Vol. 49 | No. 2 | 2010
Detection of single fluorescently labeled
proteins is useful in determining their local
mobility, local stoichiometry, fundamental
molecular interactions and molecular
function. Numerous single-molecule studies
have demonstrated the unique functions of
proteins, revealing both large and subtle details
of their biomolecular interactions. Two key
issues with such studies are the biochemical
method of fluorophore attachment and the
proper choice and use of the fluorophore itself
(for reviews, see References 1–4).
For analysis of the molecular mobility
of membrane receptors, specific localization
along with the correct determination of the
motion of the fluorescently labeled receptor
requires high signal-to-background ratios
and acquisition rates and a rigid attachment
of a minimized fluorophore (5,6). Fluores-
cently labeled receptors and ion channels for
single-molecule studies on living cells can be
obtained with fluorescent fusion proteins
(7–10), by direct covalent bonding of fluoro-
phores to cysteine or amino reactive groups,
or with protein tags that react with modified
fluorophores (11). However, while these
techniques are well-established for standard
organic fluorophores, general schemes for
direct labeling by quantum dots (QDs) are
still in their infancy (1–4).
Single-molecule tracking with QDs
displays superior performance over tracking
with large latex beads since QDs are ~10×
smaller. In addition, they typically yield
higher fluorescence signals than standard
organic dyes and fluorescent proteins due to
their improved brightness and resistance to
photobleaching. With the recent advent of
small, noncommercially available QDs, the
size limitations of standard commercial QDs
have been overcome, making them compa-
rable in size to fluorescent proteins and only
somewhat larger than the brightest fluorescing
organic dyes (12,13).
QD tracking has been limited in the
number of available labeling techniques. To
date, most QDs for tracking are conjugated
to antibodies, which unless they are directly
conjugated to primary Fab fragments, can
increase the effective radius of the QD by
≥10 nm (1,3,4). Although tracking receptors
with QD-conjugated secondary antibodies
has been successful (1,3), the desire to reduce
the common drawbacks of antibody labeling
(14–17) has led to new direct QD binding
Direct attachment of QDs to receptors
can be achieved by normal amino or cysteine
nonspecific reactions (1–4). Another possi-
bility is the biotinylation of the receptor
and addition of either streptavidin-coated
QDs or QDs conjugated with the recently
generated m-streptavidin of only 159 amino
acid residues (18,19). Yet another method is
to covalently link a QD to a fusion protein
containing a HaloTag, a modified haloalkane
dehydrogensase of 296 amino acids, which
can covalently bind a modified chloroalkane-
labeled QD (20,21).
Many other labeling systems are useful
for imaging and biological quantification of
proteins (22–24); however, with the exception
of the aformentioned techniques, they are
either noncovalent or lack suitable protocols
for QD labeling. Some systems—including
FlAsH (25), hexahistidine (13,26,27), biotin
acceptor peptide (28), and Q-tag (29)—are
so stable with organic dyes that they have
been demonstrated with single-molecule
The acyl carrier protein (ACP) system is
especially well-suited for the specific labeling
and tracking of single receptors on living
cells (30). With only 77 amino acids, ACP
is relatively smaller than comparable tags
such as the SNAP and CLIC tags (22–24)
and has reliable reactivity to coenzyme A
(CoA) bound to organic fluorophores or
to biotin (30) in a reaction catalyzed by
phosphopantetheinyl transferase (PPTase).
Covalent quantum dot receptor linkage via the acyl
carrier protein for single-molecule tracking,
internalization, and trafficking studies
Monika Zelman-Femiak1, Kun Wang1, Kira V. Gromova1,*, Petra Knaus2, and Gregory S. Harms1,3
1Bio-Imaging Center, Rudolf Virchow Center, University of Würzburg, Würzburg, Germany, 2Institute for
Chemistry and Biochemistry, Free University of Berlin, Berlin, Germany, and 3Department of Biology and Physics,
Wilkes University, Wilkes-Barre, Pennsylvania, USA
BioTechniques 49:574-579 (August 2010) doi 10.2144/000113466
Key words: quantum dot; single-molecule tracking; BMP; PTH; ACP
Supplementary material for this article is available at www.BioTechniques.com/article/113466.
