Chromosome size determines chromosome biorientation efficiency. (A) Chromosome arm ablation assay: chromosome arms of a long chromosome were cut shortly after nuclear envelope breakdown (ablation site denoted by lightning bolt) and kinetochores were followed throughout spindle assembly to determine the time of alignment with respect to other long, unaligned chromosomes in the same cell to test whether chromosome size (model 1; ablated chromosome aligns early) or chromosome identity (model 2; ablated chromosome still aligns late) determine alignment order. (B) Representative example of spindle assembly following chromosome arm ablation in PtK2 cells expressing eYFP-Cdc20. White circle indicates kinetochore on ablated chromosome, arrowheads mark ablated arms, asterisks denote unablated, unaligned long chromosomes, and lightning bolts indicate site of ablation. See also Video 7. (C) Zoom-in phase contrast images of successful chromosome arm ablation (lightning bolts) in B indicated by white box. Ablation was considered complete if a clear space was observed between the kinetochore-containing fragment and the unattached arms (arrowheads) and the chromosome fragments were mechanically uncoupled in subsequent frames. (D) Alignment time of ablated and unablated, long chromosomes in raw time and normalized such that t = 0 is the first chromosome to align following ablation and t = 1 is the last chromosome to align prior to anaphase (mean ± SD; Mann–Whitney test). (E) Control ablation scheme depicting how incomplete ablations were performed, either by chipping chromosome ends off or by nicking chromosome arms to damage DNA without changing chromosome size substantially, indicated by lightning bolts. (F) Representative example of control ablation, chipping chromosome ends. White arrowhead marks chipped chromosome end, lightning bolts denote ablation. (G) Raw time to alignment for control ablations and other unablated long chromosomes in the same cell and normalized alignment time such that t = 0 is the first chromosome to align following ablation and t = 1 is the last chromosome to align prior to anaphase (mean ± SD; Mann–Whitney test).
Figure 4.

Chromosome size determines chromosome biorientation efficiency. (A) Chromosome arm ablation assay: chromosome arms of a long chromosome were cut shortly after nuclear envelope breakdown (ablation site denoted by lightning bolt) and kinetochores were followed throughout spindle assembly to determine the time of alignment with respect to other long, unaligned chromosomes in the same cell to test whether chromosome size (model 1; ablated chromosome aligns early) or chromosome identity (model 2; ablated chromosome still aligns late) determine alignment order. (B) Representative example of spindle assembly following chromosome arm ablation in PtK2 cells expressing eYFP-Cdc20. White circle indicates kinetochore on ablated chromosome, arrowheads mark ablated arms, asterisks denote unablated, unaligned long chromosomes, and lightning bolts indicate site of ablation. See also Video 7. (C) Zoom-in phase contrast images of successful chromosome arm ablation (lightning bolts) in B indicated by white box. Ablation was considered complete if a clear space was observed between the kinetochore-containing fragment and the unattached arms (arrowheads) and the chromosome fragments were mechanically uncoupled in subsequent frames. (D) Alignment time of ablated and unablated, long chromosomes in raw time and normalized such that t = 0 is the first chromosome to align following ablation and t = 1 is the last chromosome to align prior to anaphase (mean ± SD; Mann–Whitney test). (E) Control ablation scheme depicting how incomplete ablations were performed, either by chipping chromosome ends off or by nicking chromosome arms to damage DNA without changing chromosome size substantially, indicated by lightning bolts. (F) Representative example of control ablation, chipping chromosome ends. White arrowhead marks chipped chromosome end, lightning bolts denote ablation. (G) Raw time to alignment for control ablations and other unablated long chromosomes in the same cell and normalized alignment time such that t = 0 is the first chromosome to align following ablation and t = 1 is the last chromosome to align prior to anaphase (mean ± SD; Mann–Whitney test).

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