High polar ejection force increases persistence of polar chromosomes and reduces size effect. (A) Polar ejection forces are strongest near poles. Kid overexpression will increase both chromosome centering force exerted by the spindle and, for syntelic kinetochores, kinetochore tension. Tension is set by the force balance between poleward pulling at kinetochores and anti-poleward pushing along chromosome arms. (B) Immunofluorescence of fixed PtK2 cells transiently infected with Kid OE construct (pLV Kid-HaloTag) or not infected and intensity measurements for chromosome-localized Kid normalized to Hoechst signal for one experiment (mean ± SD; Mann–Whitney test). Experiment was performed in triplicate obtaining similar results each time. (C) Schematic of the rat kangaroo chromokinesin Kid highlighting key domains, in particular the C-terminal helix-hairpin-helix domain responsible for non-specific DNA binding, which has been deleted in the Kid ΔC construct. Representative time lapse of Kid ΔC OE PtK2 cells expressing eYFP-Cdc20. See also Video 5. (D) Representative time lapse of Kid OE PtK2 cells expressing eYFP-Cdc20. Polar chromosomes are pseudocolored in magenta for first frame (00:00) and short polar chromosomes are marked with an asterisk. See also Video 6. (E) Percentage of cells with chromosomes stuck in polar attachments. Chromosomes were considered stuck if they had not reached alignment within 15 min of approaching spindle poles. (F) Number of chromosomes stuck near poles in control and Kid OE spindle assembly (mean ± SD; Mann–Whitney test). (G) Time of alignment following pole approach for stuck polar chromosomes in control and Kid OE spindles (mean ± SD; Mann–Whitney test). (H) Missegregating chromosomes at anaphase in Kid OE spindle from C. Poles are marked with asterisks and lagging chromosomes denoted by arrowheads.
Figure 3.

High polar ejection force increases persistence of polar chromosomes and reduces size effect. (A) Polar ejection forces are strongest near poles. Kid overexpression will increase both chromosome centering force exerted by the spindle and, for syntelic kinetochores, kinetochore tension. Tension is set by the force balance between poleward pulling at kinetochores and anti-poleward pushing along chromosome arms. (B) Immunofluorescence of fixed PtK2 cells transiently infected with Kid OE construct (pLV Kid-HaloTag) or not infected and intensity measurements for chromosome-localized Kid normalized to Hoechst signal for one experiment (mean ± SD; Mann–Whitney test). Experiment was performed in triplicate obtaining similar results each time. (C) Schematic of the rat kangaroo chromokinesin Kid highlighting key domains, in particular the C-terminal helix-hairpin-helix domain responsible for non-specific DNA binding, which has been deleted in the Kid ΔC construct. Representative time lapse of Kid ΔC OE PtK2 cells expressing eYFP-Cdc20. See also Video 5. (D) Representative time lapse of Kid OE PtK2 cells expressing eYFP-Cdc20. Polar chromosomes are pseudocolored in magenta for first frame (00:00) and short polar chromosomes are marked with an asterisk. See also Video 6. (E) Percentage of cells with chromosomes stuck in polar attachments. Chromosomes were considered stuck if they had not reached alignment within 15 min of approaching spindle poles. (F) Number of chromosomes stuck near poles in control and Kid OE spindle assembly (mean ± SD; Mann–Whitney test). (G) Time of alignment following pole approach for stuck polar chromosomes in control and Kid OE spindles (mean ± SD; Mann–Whitney test). (H) Missegregating chromosomes at anaphase in Kid OE spindle from C. Poles are marked with asterisks and lagging chromosomes denoted by arrowheads.

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