Assembly Characterization of Human Equilibrium Nucleoside Transporter 1 (hENT1) by Inhibitor Probe-Based dSTORM Imaging

Nucleoside transporters (NTs) play an important rolein the metabolismof nucleoside substances and the efficacy of nucleoside drugs. Itsspatial information related to biofunctions at the single-moleculelevel remains unclear, owing to the limitation of the existing labelingmethods and traditional imaging methods. Therefore, we synthesizethe inhibitor-based fluorescent probe SAENTA-Cy5 and apply directstochastic optical reconstruction microscopy (dSTORM) to conduct refinedobservation of human equilibrative nucleoside transporter 1 (hENT1),the most important and famous member of NTs. We first demonstratethe labeling specificity and superiority of SAENTA-Cy5 to the antibodyprobe. Then, we found different assembly patterns of hENT1 on theapical and basal membranes, which are further investigated to be causedby varying associations of membrane carbohydrates, membrane classicalfunctional 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 findingson the assembly features and potential assembly mechanism of hENT1promote a better understanding of its biofunction, which facilitatesfurther investigations on how NTs work in the metabolism of nucleosideand nucleoside analogues.