Visualizing kinetochore architecture

Abstract

Kinetochores are large macromolecular assemblies that link chromosomes to spindle microtubules (MTs) during mitosis. Here we review recent advances in the study of core MT-binding kinetochore complexes using electron microcopy methods in vitro and nanometer-accuracy fluorescence microscopy in vivo. We synthesize these findings in novel three-dimensional models of both the budding yeast and vertebrate kinetochore in different stages of mitosis. There is a growing consensus that kinetochores are highly dynamic, supra-molecular machines that undergo dramatic structural rearrangements in response to MT capture and spindle forces during mitosis.

Figure 1. Model the N-terminus of human Ndc80

The microtubule-binding head of human Ndc80 (PDB 2VE7), which lacked the N-terminal 80 amino acids, is shown in ribbon diagram (Ndc80 in blue, Nuf2 in gold), with an added model of the N-terminus shown in an extended conformation in stick format in magenta. Kink points correspond to the positions of prolines. Aurora B phosphorylation sites are shown in space-filling representation in red. The N-terminus model was built with PyMOL; the figure was generated with UCSF Chimera.

Figure 2. 3D models of the budding yeast kinetochore bound to a depolymerizing MT

Three dimensional models of kinetochore components were assembled to fit the Delta data of Joglekar et al. for A) metaphase and B) anaphase/telophase. The microtubule model is shown in green ribbon representation and was generated by docking the electron crystallographic structure of taxol-bound tubulin (PDB 1JFF) into the cryo-EM reconstruction of human Ndc80 bound to the microtubule (EMDB 5223); protofilament peels were generated from the structure of RB3-stathmin colchicine bound tubulin (PDB 1SA0). The yeast nucleosome (PDB 1ID3) is shown as a ribbon model in dark purple, the region of inner kinetochore complexes (such as the COMA complex) localization is shown as a light purple transparent cylinder. The Mtw1 complex is modeled as a rotationally symmetric rigid rod with dimensions based on 2D single-particle analysis and is displayed in pink. The position of the C-terminus of Spc105 is shown as an orange sphere. The globular domains of the human Ndc80 complex (PDB 2VE7) are shown in surface representation and are colored as follows: Ndc80, light blue, Nuf2, gold, Spc24, green, Spc25, red. The attachment point of Ndc80’s unstructured N-terminus is shown in magenta, and a possible extension of this protein region along the microtubule in the metaphase case is illustrated. The Ndc80-Nuf2 globular heads were oriented on the microtubule surface by docking into the cryo-EM map. The coiled-coil segments (grey) were modeled as rigid rods based on the average lengths reported by Wang et al. and the width of an idealized dimeric coiled-coil. The shorter segment was oriented with the truncated coiled-coil of the crystallized construct, which was found in two different conformers in the asymmetric unit of the crystal lattice. The more bent conformation was used in the metaphase model (A), while the straighter conformation was used in the anaphase model (B). The hinge point between the two coiled-coil segments was treated as a freely-rotatable interface, in accordance with the observations of Wang et al. The models and the figure were made with UCSF Chimera.

Figure 3. 3D model of the vertebrate kinetochore bound to a depolymerizing MT

Analogously to Figure 2, structures were assembled to fit the Delta data of Wan et al. The high-resolution structure of the vertebrate nucleosome is shown (PDB 1KX5). The probable position of the C-terminus of Knl1 is shown for reference based on in vitro data, although it was not probed in the Delta study. The position of Ndc80’s shorter coiled-coil segment was once again modeled based on the orientations observed in the crystal for the two outer molecules in each cluster; an intermediate position was generated for the central molecule.