Abstract
ABSTRACTA key step of mitosis is the congression of chromosomes to the spindle equator. Congression is driven by at least two distinct mechanisms: (1) kinetochores slide along the microtubule lattice using the plus-end directed CENP-E motor, and (2) kinetochores biorientating near the pole move to the equator through microtubule depolymerisation-coupled pulling. Here, we show that CENP-Q – a subunit of the CENP-O complex (comprising CENP-O, CENP-P, CENP-Q and CENP-U) that targets polo-like kinase (Plk1) to kinetochores – is also required for the recruitment of CENP-E to kinetochores. We further reveal a CENP-E recruitment-independent role for CENP-Q in depolymerisation-coupled pulling. Both of these functions are abolished by a single point mutation in CENP-Q (S50A) – a residue that is phosphorylated in vivo. 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.
Highlights
Chromosome congression is the process by which chromosomes align at the spindle equator, a position equidistant from both spindle poles (Kops et al, 2010; Walczak et al, 2010)
Depletion of CENP-Q causes accumulation of polar chromosomes To investigate the function of human CENP-Q, we depleted the protein in HeLa cells using small interfering RNA oligonucleotides
Quantitative immunofluorescence confirmed that the kinetochore-bound population of the CENP-Q protein was reduced by 86% (66.2) following small interfering RNA (siRNA)-mediated depletion (Fig. 1B)
Summary
Chromosome congression is the process by which chromosomes align at the spindle equator, a position equidistant from both spindle poles (Kops et al, 2010; Walczak et al, 2010) In human cells, this process takes ,15-20 min and results in the formation of the metaphase plate. Congression is coincident with the process of chromosome biorientation, that is the establishment of amphitelic (or bipolar) attachments in which sister kinetochores attach to microtubules emanating from opposite spindle poles Such amphitelic attachments are Mechanochemical Cell Biology Building, Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK. Received 25 September 2014; Accepted 3 November 2014 compatible with oscillatory motion because sisters switch between poleward and away from pole movement states (Skibbens et al, 1993) These chromosomes are able to congress because they undergo sustained movements towards the spindle equator. Cells have multiple mechanisms to position chromosomes on the metaphase plate
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