Oleg Kovtun

Biocompatible Quantum Dots for Biological Applications

Semiconductor quantum dots are quickly becoming a critical diagnostic tool for discerning cellular function at the molecular level. Their high brightness, long-lasting, size-tunable, and narrow luminescence set them apart from conventional fluorescence dyes. Quantum dots are being developed for a variety of biologically oriented applications, including fluorescent assays for drug discovery, disease detection, single protein tracking, and intracellular reporting. This review introduces the science behind quantum dots and describes how they are made biologically compatible. Several applications are also included, illustrating strategies toward target specificity, and are followed by a discussion on the limitations of quantum dot approaches. The article is concluded with a look at the future direction of quantum dots.

Visualization of the Cocaine-Sensitive Dopamine Transporter with Ligand-Conjugated Quantum Dots

The presynaptic dopamine (DA) transporter is responsible for DA inactivation following release and is a major target for the psychostimulants cocaine and amphetamine. Dysfunction and/or polymorphisms in human DAT (SLC6A3) have been associated with schizophrenia, bipolar disorder, Parkinsons disease, and attention-deficit hyperactivity disorder (ADHD). Despite the clinical importance of DAT, many uncertainties remain regarding the transporters regulation, in part due to the poor spatiotemporal resolution of conventional methodologies and the relative lack of efficient DAT-specific fluorescent probes. We developed a quantum dot-based labeling approach that uses a DAT-specific, biotinylated ligand, 2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane (IDT444), that can be bound by streptavidin-conjugated quantum dots. Flow cytometry and confocal microscopy were used to detect DAT in stably and transiently transfected mammalian cells. IDT444 is useful for quantum-dot-based fluorescent assays to monitor DAT expression, function, and plasma membrane trafficking in living cells as evidenced by the visualization of acute, protein-kinase-C (PKC)-dependent DAT internalization.

Labeling of neuronal receptors and transporters with quantum dots.

The ability to efficiently visualize protein targets in cells is a fundamental goal in biological research. Recently, quantum dots (QDots) have emerged as a powerful class of fluorescent probes for labeling membrane proteins in living cells because of breakthrough advances in QDot surface chemistry and biofunctionalization strategies. This review discusses the increasing use of QDots for fluorescence imaging of neuronal receptors and transporters. The readers are briefly introduced to QDot structure, photophysical properties, and common synthetic routes toward the generation of water-soluble QDots. The following section highlights several reports of QDot application that seek to unravel molecular aspects of neuronal receptor and transporter regulation and trafficking. This article is closed with a prospectus of the future of derivatized QDots in neurobiological and pharmacological research.

Single-Quantum-Dot Tracking Reveals Altered Membrane Dynamics of an Attention-Deficit/Hyperactivity-Disorder-Derived Dopamine Transporter Coding Variant

The presynaptic, cocaine- and amphetamine-sensitive dopamine (DA) transporter (DAT, SLC6A3) controls the intensity and duration of synaptic dopamine signals by rapid clearance of DA back into presynaptic nerve terminals. Abnormalities in DAT-mediated DA clearance have been linked to a variety of neuropsychiatric disorders, including addiction, autism, and attention deficit/hyperactivity disorder (ADHD). Membrane trafficking of DAT appears to be an important, albeit incompletely understood, post-translational regulatory mechanism; its dysregulation has been recently proposed as a potential risk determinant of these disorders. In this study, we demonstrate a link between an ADHD-associated DAT mutation (Arg615Cys, R615C) and variation on DAT transporter cell surface dynamics, a combination only previously studied with ensemble biochemical and optical approaches that featured limited spatiotemporal resolution. Here, we utilize high-affinity, DAT-specific antagonist-conjugated quantum dot (QD) probes to establish the dynamic mobility of wild-type and mutant DATs at the plasma membrane of living cells. Single DAT-QD complex trajectory analysis revealed that the DAT 615C variant exhibited increased membrane mobility relative to DAT 615R, with diffusion rates comparable to those observed after lipid raft disruption. This phenomenon was accompanied by a loss of transporter mobilization triggered by amphetamine, a common component of ADHD medications. Together, our data provides the first dynamic imaging of single DAT proteins, providing new insights into the relationship between surface dynamics and trafficking of both wild-type and disease-associated transporters. Our approach should be generalizable to future studies that explore the possibilities of perturbed surface DAT dynamics that may arise as a consequence of genetic alterations, regulatory changes, and drug use that contribute to the etiology or treatment of neuropsychiatric disorders.

A flow cytometry-based dopamine transporter binding assay using antagonist-conjugated quantum dots

Here we present the development and validation of a flow cytometry-based dopamine transporter (DAT) binding assay that uses antagonist-conjugated quantum dots (QDs). We anticipate that our QD-based assay is of immediate value to the high throughput screening of novel DAT modulators.

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.

Single Quantum Dot Tracking Reveals Serotonin Transporter Diffusion Dynamics are Correlated with Cholesterol-Sensitive Threonine 276 Phosphorylation Status in Primary Midbrain Neurons

Serotonin transporter (SERT) terminates serotonin signaling in the brain by enabling rapid clearance of the neurotransmitter. SERT dysfunction has been associated with a variety of psychiatric disorders, including depression, anxiety, and autism. Visualizing SERT behavior at the single molecule level in endogenous systems remains a challenge. In this study, we utilize quantum dot (QD) single particle tracking (SPT) to capture SERT dynamics in primary rat midbrain neurons. Membrane microenvironment, specifically membrane cholesterol, plays a key role in SERT regulation and has been found to affect SERT conformational state. We sought to determine how reduced cholesterol content affects both lateral mobility and phosphorylation of conformationally sensitive threonine 276 (Thr276) in endogenous SERT using two different methods of cholesterol manipulation, statins and methyl-β-cyclodextrin. Both chronic and acute cholesterol depletion increased SERT lateral diffusion, radial displacement along the membrane, mobile fraction, and Thr276 phosphorylation levels. Overall, this work has provided new insights about endogenous neuronal SERT mobility and its associations with membrane cholesterol and SERT phosphorylation status.

Antibody-Conjugated Single Quantum Dot Tracking of Membrane Neurotransmitter Transporters in Primary Neuronal Cultures

Single particle tracking (SPT) experiments have provided the scientific community with invaluable single-molecule information about the dynamic regulation of individual receptors, transporters, kinases, lipids, and molecular motors. SPT is an alternative to ensemble averaging approaches, where heterogeneous modes of motion might be lost. Quantum dots (QDs) are excellent probes for SPT experiments due to their photostability, high brightness, and size-dependent, narrow emission spectra. In a typical QD-based SPT experiment, QDs are bound to the target of interest and imaged for seconds to minutes via fluorescence video microscopy. Single QD spots in individual frames are then linked to form trajectories that are analyzed to determine their mean square displacement, diffusion coefficient, confinement index, and instantaneous velocity. This chapter describes a generalizable protocol for the single particle tracking of membrane neurotransmitter transporters on cell membranes with either unmodified extracellular antibody probes and secondary antibody-conjugated quantum dots or biotinylated extracellular antibody probes and streptavidin-conjugated quantum dots in primary neuronal cultures. The neuronal cell culture, the biotinylation protocol and the quantum dot labeling procedures, as well as basic data analysis are discussed.