In vivo protein architecture of the eukaryotic kinetochore with nanometer scale accuracy

Abstract

The kinetochore is a macromolecular protein machine [1] that links centromeric chromatin to the plus ends of one or more microtubules (MTs) and segregates chromosomes during cell division. Its core structure consists of eight multicomponent protein complexes, most of which are conserved in all eukaryotes. We use an in vivo two-color fluorescence microscopy technique to determine, for the first time, the location of these proteins along the budding yeast kinetochore axis at nanometer resolution. Together with kinetochore protein counts [2, 3], these localizations predict the 3D protein architecture of a metaphase kinetochore-microtubule attachment and provide new functional insights. We also find that the kinetochore becomes much shorter in anaphase as metaphase tension is lost. Shortening is due mainly to a decrease in the length of the Ndc80 complex, which may result either from intramolecular bending of the Ndc80 complex at the kink within the stalk region of the Ndc80-Nuf2 dimer [4, 5] or from a change in its orientation relative to the microtubule axis. Conformational changes within the Ndc80 and Mtw1 complexes may serve as mechanical cues for tension-dependent regulation of MT attachment and the spindle-assembly checkpoint. The geometry of the core structure of the budding yeast kinetochore reported here is remarkably similar to that found in mammalian kinetochores, indicating that kinetochore structure is conserved in eukaryotes with either point or regional centromeres.

Figure 1. Measuring the distance separating two kinetochore proteins in a budding yeast metaphase spindle

(a) A budding yeast cell in metaphase (DIC) with fluorescently labeled kinetochores (green) and spindle pole body (blue). (b) The cartoon depicts arrangement of kinetochores tagged with EGFP (white ovals with green dots) within the metaphase spindle. Tense chromatin connections (gray dotted lines) between sister kinetochores align them closely with the spindle axis. When such a cell is visualized with wide-field fluorescence microscopy, the two kinetochore clusters (each containing 16 kinetochores) appear as nearly diffraction-limited spots. (c) Metaphase spindle in a strain that has two kinetochore proteins, one protein fused to EGFP (green dots) and the other with tdTomato (red dots). When such cells are imaged simultaneously in the EGFP and tdTomato channels (lower panel), the offset between the centroids of the EGFP and tdTomato images of a kinetochore cluster can be used to determine the average distance separating the ends of the two proteins accurately. (scale bar ~ 1μm in a, ~ 500 nm in b and c; 1 pixel ~ 107 nm in b and c).

Figure 2. Ndc80 complex as an in vivo molecular ruler

(a) Structure of purified NDC80 complex [21]. An 80 amino acid long tail at the Ncd80p C-terminus separates it from the C-terminus of Nuf2p. Due to its unspecified structure, the exact distance separating the C-termini of Ndc80p and Nuf2p is unknown. (b) Distance measurements from four strains (N-Ndc80-C:Spc24-C, N-Ndc80:Nuf2-C, Ndc80-C:Spc24-C, and Ndc80-C:Nuf2-C) determine the dimension and orientation of the NDC80 complex in vivo. The non-Gaussian probability distribution fits (Supplemental Data 6) for three strains from the above list are shown [19]. Plots on the left hand side display histograms of measurements and the maximum likelihood fits for the data (solid lines). Dotted lines represent the true distance value predicted by maximum likelihood estimation. (c) Comparison of experimental distances (red bars) with the expected distances (black bars). The error bars represent the standard deviation estimated from the maximum likelihood estimation. The exact location of the C-terminus of Ndc80p is unknown. Therefore, the expected distance between the Ndc80-C: Nuf2-C and Ndc80-C:Spc24-C domains is a close estimate (marked by stars). The graphed measurement was the distance separating these two domains projected along the spindle axis (Supplemental Data 7).

Figure 3. Protein architecture of a kinetochore-microtubule attachment

(a) The average location of kinetochore proteins along the axis of the kinetochore-microtubule attachment in metaphase and late anaphase. Each colored box represents a protein complex within the kinetochore. 68% confidence intervals on the mean position for all the measurements are less than 3 nm. The exception is Spc105p-C (indicated by stars), which could not be localized using maximum likelihood estimation. The positions in this case reflect the average offset along the spindle axis, which is likely an underestimate of the actual distance. For the Mtw1 and Ctf19 complexes, we only show the spans as measured by the positions of the respective member proteins. (b) 3-D visualization of the metaphase budding yeast kinetochore-microtubule attachment as predicted by the protein localization data assuming a symmetric arrangement of kinetochore protein complexes around the cylindrical microtubule lattice. Black stars indicate the positions of fluorescent labels used in distance measurements. The configuration of the Dam1/DASH complex suggests two possibilities–a kinetochore that contains an oligomeric ring of the Dam1 complex (top), and a kinetochore that employs Dam1/DASH patches or incomplete rings (bottom). Dashed lines indicate established biochemical interactions between two protein complexes. (c) Loss of centromeric tension and changes induced the cell-cycle regulation result in a shorter kinetochore in late anaphase. A striking change occurs in the Ndc80 complex as the Nuf2p-Ndc80p dimer shows a length reduction that is 40% larger than the reduction predicted by an overall change in the orientation of the molecule with respect to the MT. The model displays a possible mechanism that relies on bending of the Ndc80-Nuf2p dimer at the kink as observed in vitro.