*K.V.G.’s present address is the Institute for Chemistry and Biochemistry, Free University of Berlin, Takustr. 6, 14195 Berlin, Germany.
Here we describe a labeling technique for the covalent linkage of quantum dots to transmembrane receptors for single-mol-
ecule tracking. Our method combines the acyl carrier protein (ACP) technique with coenzyme A (CoA)–functionalized
quantum dots to covalently attach quantum dots to ACP fusions of receptor proteins. The advantages of this approach
include: (i) the use of a smaller attachment linker than in many other quantum dot–labeling systems; (ii) the ability to
achieve a reliable 1:1 fluorophore-to-receptor labeling stoichiometry; (iii) the specificity of the method; and (iv) the cova-
lent nature of the quantum dot linkage. We demonstrate the general suitability of this technique in single-molecule track-
ing, internalization, and trafficking studies by imaging two different transmembrane receptors in living cells.
Vol. 49 | No. 2 | 2010
The advantages of tracking with ACP-CoA
and organic fluorophores were demon-
strated on G protein–coupled receptors
(GPCRs) (9,10), and AMPA receptors (31).
In particular, tracking with ACP is advanta-
geous since the receptors fused to ACP retain
biological activity. Furthermore, the covalent
binding of the CoA fluorophore by ACP is
specific and efficient. The same system was
also used for internalization studies (9,10,31).
However, until now, protocols for the direct
attachment of QDs to receptors via an ACP
tag did not exist.
Here we introduce a method for labeling
ACP tags with QDs covalently bound to CoA
in a 1:1 ratio. The QD:CoA covalent binding
occurs via straightforward thiol-reactive
chemistry in an appropriate concentration
of amine-blocking groups on the QDs. We
demonstrate the successful labeling of two
different receptors: the parathyroid hormone
receptor (PTHR), which is a GPCR, and the
bone morphogenetic protein type II receptor
(BRII). We compare the labeling and tracking
of both receptors with either our ACP-CoA-
QD technique or antibody QD conjugates.
We find that ACP-CoA-QD labeling enables
more precise tracking, longer tracking times,
and the possibility to determine all stages in the
fate of individual receptors, including behavior
at the membrane, internalization, and post-
internalization trafficking events, such as
recycling. To our knowledge, this technique
provides the shortest specific covalent tether
of a QD with a receptor.
Materials and methods
Plasmid construction of ACP-
PTHR-TC and ACP-BRII-TC1
The ACP fusion to the N terminus of the
truncated human PTHR was produced in
a similar manner as previously reported for
the N-terminally labeled GFP-PTHR (32).
The PCR products of the N-terminal signal
peptide of PTHR, ACP, and of the human
PTHR truncated to amino acids 102–270
were subcloned together by ligation after
digestion with the same restriction enzyme
into pcDNA3 (Invitrogen, Karlsruhe,
Germany) for transfection and expression in
mammalian cells. The final construct did not
include the pharmacologically unimportant
residues 61–101 but did include a two-residue
linker (from restriction sites) between the
ACP C terminus and the truncated PTHR
N terminus and a six-residue linker between
the ACP N terminus and the signal peptide
The ACP fusion to the N terminus of the
truncated type II human bone morphoge-
netic receptor (BRII-TC1) was produced in
a similar manner as above following similar
strategies (33,34). The final construct also
included a six-residue linker between the ACP
C terminus and the BRII-TC1 N terminus.
All constructs, which were subcloned into
pcDNA3, were verified by sequencing.
Human embryonal kidney cells (HEK293)
were cultured in DMEM (Sigma-Aldrich,
Taufkirchen, Germany) supplemented with
10% FCS (PAA Laboratories GmbH, Linz,
Austria) and 1% penicillin/streptomycin
(Sigma-Aldrich) solution at 37°C/5% CO2.
FuGENE 6 (Roche Applied Science, India-
napolis, IN, USA). Effectene (Qiagen,
Hilden, Germany) transfection reagents
were used for HEK293 cell transient trans-
fections. For single-molecule imaging, cells
were seeded into six-well plates containing
glass coverslips (No. 1, round, 24 mm;
Assistent, Sondheim, Germany) and trans-
fected with 0.2 μg plasmid DNA per well.
