Real-time measurements of protein dynamics using fluorescence activation-coupled protein labeling method

Article abstract of DOI:10.1021/ja200225m

We present a fluorescence activation-coupled protein labeling (FAPL) method, which employs small-molecular probes that exhibit almost no basal fluorescence but acquire strong fluorescence upon covalent binding to tag-proteins. This method enables real-time imaging of protein labeling without any washout process and is uniquely suitable for real-time imaging of protein dynamics on the cell surface. We applied this method to address the spatiotemporal dynamics of the EGF receptor during cell migration.

Full text of DOI:10.1021/ja200225m

ARTICLE
pubs.acs.org/JACS
Real-Time Measurements of Protein Dynamics Using Fluorescence
Activation-Coupled Protein Labeling Method
Toru Komatsu,†,^ Kai Johnsson,§ Hiroyuki Okuno,‡,^ Haruhiko Bito,‡,^ Takanari Inoue,|| Tetsuo Nagano,†,^
and Yasuteru Urano*,†,‡
†Graduate School of Pharmaceutical Sciences and ‡Graduate School of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku,
Tokyo, 113-0033, Japan
§Institut des Sciences et Ingꢀenierie Chimiques, Ecole Polytechnique Fꢀedꢀerale de Lausanne, CH-1015, Lausanne, Switzerland
Department of Cell Biology, Center for Cell Dynamics, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205,
United States
^JST CREST, Sanbancho-bldg., 5 Sanbancho, Chiyoda-ku, Tokyo, 102-0075, Japan
S Supporting Information
b
ABSTRACT: We present a fluorescence activation-coupled
protein labeling (FAPL) method, which employs small-molecular
probes that exhibit almost no basal fluorescence but acquire
strong fluorescence upon covalent binding to tag-proteins. This
method enables real-time imaging of protein labeling without
any washout process and is uniquely suitable for real-time
imaging of protein dynamics on the cell surface. We applied
this method to address the spatiotemporal dynamics of the EGF
receptor during cell migration.
’ INTRODUCTION
visualization and quantification, thus rendering impossible
continuous monitoring of the dynamics of a specific pool of
proteins. To overcome this generic problem, we have devel-
oped a novel fluorescence activation-coupled protein labeling
(FAPL) probe based on the SNAP-tag labeling system. A small-
molecular probe was designed so that its fluorescence is
dramatically activated (affording an extremely high signal-to-
noise ratio) concomitantly with selective labeling of a tag-
protein. Such properties have been recognized as desirable
since the first development of protein labeling probes2,13,14 but
have not been attained in current systems, for which a
washout process remains essential due to lack of specificity or
incomplete fluorescence quenching. For example, a protein
labeling probe that showed fluorescence activation upon
labeling of SNAP-tag protein has been reported,15 but the
fluorescence activation, which was based on guanine-based
quenching, was as low as 30-fold and the background fluores-
cence was not negligible. Here, we have developed FAPL
probes for the SNAP-tag protein based on the F€orster re-
sonance energy transfer (FRET)13,16 principle (Figure 1a).
The design concept has been used to obtain probes for vis-
ualization of physiological protease activities,17 but our aim
here was to achieve a sufficiently high reaction rate, high
fluorescence activation ratio, as well as high specificity toward
a generally used tag-protein without physiological functions.
Understanding protein dynamics is critical to decipher the role
of proteins in specific cellular events that underlie sophisti-
cated living systems. Detection and visualization of protein
movements, in particular, has great potential to uncover
dynamic protein functions.1 To this end, expression of
genetically encoded fusion proteins tagged with fluorescent
proteins (FPs) and, more recently, novel chemistry-based
strategies to label and monitor specific tag-proteins have
proved powerful.2ꢀ5 For example, FlAsH,2 which binds to a
tetracysteine motif, and O6-benzylguanine (BG) derivatives,6
which form a covalent bond with a tag-protein, O6-alkylguanine-
DNA-alkyltransferase (SNAP-tag), are frequently used in
protein visualization experiments. A major advantage of such
small molecule-based labeling is the enormous potential for
adjusting the functions of the probes as required by means of
chemical modification.3,7ꢀ9 A major added value is that one can
precisely control the location10,11 and timing12 of labeling
by appropriate regulation of probe delivery. For example,
selective visualization of a cell-surface pool of proteins can be
achieved by bath application of membrane-impermeable
protein labeling probes.10 However, there remains one major
caveat associated with all currently available protein labeling
techniques, namely, the inability to directly detect labeled
protein during the labeling reaction, which occurs in the
presence of an excess of unreacted or nonspecifically bound
probe. For that reason, an extensive washout procedure to
remove excess unlabeled probe is a prerequisite for protein
Received: January 10, 2011
Published: April 07, 2011
r
2011 American Chemical Society
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Figure 1. (a) Design of fluorescently activatable SNAP-tag labeling probe. Images of solution before (left) and after (right) labeling under excitation
with a UV lamp (365 nm) are shown. (b) Chemical structure of DRBG-488. (c) Fluorescence spectra of DRBG-488 (1 μM) in phosphate buffer
(100 mM, pH 7.4) before and after addition of SNAP-GST (1.5 μM). (d) The result of CBB staining and fluorometric analysis of the SDS-PAGE gel of
SNAP-GST (43 kDa, 0.5 μM) and BSA (66 kDa, 2 μM) incubated with DRBG-488 (2 μM) for 90 min.
