Biotinylated-spiperone ligands for quantum dot labeling of the dopamine D2 receptor in live cell cultures

Tomlinson, Ian; Kovtun, Oleg; Crescentini, Tiffany M.; Rosenthal, Sandra J.

doi:10.1016/j.bmcl.2019.02.024

Cited by 9 publications

(11 citation statements)

References 34 publications

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“…We examined the diffusion dynamics of a presynaptic protein partner of DAT, the D2 dopamine receptor (D2DR). Briefly, we labeled surface D2DR fused to the 3xHA epitope at the extracellular N-terminus with either biotinylated IDT772 [32] featuring modular architecture identical to that of IDT444 or a commercially available, high-affinity, biotinylated anti-HA antigen-binding fragment (Fab). Representative frames, 60-s maximum intensity projections, and reconstructed Qdot trajectories of the ligand- and anti-HA-Fab-tagged D2DRs are displayed in Fig 4a–4c (S6 File) and Fig 4d–4f (S7 File) respectively.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2019

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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.

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Section: Resultsmentioning

confidence: 99%

“…pcDNA3.1-D2DR-3xHA was acquired from the cDNA Resource Center. The IDT772 ligand was synthesized as previously described [32].…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2019

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“…Specificity in staining certain cell components is achieved by using such targeting molecules as antibodies, peptides, nucleic acid fragments, and others. External modules are attached to particles by either chemical conjugation [ 28 , 29 ], or by self-assembly, when illumina-specific adapters are added to streptavidin-biotin beads [ 30 , 31 ], or by barnase-barstar [ 32 , 33 , 34 , 35 ]. Such targeted complexes are actively used in optical microscopy, cell flow cytometry [ 36 , 37 ], and immunohistochemical [ 38 , 39 ] and enzyme immunoassay [ 40 , 41 ].…”

Section: Quantum Dotsmentioning

confidence: 99%

Photoluminescent Nanomaterials for Medical Biotechnology

et al. 2021

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Creation of various photoluminescent nanomaterials has significantly expanded the arsenal of approaches used in modern biomedicine. Their unique photophysical properties can significantly improve the sensitivity and specificity of diagnostic methods, increase therapy effectiveness, and make a theranostic approach to treatment possible through the application of nanoparticle conjugates with functional macromolecules. The most widely used nanomaterials to date are semiconductor quantum dots; gold nanoclusters; carbon dots; nanodiamonds; semiconductor porous silicon; and up-conversion nanoparticles. This paper considers the promising groups of photoluminescent nanomaterials that can be used in medical biotechnology: in particular, for devising agents for optical diagnostic methods, sensorics, and various types of therapy.

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“…Our recent research focus has been on characterizing lateral mobility and defining its signal transduction implications for the D2 subtype dopamine receptor (D2DR), a Class A GPCR [ 14 , 18 ]. D2 dopamine receptors are involved in the regulation of dopamine-dependent motor function, motivation, cognition, emotion, and neuroendocrine secretion [ 19 , 20 ].…”

Section: Introductionmentioning

confidence: 99%

Membrane Nanoscopic Organization of D2L Dopamine Receptor Probed by Quantum Dot Tracking

et al. 2021

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The role of lateral mobility and nanodomain organization of G protein-coupled receptors in modulating subcellular signaling has been under increasing scrutiny. Investigation of D2 dopamine receptor diffusion dynamics is of particular interest, as these receptors have been linked to altered neurotransmission in affective disorders and represent the primary target for commonly prescribed antipsychotics. Here, we applied our single quantum dot tracking approach to decipher intrinsic diffusion patterns of the wild-type long isoform of the D2 dopamine receptor and its genetic variants previously identified in several cohorts of schizophrenia patients. We identified a subtle decrease in the diffusion rate of the Val96Ala mutant that parallels its previously reported reduced affinity for potent neuroleptics clozapine and chlorpromazine. Slower Val96Ala variant diffusion was not accompanied by a change in receptor-receptor transient interactions as defined by the diffraction-limited quantum dot colocalization events. In addition, we implemented a Voronoї tessellation-based algorithm to compare nanoclustering of the D2 dopamine receptor to the dominant anionic phospholipid phosphatidylinositol 4,5-bisphosphate in the plasma membrane of live cells.

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Biotinylated-spiperone ligands for quantum dot labeling of the dopamine D2 receptor in live cell cultures

Cited by 9 publications

References 34 publications

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

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

Photoluminescent Nanomaterials for Medical Biotechnology

Membrane Nanoscopic Organization of D2L Dopamine Receptor Probed by Quantum Dot Tracking

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