Labeling was performed by incubating
the cells on the coverslips for 15–20 min
at 37°C in 270 µL DMEM with 1% BSA
(Sigma-Aldrich), 1.5 µM 6×His-PPTase,
and 0.3 nM CdSe/ZnS QD-CoA molecules
prepared with the QD655 Qdot Antibody
Conjugation Kit (NrQ22021MP; Molecular
Probes, Invitrogen Detection Technologies,
Eugene, Oregon, USA; Invitrogen, Karlsruhe,
Germany) and reacted with CoA (Sigma-
Aldrich) (for details of the reaction for a
1:1 QD:CoA ratio, see the Supplementary
Materials). Samples were washed three times
with 500 µL DMEM before measurements.
Experiments were conducted on cells in 300
Human PTH peptide ligand hPTH
(amino acids 1–34) purchased from Bachem
California (Torrance, CA, USA) was used
to bind the truncated 2.5 nM PTHR.
Human bone morphogenetic protein-2
(BMP-2; Sigma-Aldrich) was used to bind
the truncated BRII-TC1. Ligands diluted in
PBS were added in concentrations from 1 nM
to 10 nM, depending on the experiment.
The experimental arrangement for single-
molecule imaging has been described in detail
previously (35,36). Essentially, the samples
were mounted onto an inverted microscope
(Axiovert 200; Zeiss, Göttingen, Germany)
equipped with a 100× objective (PlanNeo-
fluor 100×, N.A. = 1.3; Zeiss), and illumi-
nated for 5 ms with the 473-nm laser line
of a diode laser (MBL-473-100; CNI Laser;
Changchun, China) with excitation intensity
of 0.5–1 kW/cm2 and synchronized with the
exposure of the Peltier-cooled electron-multi-
plying charge-coupled device (EMCCD)
camera system (CascadeII 512B; Roper Scien-
tific, Tucson, AZ, USA).
Total internal reflection
The total internal reflection (TIRF) single-QD
imaging and tracking were performed on
a Leica AM TIRF system (Leica, Wetzlar,
Germany). The TIRF microscope was custom-
equipped from the manufacturer with a 100×,
N.A. = 1.46 objective (Leica), a 405-nm,
50-mW diode laser (CUBE; Coherent, Santa
Figure 1. Schematic diagram of labeling tech-
nique. By application of the enzyme PPTase, a
single QD functionalized to CoA is transferred to
the ACP protein fused to the receptor.
Figure 2. Characterization of the ACP-CoA-QD labeling of receptors on living cells. In each panel,
fluorescent images are shown at the bottom, while the corresponding transmission image is shown
above. (A) CoA-QD labeling of HEK293 cells transfected with ACP-PTHR-TC followed by PPTase
treatment shows many QDs (bottom left). Non–PPTase-treated cells show no QDs (bottom right). (B)
CoA-QD labeling of HEK293 cells transfected with the ACP-BRII-TC1 plasmid followed by PPTase
shows many QDs (bottom left), while without PPTase treatment, there are no QDs (bottom right). (C)
Negative control of nontransfected HEK293 cells without (left) and with (right) CoA-QD and PPTase
treatment. (D) Antibody-QD labeling of transfected HEK293 cells (transmission image, top) with
the HA-BRII-TC1 plasmid with both the primary anti-HA antibody and secondary Fab antibody QD
conjugate showing many QDs (bottom). All intensity scaling is in photons/5 ms.
Vol. 49 | No. 2 | 2010
Clara, CA, USA), and the EMCCD camera
system (CascadeII 512B).
By plotting the mean square displacement
respective populations of molecules is revealed.
free diffusion model
2) versus tlag, the diffusion behavior of the
2, tlag) data sets were fitted to either the
the anomalous subdiffusion model
or the confined diffusion model
() 1 exp
where the mean-square displacement ri
is proportional to time tlag, D or Γ are the
diffusion constants, α is the anomalous power
lay coefficient, and L is the confinement length
(8). For more details about this analysis, see
the “Mobility analysis” section of the Supple-
Results and discussion
With the recent advances in general labeling
schemes for covalent bonding of both organic
dyes and QDs, we were interested in creating a
direct and simple QD labeling method using
a shorter linkage to improve tracking, inter-
nalization, and trafficking studies of single
receptors. We devised a technique that uses
the well-established system of the ACP as an
extracellular fusion protein tag for receptors
(30). However, this first required the labeling
of QDs with CoA. We used commercially
available polymer-coated CdSe/ZnS QDs
and straightforward thiol-reactive chemistry
with an appropriate concentration of amine
blocking groups on the QDs to achieve a
maximal 1:1 CoA:QD ratio (Figure 1 and
Supplementary Figure S1).
