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

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.

Characterizing the function-related specific assembly pattern of matrix metalloproteinase-14 by dSTORM imaging.

As transmembrane proteolytic enzyme, matrix metalloproteinase-14 (MMP14) regulates cell migration and cancer metastasis, but how it works at the single molecule level is unclear. Molecular localization is closely related to its function, and revealing its spatial assemble details is thus helpful to understand bio-function. Here, we apply aptamer probe and dSTORM to characterize MMP14 distribution. With demonstrating labeling properties of the probe, we investigate the specific distributed pattern of MMP14 on various cell membranes with different migratory capacities, and find that MMP14 mostly aggregate in clustering state, which becomes more significant with enhancing its hydrolysis efficiency on high-migratory cells. Lots of MMP14 are revealed to be co-localized with its substrate PTK7, and this colocalization decreases with weakening cell migration, suggesting that MMP14 may coordinate cell migration by altering its spatial relationship with substrate proteins. This work will promote a deep understanding of the roles of MMP14 in cell migration and cancer metastasis.