Strategy to find kinetochores in vivo. (A) Schematic of kinetochore states studied in this paper, not to scale. Sister chromosomes (pale blue pills) are held under tension (magenta arrows) in metaphase when kMT (magenta tubes) pulling forces are transmitted by cohesin (curved lines). Gray vertical bars: spindle-pole bodies. Tension at the kinetochores (green) can be eliminated either by the disruption of the kMTs with nocodazole or the conditional cleavage of mutant cohesin with TEV protease. This color scheme is used throughout the paper. (B) Strains used for this study, including those from previous studies (Krishnan et al., 2004; Liang et al., 2012). (C) Immunofluorescence image of a Cdc20-depleted cell, with Dam1p-GFP in green, tubulin in magenta, and DNA in blue. Owing to the merged channels, Dam1C/DASH appears white. (D) Cartoon of a mitotic yeast cell, with organelles drawn to approximate scale. The nucleus (pale blue circle), spindle (magenta lines), and kinetochores (green stars; not to scale) are colored. Cyan lines illustrate at scale a series of seven ∼100-nm cryosections. (E) Serial cryo-ET strategy. Cryo-EM images of sequential cryosections of the same cell mounted on a parallel-bar grid are shown enlarged ∼100-fold on the right.