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Introduction
Membrane receptors often play key roles in a large
number of physiological and pathophysiological conditions.
These receptors are the targets of a large number of
therapeutic drugs. Visualizing and tracking receptors stimulated
by agonists in living cells will be helpful in understanding
the molecular mechanisms of receptor activation. However,
by using classic biochemical techniques, it is impossible
to investigate receptor movement in single living cells in real
time, so it is not enough for the study of receptor regulation.
Future research on the dynamic characters of receptor
trafficking will enhance not only our understanding of
receptors in cellular function, but will also be beneficial for novel
drug design and improving treatment of the vast array of
receptor-related conditions.
Real-time microscopy with high-tempo resolution is
believed to have a great impact in the function of
proteins[1]. Recently, single or few molecule detection techniques have
made rapid progress in the research of life sciences. These
techniques have allowed us to record the behavior of
individual particles in real time[2,3]. Since the desensitization and
internalization of receptors have been studied in real-time
imaging in living cells with minute temporal
resolution[4], the challenge now is to investigate the dynamics of receptor
movement in real time or rapidly-occurring events in single
living cells with millisecond resolution. For this goal, good
labeling detection methods are important.
The α1-adrenergic receptors (AR) are members of the G
protein-coupled receptor (GPCR) family. The
α1-AR play a major role in mediating contraction and growth responses in
vascular smooth muscle cells[5]. Three genes encoding
unique receptor subtypes,
α1A-, α1B-, and
α1D-AR, have been cloned and pharmacologically
characterized[6]. According to a past investigation,
α1A -AR express not only on the cellular surface, but also in the
cytoplasm[7]. We then labeled
α1A-AR by the use of a monoclonal anti-FLAG(a kind
of tag) antibody and Cy3-conjugated goat anti-mouse IgG
(Cy3-IgG), and recorded the trajectory of their transport
process by the use of high-tempo resolution
fluorescence imaging techniques stimulated by agonist, phenylephrine (PE).
Using this method, we were able to specifically detect
surface receptors and record the behavior of individual
particles of receptors with 50 ms exposure time in real time in
single living cells. We first obtained trajectories of α1A-AR internalization stimulated by agonist in real time and
analyzed its dynamic, which was difficult to explore by
use of classic biochemical techniques or laser scanning confocal
microscope with minute temporal resolution.
In this study, we developed an approach for labeling the
receptors on the living cell surface and tracked the
internalized receptors in single living cells by the use of high-tempo
resolution fluorescence imaging techniques. This method
can provide new insights into the investigation of
mechanisms and dynamic properties of receptor transport.
Materials and methods
Cell culture, transfection, and selection of stably-expressed
cells FLAG-tagged human α1A-AR in the pDoubleTrouble
vector (pDT; α1A-AR/pDT) was kindly provided by Prof
Kenneth P. MINNEMAN (Emory University, Atlanta,GA,
USA). The human embryonic kidney 293A cells (HEK293A)
were propagated in DMEM(Dulbecco's Modified Eagle's
Medium) (Invitrogen, Carlsbad , CA, USA) with 10%
(v/v) fetal bovine serum (FBS) at 37
oC in a 5% CO2 environment.
Using Lipofectamine 2000 transfection reagent (Invitrogen,
Carlsbad, CA USA) according to the manufacturer's recommendations, the cells were transfected with an
α1A-AR constructor for 24 h. Cells stably expressing
α1A-AR were selected with 800 µg/mL G418 (Sigma, St Louis, MO, USA),
and the expression of α1A-AR was determined by radioligand
binding assays as indicated
previously[8]. HEK293A-α1A cells
were plated at a density of 1×105 cells/mL on coverslips and
maintained in DMEM with 10% FBS for 1_2 d before the
experiments.
Living cell labeling by Cy3-IgG
Living cells were incubated for 10 min at 37
oC in an anti-FLAG monoclonal antibody (12.5 µg/mL, Sigma, USA) and Cy3-IgG (3.75 µg/mL,
dye/protein »3.9; Jackson ImmunoResearch, West Grove,
PA, USA), sequentially. Before the fluorescence experiments,
the cells were washed in PBS (pH 7.4) 3 times.
