ABCB1 Can Actively Pump‐out the Background‐Free Tame Fluorescent Probe CO‐1 From Live Cells

Abstract

Cell labelling using a small fluorescent probe is an important technique in biomedical sciences. We previously developed a biocompatible and membrane‐permeable probe, CO‐1, which has low nonspecific binding affinity towards nontarget molecules. Although this background‐free tame probe has been utilized for labelling of various intracellular biomolecules in live cells, the probes’ backgroung‐free staining mechanism was not fully understood. Here, we propose that Gating‐Oriented Live‐cell Distinction (GOLD) mechanism occurs when ABCB1 transporter removes unbound CO‐1 molecules from mammalian cells and, in a minor role, DIRC2 pumps CO‐1 out from lysosomes. We also showed that solute carrier transporters were not involved in carrying CO‐1 inside of cells. The role of reporters in assisting the probes’ influx‐efflux was analyzed by the combination of CRISPR library sceenings and inhibitors test. In summary, tame probe CO‐1 cellular staining occurs in a dual mechanism where the probe moves freely through the cells membrane, but its washable property can be directly related to the action of ABCB1 transporter.

Chemical probes based on GOLD: The discovery and elucidation of mechanism

“…52 Clone cells, highly expressing each one of the target transporters, also did not have increased fluorescent intensity after CO-1 staining. 52 The results implicated that SLC was not responsible for the cellular uptake of this tame probe. On the other hand, an ABC-CRISPRi (bright sorting) screening resulted in the enrichment of only two transporters.…”

“…52 ABCB1 inhibitor blocked the exit of CO-1 from Hela cells, resulting in high fluorescence intensity in both fluorescent imaging and flow cytometry analysis. 52 ABCB1 is located on the cell membrane of Hela cells and is most likely to export CO-1 from the cytosol. 52 DIRC2 contribution was much lower, but it could not be excluded.…”

“…52 ABCB1 is located on the cell membrane of Hela cells and is most likely to export CO-1 from the cytosol. 52 DIRC2 contribution was much lower, but it could not be excluded. The analysis of highmagnification images showed that in the presence of DIRC2 inhibitors, the fluorescence intensity was 11%-21% higher than the negative control.…”

“…The analysis of highmagnification images showed that in the presence of DIRC2 inhibitors, the fluorescence intensity was 11%-21% higher than the negative control. 52 DIRC2 is localized in the lysosomal membrane, and it is important for lysosomal biogenesis. 77 Co-staining analysis showed the overlap of CO-1 with the Lysotracker label in the presence of DIRC2 inhibitors.…”

“…52 Mostly likely DIRC2 contributes to the removal of CO-1 from the lysosomes. 52 In short, CO-1 can passively enter cells without the help of SLC transporters, but the dye removal is accelerated by the action of two transporters. ABCB1 is mainly responsible for the exclusion of CO-1 from the cytosol, while DIRC2 plays a minor role in cleaning the dye from the lysosomal compartments.…”