Lowering microtubule occupancy at a kinetochore slows down the process of Mad1 loss. (A) Schematic of how the number of attached microtubules could influence two different properties of Mad1 loss: the decision to complete loss and the speed of loss. (B) Time-lapse imaging (maximum-intensity projection) of representative Mad1 loss kinetics (EYFP-Mad1) and microtubule attachment (SiR-Tubulin) in a Hec1-RNAi PtK2 cell with decreased kinetochore–microtubule affinity (Hec1-9D-FusionRed). Scale bars = 3 µm (large) and 1 µm (zoom), and t = 0 indicates the start of Mad1 loss on the orange-circled kinetochore. (C) Individual (circles) and average (lines) K-K distance in WT (n = 21 pairs) and Hec1-9D (n = 38) cells. (D) Individual (circles) and average (lines) kinetochore–microtubule intensity normalized to cellular astral microtubule intensity in Hec1-9D (n = 22 cells) and WT (n = 14 cells). (E and F) Mean, SEM, and individual trace (E) of the orange-circled kinetochore in B of the Mad1-to-Hec1 intensity ratio with t = 0 being the Mad1 loss start, and distribution of times to reach a 50% intensity ratio (F) of initial Mad1-to-Hec1, in WT cells (n = 21 kinetochores) and Hec1-9D-expressing cells (n = 38). WT data taken from Kuhn and Dumont (2017) and acquired in a parallel experiment. (G and H) Individual (circles) and average (lines) kinetochore EYFP-Mad1 intensity in 5 µM nocodazole (G) or right before Mad1 loss start (H) in Hec1-9D and WT cells. *, P < 0.005, two-sided Mann–Whitney U test).