A diagram depicting the putative location and possible function of reported kinetochore proteins. Sister kinetochores are located on opposite sides of the centromere region of replicated chromosomes, and the centromeric heterochromatin between them is rich in α-satellite DNA, its binding protein CENP-B, and an inner centromere protein (INCENP) that might be involved in maintaining sister-chromatid cohesion. This region also appears to contain mitotic centromere-associated kinesin (MCAK), which has been shown in Xenopus extracts to be required for spindle formation and maintenance. In electron micrographs of sections cut from conventionally fixed and stained preparations, the kinetochore region itself appears to consist of four structurally differentiated domains. The inner plate is closely associated with the centromeric heterochromatin and contains CENP-C, the presence of which is required for the maintenance of a functional kinetochore, and perhaps MCAK. The zone between the inner and outer plates (the interzone) appears to contain the 3F3/2 phosphoepitope, which has been proposed to control the metaphase–anaphase (M–A) transition by sensing tension, and MCAK might also be located in this region. Kinetochore microtubule (MT) plus-ends attach to and terminate at various levels within the outer plate, which has been reported to contain CENP-F (also called mitosin), CENP-E^–, ZW10 and possibly cytoplasmic dynein and its associated dynactin complex. The fibrous corona extends from the outer plate and is apparent only on unattached kinetochores. It contains CENP-E, ZW10 and cytoplasmic dynein (reviewed in Ref. ), the latter possibly being involved in MT attachment and poleward force production. Unattached (but not fully attached) kinetochores also contain Mad2 and Bub1 proteins, which are known to play important roles in regulating the M–A transition. A question mark indicates that the location of a protein is inferred from immunofluorescence microscopy rather than determined by immunoelectron microscopy.

Spindle structure and chromosome behaviour. The mitotic spindle in vertebrate cells comprises two overlapping arrays of polar microtubules (MTs), some of which have been released from the centrosome, and all of which are oriented with their ‘plus’ (+) ends distal and their minus-ends proximal to their poles. This diagram illustrates the typical sequence of events that chromosomes in vertebrate cells experience during mitosis. Initially, one kinetochore on the chromosome becomes attached to, and glides rapidly poleward (long arrow) along the surface of a single MT (a). During this poleward motion, the attaching kinetochore on the now mono-oriented chromosome acquires additional MTs that terminate in its outer plate (b). It then begins to oscillate between poleward and away from the pole states of motion (short double arrows) around a position between the pole and the spindle equator. When the unattached kinetochore on this chromosome encounters a MT growing from the distal pole, the chromosome moves to the spindle equator in a process known as congression (c). As a result of congression, the chromosome adopts an average position near the spindle equator around which it oscillates, and, over time, the sister kinetochores acquire similar numbers of MTs (d). During anaphase, the chromatids separate (e), and, although the single kinetochore on each still exhibits a modified form of directionally unstable behaviour, there is net movement of each towards their respective spindle poles. Unattached kinetochores (red label) stain strongly for proteins such as CENP-E and cytoplasmic dynein/dynactin, which are involved in attachment and movement, and Mad2, Bub1 and the 3F3/2 epitope, which are involved in the checkpoint controlling the metaphase–anaphase transition. Modified, with permission, from Ref. .

Attached kinetochores switch abruptly between a state of constant-velocity poleward motion and a neutral state, which allows motion away from the pole also at a constant velocity. Both of these motility or activity states occur in association with the plus-ends of relatively stationary kinetochore microtubules (K-MTs). An attached kinetochore moves poleward at 1.5–2.5 μm min⁻¹, and, during this time, it pulls on its associated MTs, which stretches the centromere region from its rest length (a). The force for poleward motion probably involves MT motors (e.g. dynein) located in the kinetochore corona or outer plate (see ) and the dissociation of the terminal tubulin subunits on the MT plus-ends, which might splay during this process. These motors might also regulate velocity and maintain the MT–kinetochore attachment as the K-MT plus-ends shorten. Once the tension (stretch) on the kinetochore reaches a critical level, and/or when the kinetochore runs out of a crucial component involved in poleward force production, it switches into a neutral state (b) that allows it to be transported away from the pole. This motion away from the pole is associated with, and allowed by, the elongation of K-MTs. On a mono-oriented chromosome, it is powered probably by ejection forces, produced by MTs associated with the proximal spindle pole, that push the chromosome and its arms away from the pole. On a bi-oriented chromosome, motion away from the pole is thought to be powered by the proximal ejection force and also the poleward movement of the opposing sister kinetochore. During motion away from the pole, K-MTs elongate as GTP–tubulin subunits add to a stabilizing cap of GTP–tubulins at their growing plus-ends within the kinetochore. Beneath the cap, the GTP is hydrolysed to GDP in the MT lattice. Movement away from the pole reduces tension (stretch) on the centromere. A combination of active MT plus-end-directed motors, and/or an unknown attachment molecule, might maintain attachment to the growing ends of MTs and also regulate the velocity of motion away from the pole. Abbreviations: OP, kinetochore outer plate; IP, kinetochore inner plate.

(a) In PtK1 cells, the unattached kinetochore on a single mono-oriented chromosome is sufficient to inhibit the onset of anaphase for many hours. However, anaphase occurs shortly after this unattached kinetochore is destroyed by laser microsurgery. Thus, unattached kinetochores produce a ‘wait-anaphase’ signal. (b) PtK1 cells containing two independent spindles can be created by fusing neighbouring cells. In these cells, even multiple unattached kinetochores on the mono-oriented chromosomes on one spindle do not prevent anaphase onset in an adjacent spindle when all of its kinetochores become attached. In addition, after a variable delay, anaphase in this spindle induces anaphase in the spindle with mono-oriented chromosomes and unattached kinetochores.