Other search tools

About this data

The publication data currently available has been vetted by Vanderbilt faculty, staff, administrators and trainees. The data itself is retrieved directly from NCBI's PubMed and is automatically updated on a weekly basis to ensure accuracy and completeness.

If you have any questions or comments, please contact us.

Results: 1 to 10 of 42

Publication Record

Connections

Antibody-Conjugated Single Quantum Dot Tracking of Membrane Neurotransmitter Transporters in Primary Neuronal Cultures.
Bailey DM, Kovtun O, Rosenthal SJ
(2017) Methods Mol Biol 1570: 165-177
MeSH Terms: Algorithms, Animals, Fluorescent Antibody Technique, Immunoconjugates, Models, Theoretical, Molecular Imaging, Neurons, Neurotransmitter Transport Proteins, Primary Cell Culture, Quantum Dots, Rats, Single Molecule Imaging, Statistics as Topic
Show Abstract · Added April 3, 2018
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.
0 Communities
2 Members
0 Resources
MeSH Terms
Electron Microscopy of Living Cells During in Situ Fluorescence Microscopy.
Liv N, van Oosten Slingeland DS, Baudoin JP, Kruit P, Piston DW, Hoogenboom JP
(2016) ACS Nano 10: 265-73
MeSH Terms: Animals, Bioreactors, Cell Line, Cells, Immobilized, Chlorocebus aethiops, Endocytosis, Fibroblasts, Microscopy, Electron, Scanning, Optical Imaging, Quantum Dots
Show Abstract · Added February 4, 2016
We present an approach toward dynamic nanoimaging: live fluorescence of cells encapsulated in a bionanoreactor is complemented with in situ scanning electron microscopy (SEM) on an integrated microscope. This allows us to take SEM snapshots on-demand, that is, at a specific location in time, at a desired region of interest, guided by the dynamic fluorescence imaging. We show that this approach enables direct visualization, with EM resolution, of the distribution of bioconjugated quantum dots on cellular extensions during uptake and internalization.
0 Communities
1 Members
0 Resources
10 MeSH Terms
Single-quantum-dot tracking reveals altered membrane dynamics of an attention-deficit/hyperactivity-disorder-derived dopamine transporter coding variant.
Kovtun O, Sakrikar D, Tomlinson ID, Chang JC, Arzeta-Ferrer X, Blakely RD, Rosenthal SJ
(2015) ACS Chem Neurosci 6: 526-34
MeSH Terms: Amphetamine, Attention Deficit Disorder with Hyperactivity, Cell Membrane, Central Nervous System Stimulants, Diffusion, Dopamine Plasma Membrane Transport Proteins, HEK293 Cells, Humans, Microscopy, Confocal, Microscopy, Fluorescence, Mutation, Quantum Dots, Time-Lapse Imaging
Show Abstract · Added September 28, 2015
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.
0 Communities
3 Members
0 Resources
13 MeSH Terms
Quantum dot approaches for target-based drug screening and multiplexed active biosensing.
Kovtun O, Arzeta-Ferrer X, Rosenthal SJ
(2013) Nanoscale 5: 12072-81
MeSH Terms: Animals, Binding, Competitive, Biosensing Techniques, Drug Evaluation, Preclinical, Endpoint Determination, Humans, Molecular Targeted Therapy, Quantum Dots, Surface Properties
Show Abstract · Added May 27, 2014
Biomolecule detection using quantum dots (Qdots), nanometer-sized semiconductor crystals, effectively addresses the limitations associated with conventional optical and biochemical techniques, as Qdots offer several key advantages over traditional fluorophores. In this minireview, we discuss the role of Qdots as a central nanoscaffold for the polyvalent assembly of multifunctional biomolecular probes and describe recent advances in Qdot-based biorecognition. Specifically, we focus on Qdot applications in target-based, drug screening assays and real-time active biosensing of cellular processes.
0 Communities
2 Members
0 Resources
9 MeSH Terms
Quantum dot-based single-molecule microscopy for the study of protein dynamics.
Chang JC, Rosenthal SJ
(2013) Methods Mol Biol 1026: 71-84
MeSH Terms: Calibration, Cell Survival, HeLa Cells, Humans, Microscopy, Proteins, Quantum Dots
Show Abstract · Added May 27, 2014
