Figure 6. Zn 2 promotes reversible... | Rockefeller University Press

Figure 6.

Zn2+promotes reversible detachment of DCX, tau, and MAP2C from microtubules in situ. (A) Representative micrographs of COS-7 cells expressing mCherry-α-tubulin (magenta, left) and either EGFP-MAP7, EGFP-DCX, EGFP-Tau, or EGFP-MAP2C, before treatment (“baseline,” top), after 5-min treatment with 20 µM ZnCl2 and 2.5 µM PTO (middle), or after subsequent treatment with 100 µM TPEN (bottom). Scale bar = 10 µm. (B–D) Mean tubeness (±SEM) of (B) EGFP-DCX, (C) EGFP-tau, or (D) EGFP-MAP2C normalized to baseline (gray dashed line), for the indicated treatments. Letters indicate statistically different groups (see Tables S1, S2, and S3). One-way ANOVA with post-hoc Tukey HSD. Zn2+ chelation rescue (100 µM TPEN) was measured with paired t test. (E) Representative time trace of microtubule morphology of EGFP-DCX in COS-7 cells as measured by “Tubeness” analysis (see Materials and methods and Fig. S4). Cells were treated with 20 µM ZnCl2 at 0 s and 100 µM TPEN at 300 s. (F) Mean tubeness (±SEM) of the indicated microtubule-associated proteins in COS-7 cells treated with 20 µM ZnCl2 and 2.5 µM PTO for 5 min, was normalized to baseline. Letters indicate statistically different groups (see Table S4). One-way ANOVA with post-hoc Tukey HSD. (G) Representative immunofluorescence micrographs of methanol-fixed primary rat hippocampal neurons depolarized with 50 mM KCl in the absence (top) or presence (bottom) of 100 µM ZnCl2. Fixed cells were incubated with anti-β-tubulin monoclonal antibodies (magenta, left), anti-tau polyclonal antibodies (green, center), and channels are shown merged (right). Scale bar = 10 µm. (H) Fluorescence intensity of AlexaFluor 488 secondary antibody-labeled tau decorating linear tubulin-positive regions across a 1 mm2 area of neurons depolarized with 50 mM KCl in the absence (blue) or presence (magenta) of 100 µM ZnCl2 (see Fig. S5 A for the stitched image of entire 1 mm2 area; 50 mM KCl: n = 4,330 linear segments from two biological replicates; 50 mM KCl and 100 µM ZnCl2: n = 4,820 linear segments from two biological replicates). Unpaired t test assuming unequal variances. All experiments were performed in the absence of extracellular Ca2+, except where indicated. **** P < 0.0001.

Zn ²⁺ promotes reversible detachment of DCX, tau, and MAP2C from microtubules in situ. (A) Representative micrographs of COS-7 cells expressing mCherry-α-tubulin (magenta, left) and either EGFP-MAP7, EGFP-DCX, EGFP-Tau, or EGFP-MAP2C, before treatment (“baseline,” top), after 5-min treatment with 20 µM ZnCl₂ and 2.5 µM PTO (middle), or after subsequent treatment with 100 µM TPEN (bottom). Scale bar = 10 µm. (B–D) Mean tubeness (±SEM) of (B) EGFP-DCX, (C) EGFP-tau, or (D) EGFP-MAP2C normalized to baseline (gray dashed line), for the indicated treatments. Letters indicate statistically different groups (see Tables S1, S2, and S3). One-way ANOVA with post-hoc Tukey HSD. Zn²⁺ chelation rescue (100 µM TPEN) was measured with paired t test. (E) Representative time trace of microtubule morphology of EGFP-DCX in COS-7 cells as measured by “Tubeness” analysis (see Materials and methods and Fig. S4). Cells were treated with 20 µM ZnCl₂ at 0 s and 100 µM TPEN at 300 s. (F) Mean tubeness (±SEM) of the indicated microtubule-associated proteins in COS-7 cells treated with 20 µM ZnCl₂ and 2.5 µM PTO for 5 min, was normalized to baseline. Letters indicate statistically different groups (see Table S4). One-way ANOVA with post-hoc Tukey HSD. (G) Representative immunofluorescence micrographs of methanol-fixed primary rat hippocampal neurons depolarized with 50 mM KCl in the absence (top) or presence (bottom) of 100 µM ZnCl₂. Fixed cells were incubated with anti-β-tubulin monoclonal antibodies (magenta, left), anti-tau polyclonal antibodies (green, center), and channels are shown merged (right). Scale bar = 10 µm. (H) Fluorescence intensity of AlexaFluor 488 secondary antibody-labeled tau decorating linear tubulin-positive regions across a 1 mm² area of neurons depolarized with 50 mM KCl in the absence (blue) or presence (magenta) of 100 µM ZnCl₂ (see Fig. S5 A for the stitched image of entire 1 mm² area; 50 mM KCl: n = 4,330 linear segments from two biological replicates; 50 mM KCl and 100 µM ZnCl₂: n = 4,820 linear segments from two biological replicates). Unpaired t test assuming unequal variances. All experiments were performed in the absence of extracellular Ca²⁺, except where indicated. **** P < 0.0001.