Structural organization of the kinetochore-microtubule interface

Abstract

Successful mitosis depends on the stable, yet regulated attachment of chromosomes to spindle microtubules. The kinetochore, a large macromolecular structure assembled at sites of centromeric heterochromatin, is responsible for generating and regulating these essential attachments. Over the last several years, concerted experimental efforts have brought the structural view of the kinetochore-microtubule interface more clearly into focus. Here, we review important recent advancements and discuss several unresolved questions regarding how kinetochores dynamically bridge mitotic chromosomes to spindle microtubules.

Figure 1. Schematic view of kinetochores

A) During mitosis, sister chromatids are held together at centromeres by a cohesion complex (dark blue circles). Kinetochores (orange) assemble on centrometic chromatin and create a contact with microtubules (green). The plus (+) and minus (-) ends of microtubules are indicated. B) A close-up of kinetochores showing some of its components required for end-on microtubule binding. With the exception of the CENP-T/W complex (abbreviated as W and T) and CENP-C (abbreviated as C), all subunits of the constitutive centromere associated network (CCAN) have been omitted. CENP-T/W associates with histone H3-containing nucleosomes (H3), whereas CENP-C associates with nucleosomes containing the H3 variant CENP-A. The N-terminal region of CENP-T is an extended, largely disordered polypeptide chain that makes contacts with the 4-subunit Mis12 complex (MIS12-C) and with NDC80-C [11]. The N-terminal region of CENP-C is probably also disordered and makes contacts with MIS12-C [9,10]. The Knl1 complex (KNL1-C), which comprises Knl1 and Zwint-1 (Zwi), might contain a microtubule-binding site in the N-terminal region of Knl1 [28,56]. The C-terminal region of Knl1 interacts directly with the MIS12-C [70]. The NDC80-C is a tetramer. The Spc24 and Spc25 subunits interact with the MIS12-C, whereas the Hec1/Ndc80 and Nuf2 subunits face the microtubule.

Figure 2. The “toe” and the “toe-print”, part I

A) Cartoon showing the CH domains from a pair of NDC80-C bound to the α-tubulin/β-tubulin dimer [37]. The model was created by fitting the high-resolution structures of the NDC80^Bonsai complex [30] and of the α-tubulin/β-tubulin dimer [43] in a cryo-EM 3D reconstruction of NDC80^Bonsai-decorated microtubules [37]. The lower NDC80-C contacts microtubules at the intra-dimer interface. The upper NDC80-C docks at the inter-dimer interface. Changes in the relative orientation at these interfaces might modify the binding affinity for NDC80-C [37]. B) Close-up of the area boxed in A and showing residues in the “toe” and “toe-print” discussed in the text. K146 and K166 from Hec1 form a tight pair that faces a negative patch on β-tubulin. The cartoon models were created with PyMol (www.pymol.org) and assembled in Adobe Illustrator.

Figure 3. The “toe” and the “toe-print”, part II

A) The view was rotated ~180° relative to the view in Fig. 2A. B) Close-up of the area boxed in A. The C-terminal tail of β-tubulin was invisible in the cryo-EM reconstructions. A hypothetical path for the C-terminal tail of tubulin (so-called E-hook) is shown as a dotted green line. K89 and K115 of Hec1 are potentially positioned for an interaction with the E-hook.

Figure 4. Hypothetical binding mechanisms

A) The arrow indicates longitudinal interactions between two NDC80 complexes. Two possible implementations of this configuration that incorporate binding cooperativity are shown in panels A’ and A”. The model in A” recapitulates structural findings that the N-terminal tail of Hec1 packs between two NDC80 complexes in the longitudinal direction [37]. In A’, the interaction between NDC80 complexes does not involve the N-terminal tails, which are rather engaged directly in microtubule binding. B) The arrow indicates lateral interactions between two NDC80 complexes. B’) A possible implementation in which the C-terminal tails form contacts with NDC80 complexes on laterally neighboring protofilaments. There is no experimental evidence for this model. C) Embedding of individual NDC80 complexes within kinetochores (details not shown, see Fig. 1) allows multiple NDC80 complexes to form interactions with microtubules without any additional contacts between NDC80 complexes at the microtubule-binding interface. D) Molecular cross-linking of NDC80 complexes, as it might be implemented by factors such as the Dam1 complex or the SKA complex.