Which phase of mitosis do the kinetochores connect to each individual chromatid?

Role in Mitotic Spindle Checkpoint

Kinetochores are important elements of a mitotic checkpoint. Failure of kinetochores to bind to spindle microtubules, or incorrect association such as when both sister kinetochores attach to microtubules from the same spindle pole, results in mitotic delay or arrest. Some proteins, for example, mitotic arrest-deficient protein 2 (MAD2), may monitor microtubule binding to kinetochores. Others respond to the ‘tension’ imposed on the kinetochore by the spindle microtubules by altering their phosphorylation state. These proteins are phosphorylated in misaligned kinetochores (and can be detected using an antibody, 3F3/2, that recognizes such epitopes) but dephosphorylated when kinetochores are correctly attached to the mitotic spindle. At present, we have an incomplete understanding of the pathway(s) through which kinetochores influence the spindle checkpoint.

4.2 Time

Kinetochores can be purified by incubating clarified lysate with dynabeads for as little as 15 min (Fig. 2B and C). These kinetochore particles still contain all the components, but they are comparatively less concentrated than kinetochores purified following longer incubations (Fig. 2B and C). Increasing incubation time to up to an hour results in complete purification of the KMN complex and outer components such as Dam1 and Stu2 (Fig. 2C). If the kinetochore purification is being performed to assess functionality of outer kinetochore components, performing the immunoprecipitation for 3 h enriches for the most outer kinetochore components (Fig. 2C).

Abstract

The kinetochore mediates chromosome segregation at cell division. It is the macromolecular machine that links chromosomes to spindle microtubules, and is made of more than 100 protein species in mammalian cells. Molecular tools are presently revealing the biochemical interactions and regulatory mechanisms that ensure proper kinetochore function. Here, we discuss two approaches for imaging and physically probing kinetochores despite mitotic cell rounding and rapid kinetochore dynamics. First, we describe how mild spindle compression can improve kinetochore imaging and how stronger compression can mechanically perturb the spindle and kinetochores. Second, we describe how simultaneously imaging two-colored kinetochore reporter probes at subpixel resolution can report on kinetochore structural dynamics under cellular forces. We hope that the experimental details we provide here will make these two approaches broadly accessible and help move forward our understanding of kinetochore function—and make these approaches adaptable to the study of other cellular structures.

2.3 Kinetochore-Derived Microtubule Growth

Kinetochore-derived MT growth has been proposed to enhance the encounter of kinetochores and spindle MTs (Fig. 6.1C). MTs have been observed to grow at or near kinetochore regions and later incorporate into the mitotic spindle (Khodjakov et al., 2003). The mechanism of MTs emerging directly from/around kinetochores is not completely understood. The chromosomal passenger complex (CPC) at the centromere has been shown to stimulate the pathway, possibly through Aurora B-mediated MT stabilization (Sampath et al., 2004). Ran-GTP is also found to be required for kinetochore-mediated MT organization (Tulu et al., 2006).

Abstract

Kinetochores are multifunctional supercomplexes that link chromosomes to dynamic microtubule tips. Groups of proteins from the kinetochore are arranged into distinct subcomplexes that copurify under stringent conditions and cause similar phenotypes when mutated. By coexpressing all the components of a given subcomplex from a polycistronic plasmid in bacteria, many laboratories have had great success in purifying active subcomplexes. This has enabled the study of how the microtubule-binding subcomplexes of the kinetochore interact with both the microtubule lattice and dynamic microtubule tips. Here we outline methods for rapid cloning of polycistronic vectors for expression of kinetochore subcomplexes, their purification, and techniques for functional analysis using total internal reflection fluorescence microscopy (TIRFM).

4.3 Kinetochore and its unusual protein composition

The kinetochore is a complex structure that establishes the attachment of spindle microtubules to chromosomes and is thus essential for faithful chromosome segregation. Under the transmission electron microscope, the kinetochore in vertebrate animals appears as a trilaminar stack of plates situated on opposite sides of the centromere of mitotic chromosomes (Brinkley and Stubblefield, 1966). The outer plate contains microtubule-interacting proteins (such as CENP-E, etc.) and the SAC proteins, whereas the inner plate is immediately adjacent to the centromere and is organized on a chromatin structure containing nucleosomes with a specialized histone named CENP-A, which substitutes histone H3 in the centromere region, auxiliary proteins, and centromere DNA (Chan et al., 2005). Electron microscopic studies show that the mitotic chromosomes of trypanosomes also possess an electron-dense structure typical of the canonical kinetochore found in other eukaryotic organisms (Ogbadoyi et al., 2000). However, the observed number of kinetochore appears not to exceed 8 (Solari, 1995), but there are more than 100 chromosomes. This suggests either a nonconventional function of the kinetochore or the existence of alternative chromosome segregation mechanism(s) (Ogbadoyi et al., 2000). It is also very likely that those chromosomes without detectable kinetochores may contain smaller kinetochores whose ultrastructure is not readily visible by electron microscopy.

