CLEM characterization of microtubule density near kinetochores during early prometaphase. (A) Maximum-intensity projection of an early prometaphase cell coexpressing Hec1-tdTomato and Mad2-Venus. Notice highly variable concentration (red vs. yellow) of Mad2 at various kinetochores. See Video 5 for the full volume of this cell with individual kinetochores marked. (B and C) LM and correlative EM images of individual kinetochores with intermediate (B) or low (C) amounts of Mad2-Venus. Orange lines demark positions of microtubules traced in EM. Yellow arrows denote end-on–attached microtubules, and blue arrows point at laterally interacting microtubules. (D) Tukey box plot presenting the number of microtubules (MTs) within the 250-nm area adjacent to the kinetochore plate (60 kinetochores from two cells; mean = 35). (E) Distribution of angles between microtubules and the kinetochore plate. (F) Scatterplot presenting the number of end-on–attached microtubules versus the amount of Mad2 at individual kinetochores. Mad2 intensity is normalized so that the background is 0 and the mean value is 1. Regression line slop coefficient = 0.0669 (R2 = 0.01).
