Zn ²⁺ inhibits KIF5A movement in situ in a dose dependent manner. (A) Schematic of the components of the peroxisome dispersion assay. (B) Representative plots of mean change in peroxisome distance from the cell center over time, for COS-7 cells expressing KIF5A(1-560)-mNG-FRB and PEX3-mRFP-2xFKBP, treated at 0 s with 1 µM PTO and various concentrations of Zn²⁺ (none, purple; 2 µM, blue; 3 µM, teal; or 5 µM, lime), and 100 nM Zotarolimus (“Zota”) at 300 s. (C) Representative micrographs of COS-7 cells expressing KIF5A(1-560)-mNG-FRB (green, top), PEX3-mRFP-2xFKBP (magenta, middle), or merged channels (bottom), at 0 s (“baseline”) or 25 min after treatment with 100 nM Zotarolimus (“post-Zota”) in the presence of the indicated Zn²⁺ concentrations. Scale bar = 10 µm. (D–F) Fluorescent intensity averages of PEX3-mRFP-2xFKBP in COS-7 cells co-expressing KIF5A(1-560)-mNG-FRB. Cells were imaged and aligned by their geometric centers after the following treatments and fixation: (D) 2 µl DMSO for 5 min, then an additional 2 µl DMSO for 25 min (n = 42 cells); (E) 1 µM PTO for 5 min, then 100 nM Zotarolimus for 25 min (n = 44 cells); (F) 10 µM ZnCl₂ and 1 µM PTO for 5 min, then 100 nM Zotarolimus for 25 min (n = 46 cells). Black dotted line indicates the area within 10 µm of the center. (G) Box plots and individual points representing the proportion of PEX3-mRFP-2xFKBP fluorescence within 10 µm of the geometric cell center, for the each of the conditions in (D–F). One-way ANOVA with post-hoc Tukey HSD. All experiments were performed in the absence of extracellular Ca²⁺. **** P < 0.0001, ** P < 0.01.