Despite the presence of canonical kinetochore structure in trypanosomes, the protein components of the kinetochore were largely unknown (Berriman et al., 2005). Only a few kinetochore protein homologs were found based on sequence homology (Berriman et al., 2005), which include the inner kinetochore protein MCAK (Kinesin-13 family kinesins) and the outer kinetochore protein TOG/MOR1. Surprisingly, the TOG/MOR1 homolog was found to be localized to the cytosol without enrichment in the nucleus in procyclic trypanosomes (our unpublished data), suggesting that it is either not a kinetochore component in trypanosomes or not a bona fide homolog of TOG/MOR1. Recently, 19 candidate kinetochore proteins were identified in trypanosomes by using localization-based screening and proteomics approach, and RNAi-mediated knockdown of these kinetochore proteins resulted in severe chromosome mis-segregation (Akiyoshi and Gull, 2013b). All of the 19 kinetochore proteins bear no detectable sequence homology to the kinetochore proteins in other eukaryotes, suggesting that trypanosomes employ a chromosome segregation mechanism involving novel kinetochore components (Akiyoshi and Gull, 2013b).

4.16.4.1 The Tensed K-Fiber Hypothesis

Kinetochores experience pulling force throughout mitosis. This was long evident from their morphology during anaphase. Pulling force on metaphase chromosomes was conclusively shown by laser ablation of one sister kinetochore in a metaphase pair, which causes its sister to move rapidly towards the pole98 (Figure 4(b)). Strong evidence for tension on metaphase kinetochores was also provided by measuring the distance between sister kinetochores before and after depolymerizing microtubules. Loss of microtubules decreased this distance, which was interpreted as due to relaxation of an elastic connection71 (Figure 4(c)). How is this tension on metaphase kinetochores generated, and more important for our force map, how is it balanced? Tension in one spindle component must be balanced by compression in another. Pulling forces are thought to be generated at the interface between kinetochores and plus-ends, presumably by microtubule depolymerization as discussed above, and also perhaps activity of minus-end directed motors such as dynein (Figure 3(e)). Poles might also generate pulling forces at the minus-end of the k-fiber using depolymerization or plus-end-directed motors,99–101 but there is little direct evidence for this possibility. Tension at metaphase kinetochores must be balanced by compression elsewhere.94 Early ideas were influenced by spindle shape. K-MTs are approximately straight in some systems, while nK-MTs are curved. nK-MTs can also splay outwards more at metaphase than anaphase.102 Together, these observations suggested that the compressive load needed to balance tension at kinetochores is born by nK-MTs (Figure 4(a), left). This view seems logical, and is widely assumed to hold for all spindles. Pulling on poles by A-MTs may help balance kinetochore tension in some systems. However, A-MTs often make only transient and weak interactions with the cortex. In most systems, it appears that forces balance within the main body of the spindle.

6.1 Image acquisition and data analysis

Kinetochore pair tracking was performed on U2OS cells stably expressing EGFP-CENP-A, generated by FuGENE 6 (Roche Diagnostics) plasmid transfection followed by 1 mg/mL G418 selection. Cells were treated with DMSO, Taxol, or UMK57 in FluoroBrite DMEM for 1 h. Coverslips were then mounted in a rose chamber and maintained at 37 °C during image acquisition. Live cell imaging was performed using a QuorumWaveFX-X1 spinning disk confocal system on a Nikon Eclipse Ti microscope, equipped with an ILE laser source (Andor Technology) and a Hamamatsu ImageEM camera. A Plan Apo VC 60 ×,1.4 NA, oil immersion objective (Nikon) was used to image kinetochore oscillations. Mitotic cells were identified by DIC and fluorescence microscopy. z-Stacks with a 0.5 μm step size (13 slices) were acquired every 5 s.

Bi-oriented kinetochore pairs moving around the equator were selected for quantification. Kinetochore pairs were tracked manually in three dimensions using the Fiji plugin TrackMate (Tinevez et al., 2017). Poleward and antipoleward velocity measurements were calculated for trajectories between directional switches [v = |(x2 − x1)/(t2 − t1)|], where x2 and x1 are the relative positions of a given kinetochore at time points t2 and t1. Deviation from average position (DAP) was calculated as the standard deviation of the distances between a kinetochore and its average position at every time point (Bissonette & Stumpff, 2014). Switch rate was calculated as the number of times a kinetochore changed direction during oscillatory movement divided by the amount of time the kinetochore was filmed. Kymographs were generated in Fiji using the KymographBuilder plugin.

Kinetochore Domain

At metaphase, the kinetochore, a proteinaceous multidomain structure, is assembled on the outer surface of the centromere, promoting attachment of the chromosome to spindle microtubules and movement during anaphase.

Conclusions

The KT is an incredible molecular super-structure that possesses “brains” and “brawn”. In the brains department, it orchestrates a biochemical SAC signal that helps ensure anaphase only occurs once every chromosome is best-positioned for equal segregation of the genome. In the brawn department, the KT grabs onto MTs in a manner that is robust enough to maintain association with KT-MTs throughout cell division while still allowing MT polymerization and depolymerization forces to be harnessed for chromosome movements. Much has been learned about the molecular composition, structural organization, and major functions of the KT in the past few decades, but the coming years hold significant promise as vigorous investigations continue - especially at the interface of mechanobiology and biochemical pathways. It is an exciting time to study kinetochore biology and we hope that this article will provide an accessible entry-point to the field.