Dopamine transporter is enriched in filopodia and induces filopodia formation

Highlights

• DAT is accumulated in filopodia containing myosin X.

• DAT mutants with disrupted outward-facing conformation are not enriched in filopodia.

• DAT induces filopodia formation..

• We proposed that a concave shape of DAT mediates its accumulation in curved membranes.

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 disk 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.

Introduction

Dopamine (DA) signaling in the central nervous system is involved in various aspects of locomotor activity, emotions, reward, and affect (Iversen and Iversen, 2007). Dysfunctions of the DA system are implicated in a spectrum of abnormalities, such as Parkinson's disease, schizophrenia, bipolar disorder, attention deficit hyperactivity disorder, and psychostimulant drug abuse (Hyman et al., 2006, Ueno, 2003). In the mouse brain the somatodendritic compartments of DA neurons are located in the midbrain, in particular in the substantia nigra and ventral tegmental area. During development these neurons project axons mainly to striatum to form a highly elaborate axonal network. The amplitude and duration of the dopaminergic neurotransmission are controlled by the regulation of extracellular DA concentrations via reuptake by the plasma membrane DA transporter (DAT) (Gether et al., 2006, Schmitz et al., 2003). DAT is expressed exclusively in DA neurons with the highest concentration in striatal axons. The mechanisms responsible for targeting of DAT, that is synthesized in the somatodendritic compartments of DA neurons in the midbrain, to the axonal processes of these neurons in the striatum and maintaining high levels of DAT in axons are unknown.

DAT functions in the plasma membrane, and therefore, subcellular localization and trafficking of DAT are important for its activity. For instance, regulation of DAT surface levels by endocytosis has been proposed (Melikian, 2004). In addition, DAT activity is regulated by its sub-compartmentalization in specialized domains of the plasma membrane (Adkins et al., 2007, Foster et al., 2008, Hong and Amara, 2010). We have previously demonstrated DAT localization in filopodia-like membrane protrusions in non-neuronal cells and primary mesencephalic cultures of DA neurons (Rao et al., 2012, Sorkina et al., 2009). Interestingly, DAT was less mobile in these membrane protrusions than in other regions of the plasma membrane (Rao et al., 2012, Sorkina et al., 2009). We then hypothesized that targeting to membrane protrusions serves to retain functional DAT at the cell surface.

In the present study we addressed three main questions. First, are DAT-containing structures actually the filopodia or other types of membrane protrusive structures? Filopodia are broadly defined as thin (0.1–0.3 μm), finger-like structures that are filled with tight parallel bundles of filamentous (F)-actin (Mattila and Lappalainen, 2008). In neurons filopodia have important roles in axonal outgrowth, branching, cell migration, dendritic spine formation and interneuron communication (Gallo, 2011, Gallo, 2013). Second, is DAT enriched in the filopodia-like protrusions? In other words, is concentration of DAT higher in these protrusions than in other areas of the membrane? Third, if DAT is enriched in filopodia-like protrusions, is it targeted to pre-existing protrusions or capable to induce de novo formation of membrane protrusions and maintain these structures? To address these questions, the localization of DAT and its mutants was analyzed using fluorescence microscopy imaging in comparison with subcellular distribution of other membrane proteins, F-actin and resident filopodia proteins.