Figure 4. Zn 2 does not cause motor... | Rockefeller University Press

Figure 4.

Zn2+does not cause motor proteins to detach from microtubules in situ and in vitro. (A and B) Representative oblique illumination micrographs of COS-7 cells expressing KIF5A(1-560)-mNG-FRB (green, left) and mCherry-α-tubulin (magenta, middle), and merged channels (right). Cells were imaged at baseline (top) and 5 min after treatment (bottom) with (A) 20 µM ZnCl2 and 2.5 µM pyrithione (PTO), or (B) 2.5 µM PTO alone. Scale bar = 10 µm. (C) Ratio of edge-to-center fluorescence of KIF5A(1-560)-mNG -FRB in COS-7 cells as treated in A and B (Zn2+-treated, n = 16 cells from three individual biological replicates; PTO control, n = 13 cells from three individual biological replicates). Two-tailed paired t tests. (D) Representative timelapse plot of edge-to-center fluorescence ratio of KIF5A(1-560)-mNG-FRB (green) and mCherry-α-tubulin (magenta) in COS-7 cells, treated as in A. (E) Representative confocal micrographs of COS-7 cells expressing KIF5A(1-560)-EGFP (green, left) and mCherry-α-tubulin (magenta, middle left), merged channels (middle right), and “Tubeness” transformation (Fiji Plugin) of the KIF5A(1-560)-EGFP channel (right). Cells were imaged at baseline (top) and 5 min after treatment (bottom) with 20 µM ZnCl2 and 2.5 µM PTO. Zoomed insets (i–iv, represented by white boxes) from the KIF5A(1-560)-EGFP and Tubeness transformation are shown at the far right. Scale bar = 10 µm. (F) Tubeness of KIF5A(1-560)-EGFP in COS-7 cells as treated in E, normalized to baseline (n = 24 cells from three individual biological replicates). Z test to (x = 1). All in situ experiments were performed in the absence of extracellular Ca2+. (G) Cartoon representation of the dynein (left) and kinesin-1 (right) fragments used for in vitro binding assays. The full-length yeast dynein complex (left; heavy chain in purple with associated light, light-intermediate, and intermediate chains shown in gray) and full-length kinesin heavy chain and its associated fragments (right), and, along with the relevant fragments, are depicted. The SRS domain is represented as a gray oval, with EGFP shown in green. (H) Representative TIRF micrographs of coverslip-mounted taxol-stabilized microtubules (left, interference reflection micrographs), raw fluorescence of EGFP-labeled motor MTBDs (center), and pseudocolored fluorescence of motors MTBDs (right), for both dynein (left panels) and kinesin-1 (right panels), in the presence of either 0 ZnCl2 (top) or 100 µM ZnCl2 (bottom). (I) Dot plot of in vitro microtubule-localized fluorescent intensity (normalized to each replicate’s respective average fluorescence for the 0 Zn2+ control) of EGFP-SRS-DyneinMTBD (purple), EGFP-SRS-kinesinMTBD (blue) in the presence of 0 Zn2+ (“Dynein,” n = 82 microtubules, “kinesin,” n = 47 microtubules) or 100 μM ZnCl2 (“Dynein,” n = 74 microtubules, “kinesin,” n = 37 microtubules). Each dot represents a unique microtubule; each color indicates a separate replicate. **** P < 0.0001, n.s. not significant.

Zn ²⁺ does not cause motor proteins to detach from microtubules in situ and in vitro. (A and B) Representative oblique illumination micrographs of COS-7 cells expressing KIF5A(1-560)-mNG-FRB (green, left) and mCherry-α-tubulin (magenta, middle), and merged channels (right). Cells were imaged at baseline (top) and 5 min after treatment (bottom) with (A) 20 µM ZnCl₂ and 2.5 µM pyrithione (PTO), or (B) 2.5 µM PTO alone. Scale bar = 10 µm. (C) Ratio of edge-to-center fluorescence of KIF5A(1-560)-mNG -FRB in COS-7 cells as treated in A and B (Zn²⁺-treated, n = 16 cells from three individual biological replicates; PTO control, n = 13 cells from three individual biological replicates). Two-tailed paired t tests. (D) Representative timelapse plot of edge-to-center fluorescence ratio of KIF5A(1-560)-mNG-FRB (green) and mCherry-α-tubulin (magenta) in COS-7 cells, treated as in A. (E) Representative confocal micrographs of COS-7 cells expressing KIF5A(1-560)-EGFP (green, left) and mCherry-α-tubulin (magenta, middle left), merged channels (middle right), and “Tubeness” transformation (Fiji Plugin) of the KIF5A(1-560)-EGFP channel (right). Cells were imaged at baseline (top) and 5 min after treatment (bottom) with 20 µM ZnCl₂ and 2.5 µM PTO. Zoomed insets (i–iv, represented by white boxes) from the KIF5A(1-560)-EGFP and Tubeness transformation are shown at the far right. Scale bar = 10 µm. (F) Tubeness of KIF5A(1-560)-EGFP in COS-7 cells as treated in E, normalized to baseline (n = 24 cells from three individual biological replicates). Z test to (x = 1). All in situ experiments were performed in the absence of extracellular Ca²⁺. (G) Cartoon representation of the dynein (left) and kinesin-1 (right) fragments used for in vitro binding assays. The full-length yeast dynein complex (left; heavy chain in purple with associated light, light-intermediate, and intermediate chains shown in gray) and full-length kinesin heavy chain and its associated fragments (right), and, along with the relevant fragments, are depicted. The SRS domain is represented as a gray oval, with EGFP shown in green. (H) Representative TIRF micrographs of coverslip-mounted taxol-stabilized microtubules (left, interference reflection micrographs), raw fluorescence of EGFP-labeled motor MTBDs (center), and pseudocolored fluorescence of motors MTBDs (right), for both dynein (left panels) and kinesin-1 (right panels), in the presence of either 0 ZnCl₂ (top) or 100 µM ZnCl₂ (bottom). (I) Dot plot of in vitro microtubule-localized fluorescent intensity (normalized to each replicate’s respective average fluorescence for the 0 Zn²⁺ control) of EGFP-SRS-Dynein^MTBD (purple), EGFP-SRS-kinesin^MTBD (blue) in the presence of 0 Zn²⁺ (“Dynein,” n = 82 microtubules, “kinesin,” n = 47 microtubules) or 100 μM ZnCl₂ (“Dynein,” n = 74 microtubules, “kinesin,” n = 37 microtubules). Each dot represents a unique microtubule; each color indicates a separate replicate. **** P < 0.0001, n.s. not significant.