Middle and peripheral KT pairs display similar inter- and intra-KT distances. (A) Example of a metaphase PtK1 cell immunostained for α-tubulin (purple), ACA (red), and Hec1 (green). The white arrow in A indicates the position and direction of the line scan for the fluorescence intensity profiles displayed in B and C. Bar, 5 µm. (B and C) Fluorescence intensity profiles obtained from a line scan along the arrow traced in A. The inter- and intra-KT distances were obtained by measuring the distance between the ACA peaks (B) and the Hec1 and ACA peak (C), respectively. This method was used to measure the inter- and intra-KT distances in two pairs of middle sister KTs and two pairs of peripheral sister KTs in each of 57 cells. The data obtained from these measurements are reported in D and E. (D) Distribution of inter-KT distances in metaphase PtK1 cells (1.90 ± 0.44 µm and 2.01 ± 0.40 µm for middle and peripheral sister KTs; n = 57 cells). (E) Distribution of intra-KT distances in metaphase PtK1 cells (0.11 ± 0.04 µm for both middle and peripheral sister KTs; n = 57 cells).