Dopamine transporter is enriched in filopodia and induces filopodia formation

Abstract

Dopamine transporter (DAT, SLC6A3) controls dopamine (DA) neurotransmission by mediating re-uptake of extracellular DA into DA neurons. DA uptake depends on the amount of DAT at the cell surface, and is therefore regulated by DAT subcellular distribution. Hence we used spinning disc confocal microscopy to demonstrate DAT localization in membrane protrusions that contained filamentous actin and myosin X (MyoX), a molecular motor located in filopodia tips, thus confirming that these protrusions are filopodia. DAT was enriched in filopodia. In contrast, R60A and W63A DAT mutants with disrupted outward-facing conformation were not accumulated in filopodia, suggesting that this conformation is necessary for DAT filopodia targeting. Three independent approaches of filopodia counting showed that DAT expression leads to an increase in the number of filopodia per cell, indicating that DAT can induce filopodia formation. Depletion of MyoX by RNA interference resulted in a significant loss of filopodia but did not completely eliminate filopodia, implying that DAT-enriched filopodia can be formed without MyoX. In cultured postnatal DA neurons MyoX was mainly localized to growth cones that displayed highly dynamic DAT-containing filopodia. We hypothesize that the concave shape of the DAT molecule functions as the targeting determinant for DAT accumulation in outward-curved membrane domains, and may also allow high local concentrations of DAT to induce an outward membrane bending. Such targeting and membrane remodeling capacities may be part of the mechanism responsible for DAT enrichment in the filopodia and its targeting to the axonal processes of DA neurons.

Synergy between intrinsically disordered domains and structured proteins amplifies membrane curvature sensing

“…Having characterized the molecular mechanism of curvature sensing by disordered domains in vitro, we next asked whether disordered domains are capable of sensing membrane curvature in the crowded, heterogeneous environment of the cellular plasma membrane. Here we utilized a recently developed assay 31 to quantify the partitioning of transmembrane fusion proteins between filopodia, which have an average diameter of approximately 100–200 nm, 32 and the comparatively flat plasma membrane surface (Fig. 4a ).…”

“…Such domains would presumably work against the ability of IBAR to sense concave membrane curvature. Finally, disordered domains are ubiquitous among transmembrane proteins 47 , such as G protein-coupled receptors 48 and the components of highly curved synaptic vesicles 49 , suggesting that disordered domains may help transmembrane proteins sense membrane curvature, in addition to recent reports of curvature sensing by specific transmembrane protein structural features 31 , 50 , 51 .…”

Targeting of dopamine transporter to filopodia requires an outward-facing conformation of the transporter

“…RFP-HA-DAT (Addgene plasmid # 90265) was also previously described 24 . To generate R60A mutation in the template of YFP-DAT (no HA tag, Addgene plasmid # 90228) 39 , the 1622 bp DAT sequence (begins with bp 242 in DAT cDNA) in YFP-HA-R60A was replaced by the corresponding sequence from the YFP-DAT construct using PflMI (5’ end) and SmaI (3’ end) restriction sites.…”

Membrane curvature in cell biology: An integration of molecular mechanisms

“…Furthermore, some transmembrane proteins that localize preferentially to filopodia are shaped to induce negative curvature, such as the dopamine transporter ( Fig. 2 B ; Caltagarone et al, 2015 ).…”