Zn ²⁺ inhibits organellar motility with nanomolar IC ₅₀ in HeLa cells and neurons. (A) Schematic of the IC₅₀ assay in HeLa cells (top) showing cytosolic localization of co-transfected GZnP2 sensor and either lysosomal (left, magenta) or mitochondrial (right, red) mCherry. Representative micrographs of HeLa cells (bottom) illustrating the localization of GZnP2 sensor in the cytoplasm and either LAMP1-mCherry (magenta, top) or mito-mCherry (red, bottom). Scale bar = 20 µm. (B and C) Representative plots of [Zn²⁺] (B; determined using GZnP2; see Materials and methods) and relative lysosomal motility (C; via LAMP1-mCherry) in a HeLa cell treated as indicated (100 µM TPEN at t = 120 s; a 2-min duration washout at t = 300 s; 100 µM ZnCl₂ at t = 420 s; and 5 µM pyrithione at t = 1,680 s). (D and E) Representative plot of relative motility versus [Zn²⁺] used to calculate IC₅₀ values Zn²⁺ inhibition of (D) LAMP1-mCherry or (E) mito-mCherry motility. (F) Schematic of the IC₅₀ assay in primary hippocampal neurons showing cytosolic localization of co-transfected GZnP2 sensor (green) and LAMP1-mCherry tagged lysosomes (magenta). (G) Overlaid plots of KymoButler-analyzed lysosome speed versus [Zn²⁺] used to calculate IC₅₀ values for Zn²⁺ inhibition of lysosomes in neuron axons. The colors represent individual experiments in different axons. (H) Mean IC₅₀ values (±SEM) for LAMP1-mCherry (“LAMP1”) in HeLa cells (magenta; n = 8 cells from six individual replicates) or primary rat hippocampal neuron axons (cyan; n = 7 neuron axons from individual replicates) and mito-mCherry in HeLa cells (“Mito,” red; n = 7 cells from five individual replicates. All experiments were performed in the absence of extracellular Ca²⁺.