Mechanisms of force generation by end-on kinetochore-microtubule attachments

Abstract

Generation of motile force is one of the main functions of the eukaryotic kinetochore during cell division. In recent years, the KMN network of proteins (Ndc80 complex, Mis12 complex, and KNL-1 complex) has emerged as a highly conserved core microtubule-binding complex at the kinetochore. It plays a major role in coupling force generation to microtubule plus-end polymerization and depolymerization. In this review, we discuss current theoretical mechanisms of force generation, and then focus on emerging information about mechanistic contributions from the Ndc80 complex in eukaryotes and the microtubule-binding Dam1/DASH complex from fungi. New information has also become available from super-resolution light microscopy on the protein architecture of the kinetochore-microtubule attachment site in both budding yeast and humans, which provides further insight into the mechanism of force generation. We briefly discuss potential contributions of motors, other microtubule-associated proteins, and microtubule depolymerases. Using the above evidence, we present speculative models of force generation at the kinetochore.

Figure 1

(a) Morphology of polymerizing and depolymerizing MT plus-ends in vitro. Disassembling MT plus-ends allow two mechanisms of force generation coupled to the disassembly. (b) Biased diffusion models - proteins weakly bound to the MT lattice diffuse along the lattice in the absence of external force. However, the shortening plus-end acts as a moving boundary and biases the diffusive movement towards the minus end. (c) Forced walk models – A ring around the lattice serves as a barrier to the relaxation of curling protofilaments. Continuous outwards curling pushes the ring towards the minus end. (d) A plus-end growing against a rigid barrier generates force by thermal ratchet mechanism. Two types of forced walk couplers based on EM and in vitro data have been proposed (e and f). (e) Morphology of kMT plus-ends observed in ultrathin sections. Averaging over many protofilaments reveals the presence of fibrillar structures (orange) of unknown identity that bind on the inside of the curling protofilament (blue). kMT protofilaments (blue) display an intermediate curvature that may be indicative of incomplete relaxation of strain. If the fibrils bound to each protofilament are responsible for this curvature, then they can convert the strain into a minus-end directed force. (f) Dam1/DASH rings in vitro act as force couplers that convert the strain energy efficiently into a pushing force or work.

Figure 2. Protein architecture and force generation in HeLa and yeast kinetochores

(a) Proposed models for force generation at the vertebrate kinetochore. These schematics show the two possible arrangements of the Ndc80 complex binding to the MT lattice in HeLa kinetochores. The upper model is deduced from distance measurement data obtained by Wan et al., while the lower model is based on the tilted orientation of the Ndc80 bound to MT lattice observed in vitro. (b) Kinetochore architecture obtained from live budding yeast cells. Of particular interest are the positions of Dam1/DASH complex (green ellipsoids) and Ndc80 complex (orange fibrils). The first version depicts the simplest arrangement representing average distances. Other arrangements are also possible, and the second cartoon depicts a proposed model for persistent kMT attachment through the Ndc80 complex. The Mtw1 complex (blue fibrils) is assumed to attain various orientations with respect to the kinetochore-MT axis and allow the Ndc80 complex (orange fibrils) to reach significant distances downstream from the kMT plus-end. Color key: NDC80 – ORANGE, KNL-1/Spc105 – RED, Mis12/MTW1 – BLUE, Dam1/DASH – GREEN