Mad1 loss begins rapidly after end-on attachment initiation and before a full k-fiber forms. (A) Time-lapse imaging (maximum-intensity projection) of a representative kinetochore pair’s SAC satisfaction kinetics (EYFP-Mad1; Mad1 loss start at t = 0), attached microtubules’ geometry and intensity (SiR-tubulin), and centromere tension (CenpC-mCherry) in a PtK2 cell. Dashed arrows illustrate analysis shown in B. Bars, 1 µm. See also Video 6. (B) Microtubule attachment geometry (and occupancy) analysis as an end-on attachment forms, corresponding with images in A. A negative value indicates that the kinetochore is near the end of its lateral microtubule track. (C–E) Concurrent quantification (mean and SEM) of the Mad1/CenpC intensity ratio (C), microtubule attachment geometry (Tubout–Tubin; D), and tension (K–K distance) around SAC satisfaction (E), with t = 0 indicating the start of Mad1 loss on K2 (n = 21 kinetochores). Boxed numbers map to images in A. Mad1 loss starts rapidly after end-on attachment initiation, when less than a full complement of microtubules is bound. *, P < 0.01; Wilcoxon signed-rank test. AU, arbitrary unit.
