providing pGEX-PAK-
CRIB, A. Miyawaki for Venus and SECFP plasmids, and
M. Matsuda for pRaichu-Rac1. We also wish to thank T.
Umemiya, K. Azuma, K. Matsuno, and T. Nakajo for tech-
nical assistance. This work was supported in part by a Grant-
in-Aid for Scientific Research Priority Area 12215018 from
the Ministry of Education, Culture, Sports, Science and
Technology, Japan (to K. H.), a Grant-in-Aid for Scientific
Research 13670163 from the Japan Society for the Promo-
tion of Science (to K. H.) and Saitama Medical University
grant 21-1-117 from Saitama Medical University (to M. H.).
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