Department of Chemistry, Vanderbilt University, Nashville, Tennessee, United States of America.
Vanderbilt Institute of Nanoscale Science and Engineering, Vanderbilt University, Nashville, Tennessee, United States of America.
PLoS One. 2019 Nov 21;14(11):e0225339. doi: 10.1371/journal.pone.0225339. eCollection 2019.
The presynaptic dopamine transporter mediates rapid reuptake of synaptic dopamine. Although cell surface DAT trafficking recently emerged as an important component of DAT regulation, it has not been systematically investigated. Here, we apply our single quantum dot (Qdot) tracking approach to monitor DAT plasma membrane dynamics in several heterologous expression cell hosts with nanometer localization accuracy. We demonstrate that Qdot-tagged DAT proteins exhibited highly heterogeneous membrane diffusivity dependent on the local membrane topography. We also show that Qdot-tagged DATs were localized away from the flat membrane regions and were dynamically retained in the membrane protrusions and cell edges for the duration of imaging. Single quantum dot tracking of wildtype DAT and its conformation-defective coding variants (R60A and W63A) revealed a significantly accelerated rate of dysfunctional DAT membrane diffusion. We believe our results warrant an in-depth investigation as to whether compromised membrane dynamics is a common feature of brain disorder-derived DAT mutants.
突触前多巴胺转运体介导突触多巴胺的快速重摄取。尽管细胞表面 DAT 转运最近被认为是 DAT 调节的一个重要组成部分，但它尚未得到系统的研究。在这里，我们应用我们的单量子点 (Qdot) 跟踪方法来监测几种异源表达细胞宿主中 DAT 质膜动力学，具有纳米级定位精度。我们证明，Qdot 标记的 DAT 蛋白表现出高度异质的膜扩散性，这取决于局部膜形貌。我们还表明，Qdot 标记的 DAT 位于远离平坦膜区域的位置，并在成像过程中动态保留在膜突起和细胞边缘。野生型 DAT 和其构象缺陷编码变体 (R60A 和 W63A) 的单量子点跟踪显示，功能失调的 DAT 膜扩散率显著加快。我们相信我们的结果值得深入研究，即膜动力学受损是否是源自大脑紊乱的 DAT 突变体的共同特征。