Figure 2. Kinetic study of SNAP-tag labeling with DRBG-488. (a) Fluorescence increase of DRBG-488 (2 μM) in labeling of SNAP-tag at various
protein concentrations (50ꢀ250 nM). (b) Fluorescence increase of DRBG-488 (1ꢀ5 μM) in labeling of SNAP-tag (50 nM). (c) Plot of probe
concentration versus calculated k0 (= k[S]), showing a linear relationship.
By careful design, we have developed FAPL probes that meet
these criteria, allowing practical labeling of general-tag protein in
real-time without any washout. We confirmed that these probes
are versatile research tools to study protein dynamics.
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’ RESULTS AND DISCUSSION
The SNAP-tag forms a covalent bond with BG derivatives by
nucleophilic attack at the active site cysteine, resulting in release
of the guanine moiety.6 In designing our probes, we had to decide
which position of the guanine moiety would be most suitable for
attachment of the fluorescence quencher, so we examined the
structureꢀactivity relationship in the labeling reaction. Crystal-
lographic studies of O6-benzylguanine-bound SNAP-tag protein
have shown that the N-1, N-3, N-7, and O-6 positions of O6-
alkylguanine should be preserved for substrate recognition of the
enzyme.6,18,19 Therefore we compared the reaction rates of C-8
and N-9 modified O6-benzylguanine derivatives and found that
modification at the C-8 position was superior from the viewpoint
of fast labeling of SNAP-tag (Figure S1 and S2 in the Supporting
Information). On the basis of this result, we designed and
synthesized a fluorescently activatable SNAP-tag labeling probe
in which the fluorophore is modified at the benzyl moiety and the
quencher is modified at the guanine moiety via a linker attached
at the C-8 position. The synthesis was done via a fully protected
benzylguanine scaffold with appropriate linkers for fluorophores
and quenchers. From this synthetic intermediate, one can modify
the components in the order of quencher motif to fluorophore
motif (Scheme S1 in the Supporting Information) or vice versa
(Scheme S2 in the Supporting Information). Both synthetic
routes gave similar yields in this case. Here, we selected disperse
red-1 as a quencher and chose two fluorophores, Alexafluor-488
and fluorescein, to synthesize probes named DRBG-488 and
DRBGFL, respectively (Figure 1b). Both fluorophores show
fluorescence at similar wavelengths (around 500 nm), but the
design goals are different in terms of probe delivery. In the former
probe, Alexafluor-488 has multiple negative charges which
render the probe completely membrane-impermeable,9 while
in the latter, fluorescein can be modified with diacetyl pro-
tection (DRBGFL-DA) to obtain a cell-permeable probe.6 Both
DRBG-488 and DRBGFL showed almost no fluorescence,
demonstrating high FRET efficiency (>99%), and were comple-
tely stable in the absence of SNAP-tag protein. However, after
SNAP-tag protein was labeled, fluorescence rapidly appeared,
with a fluorescence activation ratio of over 300 (Figure 1c and
Figure S4 in the Supporting Information). We observed no
background labeling of excess amounts of other proteins in the
system, confirming that the labeling and fluorescence activation
were completely selective for SNAP-tag protein (Figure 1d). The
labeling reaction of DRBG-488 proceeds via pseudofirst order
kinetics, and the time required to achieve 50% labeling of protein
(τ1/2) was calculated to be about 6 min when we used 5 μM
DRBG-488 (Figure 2). Among protein labeling probes that have
so far been reported to achieve fluorescence activation,2,13,14 this
labeling system is far superior in speed and selectivity of labeling
as well as in the ratio of fluorescence activation and is therefore
expected to offer great advantages in practical use.