Calcium response To detect the response to PE by
HEK293A-α1A cells, Fluo-3 (Molecular Probes Inc, Eugene,
OR, USA) was used to indicate the increase of calcium
signal with PE stimulation. The cells were incubated with
medium containing 4 µmol/L Fluo-3 for 60 min at 37
oC before the experiments. After washing 3 times with PBS, the cells
were observed by the epi-fluorescence microscope. PE 10
µmol/L was added as a PE pulse, and at the same time, the
fluorescence from Fluo-3 was detected. The microscope
system used to measure Fluo-3 fluorescence was the same
that was used for the epi-flourescence imaging, and a 488 nm
laser beam (Model 163C, Spectra-Physics, Millennia IIsUSA),
a ×40 objective (NA=0.6, Nikon, Tokyo, Japan), and a
dichroic mirror (505 nm long-pass, Chroma Technology,
Rockingham, VT, USA) were used. The emission
fluorescence through a 535/50 nm band-pass filter was collected by
the same CCD(Charge Coupled Device).
Drug treatments To study the internalization of
α1A-AR under PE stimulation, the cells were incubated with medium
containing 10 µmol/L PE (Sigma, USA) for 20 min before the
experiments. The concentration of PE was maintained in the
cell culture throughout the experiments or PE was added to
the cultures transiently during observation.
Immunofluorescence Living
HEK293A-α1A cells were incubated for 10 min at 37
oC in an anti-FLAG monoclonal antibody (12.5 µg/mL, Sigma, USA) and FITC(Fluorescein
isothiocynate)-conjugated goat anti-mouse IgG (Jackson
ImmunoResearch, USA), sequentially. Then the cells were
incubated with medium containing 10 µmol/L PE (Sigma,
USA) for 20 min. After that, the cells were fixed for 15 min in
4% paraformaldehyde in PBS and permeabilized with 0.2%
Triton X-100. Following washes with PBS, the cells were
incubated with TRITC (Tetramethyl Rhodamine
Isothiocyanate)-labeled phalloidin (Sigma) for 25 min and then washed and
mounted in Vectashield (Vector Laboratories, Burlingham,
CA, USA). Images were acquired using a confocal
laser-scanning microscope.
Laser confocal microscopy The samples were imaged
with a Spectra-Physics (USA) laser scanning confocal
microscope with a Plan-Apo ×60 oil immersion objective lens
(Leica, Wetzlar, Germany). The software used to collect the
images was the Leica TCS NT, version 1.6.587 (Germany).
The images were transferred to a computer for reduction and
analysis with Adobe Photoshop version 4.0 (Adobe Systems,
Mountain View, CA, USA). The setting on the laser was
constant for all experiments. However, both FITC and TRITC
signals were digitally enhanced by adjusting the
photomultiplier tube (PMT). The initial adjustment of the PMT
allowed us to minimize the background signal while
maximizing the fluorescent signals of interest.
Epi-fluorescence imaging The cells were excited with a
532 nm laser beam (Millennia IIs, Spectra-Physics, USA)
which was reflected to the sample by a dichroic mirror (575
nm long-pass, Chroma Technology, USA). The fluorescent
emission from Cy3 was collected by a ×100 objective
(NA= 1.40, oil, Nikon, Japan) and mounted on an inverted
fluorescence microscope (TE300, Nikon, Japan). Through a 605/40
nm band-pass filter or a long-pass 565 nm filter (Chroma
Technology, USA), the scattering light was blocked and Cy3
fluorescent images were obtained with a back-illuminated,
frame-transfer CCD (Cascade 512B, Roper Scientific, Tucson,
Arizona, USA). By using only part of the CCD, the frame
rate could be increased and we successfully measured high
quality point-spread functions at 50 ms time resolution.
Image acquisition, storage, and display were performed using
MetaView software (Universal Imaging Co, Downingtown,
PA, USA) and WinView32 software (Roper Scientific, USA).
The whole observation was conducted at room temperature.
Image analysis Each complex of
α1A-AR and labeling antibody forms a diffraction-limited image. The pixel data of
each spot was least-squares fitted by a 2-D Gaussian
function to obtain the center value[9] with a precision less than 10
nm[10]. The whole fitting was via a user-defined program in
Matlab (MathWorks Inc, Natick, MA, USA).
Statistical analysis Data are given as mean±SEM of at
least 3 individual experiments. Comparisons between means
were performed by using Student's t-test with SPSS
software (SPSS Inc,Chicago, IL, USA ).