We next tested if the CoA-QDs would
specifically and covalently bind to ACP
fused to the extracellular domains of receptor
proteins upon enzymatic reaction with
PPTase (Figure 1). For ACP extracellular
receptor fusions, we selected two receptor
candidates that are truncation mutants: a
truncation of the PTHR (32) and a truncation
of the BRII (33,34), called ACP-PTHR-TC
and ACP-BRII-TC1, respectively (33,34).
ACP-BRII-TC1 resembles the BRII lacking
almost the entire cytoplasmic domain,
with the extracellular fusion of ACP at the
very N terminus of the truncated receptor.
ACP-PTHR-TC resembles the PTH type 1
receptor except that it lacks transmembrane
domains III to VII and the long 150-amino
acid C terminus and has the ACP sandwiched
in the N-terminal ligand binding region.
CoA-QD labeling of HEK293 cells
transiently transfected with low levels of either
ACP-PTHR-TC or ACP-BRII-TC1 marked
the receptors with an efficiency and intensity
that is well-suited for tracking them on the
plasma membrane (Figure 2, A and B). We
verified the specificity of the ACP reaction
by applying CoA-QDs without the enzyme
Figure 3. Tracking of the surface mobility and internalization of ACP-PTHR-TC in live HEK293 cells. (A) Transmission (upper) and TIRF images (lower) of
the CoA-QD-labeled ACP-PTHR-TC on HEK293 cells (left). Two traces in a magnified portion of the left image are shown in red and green (right). (B)
Traces and plots of mean square displacement (MSD) versus time lag (the time between subsequent recordings) for each track of the two individual
QD-receptor clusters from panel A. Before ligand addition, the receptor (i) was freely but slowly mobile (0.003 ± 0.001 µm2/s) and became more mo-
bile (0.021 ± 0.005 µm2/s) 2 min after addition of 2.5 nM PTH. Zero, 5, and 10 min after PTH addition, the receptor (ii) displayed extremely confined
motion (0.0002 ± 0.0001 µm2/s, 0.00083 ± 0.00008 µm2/s, and 0.00005 ± 0.00001 µm2/s, respectively). (C) The average motion was determined
by probability analysis (see the “Materials and methods” section) and plotted as MSD versus time lag. The motion before PTH addition (t = 0 min) is
indicated by the solid line with dark circles and after PTH addition by the dotted line with open circles. (D) Four images acquired at 2, 5, 10, and 15
min after ligand application display the internalization behavior for the zoomed image from panel A. (E) Temporal statistics from more than 10 cells for
the internalization of ACP-PTHR-TC after addition of 2.5 nM PTH. (F) Trafficking of ACP-PTHR-TC receptor from the membrane into the cell. Overlay
fluorescence and transmission image of four QD-receptor complexes with one trace showing two consecutive steps from the membrane (first image).
Zoomed images (right three) of the QD-receptor complex at the membrane in the first image of the sequence with the same trafficked receptor complex
(second image) and 2 and 3 min later (third and fourth images). Intensity scaling in panels A and D is in photons/5 ms.
Vol. 49 | No. 2 | 2010
PPTase on the same transfected HEK293
cells used in Figure 2, A and B. The CoA-QDs
never attached and always fully washed away
(Figure 2, A and B). Clearly, the signals came
only from the labeled receptors as nonstained
cells never displayed the bright spots of the
QDs under these conditions and since
nontransfected cells never bound CoA-QD
even with PPTase treatment (Figure 2C).
We also sought to compare the efficiency
of CoA-QD labeling by subjecting the same
transfected cells to labeling with antibody-
conjugated QDs. As the BRII-TC1 receptor
was also available with an extracellular HA tag
(33,34), we compared the HA-tagged receptor
stained with mouse anti-HA primary and
antibody to the CoA-QD-ACP–labeled
receptor. We observed that the HA labeling
exhibited, on average, the same number of
labeled receptors per cell as the ACP-CoA-
QD method (Figure 2, compare panel D with
We then tracked the covalently CoA-QD-
tagged ACP-PTHR-TC on living cells.