Real-time microscopic visualization of single molecules in living cells provides a molecular perspective of cellular dynamics, which is difficult to be observed by conventional ensemble techniques. Among various classes of fluorescent tags used in single-molecule tracking, quantum dots are particularly useful due to their unique photophysical properties. This chapter provides an overview of single quantum dot tracking for protein dynamic studies. First, we review the fundamental diffraction limit of conventional optical systems and recent developments in single-molecule detection beyond the diffraction barrier. Second, we describe methods to prepare water-soluble quantum dots for biological labeling and single-molecule microscopy experimental design. Third, we provide detailed methods to perform quantum dot-based single-molecule microscopy. This technical section covers three protocols including (1) imaging system calibration using spin-coated single quantum dots, (2) single quantum dot labeling in living cells, and (3) tracking algorithms for single-molecule analysis.
0 Communities
1 Members
0 Resources
7 MeSH Terms
Imaging of endothelial progenitor cell subpopulations in angiogenesis using quantum dot nanocrystals.
Barnett JM, Penn JS, Jayagopal A
(2013) Methods Mol Biol 1026: 45-56
MeSH Terms: Acetylation, Animals, Bone Marrow Cells, Choroidal Neovascularization, Endothelial Cells, Immunomagnetic Separation, Intracellular Space, Lipoproteins, LDL, Molecular Imaging, Neovascularization, Pathologic, Quantum Dots, Rats, Retina, Stem Cells
Show Abstract · Added October 9, 2013
Over the last decade, research has identified a class of bone marrow-derived circulating stem cells, termed endothelial progenitor cells (EPCs), that are capable of homing to vascular lesions in the eye and contributing to pathological ocular neovascularization (NV). In preclinical and biological studies, EPCs are -frequently identified and tracked using a intracellularly loaded fluorescent tracer, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbo cyanine perchlorate-labeled acetylated LDL (DiI-acLDL). However, this method is limited by photobleaching and insufficient quantum efficiency for long-term imaging applications. We have developed a method for conjugation of high quantum efficiency, photostable, and multispectral quantum dot nanocrystals (QD) to acLDL for long-term tracking of EPCs with improved signal-to-noise ratios. Specifically, we conjugated QD to acLDL (QD-acLDL) and used this conjugated fluorophore to label a specific CD34(+) subpopulation of EPCs isolated from rat bone marrow. We then utilized this method to track CD34(+) EPCs in a rat model of laser-induced choroidal neovascularization (LCNV) to evaluate its potential for tracking EPCs in ocular angiogenesis, a critical pathologic feature of several blinding conditions.
1 Communities
1 Members
0 Resources
14 MeSH Terms
Single quantum dot imaging in living cells.
Chang JC, Rosenthal SJ
(2013) Methods Mol Biol 991: 149-62
MeSH Terms: HeLa Cells, Humans, Microscopy, Fluorescence, Quantum Dots
Show Abstract · Added May 27, 2014
Direct visualization of biological processes at single-molecule level provides a detailed perspective which conventional bulk measurements are hard to achieve. Among various classes of fluorescent tags used in single-molecule tracking, quantum dots are particularly useful due to their unique photophysical properties. In this chapter, we describe the principles, methodologies, and experimental protocols for qdot-based single-molecule imaging. The first half provides an overview of fluorescent microscopy and advances in single-molecule tracking using quantum dots. The remainder of this chapter describes methods to carry out qdot-based single-molecule experiments. Detailed protocols including qdot labeling, microscopy setup, and single-molecule analysis using appropriate computational programs are given.
0 Communities
1 Members
0 Resources
4 MeSH Terms
Quantum dot conjugates of GABA and muscimol: binding to α1β2γ2 and ρ1 GABA(A) receptors.
Gussin HA, Tomlinson ID, Cao D, Qian H, Rosenthal SJ, Pepperberg DR
(2013) ACS Chem Neurosci 4: 435-43
