Fig. 7.

Axial positions of different kinetochore proteins along the inter sister kinetochore axis. a Native chromosomes released from syncytial embryos coexpressing a red and a green fluorescent CKC component were labeled with a DNA stain (DNA). The appearance of the native chromosomes with red (Cenp-Ar) and green (Cenp-Ag) fluorescent Cenp-A/Cid is illustrated at low magnification. Bar = 5 μm. b The fourth chromosome indicated by the white arrows in a is shown at high magnification. Bar = 0.5 μm. c A fourth chromosome from an embryo expressing red fluorescent Cenp-A/Cid (Cenp-Ar) and green fluorescent Nuf2 (Nuf2g) is shown at the same magnification as in b. These high magnification views, b and c, display the pixel resolution as acquired. d For an estimation of the spatial separation between a red and a green fluorescent CKC component, signal intensities were quantified along the axis running through the two sister kinetochores, as illustrated by the white line in c. The displayed intensity profiles are from the chromosome shown in c. The spatial separation (d_RGintra) was calculated by halving the difference between the distances separating the red (d_RRinter) and green (d_GGinter) signal maxima of the sister kinetochores, as indicated by the equation. e Scheme summarizing the positions of the analyzed CKC components along the spindle axis. Double arrows represent the spatial separation as revealed by the pairwise analyses outlined in a–d with hundreds of native chromosomes (see also Table 1). Arrows above the upper dashed line represent data from experiments comparing the distance between red fluorescent Cenp-A/Cid and various green fluorescent CKC components. Analogous analyses of the distance between various green fluorescent CKC components and red fluorescent Spc25 are represented by the arrows between the dashed lines. The arrows below the lower dashed lines represent experiments after expression of Cenp-C versions labeled with both a green and a red fluorescent protein at the N and C termini. Color coding specifies the green fluorescent component in all these pairwise analyses. The vertical lines indicate the position of CKC components as revealed by pooling all d_RRinter and d_GGinter values obtained for a given CKC component during the pairwise analyses (see Table 2). The numbers indicate the spatial separation (nm) from the innermost centromere component Cenp-A/Cid, which was set to zero. In case of Cenp-C, the N terminus (N) and the C terminus (C) were mapped. f–i Histogram curves illustrate the distribution of the d_RGintra values obtained in the pairwise analyses with chromosomes from embryos expressing red fluorescent Cenp-A/Cid and green CKC components, in f, as well as in the analogous analyses with red fluorescent Spc25 in combination with green CKC components, in g, or with Cenp-C versions carrying red and green fluorescent proteins at N and C termini, in h. Moreover, i the distribution of the d_inter values, i.e., all the d_RRinter and d_GGinter measurements obtained for a given CKC components (see Table 2) are displayed as well