Results
α1A-AR were labeled specifically on the surface of living
cells The specific detection of
α1A-AR on the surface of living
HEK293A-α1A cells was achieved by the use of a
monoclonal primary antibody and Cy3-IgG (Figure 1A). Each
complex of α1A-AR and labeling antibody forms a
diffraction-limited image. The individual spots are the images of the
complex of α1A- AR and labeling antibody on the cell surface,
and each spot may contain more than 1
α1A-AR. Under the same brightness, the unspecific adsorption of the primary
antibody and Cy3-IgG in HEK293A cells (Figure 1B), and
without the primary antibody, the unspecific adsorption of
Cy3-IgG in HEK293A-α1A cells (Figure 1C) were very low.
The histograms of the different fluorescence intensity are
shown in Figure 1D, among which, α1A-AR labeled
specifically by the primary antibody and Cy3-IgG in
living HEK293A-α1A-cells are shown in red, the unspecific adsorption of the
primary antibody and Cy3-IgG in HEK293A cells are shown
in blue, and without the primary antibody, the unspecific
adsorption of Cy3-IgG in HEK293A-α1A cells is shown in
green (Figure 1D). So by this method, we can specifically
label the receptors on the living cell surface.
Labeling with the antibody does not affect the activity of
α1A-AR To check the biological activity of
α1A-AR after
labeling with the antibody, 10 µmol/L PE was added to label
HEK293A-α1A cells to induce a calcium response which was
indicated by Fluo-3 (Figure 2A). Two
Ca2+ transient charac-teristics, the amplitudes of
Ca2+ transient increase (presented as a ratio of fluorescence/background fluorescence,
F/F0), and the rate of the
Ca2+ transient upstroke rise (presented as
the change of relative fluorescence intensity per second,
dFI), are obtained from the initial time course of Fluo-3
fluorescence increase in whole cells. Three independent
experiments were measured individually with 20 ms
exposure time per frame. The
F/F0 of labeled cells was
1.86±0.15, and that of unlabeled cells was
1.83±0.07 (mean±SEM). The dFI of
labeled cells and unlabeled cells were
1.70±0.41 and 1.88±
0.26 (mean±SEM), respectively (Figure 2B). These data
demonstrate that labeling with the antibody does not affect
the activity of α1A-AR.
α1A-AR internalize under stimulation of agonist,
PE With the extrinsic fluorescence labeling method, we
monitored individual HEK293A-α1A cells by epi-fluorescence
microscopy. After incubation with PE for 20 min, part of the
particles appeared within the cells (Figure 3B). On the
contrary, if there was a lack of PE stimulation, the
fluorescence spots indicated that α1A-AR stayed on the cell surface
after the cells were immersed in DMEM for 20 min (Figure
3A). To estimate the ratio of internalized
α1A-AR against total α1A-AR, we counted the number of intracellular fluorescent spots
versus the number of whole spots in a focus plane among 39
cells that exhibited internalization from 7 separate
experi-ments. The ratio of α1A-AR internalization within 20 min was
41%±14%. By laser confocal microscopy, we observed that
living HEK293A-α1A cells were labeled with FITC-IgG (green)
and the F-actins were labeled with TRITC-phalloidin (red).
After the cells were stimulated with PE for 20 min, apparent
colocalization was found between α1A-AR and F-actins
(Figure 3C).
Tracking of α1A-AR stimulated by PE in single living
cells in real time An analysis of
α1-AR trajectories in cells in response to PE provides information about the dynamic
properties and mechanisms of receptor transport. To
measure the displacement of α1-AR in living cells, 50 ms
exposure time of stack frames was chosen and a 7×7 pixel array of
each diffraction-limited spot was fitted to a 2-D Gaussian
peak to increase precision of spatial
localization[9]. After 40 min stimulation of 10
μmol/L PE, trajectories of approximate linear motion in
HEK293A-α1A (Figure 4) cells were recorded.
The 4 different colored lines in Figure 4A indicate the
representative trajectories. The arrows in Figure 4B indicate the
beginning points of the representative trajectories. To
display the details of displacement of the labeled
α1A-AR, we enlarged the trajectory plots of
HEK293A-α1A cells (Figure 4C_4F). For all the trajectories of active transport, the
velocity of the representative α1-AR trajectories was from 0.10 to
0.50 µm/s, as shown in Figure 4.
