Dopamine transporter is enriched in filopodia and induces filopodia formation
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.
Section snippets
Reagents
Antibodies were purchased from the following sources: mouse monoclonal antibody to hemagglutinin epitope HA11 (16B12, cat# MMS-101P) and HA11 conjugated with Alexa488 (HA11-A488, cat# A488-101L) were from Covance (Berkley, CA); rabbit polyclonal myosin X (MyoX) antibody (working dilution 1:100–500 and 1:1000 in immunofluorescence and western blotting experiments, respectively) from Sigma Aldrich (St. Louis, MO) (cat# HPA024223); rabbit polyclonal antibody to α-actinin from Cell Signaling
DAT-containing membrane protrusions are filopodia
Previous studies demonstrated localization of DAT in filopodia-like membrane protrusions in non-neuronal cells and cultured post-natal dopaminergic neurons (Rao et al., 2012, Sorkina et al., 2009). To test whether these protrusions containing DAT are “classical” filopodia, DAT localization was compared with that of filamentous (F)-actin and a marker of filopodia, unconventional myosin, MyoX (also known as myosin 10) (Berg and Cheney, 2002). PAE cells were used as the main experimental model
Discussion
In this study we used fluorescence microscopy imaging of living and fixed cells to demonstrate that finger-like membrane protrusions containing DAT are the “conventional” filopodia, and to analyze the mechanisms of DAT targeting to these filopodia. The filopodia nature of DAT containing protrusions was evident from the high extent of DAT co-localization with the actin-associated motor MyoX in various types of cells heterologously expressing DAT (Figs. 1, S1 and 5). MyoX was located in the tips
Acknowledgments
We thank Drs. Cheney, Lichius, Roy, Umasanker and Verkhusha for generous gifts of the reagents. This work was supported by the NIH/NIDA grants DA014204 (J. C, S. M. and A.S.) and T32DA031111 (J. C.). The authors have no conflict of interest to declare.
References (38)
- et al.
Membrane shape modulates transmembrane protein distribution
Dev. Cell
(2014) Mechanisms underlying the initiation and dynamics of neuronal filopodia: from neurite formation to synaptogenesis
Int. Rev. Cell Mol. Biol.
(2013)- et al.
Neurotransmitter transporters: molecular function of important drug targets
Trends Pharmacol. Sci.
(2006) - et al.
Membrane cholesterol modulates the outward facing conformation of the dopamine transporter and alters cocaine binding
J. Biol. Chem.
(2010) - et al.
Dopamine: 50 years in perspective
Trends Neurosci.
(2007) Neurotransmitter transporter trafficking: endocytosis, recycling, and regulation
Pharmacol. Ther.
(2004)- et al.
Multiple molecular determinants in the carboxyl terminus regulate dopamine transporter export from endoplasmic reticulum
J. Biol. Chem.
(2004) - et al.
Neuronal myosin-X is upregulated after peripheral nerve injury and mediates laminin-induced growth of neurites
Mol. Cell. Neurosci.
(2013) - et al.
Oligomerization of dopamine transporters visualized in living cells by fluorescence resonance energy transfer microscopy
J. Biol. Chem.
(2003) - et al.
Myosin X transports Mena/VASP to the tip of filopodia
Biochem. Biophys. Res. Commun.
(2004)
Oligomerization and trafficking of the human dopamine transporter. Mutational analysis identifies critical domains important for the functional expression of the transporter
J. Biol. Chem.
Membrane mobility and microdomain association of the dopamine transporter studied with fluorescence correlation spectroscopy and fluorescence recovery after photobleaching
Biochemistry
F-actin dynamics in Neurospora crassa
Eukaryot. Cell
Myosin-X is an unconventional myosin that undergoes intrafilopodial motility
Nat. Cell Biol.
Myosin-X is a molecular motor that functions in filopodia formation
Proc. Natl. Acad. Sci. U. S. A.
Effects of 6-hydroxydopamine on primary cultures of substantia nigra: specific damage to dopamine neurons and the impact of glial cell line-derived neurotrophic factor
J. Neurochem.
Phorbol ester induced trafficking-independent regulation and enhanced phosphorylation of the dopamine transporter associated with membrane rafts and cholesterol
J. Neurochem.
The cytoskeletal and signaling mechanisms of axon collateral branching
Dev. Neurobiol.
Myo10 is a key regulator of TNT formation in neuronal cells
J. Cell Sci.
Cited by (11)
Enzymatic trans-bilayer lipid transport: Mechanisms, efficiencies, slippage, and membrane curvature
2021, Biochimica et Biophysica Acta - BiomembranesCitation Excerpt :There are two ways by which this could be accomplished. First, either the transmembrane protein reshapes the membrane [46,47] or second, the protein is sorted into membrane regions which are preferentially curved [48,49]. Since flippases transition between different conformations during the transport cycle, membrane curvature could stabilize certain conformations over the other.
Trimerization of dopamine transporter triggered by AIM-100 binding: Molecular mechanism and effect of mutations
2019, NeuropharmacologyCitation Excerpt :While the ability of AIM-100 to stabilize DAT trimers or oligomers appears to depend on the conformation, OF or IF, of DAT (Sorkina et al., 2018), the molecular mechanisms that underlie the stabilization of the trimers remain unknown. The breakthrough in structural (Penmatsa et al., 2013, 2015; Wang et al., 2015), in silico (Cheng and Bahar, 2015; Cheng et al., 2015, 2017, 2018; Gur et al., 2017; Kaya et al., 2018; Khelashvili et al., 2015; LeVine et al., 2018) and live-cell (Adkins et al., 2007; Caltagarone et al., 2015; Sorkina et al., 2003, 2013) studies of DAT dynamics and function now permit us to gain a better understanding of the link between DAT structural dynamics, oligomerization, and function. Here we explore the propensity of DAT to trimerize in the absence and presence of AIM-100, as well as its implication on DAT structural dynamics using a combination of computational and biochemical methods, and live-cell imaging assays.
Plasma membrane phospholipid phosphatase-related proteins as pleiotropic regulators of neuron growth and excitability
2022, Frontiers in Molecular NeuroscienceMethodological Nuances of Measuring Membrane Protein Nanoscopic Organization: A Case of Dopamine Transporter
2022, Journal of the Electrochemical SocietyA Novel Biotinylated Homotryptamine Derivative for Quantum Dot Imaging of Serotonin Transporter in Live Cells
2021, Frontiers in Cellular NeurosciencePrinciples and Applications of Biological Membrane Organization
2020, Annual Review of Biophysics