Long chromosomes align less efficiently than short chromosomes and experience higher spindle pushing force. (A) Chromosome spread of PtK2 cell line expressing eYFP-Cdc20. Red arrowheads indicate chromosomes classified as short. (B) Long (blue, top) and short (pink, bottom) chromosomes in a live mitotic PtK2 cell. Chromosomes were classified by phase contrast microscopy with the help of temporal tracking information. (C) Spindle assembly schematic depicting aligned, or congressed, chromosomes oscillating within the central gray box of the metaphase plate and unaligned chromosomes, in various attachment states. (D and E) Alignment for D and E is defined by K-K stretch and oscillatory movement within the spindle center as indicated here. (D) Representative time-lapse imaging of spindle assembly of cells shown in B showing that some chromosomes align soon after onset of mitosis (pink box indicating oscillatory area of the metaphase plate) while others move to poles, leading to a delay in alignment. Two short chromosomes are highlighted in pink and three long chromosomes are highlighted in blue. See also Video 1. (E) Percent of long and short chromosomes, which are early aligning (the first three to begin oscillating in a given spindle) or late aligning (the last three). n denotes the number of chromosomes counted while N denotes number of cells (Fisher’s exact test). (F) For bioriented attachments, poleward pulling by sister k-fibers produces opposing force that generates kinetochore tension, while for syntelic errors, poleward pulling is counteracted by polar ejection force along chromosome arms. (G and H) To assess whether polar ejection force scales with chromosome size, live imaging was performed on STLC-treated monopolar spindles in PtK2 cells expressing eYFP-Cdc20 and tubulin-mCherry (G) (see also Video 2) and the distance of kinetochores (kts) from the pole was used to evaluate the magnitude of pushing force (mean ± SD) (H) (unpaired t test).