MeSH Terms: Animals, Binding Sites, Female, Humans, Muscimol, Protein Binding, Protein Subunits, Quantum Dots, Receptors, GABA-A, Receptors, GABA-B, Xenopus laevis, gamma-Aminobutyric Acid
Show Abstract · Added May 27, 2014
GABAA receptors are ligand-gated ion channels that mediate inhibitory synaptic signaling in the CNS. Fluorescent probes with the ability to target these receptors can provide insights into receptor location, distribution and dynamics in live cells, while revealing abnormalities in their distribution and dynamics that could occur in a variety of diseases. We have developed fluorescent probes of GABAA receptors that are composed of a CdSe/ZnS core-shell nanocrystal (quantum dot; qdot) conjugated to pegylated derivatives of the GABA receptor agonists GABA and muscimol (GABA-qdots and muscimol-qdots, respectively). Quantitative fluorescence imaging was used to analyze the binding activity of these conjugates to α1β2γ2 GABAA and ρ1 GABAA receptors expressed in Xenopus oocytes. The selectivity of these conjugates for α1β2γ2 GABAA and ρ1 GABAA receptors was determined by their ability to compete with the antagonists bicuculline and methyl-(1,2,3,6-tetrahydropyridin-4-yl)phosphinic acid (TPMPA). Both GABA- and muscimol-qdots exhibited robust binding to both α1β2γ2 and ρ1 GABAA receptors. At α1β2γ2 receptors, pretreatment with bicuculline reduced conjugate binding by ≥8-fold on average, an extent far exceeding the reduction produced by TPMPA (~30%). Conversely, at ρ1 receptors, pretreatment with TPMPA inhibited binding by ~10-fold, an extent greatly exceeding the change produced by bicuculline (~50% or less). These results indicate specific binding of muscimol-qdots and GABA-qdots to α1β2γ2 GABAA and ρ1 GABAA receptors in a manner that preserves the respective pharmacological sensitivities of these receptors to TPMPA and bicuculline, and encourage the use of qdot-conjugated neurotransmitter analogs as labeling agents at GABAA receptors.
0 Communities
1 Members
0 Resources
12 MeSH Terms
Visualization of lipid raft membrane compartmentalization in living RN46A neuronal cells using single quantum dot tracking.
Chang JC, Rosenthal SJ
(2012) ACS Chem Neurosci 3: 737-43
MeSH Terms: Animals, Cell Compartmentation, Cell Line, Transformed, G(M1) Ganglioside, Membrane Microdomains, Neurons, Quantum Dots, Rats
Show Abstract · Added May 27, 2014
Lipid rafts are cholesterol-enriched subdomains in the plasma membrane that have been reported to act as a platform to facilitate neuronal signaling; however, they are suspected to have a very short lifetime, up to only a few seconds, which calls into question their roles in biological signaling. To better understand their diffusion dynamics and membrane compartmentalization, we labeled lipid raft constituent ganglioside GM1 with single quantum dots through the connection of cholera toxin B subunit, a protein that binds specifically to GM1. Diffusion measurements revealed that single quantum dot-labeled GM1 ganglioside complexes undergo slow, confined lateral diffusion with a diffusion coefficient of ∼7.87 × 10(-2) μm(2)/s and a confinement domain about 200 nm in size. Further analysis of their trajectories showed lateral confinement persisting on the order of tens of seconds, comparable to the time scales of the majority of cellular signaling and biological reactions. Hence, our results provide further evidence in support of the putative function of lipid rafts as signaling platforms.
0 Communities
1 Members
0 Resources
8 MeSH Terms
Labeling of neuronal receptors and transporters with quantum dots.
Chang JC, Kovtun O, Blakely RD, Rosenthal SJ
(2012) Wiley Interdiscip Rev Nanomed Nanobiotechnol 4: 605-19
MeSH Terms: Animals, Fluorescent Dyes, Humans, Molecular Imaging, Molecular Probes, Neurons, Neurotransmitter Transport Proteins, Protein Transport, Quantum Dots, Receptors, Neurotransmitter
Show Abstract · Added July 10, 2013
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.
Copyright © 2012 Wiley Periodicals, Inc.
0 Communities
3 Members
0 Resources
10 MeSH Terms