Novel Fluorescent Ligands Enable Single-Molecule Localization Microscopy of the Dopamine Transporter

Featured Paper of the Month – February 2021

The dopamine transporter (DAT) functions to control dopaminergic neurotransmission and is a target for therapeutic agents, including ADHD medications, as well as abused substances, such as cocaine. Here, we develop new fluorescently labeled ligands as promising new tools for studying DAT localization and regulation with single-molecule resolution. Visualization with confocal or TIRF microscopy revealed selective binding of these fluorescent ligands to DAT. Single particle tracking experiments were performed that enabled quantification and categorization of the dynamic behavior of DAT into four distinct motion classes (immobile, confined, Brownian, and directed). Moreover, we show that these ligands can be used in direct stochastic optical reconstruction microscopy (dSTORM) experiments permitting further analyses of DAT distribution on the nanoscale.

Publication Information

Guthrie, Daryl A; Herenbrink, Carmen Klein; Lycas, Matthew Domenic; Ku, Therese; Bonifazi, Alessandro; DeVree, Brian T; Mathiasen, Signe; Javitch, Jonathan A; Grimm, Jonathan B; Lavis, Luke; Gether, Ulrik; Newman, Amy Hauck

Novel Fluorescent Ligands Enable Single-Molecule Localization Microscopy of the Dopamine Transporter Journal Article

In: ACS Chemical Neuroscience, vol. 11, no. 20, pp. 3288–3300, 2020.

Abstract | Links

@article{Guthrie:2020aa,

title = {Novel Fluorescent Ligands Enable Single-Molecule Localization Microscopy of the Dopamine Transporter},

author = {Daryl A Guthrie and Carmen Klein Herenbrink and Matthew Domenic Lycas and Therese Ku and Alessandro Bonifazi and Brian T DeVree and Signe Mathiasen and Jonathan A Javitch and Jonathan B Grimm and Luke Lavis and Ulrik Gether and Amy Hauck Newman},

url = {https://pubmed.ncbi.nlm.nih.gov/32926777/},

doi = {10.1021/acschemneuro.0c00397},

year  = {2020},

date = {2020-10-21},

booktitle = {ACS Chemical Neuroscience},

journal = {ACS Chemical Neuroscience},

volume = {11},

number = {20},

pages = {3288--3300},

publisher = {American Chemical Society},

abstract = {The dopamine transporter (DAT) is critical for spatiotemporal control of dopaminergic neurotransmission and is the target for therapeutic agents, including ADHD medications, and abused substances, such as cocaine. Here, we develop new fluorescently labeled ligands that bind DAT with high affinity and enable single-molecule detection of the transporter. The cocaine analogue MFZ2-12 (1) was conjugated to novel rhodamine-based Janelia Fluorophores (JF549 and JF646). High affinity binding of the resulting ligands to DAT was demonstrated by potent inhibition of $[$3H$]$dopamine uptake in DAT transfected CAD cells and by competition radioligand binding experiments on rat striatal membranes. Visualization of binding was substantiated by confocal or TIRF microscopy revealing selective binding of the analogues to DAT transfected CAD cells. Single particle tracking experiments were performed with JF549-conjugated DG3-80 (3) and JF646-conjugated DG4-91 (4) on DAT transfected CAD cells enabling quantification and categorization of the dynamic behavior of DAT into four distinct motion classes (immobile, confined, Brownian, and directed). Finally, we show that the ligands can be used in direct stochastic optical reconstruction microscopy (dSTORM) experiments permitting further analyses of DAT distribution on the nanoscale. In summary, these novel fluorescent ligands are promising new tools for studying DAT localization and regulation with single-molecule resolution.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}