Abstract：
Nucleoside transporters (NTs) play an important role in the metabolism of nucleoside substances and the efficacy of nucleoside drugs. Its spatial information related to biofunctions at the single-molecule level remains unclear, owing to the limitation of the existing labeling methods and traditional imaging methods. Therefore, we synthesize the inhibitor-based fluorescent probe SAENTA-Cy5 and apply direct stochastic optical reconstruction microscopy (dSTORM) to conduct refined observation of human equilibrative nucleoside transporter 1 (hENT1), the most important and famous member of NTs. We first demonstrate the labeling specificity and superiority of SAENTA-Cy5 to the antibody probe. Then, we found different assembly patterns of hENT1 on the apical and basal membranes, which are further investigated to be caused by varying associations of membrane carbohydrates, membrane classical functional domains (lipid rafts), and associated membrane proteins (EpCAM). Our work provides an efficient method for labeling hENT1, which contributes to realize fine observation of NTs. The findings on the assembly features and potential assembly mechanism of hENT1 promote a better understanding of its biofunction, which facilitates further investigations on how NTs work in the metabolism of nucleoside and nucleoside analogues.
摘要：
核苷转运体(NTs)在核苷类物质的代谢和核苷类药物的疗效中起着重要作用。其在单分子水平上与生物功能相关的空间信息仍不清楚，由于现有标记方法和传统成像方法的局限性。因此,我们合成了基于抑制剂的荧光探针SAENTA-Cy5，并应用直接随机光学重建显微镜（dSTORM）进行人平衡核苷转运蛋白1（hENT1）的精细观察，NTs中最重要和最著名的成员。我们首先证明了SAENTA-Cy5对抗体探针的标记特异性和优越性。然后，我们在根尖和基底膜上发现了不同的hENT1组装模式，进一步研究是由膜碳水化合物的不同关联引起的，膜经典功能结构域（脂筏），和相关膜蛋白（EpCAM）。我们的工作为标记hENT1提供了一种有效的方法，有助于实现对NTs的精细观察。关于hENT1的组装特征和潜在组装机制的发现促进了对其生物功能的更好理解，这有助于进一步研究NTs如何在核苷和核苷类似物的代谢中发挥作用。