Figure 3. (a) Comparison of FAPL probe (DRBG-488, bottom) and
conventional protein labeling probe (BG-Alexa488, top). (N) SNAP-
EGFR-expressing COS7 cells were incubated with conventional protein
labeling probe BG-Alexa488 (1 μM) or FAPL probe DRBG-488
(2 μM). For BG-Alexa488, cells were washed with PBS three times
before fluorescence images were taken. The quantitative fluorescence
values are summarized in Figure S6 in the Supporting Information. (b)
Overlay of confocal fluorescence images of MDCK cells expressing
SNAP-EGFR-CFP after 1 h of labeling with DRBG-488 (2 μM). Green
= YFP channel (DRBG-488), red = CFP channel (ECFP). A vertically
scanned fluorescence image taken at the position indicated by the white
line is also shown, along with a line graph of fluorescence intensity. (c)
Overlay of confocal fluorescence and DIC images of MDCK cells
expressing cytosolic SNAP-tag after 3 h of labeling with DRBGFL-
DA. A vertically scanned fluorescence image taken at the position
indicated by the white line is also shown. (d) CBB-staining and
fluorescence images of cell lysate from (N) SNAP-EGFR-expressing
cells labeled with DRBG-488. A red arrow indicates the expected
molecular weight of (N) SNAP-EGFR.
both normal and tumor cells.21ꢀ24 We therefore sought to scrutinize
the relationship between its dynamics and biological functions.
We first confirmed that incubation with cell-impermeable
DRBG-488 allowed immediate visualization of (N) SNAP-
EGFR without any washout process (Figure 3a and Figure S5
and Supplementary Movie 1 in the Supporting Information).
This is in sharp contrast with conventional protein labeling with a
nonactivatable dye (BG-Alexa488), for which a lengthy post-
incubation washout period was required before effective visuali-
zation was possible. The labeling reaction was completely
selective for SNAP-tagged EGF receptor (Figure 3d) and
occurred specifically on the cell surface (Figure 3b and Figure
S7 in the Supporting Information). Immunofluorescence stain-
ing indicated that our method could visualize SNAP-tagged EGF
receptor expressed at a level comparable to that of physiological
To establish the utility of these probes for live cell imaging, we
applied DRBG-488 and DRBGFL-DA to label and visualize SNAP-
tag expressed on the cell surface or in cytosol, respectively. As a
model extracellular SNAP-tag protein, we prepared a SNAP-tagged
EGF receptor, named (N) SNAP-EGFR, in which SNAP-tag
protein was inserted after the signal sequence, resulting in a
N-terminal SNAP-fusion of the mature EGF receptor.20 The EGF
receptor is a cell-surface tyrosine-kinase receptor for growth factors
such as EGF and transforming growth factor (TGF)-R and has well-
established roles in proliferation, differentiation, and migration of
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Figure 4. (a) The concept of a membrane trafficking study with use of FAPL probes. (b) The costaining image of (N) SNAP-EGFR-expressing COS7
cells labeled with DRBG-488 (2 μM, green) and lysosome marker, lysotracker (50 nM, red), in the presence of EGF (100 nM). (c) Confocal
fluorescence images of SNAP-EGFR-CFP-expressing MDCK cells labeled with DRBG-488 (2 μM) for 2 h. Fluorescence images at CFP channel (top,
ECFP) and YFP channel (bottom, DRBG-488) are shown. EGF (100 ng/mL) was added at 5 min, and fluorescence images were taken every 1 min.
Formation of small vesicles right after EGF stimulation (white arrowhead) and accumulation to lysosomes (arrow) were more clearly visualized in FAPL-
based imaging. See Supplementary Movie 2 in the Supporting Information for details.
Figure 5. Application of FAPL probe for cell migration study. (a) Fluorescence images of SNAP-EGFR (labeled with DRBG-488) during MDCK cell
migration induced by uniform EGF stimulation. Vertical line (dotted) represents the start position of migration, and horizontal line represents the axis
on which kymograph analysis in part b was performed. See Supplementary Movie 3 in the Supporting Information for details. (b) The result of
kymograph analysis of SNAP-EGFR (green) and membrane marker CFP-CAAX (red). Internalization of SNAP-EGFR was observed during cell
migration, mainly from the rear of the cell (dotted parallelogram). Ruffle formation was also observed during 10ꢀ30 min (white arrowhead). One
representative image is shown as an overlay of SNAP-EGFR (green) and CFP-CAAX (red) fluorescence (t = 18 min). (c) Fluorescence intensities of
newly formed SNAP-EGFR vesicles at the front and rear of cells, with respect to direction of motion. On the kymograph, the region of interest (ROI, 2.5
mm ꢁ 10 min) for measurement of fluorescence intensities was set inside cells adjacent to the membrane at the front and rear of cells. Fluorescence
intensities were normalized to the average fluorescence intensity at 0ꢀ10 min. Repeated-measures ANOVA showed a significant difference between the
rear and front sides (/, P < 0.05). Data are averages of 10 cells that showed typical migration. Error bar represents the standard error (SE).
