Expansion of the outer kinetochore in taxol-treated cells. (A) Representative kinetochores from metaphase and taxol-treated cells. Positions of CenpA-GFP and Hec1 centroids are marked by green and red dots, respectively, and white dots on the dual-color image. Blue arrows denote orientation of K fibers. Notice variability in the shape of gold-particle clusters between sister kinetochores in immuno-EM images. Delta values for each kinetochore and interkinetochore stretch (Hec1–Hec1) are shown. (B and B″) Averaged images of aligned kinetochores in metaphase (n = 90, four cells) and taxol-treated (n = 60, three cells). (B) Delta and FWHM_Hec1 values directly measured in the averaged fluorescent images are indicated. Notice a close match between these values and mean values shown in Fig. 1 D. Centroids CenpA-GFP and Hec1 fluorescence are indicated by white crosses. (B′) Cumulative distribution of gold particles in immuno-EM images of control and taxol-treated metaphase kinetochores. Notice the difference in shape and size of the Hec1 distributions. Dashed boundaries enclose 90% of gold particles. Red and green crosses denote centroids of Hec1 and CenpA-GFP fluorescence (B″) Positions of CenpA-GFP centroids (green dots) relative to fixed centroids of Hec1 (red crosses) and the orientation of the microtubule bundles (yellow lines). CenpA centroids are tightly clustered during metaphase but are spread in taxol-treated cells.