Caltagarone John, Ma Shiqi, Sorkin Alexander
Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, United States.
Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, United States.
Mol Cell Neurosci. 2015 Sep;68:120-30. doi: 10.1016/j.mcn.2015.04.005. Epub 2015 Apr 30.
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.
多巴胺转运体（DAT，SLC6A3）通过介导细胞外多巴胺重新摄取到多巴胺能神经元中来控制多巴胺（DA）神经传递。多巴胺摄取取决于细胞表面DAT的数量，因此受DAT亚细胞分布的调节。因此，我们使用转盘共聚焦显微镜来证明DAT定位于含有丝状肌动蛋白和肌球蛋白X（MyoX）的膜突起中，肌球蛋白X是一种位于丝状伪足尖端的分子马达，从而证实这些突起是丝状伪足。DAT在丝状伪足中富集。相比之下，具有外向构象破坏的R60A和W63A DAT突变体没有在丝状伪足中积累，这表明这种构象对于DAT靶向丝状伪足是必需的。三种独立的丝状伪足计数方法表明，DAT表达导致每个细胞丝状伪足数量增加，表明DAT可以诱导丝状伪足形成。通过RNA干扰耗尽MyoX导致丝状伪足显著减少，但并未完全消除丝状伪足，这意味着不含MyoX也可以形成富含DAT的丝状伪足。在培养的新生多巴胺能神经元中，MyoX主要定位于生长锥，生长锥显示出含有高度动态DAT的丝状伪足。我们假设DAT分子的凹形作为DAT在向外弯曲的膜结构域中积累的靶向决定因素，并且还可能允许高局部浓度的DAT诱导向外的膜弯曲。这种靶向和膜重塑能力可能是负责DAT在丝状伪足中富集及其靶向多巴胺能神经元轴突过程的机制的一部分。
