Supplemental Fig. 1 Inhibition of the Ndc80 complex prevents chromosome segregation upon anaphase induction. Cells were pretreated with Monastrol (35 μM) to prevent spindle pole separation and with MG132 (25 μM) to block mitotic exit. At time 00:00, cells (n = 4) were treated with flavopiridol (10 μM) to induce a forced mitotic exit that is independent of proteasome activity to mimic anaphase onset. After 5 min of flavopiridol treatment, chromosomes have moved toward the spindle pole in the uninjected control cell (top row). The bottom row shows a cell injected with anti-Ndc80 complex antibodies (n = 4) before flavopiridol treatment. The presence of anti-Ndc80 complex antibodies prevented chromosome movement toward the spindle poles after flavopiridol treatment. Bar = 10 μm (JPG 58 KB)

Supplemental Fig. 2 Inhibition of dynein/dynactin by microinjection of antibody to Zwilch causes a similar phenotype as inhibition of dynein/dynactin by dynamitin injection. a Dynein/dynactin accumulation at kinetochores is impaired by microinjection of anti-Zwilch. Cells were pretreated with MG132 (25 μM) to block mitotic exit. 45 min after injection, cells were treated with nocodazole (50 ng/ml) for 10 min to disassemble the microtubules and enhance dynein/dynactin association with the kinetochores. The cell (n = 5) in the top row was injected with anti-Zwilch antibodies. The cell in the bottom row is a control noninjected cell. Cells were labeled with anti-rabbit IgG to detect injected antibody. Cells were labeled with mouse monoclonal antibody to the p150^Glued subunit of the dynactin complex. Chromosomes can be seen in the phase-contrast image. In uninjected cells, p150^Glued was found concentrated at kinetochores. Injection of anti-Zwilch antibody caused loss of p150^Glued concentration at kinetochores. Images represent one confocal plane. Bar = 10 μm. b Cells (n = 9) were treated with MG132 and monastrol and then injected with anti-Zwilch antibody and anti-Ndc80 complex antibodies. In the depicted example, anti-Zwilch was injected just before the first image at time 00:00, and the anti-Ndc80 complex antibody was injected approximately 10 min later. With time, chromosomes lose attachment and orientation toward the spindle poles and become localized near the cell periphery (arrows). This response is similar to that obtained when anti-Ndc80 complex antibodies and dynamitin are coinjected (see Fig. 4). Bar = 10 μm. The complete video sequence for this cell is available in Movie 10 (JPG 54 KB)