EGF receptor (Figure S6 in the Supporting Information). We
confirmed that labeling occurred with pseudofirst order kinetics,
just as in the in vitro experiments, and the calculated kinetic
constant was about half of that in the in vitro experiments
(Figures S8 and S9 in the Supporting Information). We assume
this difference might arise from the structural difference of
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Figure 6. (a) A schematic view of real-time pulse-chase labeling to monitor protein newly delivered to the cell surface. (b) The result of real-time pulse
(BG-Alexa546)-chase (DRBG-488) labeling of (N) SNAP-EGFR-expressing HEK293 cells. (c) Fluorescence ratio (green/red) increase of control cells
(black) and ATP-starved (red) cells in real time pulse-chase labeling. Error bar represents SE (n = 5 for the control and n = 3 for FCCP-treated cells).
(d) The result of pulse-chase labeling of HEK293 cells pretreated and coincubated with protein synthesis inhibitors. Pulse-chase labeling was performed
with pretreatment (90 min) and coincubation with cycloheximide (70 μM), anisomycin (20 μM), or rapamycin (20 μM). Cells were washed and fixed,
and the fluorescence ratiometric image (green/red) was taken with a confocal microscope.
soluble SNAP-tag protein and (N) SNAP-EGFR and can be
optimized in future studies. However, the speed of labeling was
still fast enough to study cell-surface protein dynamics, which
usually occurs in time scales of hours.23 Similarly, membrane-
permeable DRBGFL-DA could readily label cytosolic SNAP-tag
protein, producing a rapid and substantial rise in cytosolic
fluorescence (Figure 3c). Thus, our FAPL probes should be
applicable to a wide range of target proteins regardless of their
location, i.e., either inside or outside of cells. In both cases,
protein visualization was achieved simply by incubating cells with
probes, requiring no washout process.
We expected that the use of membrane-impermeable
DRBG-488 to visualize protein labeling only on the cell surface
would allow us to design a system to visualize protein translo-
cation across the cell membrane, such as endocytosis or
exocytosis (Figure 4a).25 These cellular events are important
to control surface concentrations of receptors and to mediate
cellular responses to physiological ligands.24 Since these events
occur dynamically, a real-time protein imaging system should
be highly informative. In the continued presence of the FAPL
probe, surface proteins would be continuously labeled and
protein internalization would be measurable as a net increase
of fluorescent vesicles inside cells (Figure 4b). To test this idea,
we prepared SNAP-EGFR-CFP, in which both a SNAP-tag and
a fluorescent protein are attached to the EGF receptor. In
FAPL-based imaging, a signal was observed only on the plasma
membrane before EGF stimulation, so internalization of en-
docytosed vesicles after EGF stimulation could be directly
visualized and easily quantitated.22 The image was much
clearer than that of fluorescent protein (FP)-based imaging,
since there was no contaminating signal from pre-existing
internal proteins awaiting delivery to the cell surface
(Figure 4c and Supplementary Movie 2 in the Supporting
Information).
Next, we took advantage of the unique features of the FAPL
method to study EGF receptor dynamics during EGF-induced
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cell migration. It is known that derangement of endocytosis of
certain receptors results in impaired migration, which strongly
suggests the importance of controlled endocytosis for proper cell
migration.25,26 Several reports have suggested that EGF receptor
distribution is polarized toward the leading edge after the onset of
migration,24,27 but it has been unclear how such a gradient could
be maintained. We found that, as cells began to migrate upon
EGF stimulation, a dramatic increase of vesicular internalization
of the receptors was observed and newly formed vesicles were
found in significantly greater numbers at the rear than at the front
with respect to the migration direction (P < 0.05, ANOVA)
(Figure 5 and Supplementary Movie 3 in the Supporting
Information). This implies that endocytosis of the EGF receptor
in stimulated cells may be slowed down at the leading edge as
compared to the rear, and this would help maintain an increased
surface distribution of EGF receptor at the leading edge. Alter-
natively, facilitated endocytosis of integrins and related proteins
associated with Rho signaling at the rear of cells have been
reported,28,29 and a similar mechanism may govern the polarized
sorting of EGF receptor during directional migration.