Although there is a previous report on the
diffusion of the whole PTHR receptor (37),
it was not certain what type of mobility this
extremely truncated receptor might have
and what role it might play. Using TIRF
microscopy, we imaged the receptors on the
basolateral surface of cells (Figure 3A). Upon
tracking, we easily identified long (>50 step)
traces of individual QD-receptor complexes,
primarily indicating free diffusion both before
and after PTH addition, followed by quick
internalization (Figure 3B, ia and ib). A small
subpopulation of QD-receptor complexes
displayed restricted mobility both before and
after ligand addition and usually endured a long
time prior to internalization, if they were inter-
nalized at all (Figure 3B, iia–iic). The motion
of all QD-receptor complexes was analyzed
before and after ligand addition to determine
the average motion (Figure 3C). Prior to PTH
application, the QD receptor complexes
diffused in a partially confined manner on
the membrane with a diffusion coefficient
of 0.014 ± 0.002 µm2/s and a confinement
coefficient c = 0.739 ± 0.041. Slow motion
was exhibited by the QD-receptor complexes
that remained at the membrane >10 min
after PTH stimulation; average motion was
determined to be just above the ascertained
positional accuracy, yielding a value of 0.00033
± 0.00005 µm2/s.
Since the tether should be ~1 order of
magnitude shorter for the ACP-CoA-QD
labeling in comparison to the anti-HA/anti-
mouse-Fab-QD labeling, we decided to compare
the movement of the receptors for any obvious
differences. We tracked the ACP-BRII-TC1
and HA-BRII-TC1 on cells both before and
after addition of BMP-2. Before and after
ligand application, the ACP-BRII-TC1 and
HA-BRII-TC1 receptors demonstrated freely
diffusing behavior (Figure 4, A and B) until just
before the receptors internalized, whereupon
the motion either significantly slowed
Dlat, ACP, 10 min after = 0.0004 ± 0.0002 µm2/s or
became confined to regions of ~270 ± 20 nm
in diameter. However, the diffusion trajectories
from the HA-tagged and labeled receptors
always appeared to display a larger motion, but
this motion did not result in a significantly larger
diffusion coefficient of the average mobility
behavior (Figure 4B), where before BMP-2
addition Dlat, ACP, before = 0.014 ± 0.002 µm2/s
and Dlat, HA, before = 0.018 ± 0.004 µm2/s.
The internalization behavior for both
ACP-QD–labeled receptors was then
examined. In each case, the TIRF and
widefield images of the basolateral surface
of cells were analyzed for instances when a
QD-receptor complex would internalize and
disappear from the membrane in longer time-
lapse sequences. An example of each recep-
tor’s time-lapse internalization is displayed
in Figure 3D for the ACP-PTHR-TC and in
Figure 4C for the ACP-BRII-TC1. Although
all four QD-receptor complexes in the example
shown for ACP-PTHR-TC (Figure 3D) inter-
nalized, on average only 60% of receptors fully
internalized with a single exponential rate of
~0.1 QD-receptor complex per minute (Figure
3E). The ACP-BRII-TC1 QD-receptors
were nearly fully internalized (greater than
80%) with apparent bi-exponential rates of
0.1 and 1 QD-receptor complex per minute
(80% and 20%, respectively; Figure 4D).
Recycled QD-receptors appeared in all cases
(Figure 4C and Supplementary Figure S2),
but the recycled receptors were not included
in the determination of internalization rate.
The technique is also suitable for longer and
larger scale trafficking events by imaging wider
fields and increased depths with slightly lower
magnification and numerical aperture objec-
tives. Indeed, in many cases, the receptor at
the membrane could be tracked for internal-
ization into the cells (Figure 3F).
Here, we demonstrated our labeling
technique on decoy versions of the PTH
and BRII transmembrane receptors, which
each carry the ligand binding site but lack the
intracellular domain essential for signaling.
The labeling was efficient and specific.
