In addition to its high-resolution imaging capabilities, atomic force microscopy (AFM) has emerged as a powerful tool for measuring the nanomechanical properties and interaction forces of biomolecular complexes.While most of these types of AFM studies are performed on isolated molecules, for true biological relevance, these studies are best performed on living cell systems.
Furthermore, it now seems important to combine AFM with other techniques such as light microscopy to collect both types of data simultaneously.So far, few studies have been based on the combination of atomic force microscopy and inverted light microscopy.
In the present study, we investigated the potential application of force measurements in monitoring the response and binding properties of dopamine D1 receptors following dopamine stimulation.We use a fully integrated AFM and epifluorescence system to correlate fluorescence imaging and AFM force measurements.
Dopamine (DA, 4-(2-aminoethyl) benzo-1,2-diol) is a major neurotransmitter belonging to the catecholamine family, based on an aromatic amino acid called tyrosine and a precursor to two major hormones: the adrenal glands and norepinephrine.
In the peripheral nervous system, dopamine’s primary role is to regulate cardiovascular functions such as stimulants, hormone turnover, renal function, vascular flow, and gastrointestinal motility.In the central nervous system (CNS), dopamine is involved in the control of motor function, cognition, mood, food intake and endocrine regulation.Dysfunction of dopaminergic neurotransmission in the central nervous system is associated with a variety of neuropsychiatric disorders, including Tourette’s syndrome, Parkinson’s disease, schizophrenia, paranoia, and attention deficit hyperactivity disorder (ADHD).Dopamine receptors are divided into D1 to D5, with D1 and D2 receptors accounting for the largest proportion.For the treatment of these diseases, identifying dopaminergic drugs without side effects is one of the greatest challenges in neuronal research and drug discovery.
In this particular study, we used the YFP-labeled transmembrane D1 receptor to study the specific city of interaction between this receptor and the dopamine-modified AFM tip, using our integrated tools for force spectroscopy and optical imaging characterization.
SH-SY5Y cells were transfected with YFP-tagged dopamine D1 receptor (DRD1-EYFP).Transfection was performed by nucleofection (Nucelofector, AMAXA) using cell suspension (106 cells/ml), 4 µg plasmid DNA, and 100 µl transfection buffer (AMAXA).Subsequently, cells were seeded on sterile coverslips (18×18 mm) in 6-well plates.Adherent cells expressing DRD1-EYFP were stimulated with 10-50 µM dopamine hydrochloride 48 hours after transfection.
Brightfield (BF), DIC and epifluorescence images were acquired on a Zeiss Axio Observer inverted microscope equipped with an AxioCam MRC camera and AxioVision software.All AFM images were recorded on an AFM fully integrated with the light microscope using a DNP/MSCT cantilever.
All experiments were performed in Contact and TappingMode™ using PBS buffer and the softest DNP and MSCT type cantilevers (nominal spring constants of 0.06 N/m and 0.01 N/m, respectively).Dopamine-functionalized cantilevers were prepared as previously described.
Briefly, polyethylene glycol (PEG) derivatives with amino-reactive and thiol-reactive ends were used as linkers and inert backfill molecules, so that only dopamine could contribute to the observed binding interactions.Calculate the spring constant in the fluid on the rigid support (glass bottom of the Petri dish) and manually update the deflection sensitivity in the software.In force-volume mode, with scan rates between 3 and 3.5 Hz and a retraction delay of 10 ms, a total of 3072 force curves were recorded.The scanning area is 2×2 µm.
Overexpressed YFP-tagged D1 receptors were predominantly located in the plasma membrane and were randomly distributed in neuronal SH-SY5Y cells.Based on the fluorescence data, the receptor was internalized into the cytoplasm following dopamine stimulation.To support this hypothesis and test the potential application of AFM, we followed the binding of DA to the membrane-bound D1 receptor and its subsequent internalization.
First, as shown in Figure 1, DNP cantilevers were functionalized with dopamine analogs.Next, live cells were exposed to several DA concentrations ranging from 10 to 50 μM to stimulate D1 membrane receptors.
Figure 1. Studying ligand-receptor interactions on living cells.The AFM tip has been functionalized to retain the dopamine (DA) chemical moiety that interacts with the D1 receptor.Furthermore, the length of the spacer minimizes the effect of the tip on the interaction itself.
Epifluorescence images and time-lapse movies were recorded to detect changes in the fluorescent signal of D1 receptor distribution to study the internalization and binding properties of YFP-labeled dopamine D1 receptors.
Figure 2A shows the experiment at t0: at this time point, no dopamine was applied, so D1 receptors were not stimulated.Thus, YFP-labeled D1 receptors remained membrane bound and displayed uniform fluorescent staining on the cell surface.After DA stimulation of D1 receptors, the fluorescence distribution changed significantly.Figure 2B shows a typical example of images recorded at t10min.For all DA concentrations, no membrane fluorescence was detected after 10 min of incubation.
Post time: Oct-17-2022