(AMAXA). Subsequently, cells wereseeded on sterile cover slips (18x18mm) in 6-well plates. 48 h aftertransfection adherent cells expressing
the DRD1-EYFP were stimulated with10–50 μM Dopamine hydrochloride.

AFM tip

USING AFM TO TRACKDOPAMINE BINDING TOMEMBRANE-BOUND D1 RECEPTORSThe overexpressed YFP-tagged
D1 receptors localize mainly in theplasma membrane and show a random
distribution in neuronal SH-SY5Ycells. Based on ﬂuorescence data thereceptors internalize into the cytoplasm
upon stimulation with dopamine. Inorder to support this hypothesis and totest the potential application of AFM, weused the BioScope II force spectroscopy
capabilities to track binding of DA toPAGE 2O-CH3

H3C-ODAHOOH

Plasmamembrane

All experiments were performedin contact and TappingMode™ using
PBS buffer and the softest DNP- andMSCT-type cantilevers (nominalspring constant 0.06 N/m and 0.01
N/m, respectively.). Dopaminefunctionalized cantilevers wereprepared as previously described.11
Brieﬂy, a polyethylene-glycol (PEG)
derivative, having an amino-reactive
end and a thiol-reactive end, was used
as a linker and as an inert back-ﬁlling
molecule so that only dopamine could
contribute to the observed bindinginteractions. Spring constants werecalculated in ﬂuid on a stiff support(glass bottom of the Petri dish) using
the thermal tune option of the Veeco
NanoScope® Controller and deﬂection
sensitivities were manually updated
using the integrated PicoForce software
capabilities. A total of 3072 force
curves were recorded in force volume
mode, at scan rate between 3 and3.5 Hz, and with a retract delay of 10
ms. The scan area was 2x2 μm.

O-CH3

H3C-O

Brightﬁeld (BF), DIC andepiﬂuorescence images wereacquired on a Zeiss Axio Observer
inverted microscope equipped with
an AxioCam MRC camera and theAxioVision software. All AFM images
were recorded on a Veeco BioScope™ II
that has been fully integrated with theoptical microscope, and using DNP/
MSCT cantilevers.

Receptorcytosol
Figure 1: Studying ligand-receptor interactions on a living cell. The AFM tip has been functionalized insuch a way that the chemical moieties of dopamine (DA) involved in the interaction with its D1-receptor arepreserved. In addition, the length of the spacer minimizes the effect of the tip on the interaction itself.

membrane-bound D1 receptors andtheir subsequent internalization.
First, DNP cantilevers werefunctionalized with a dopamine
analog as illustrated in Figure 1. In
the next step live cells were exposed
to several DA concentrations from10 to 50 μM in order to stimulate
the D1 membrane receptors.
Both epiﬂuorescence images and timelapse movies were recorded to detectchanges in the ﬂuorescent signal of D1
receptor distribution and consequently,
to investigate the internalization andbinding properties of the YFP-tagged
dopamine D1-receptor.
Figure 2A shows the experiment at
t0: at this time point, the dopamine
has not been applied and thus, D1
receptors are not stimulated. As aresult, the YFP-tagged D1 receptors
remain membrane-bound revealinga homogeneous ﬂuorescent staining
across the cell surface. A signiﬁcantchange in the ﬂuorescent distribution

AFM AND NEURODEGENERATIVE DISEASES (PART II)

is achieved upon stimulation of theD1-receptor by DA. Figure 2B shows atypical example of the images recorded
at t10min. For all DA concentrations, nomembrane ﬂuorescence was detected
after 10 minutes incubation.
Instead, multiple small intracellularﬂuorescent vesicles variable in size andbrightness were observed indicating
possible receptor internalization(Figure 2B). This observation
points to an active binding of theligand DA to the D1 receptor in theSH-SY5Y cell model. Note that weak
ﬂuorescence signals required us to
use long exposure times to obtain
the ﬂuorescence images. In order toavoid possible bleaching effects weclosed the ﬂuorescence shutter during
the application of DA and opened it
just before acquiring the image.
In addition to ﬂuorescence imaging,
DA – D1-receptor unbinding forces wererecorded using AFM force volume modeon living cells at different time points inFigure 2: Correlating ﬂuorescence imaging and AFM force measurements. The AFM tip was tagged with a DA analog to follow the location of YFP-tagged
D1-receptors before and after applying free DA. Before stimulation by DA, receptors are cell membrane-bound and thus, ﬂuorescence is distributed all overthe cell surface (A) and force curves exhibit a high percentage of speciﬁc unbinding events (C). 10 minutes after DA application, all the ﬂuorescence isconcentrated into small dots (B) and no speciﬁc events are recorded in force volume proving that all the receptors have been internalized into the cytosol.

the presence and absence of dopamine.
Force volume imaging of a single cellwith a DA-functionalized tip at time
point t0 (no stimulation of D1-receptors)
resulted in a signiﬁcant portion (13.12%)
of force curves exhibiting a speciﬁc
unbinding event (Figure 2C).

1 hour of DA stimulation. Comparisonof images and time-lapse movies
conﬁrmed that the observed changeof ﬂuorescence pattern is a directconsequence of the internalization
process.

Studies report the pharmaceuticalcharacteristics of dopamine, as well as
many of its agonists and antagonists,
to its potential receptors.12-15 Dependingon the receptor type, the dissociation
constants were found to be between
880 and 2980 nM.12

RESULTS

In parallel to optical observations,the force measurements were also
performed at time point t10min (afterstimulation of D1-receptors by DA). As
shown in Figure 2D, all recorded force
curves exhibited no speciﬁc unbinding
event, thus supporting our observations
by ﬂuorescence imaging of theinternalization of the D1-receptors
into the cytosol. We did not ﬁnd thisphenomenon to be reversible even after
PAGE 3In our studies, DA – DI-receptor
interactions exhibited a single
unbinding force centered around 223± 82 pN. Interestingly, the mean peakvalue was stable when measured atthe edge of the cell, whereas strongest
variations and higher values wereobserved for measurements taken near
the cell nucleus. To date, the interaction
mechanism between dopamine andits receptor is not fully understood.

AFM AND NEURODEGENERATIVE DISEASES (PART II)

Dynamic force spectroscopy can also
be used for determination of kinetic
parameters. In our working setup, all
AFM scans were performed under thesame conditions, but it is also possible
to vary the scanning parameters andplot the unbinding force as a functionof the loading rate. Curve progression
can provide information as to whetherthe if kinetics of the unbinding process
is more dependent on the inner or