Zn ²⁺ influx arrests axonal transport of mitochondria, lysosomes, and NPY ⁺ vesicles. (A) Representative kymographs (left) and measured tracks (identified using KymoButler; right) of mito-mCherry motility along axons from primary cultured rat hippocampal neurons prior to (“baseline”; top) or after initiation of Zn²⁺ influx by high K⁺ depolarization (middle-top), following washout and addition of 100 µM TPEN (middle-bottom), and then washout and addition of 100 µM Zn²⁺ and 2.5 µM PTO (bottom). (B) Mean instantaneous speed (±SEM) of mitochondria moving retrograde (blue) or anterograde (green) across baseline, Zn²⁺ influx by high K⁺ depolarization, TPEN, and Zn²⁺ + PTO treatment (60-s binned, representing 7 axons). (C) Mean speed (±SEM) of mitochondria moving in the indicated directions for each condition (n = 7 axons from three independent replicates). (D) Proportions of mitochondria moving in the indicated directions across baseline, depolarization with Zn²⁺, TPEN, and Zn²⁺ + PTO treatment (60-s binned, representing 7 axons). (E) Mean proportions of mitochondrial motility for each condition. (F) Mean instantaneous speed (±SEM) of lysosomes moving retrograde (blue) or anterograde (green) across baseline, Zn²⁺ influx by high K⁺ depolarization, following washout and addition of 100 µM TPEN, and then washout and addition of 100 µM Zn²⁺ and 2.5 µM PTO (bottom; 60-s binned, representing 6 axons). (G) Mean speed (±SEM) of lysosomes moving in the indicated directions for each condition (n = 6 axons from three independent replicates). (H) Proportions of lysosomes moving in the indicated directions across baseline, Zn²⁺ influx by high K⁺ depolarization, TPEN, and Zn²⁺ + PTO treatment (60-s binned, representing 6 axons). (I) Mean instantaneous speed (±SEM) of NPY⁺ vesicles moving retrograde (blue) or anterograde (green) across baseline, 100 µM Zn²⁺ and 2.5 µM PTO, and addition of 100 µM TPEN after washout (30-s binned, representing 5 axons). (J) Mean speed of NPY⁺ vesicles moving in the indicated directions for each condition (n = 5 axons from five independent replicates). (K) Proportions of NPY⁺ vesicles moving in the indicated directions across baseline, depolarization with Zn²⁺, TPEN, and Zn²⁺ + PTO treatment (30-s binned, representing 5 axons). All experiments were performed in the absence of extracellular Ca²⁺. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, n.s. not significant.