Single Quantum Dot Tracking Unravels Agonist Effects on the Dopamine Receptor Dynamics.

D2 dopamine receptors (DRD2s) belong to a family of G protein-coupled receptors that modulate synaptic dopaminergic tone via regulation of dopamine synthesis, storage, and synaptic release. DRD2s are the primary target for traditional antipsychotic medications; dysfunctional DRD2 signaling has been linked to major depressive disorder, attention-deficit hyperactivity disorder, addiction, Parkinson's, and schizophrenia. DRD2 lateral diffusion appears to be an important post-translational regulatory mechanism; however, the dynamic response of DRD2s to ligand-induced activation is poorly understood. Dynamic imaging of the long isoform of DRD2 (D2L) fused to an N-terminal antihemagglutinin (HA) epitope and transiently expressed in HEK-293 cells was achieved through a combination of a high-affinity biotinylated anti-HA antigen-binding fragment (Fab) and streptavidin-conjugated quantum dots (QD). Significant reduction (∼40%) in the rate of lateral diffusion of QD-tagged D2L proteins was observed under agonist (quinpirole; QN)-stimulated conditions compared to basal conditions. QN-induced diffusional slowing was accompanied by an increase in frequency, lifetime, and confinement of temporary arrest of lateral diffusion (TALL), an intrinsic property of single receptor lateral motion. The role of the actin cytoskeleton in QN-induced diffusional slowing of D2L was also explored. The observed dynamic changes appear to be a sensitive indicator of the receptor activity status and might also spatially and temporally shape the receptor-mediated downstream signaling. This dynamic information could potentially be useful in informing drug discovery efforts based on single-molecule pharmacology.

Quantum dots reveal heterogeneous membrane diffusivity and dynamic surface density polarization of dopamine transporter.

The presynaptic dopamine transporter mediates rapid reuptake of synaptic dopamine. Although cell surface DAT trafficking recently emerged as an important component of DAT regulation, it has not been systematically investigated. Here, we apply our single quantum dot (Qdot) tracking approach to monitor DAT plasma membrane dynamics in several heterologous expression cell hosts with nanometer localization accuracy. We demonstrate that Qdot-tagged DAT proteins exhibited highly heterogeneous membrane diffusivity dependent on the local membrane topography. We also show that Qdot-tagged DATs were localized away from the flat membrane regions and were dynamically retained in the membrane protrusions and cell edges for the duration of imaging. Single quantum dot tracking of wildtype DAT and its conformation-defective coding variants (R60A and W63A) revealed a significantly accelerated rate of dysfunctional DAT membrane diffusion. We believe our results warrant an in-depth investigation as to whether compromised membrane dynamics is a common feature of brain disorder-derived DAT mutants.

Single Quantum Dot Tracking Illuminates Neuroscience at the Nanoscale.

The use of nanometer-sized semiconductor crystals, known as quantum dots, allows us to directly observe individual biomolecular transactions through a fluorescence microscope. Here, we review the evolution of single quantum dot tracking over the past two decades, highlight key biophysical discoveries facilitated by quantum dots, briefly discuss biochemical and optical implementation strategies for a single quantum dot tracking experiment, and report recent accomplishments of our group at the interface of molecular neuroscience and nanoscience.