Figure 5. Behavior of kinetochores and kinetochore microtubules. (A, A′, B, and B′) LLCPK1 cells were treated with MG132 for 1 h, injected with antibody, fixed, and stained 1 h later with or without prior cold treatment using anti-tubulin, CREST, and anti–mouse IgG (insets) antibodies versus DAPI (not depicted). Kinetochore microtubules were unaffected by anti-dynein antibody in cells retained at 37°C (A and A′) but were severely destabilized in these cells after cold treatment (B, B′) as compared with the control antibody. (C and D) HeLa cells stably expressing YFP–CENP-A, a cotransfection marker mCherryC1 with (D) or without (C) the myc-C1140 dynein tail construct, were incubated with MG132 for 45 min and imaged at one frame per 15 s for 15 min (C′ and D′) Time-lapse images of individual kinetochore pairs (boxed) followed for 3 min (C′) from a control cell and (D′) a tail-expressing cell. The maximal amplitude of the oscillation of each pair is indicated at the bottom. Arrows indicate the midline of metaphase plate. (E) Maximum amplitude of kinetochore pair oscillations from control and dynein tail–expressing cells from four and six dividing cells, respectively. (F and G) COS7 cells expressing GFP-Hec1 were injected either with a control mouse IgG (F, n = 6 cells) or dynein IC (G, n = 12 cells) antibody 30 min after MG132 treatment. Live images of kinetochores were acquired every 15 s for 6–9 min starting 30 min after injection. (F′ and G′) Time-lapse images of kinetochore pairs (F and G, boxes). (H) Analysis of kinetochore behavior in dynein IC antibody-injected versus control antibody-injected cells (n = 6 cells, 55 kinetochore pairs for each condition). (I) Models for dynein role in kinetochore microtubule attachment, orientation, and alignment. Model I: Dynein removes disassembly factor (blue oval) from kinetochore, a model not supported by the data (see text). Model II: (a) Dynein-mediated tension reorients kinetochores; (b) dynein brings microtubule ends toward core microtubule attachment sites; and (c) dynein-mediated tension persists during metaphase as indicated by role in kinetochore oscillations. Bars, 5 μM.
Figure 5.

Behavior of kinetochores and kinetochore microtubules. (A, A′, B, and B′) LLCPK1 cells were treated with MG132 for 1 h, injected with antibody, fixed, and stained 1 h later with or without prior cold treatment using anti-tubulin, CREST, and anti–mouse IgG (insets) antibodies versus DAPI (not depicted). Kinetochore microtubules were unaffected by anti-dynein antibody in cells retained at 37°C (A and A′) but were severely destabilized in these cells after cold treatment (B, B′) as compared with the control antibody. (C and D) HeLa cells stably expressing YFP–CENP-A, a cotransfection marker mCherryC1 with (D) or without (C) the myc-C1140 dynein tail construct, were incubated with MG132 for 45 min and imaged at one frame per 15 s for 15 min (C′ and D′) Time-lapse images of individual kinetochore pairs (boxed) followed for 3 min (C′) from a control cell and (D′) a tail-expressing cell. The maximal amplitude of the oscillation of each pair is indicated at the bottom. Arrows indicate the midline of metaphase plate. (E) Maximum amplitude of kinetochore pair oscillations from control and dynein tail–expressing cells from four and six dividing cells, respectively. (F and G) COS7 cells expressing GFP-Hec1 were injected either with a control mouse IgG (F, n = 6 cells) or dynein IC (G, n = 12 cells) antibody 30 min after MG132 treatment. Live images of kinetochores were acquired every 15 s for 6–9 min starting 30 min after injection. (F′ and G′) Time-lapse images of kinetochore pairs (F and G, boxes). (H) Analysis of kinetochore behavior in dynein IC antibody-injected versus control antibody-injected cells (n = 6 cells, 55 kinetochore pairs for each condition). (I) Models for dynein role in kinetochore microtubule attachment, orientation, and alignment. Model I: Dynein removes disassembly factor (blue oval) from kinetochore, a model not supported by the data (see text). Model II: (a) Dynein-mediated tension reorients kinetochores; (b) dynein brings microtubule ends toward core microtubule attachment sites; and (c) dynein-mediated tension persists during metaphase as indicated by role in kinetochore oscillations. Bars, 5 μM.

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