Chromosome congression is promoted by CENP-Q- and CENP-E-dependent pathways.
Bottom Line: Importantly, the S50A mutant does not affect the loading of Plk1 onto kinetochores and leaves the CENP-O complex intact.Thus, the functions of CENP-Q in CENP-E loading and depolymerisation-coupled pulling are independent from its role in Plk1 recruitment and CENP-O complex stabilisation.Taken together, our data provide evidence that phosphoregulation of CENP-Q plays a central function in coordinating chromosome congression mechanisms.
Affiliation: Mechanochemical Cell Biology Building, Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK.Show MeSH
Mentions: The key question is whether the polar chromosome phenotype in CENP-Q-depleted cells simply reflects the unbinding of CENP-E motor proteins from kinetochores, or whether CENP-Q makes additional contributions to chromosome congression that are independent of CENP-E recruitment. We reported above, we were in a position to answer this question because depletion of CENP-E did not affect CENP-Q binding to kinetochores, compared with depletion of CENP-Q that also removed CENP-E. We therefore determined how these differing kinetochore states affected the congression of chromosomes by tracking the fates of uncongressed kinetochore pairs by using live-cell microscopy. For this, we used HeLa K cells expressing eGFP–CENP-A (a kinetochore marker) and eGFP–centrin1 (a spindle pole marker), and collected time-lapse movies over the course of 5 min in both CENP-Q-depleted cells and CENP-E-depleted cells. Sister pairs were assigned as non-biorientated (as judged in in the first frame of the movie; supplementary material Movies 3–6) if the sister–sister axis was rotated by approximately 90° relative to the pole-to-pole axis (see schematic in Fig. 3A). Biorientation would be improbable with this geometry because kinetochores could not make end-on attachments with microtubules coming from opposite spindle poles. Similarly, kinetochore pairs positioned behind the spindle pole were classed as non-biorientated because, in this state, biorientation would be geometrically impossible. In contrast, kinetochore pairs with a sister–sister axis of ∼45° or less relative to the pole–pole axis were classified as orientated. We cannot be sure that these sisters are biorientated because it is not possible to distinguish this state from sister pairs in which one sister is mono-orientated and the second is laterally attached (Kapoor et al., 2006; schematic in Fig. 3A). The relative proportion of orientated and non-biorientated uncongressed kinetochore pairs was very similar in CENP-Q- and CENP-E-depleted cells, with around 80% occupying a non-biorientated state (Fig. 3B). The fates of these non-biorientated unaligned kinetochore pairs were also very similar, with the majority (>90%) remaining stalled and unable to progress towards the spindle equator (Fig. 3C,E).
Affiliation: Mechanochemical Cell Biology Building, Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK.