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

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.

Membrane composition influences the conformation and function of the dopamine transporter in vivo

“…Although the partitioning of DAT and other transporters into cholesterol-rich membranes represented by DRMs has been replicated by several groups (Cremona et al, 2011;Hong and Amara, 2010;Jones et al, 2012), the larger significance of this partitioning remains unclear. By super-resolution microscopy, DAT has been found associated to cholesterol-enriched, Flot1 positive nanodomains (Rahbek-Clemmensen et al, 2017), that is well correlated with the biochemical data. Flot1 depletion in cell lines has no impact on DAT-mediated uptake of DA, but in primary mesencephalic neurons, Flot1 depletion diminishes the non-exocytic release of DA through DAT after administration of the psychostimulant amphetamine (AMPH) (Cremona et al, 2011), and diminished AMPH-evoked locomotion in Drosophila larvae heterologously expressing human DAT (hDAT) (Pizzo et al, 2013).…”

The quaternary structure of human tyrosine hydroxylase: effects of dystonia‐associated missense variants on oligomeric state and enzyme activity

“…It is well established that TH is activated and stabilized by binding to 14-3-3 proteins ) and it has also been suggested that TH might be part of macromolecular assemblies with multiple protein components that are involved in dopamine synthesis and transport (Parra et al 2016). However, more recent imaging data are not compatible with such multi-protein complexes, at least in dopaminergic neurons (Rahbek-Clemmensen et al 2017).…”

“…However, more recent imaging data are not compatible with such multi‐protein complexes, at least in dopaminergic neurons (Rahbek‐Clemmensen et al . ).…”

Dual role of Miro protein clusters in mitochondrial cristae organisation and ER-Mitochondria Contact Sites

“…6). Further, we performed Density-Based Spatial Clustering of Applications with Noise (DBSCAN) which has been widely used to asses clustering of various membrane proteins 61 . The DBSCAN cluster map showed nanoscale domains formed by both GFP Miro1 and GFP Miro2 in Hela cells ( Fig.…”

“…6A). In addition, we carried out DBSCAN analysis which can reveal the extent of cluster formation from the raw localization data 61 . In WT MEFs, DBSCAN analysis revealed the previously reported formation of Mic60/Mitofilin clusters arranged in a "discontinuous rail-like distribution" 31 ( Fig.…”