Super-resolution microscopy reveals functional organization of dopamine transporters into cholesterol and neuronal activity-dependent nanodomains

Troels Rahbek-Clemmensen,Freja H Hansen,Matthew D Lycas,Trine N Jørgensen,Ulrik Gether,Mia Apuschkin,Jacob Eriksen,Simon Erlendsson,Frederik Vilhardt

doi:10.1038/s41467-017-00790-3

Troels Rahbek-Clemmensen, Freja H Hansen + Show 7 more

Open Access

https://doi.org/10.1038/s41467-017-00790-3

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Abstract

Dopamine regulates reward, cognition, and locomotor functions. By mediating rapid reuptake of extracellular dopamine, the dopamine transporter is critical for spatiotemporal control of dopaminergic neurotransmission. Here, we use super-resolution imaging to show that the dopamine transporter is dynamically sequestrated into cholesterol-dependent nanodomains in the plasma membrane of presynaptic varicosities and neuronal projections of dopaminergic neurons. Stochastic optical reconstruction microscopy reveals irregular dopamine transporter nanodomains (∼70 nm mean diameter) that were highly sensitive to cholesterol depletion. Live photoactivated localization microscopy shows a similar dopamine transporter membrane organization in live heterologous cells. In neurons, dual-color dSTORM shows that tyrosine hydroxylase and vesicular monoamine transporter-2 are distinctively localized adjacent to, but not overlapping with, the dopamine transporter nanodomains. The molecular organization of the dopamine transporter in nanodomains is reversibly reduced by short-term activation of NMDA-type ionotropic glutamate receptors, implicating dopamine transporter nanodomain distribution as a potential mechanism to modulate dopaminergic neurotransmission in response to excitatory input.

Highlights

Dopamine regulates reward, cognition, and locomotor functions
Dualcolor dSTORM imaging demonstrates that the domains are adjacent to, but not overlapping with, two other key components of dopaminergic terminals, tyrosine hydroxylase (TH) and vesicular monoamine transporter 2 (VMAT2)
For photoactivated localization microscopy (PALM) imaging, the cells were analyzed by total internal reflection microscopy (TIRF-M) enabling imaging of the plasma membrane facing the glass surface[23]

Summary

Introduction

Cognition, and locomotor functions. By mediating rapid reuptake of extracellular dopamine, the dopamine transporter is critical for spatiotemporal control of dopaminergic neurotransmission. The important insights from EM and FM are potentially limited by low antibody labeling efficiency in EM studies, and the restricted resolution of FM These approaches may have missed nanoscale heterogeneities in the subcellular distribution of the transporter that could be important for spatiotemporal control of its function. In this context it is interesting that DAT has been suggested to be sequestered into cholesterol- and glycosphingolipid-enriched plasma membrane micro domains (“membrane rafts”)[12,13,14,15], and that these have been proposed to serve as “hot spots” for regulation of transporter trafficking and function including amphetamine-induced dopamine release[12, 13]. Summarized, our data describe a dynamic nanodomain distribution of DAT that might enable the neuron to rapidly switch the transporter between different functional localizations and thereby optimize availability and activity of the transporter on the nanoscale in the presynaptic terminals

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