Discussion
The primary antibody and secondary antibody have been
well used in classic biochemical experiments, such as
Western blotting and ELISA. However, this method has been
seldom used in the research of single living cells. Here we
developed this method for tagging living cell surface
proteins with extracellular tags within a total labeling time as
short as 10 min at 37 oC. We incorporated a FLAG tag which
is a short peptide sequence (8 amino acids) into
α1A-AR
extracellular domains. Using this labeling method, we not
only recorded the trajectories in the course of internalization
of receptors in real time, but also analyzed the dynamic
properties of α1A-AR stimulated by PE. This method can provide
some new insights into the investigation of mechanisms and
dynamic properties of receptor transport. Along with the
diverse forms of commercially-available, labeled antibodies,
this method of tagging membrane receptors is a very
versatile approach that can be generally applicable to membrane
proteins.
It is known that GPCR signaling is tightly regulated by a
series of cellular proteins that promote receptor
desensitization and internalization[11]. In an early study performed in
HEK293 cells with a transient transfection of
α1A-AR/GFP(Green Fluorescent Protein), the analysis of the increase of
intracellular fluorescent intensity suggests that the
significant internalization of α1A-AR occurs under stimulation of
agonist PE after 20 min[12]. However, the authors could not
distinguish the internalized receptors from the intracellular
α1A-AR/GFP because it is very hard to distinguish
intracellular pools of GFP-tagged membrane proteins from
extracellular pools in living cells, so the internalization process of
α1A-AR could not be investigated directly. It is also difficult
for tracking single GFP particles in living cells because the
intensity of GFP fluorescence is too weak to detect single
GFP molecules. Auto-fluorescence of the living cell also
increases the difficulty of detecting the single GFP molecule.
Here we developed an approach for labeling the receptors
on the living cell surface and for tracking the internalized
receptors in living cells. With this approach, we were able to
investigate the dynamics of receptor movement in real time
or rapidly-occurring events in living cells with a millisecond
resolution. The labeling is restricted to cell surface proteins
because the antibody is not permeable through the cell
membrane. By this method, we were able to specifically
detect surface receptors, and it was concluded from the
coherence of the calcium response among the labeled cells
and control that the labeling with the antibody did not impair
the receptors' activity. So this kind of specific labeling
method, which labels the receptors on the living cell surface,
can provide a convenient means in the real-time detection of
the behavior of receptors.
Furthermore, using this labeling method, we recorded the
trajectories in the course of internalization of receptors in
real time. The ratio of α1A-AR internalization within 20 min
was 41%±14% (mean±SD). This result is a little higher than
Chalothorn's study mentioned above[12]. The difference
between the 2 experiments could be explained by the
different labeling methods. α1A-AR localize not only on the cell
surface, but also intracellularly[7]. The internalized
a1A-AR/GFP cannot be distinguished from the intracellular or
newly-produced α1A-AR/GFP as in previous studies. Thus, the
newly-produced α1A-AR/GFP and the
α1A-AR/GFP transported to the membrane may interrupt the detection of
internalization quantity. On the contrary, the extrinsic labeling
we used could avoid the influence of intracellular
α1A-AR and reduce the interference from the cell
metabolism[2].
An analysis of α1-AR trajectories in cells in response to
PE provides information about the dynamic properties, and
we can even study the mechanisms of receptor transport.
Stimulated with 10 µmol/L PE for 20 min, apparent
colocalization was found between α1A-AR and F-actin, which
was observed by laser confocal microscopy (Figure 3C). It
indicated that the internalization of
a1A-AR was closely
related to the cytoskeleton. After 40 min stimulation of 10
µmol/L PE, trajectories of approximate linear motion in
HEK293A-α1A cells were recorded. We can calculate the
velocity of the representative α1-AR trajectories which
ranged from 0.10 to 0.50 µm/s. So using this labeling method,
we can not only record the trajectories in the course of
internalization of receptors in real time, but also analyze the
dynamic properties of α1A-AR. In this research, we first
obtained trajectories of α1-AR internalization stimulated by
an agonist in real time that was difficult to explore by
using classic biochemical techniques or a laser scanning confocal
microscope with minute temporal resolution.
In conclusion, the specific labeling method of the living
cell surface provides a convenient means of real-time
detection of the behavior of surface receptors. By this method,
we were able to specifically detect α1-AR, record the
behavior of individual particles of receptors within 50 ms exposure
time in real time in single living cells, and analyze its dynamic
properties. This work can provide some new insights into
the investigation of mechanisms and dynamic properties of
receptor transport.
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