Finally, we demonstrated that the FAPL labeling system is also
applicable to study exocytosis, namely, the surface delivery of
newly synthesized proteins. Together with endocytosis, exocy-
tosis of membrane proteins is an important process to maintain
cell surface protein levels.29,30 To distinguish newly plasma
membrane-inserted proteins from pre-existing ones, pulse-chase
labeling, where new and old proteins are labeled with different
dyes, is classically performed.11 However, because dye washout is
essential, conventional pulse-chase labeling can only measure the
total amount of secreted proteins accumulated during a labeling
period on the scale of 1 h. In contrast, the FAPL method, which
does not require washout, can in principle enable us to monitor
changes of secreted protein in real time (Figure 6a). Indeed,
optimization of pulse-chase labeling conditions allowed us to
observe a time-dependent increase in chase fluorescence
(DRBG-488), in contrast to the constant pulse fluorescence
(BG-Alexa546) (Figure 6b,c and Supplementary Movie 4 in the
Supporting Information). This increase was completely blocked
by ATP starvation and partly inhibited by addition of protein
synthesis inhibitors, such as cycloheximide, anisomycin, and
rapamycin (Figure 6d), thus confirming that a substantial
proportion of the live-imaged surface-delivery events consisted
of insertion of newly synthesized (N) SNAP-EGFR into the
plasma membrane. The fact that protein synthesis inhibitors did
not completely block the fluorescence increase might arise from
the presence of fast (protein-synthesis-independent) recycling
loops of EGF receptor, which are likely to contribute to the
observed changes in cell surface protein levels as well.23
been growing to a powerful technique for analysis of protein
functions.1 Recently, small molecule-based protein labeling
technique has also proved useful in the study, while one short-
coming was the requirement of washout of excess probes, a step
that has precluded continuous monitoring of protein trafficking
processes with all previously available probes.
To overcome the problem, we have designed and synthesized
new protein labeling probes, which exhibit a strikingly large
fluorescence activation coupled to the labeling reaction, and we
thus obviated the need for any washout step in protein dynamics
study. The key advantages of our method are the almost
complete quenching of fluorescence in the unreacted probe
molecules and the fast and highly selective labeling reaction with
a tag-protein that can be generally used in a wide variety of
experimental platforms. Indeed, the method enabled us to study
the removal and insertion of cell surface receptors as real-time
events and enabled us to understand the spatiotemporal dy-
namics of EGF receptor endocytosis during cell migration.
Further, because our FAPL method can be applied to any target
protein regardless of location inside or outside the cells, it should
make feasible a variety of novel imaging techniques, such as
superhigh-resolution imaging of target proteins inside living
cells32 and high-throughput screening or in vivo imaging17 where
washout is undesirable or impossible. The probe design can be
easily adjusted as required by means of chemical modification,
and thus, we believe this method represents a versatile tool to
analyze dynamic protein behaviors in living systems.
’ ASSOCIATED CONTENT
S
Supporting Information. Structures of key compounds
b
used in the study; experimental conditions; supplementary
methods for chemical synthesis and characterization of com-
pounds; Schemes S1ꢀS3; supplementary experiments and data;
Figures S1ꢀS9; supplementary movies (Supplementary Movies
1ꢀ4; avi and mpg files); supplementary references; and personal
acknowledgement. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
uranokun@m.u-tokyo.ac.jp
’ ACKNOWLEDGMENT
We thank Yayoi Kondo for technical support in DNA and
cellular experiments and Dr. Masanori Osawa for technical
support in soluble protein preparation. We also thank W. R. S.
Steele for careful proofreading of the paper. This work was
financially supported by the Ministry of Education, Culture,
Sports, Science and Technology of Japan (Grant Nos. 20117003
and 19205021 to Y.U., and Specially Promoted Research 22000006
to T.N. and a Global COE Program “Center of Education and
Research for Chemical Biology of the Diseases”). T.N. was also
supported by the Hoh-ansha Foundation. T.K. was a recipient of a
fellowship from Japanese Society for the Promotion of Science.
Mis-translocation of EGF receptors is often observed in
diseases showing uncontrollable cell growth, such as polycystic
kidney disease (PKD) or multiple tumors,31 so the study of EGF
receptor surface-display mechanisms and the screening-based
development of potential regulatory molecules will be helpful to
investigate the pathogenesis and treatment of these diseases. The
ease of use of our labeling method should make it an invaluable
tool for these purposes, and studies along these lines are
under way.
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