Furthermore, the excellent stability of the
QDs made it possible to analyze internal-
ization of the receptors with minute time-
scale time-lapse imaging to detect trafficking
of individual receptors from the membrane
into the cell and to also observe recycling
The tracking of the receptors with the
ACP-QD labeling technique in comparison
to antibody-QD labeling displayed slightly
less mobility, which we interpret as the effect
of the shorter linker length between the
receptor and QD. Although we expected high
and free diffusion mobility of the truncated
receptors, especially in comparison to studies
Figure 4. Tracking and internalization of the BRII-TC1 receptor on HEK293 cells. (A) Comparison of
tracking from QD-receptors with ACP-CoA-QD labeling (upper) and HA/anti-HA/anti-mouse-Fab-QD
labeling (lower) with MSD versus time lag plotted for each track after addition of 1 nM BMP-2. (B)
The average motion was determined by probability analysis (see the “Materials and methods” sec-
tion) and plotted as the MSD versus time lag of all ACP-BMPII-TC1 (top graph) and HA-BMPII-TC1
(lower graph) receptors before (dotted line with open circles) and after (solid line with dark circles)
ligand addition. (C) Transmission (first image) and TIRF images (right four) of the CoA-QD ACP-BRII-
TC1 on HEK293 cells acquired at 0, 7, and 15 min after 1 nM BMP-2 addition (right three magni-
fied images are of the same region at t = 0, 7, and 15 min) to display the internalization behavior.
Intensity scaling is in photons/5 ms. (D) Temporal statistics from >10 cells for the internalization of
ACP-BRII-TC1 after 1 nM BMP-2 addition.
performed on their full-length counterparts
(37), we observed only moderate but free
diffusion. However, before BMP-2 addition,
the ACP-BRII-TC1 receptor remained
freely diffusive, which was consistent
with the observation that it does not bind
to other receptors before ligand addition
(33,34,38). The incomplete internalization
of the ACP-PTHR-TC receptor after ligand
addition agrees with previously reported
truncation mutants of this receptor (39).
The nearly complete internalization of the
ACP-BRII-TC1 receptor can be explained by
the binding of the receptor to the epidermal
growth factor pathway substrate 15 related
(EPS15R) protein, a substrate of tyrosine
kinase activity of the epidermal growth
factor receptor and a constitutive resident
in clathrin-coated pits (CCPs), which are
the main internalization route of BRII (33).
We suggest that binding to EPS15R leads the
ligand-bound BRII-TC1 into CCPs, which
causes reduced mobility of the receptor,
confinement to a region the size of CCPs,
and the nearly full internalization of this
In the future, we expect to show more direct
applications with this labeling technique on
full-length and specific signaling mutations of
membrane receptor proteins to not only track
mobility at the membrane, but also to obtain
more precise information on internalization
dynamics and trafficking. We plan to take
advantage of the recently reported 12–amino
acid CoA binding domain (40,41), as well
as newly described smaller and biologically
benign QDs and nanoparticles (12,13,18),
which will make this technique perhaps the
smallest, brightest, most specific, and most
stable covalent labeling for tracking, internal-
ization, and trafficking studies of individual
receptors. In combination with recently
described methods for 3-D tracking (42),
this would make it possible to determine the
entire fate of individual receptors.
We thank Kai Johnsson (École Polytechnique
Fédérale de Lausanne [EPFL], Lausanne,
Switzerland) for providing us with the ACP
and PPTase plasmid constructs. We thank
Martin Lohse (University of Würzburg,
Würzburg, Germany) and Jean-Pierre
Vilardaga (University of Pittsburgh, Pitts-
burgh, PA, USA) for the N-GFP-PTHR
construct, and Asja Guzman (Free University
of Berlin, Berlin, Germany) and Mike
Friedrich (University of Würzburg) for criti-
cally reading the manuscript. This work was
supported by the German Science Foundation
(grant nos. FZ-82, GK 1048, and 1342 to
G.S.H., and grant no. SFB 449, to P.K.) and
the Bavarian State Ministry for Research and
Education through the Bio-Imaging Center of
the University of Würzburg (G.S.H.).
The authors declare no competing interests.
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Received 1 April 2010; accepted 18 June 2010.
Address correspondence to Gregory Harms,
Bio-Imaging Center, Rudolf Virchow Center,
University of Würzburg, Josef-Schneider-Str.
2, Haus D15, D-97080, Würzburg, Germany.
The International Journal of Life Science Methods
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