Quantification of lysosomal labile Zn2+ and monitoring of Zn2+ efflux using a small-molecule–protein hybrid fluorescent probe

Lysosomal labile Zn2+ levels have been unclear. By targeting a small-molecule fluorescent Zn2+ probe, ZnDA-3H, to lysosomes via VAMP7-Halo, the lysosomal labile Zn2+ concentration was determined to be 1.9 nM in HeLa cells. Furthermore, ZnDA-3H enabled direct visualization of the Zn2+ efflux from the lysosomes to cytosol upon TRPMLs activation. A green fluorescent Zn2+ probe, ZnDA-3H, was applied to visualize lysosomal Zn2+ by labeling to VAMP7-Halo. Lysosomal [Zn2+] was quantified by ratiometric imaging using ZnDA-3H and a red fluorescent HaloTag TMR ligand. Zn2+ efflux from the lysosomes to cytosol upon TRPMLs activation could be monitored using ZnDA-3H. [Display omitted] •A green fluorescent Zn2+ probe, ZnDA-3H, was applied to visualize lysosomal Zn2+.•HaloTag-conjugated ZnDA-3H showed small changes in Kd in the physiological pH range.•Lysosomal [Zn2+] was determined using ZnDA-3H via labeling to VAMP7-Halo.