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Chromosome congression is promoted by CENP-Q- and CENP-E-dependent pathways.

Bancroft J, Auckland P, Samora CP, McAinsh AD - J. Cell. Sci. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Mechanochemical Cell Biology Building, Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK.

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Related in: MedlinePlus

Fates of unaligned kinetochore pairs. (A) Schematic representing the orientation of unaligned kinetochore pairs within the mitotic spindle. The black dotted line represents the pole-to-pole axis, dotted green lines on chromosomes 4 and 5 represent the kinetochore sister–sister axis, the ∼90° angle between the sister–sister axis of chromosome 4 and the spindle pole-to-pole axis indicates non-biorientation. The reduced angle (≤45°) of the sister–sister axis relative to spindle axis of chromosome 5 indicates biorientation. Chromosomes 1, 2 and 3 are behind the pole and are therefore non-biorientated. Chromosome 6 is mono-orientated by the pole proximal kinetochore and laterally attached to an adjacent K-fibre by the pole distal kinetochore. Discriminating this chromosome from biorientated chromosomes (e.g. number 5) was not possible in our assay. (B) The orientation state of unaligned kinetochore pairs in CENP-Q- (n = 217 kinetochores) and CENP-E-siRNA- (n = 211 kinetochores) treated HeLa K cells stably expressing eGFP–CENP-A and eGFP–centrin1. Pink represents non-biorientated kinetochores (classes 1, 2, 3 and 4), and orange orientated kinetochores (classes 5 and 6). n≥3 independent experiments. (C) The fates of non-biorientated kinetochore pairs over 5 min in live HeLa K cells (stably expressing eGFP–CENP-A and eGFP–centrin1) after 48 h of treatment with CENP-Q or CENP-E siRNA. (D) The fates of orientated kinetochore pairs over 5 min in live HeLa K cells (stably expressing eGFP–CENP-A and eGFP–centrin1) after 48 h of treatment with CENP-Q or CENP-E siRNA. (E) Example frames from movies of kinetochore fates in HeLa K cells (stably expressing eGFP–CENP-A and eGFP–centrin1) after 48 h of treatment with CENP-Q or CENP-E siRNA. Orange chevrons indicate pairs that are orientated with the spindle axis, blue chevrons indicate pairs that are non-biorientated and white chevrons indicate where the designation of state is unclear. Yellow stars indicate the spindle pole when visible. The first column shows an example of a biorientated kinetochore pair in a CENP-Q-depleted cell, the pair does not congress but switches to a non-biorientated state (supplementary material Movie 3). The second column shows an example of an orientated kinetochore pair that fails to congress in CENP-Q-depleted cells (supplementary material Movie 4). In contrast, the third column shows an example of an orientated sister pair in CENP-E-depleted cells that is still able to congress to the metaphase plate (supplementary material Movie 5). The forth column shows an unaligned non-biorientated sister pair that cannot move to the metaphase plate in a CENP-E-depleted cell (supplementary material Movie 6). t = 0, first frame of the movie. The dashed lines indicate the metaphase plate periphery.
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f03: Fates of unaligned kinetochore pairs. (A) Schematic representing the orientation of unaligned kinetochore pairs within the mitotic spindle. The black dotted line represents the pole-to-pole axis, dotted green lines on chromosomes 4 and 5 represent the kinetochore sister–sister axis, the ∼90° angle between the sister–sister axis of chromosome 4 and the spindle pole-to-pole axis indicates non-biorientation. The reduced angle (≤45°) of the sister–sister axis relative to spindle axis of chromosome 5 indicates biorientation. Chromosomes 1, 2 and 3 are behind the pole and are therefore non-biorientated. Chromosome 6 is mono-orientated by the pole proximal kinetochore and laterally attached to an adjacent K-fibre by the pole distal kinetochore. Discriminating this chromosome from biorientated chromosomes (e.g. number 5) was not possible in our assay. (B) The orientation state of unaligned kinetochore pairs in CENP-Q- (n = 217 kinetochores) and CENP-E-siRNA- (n = 211 kinetochores) treated HeLa K cells stably expressing eGFP–CENP-A and eGFP–centrin1. Pink represents non-biorientated kinetochores (classes 1, 2, 3 and 4), and orange orientated kinetochores (classes 5 and 6). n≥3 independent experiments. (C) The fates of non-biorientated kinetochore pairs over 5 min in live HeLa K cells (stably expressing eGFP–CENP-A and eGFP–centrin1) after 48 h of treatment with CENP-Q or CENP-E siRNA. (D) The fates of orientated kinetochore pairs over 5 min in live HeLa K cells (stably expressing eGFP–CENP-A and eGFP–centrin1) after 48 h of treatment with CENP-Q or CENP-E siRNA. (E) Example frames from movies of kinetochore fates in HeLa K cells (stably expressing eGFP–CENP-A and eGFP–centrin1) after 48 h of treatment with CENP-Q or CENP-E siRNA. Orange chevrons indicate pairs that are orientated with the spindle axis, blue chevrons indicate pairs that are non-biorientated and white chevrons indicate where the designation of state is unclear. Yellow stars indicate the spindle pole when visible. The first column shows an example of a biorientated kinetochore pair in a CENP-Q-depleted cell, the pair does not congress but switches to a non-biorientated state (supplementary material Movie 3). The second column shows an example of an orientated kinetochore pair that fails to congress in CENP-Q-depleted cells (supplementary material Movie 4). In contrast, the third column shows an example of an orientated sister pair in CENP-E-depleted cells that is still able to congress to the metaphase plate (supplementary material Movie 5). The forth column shows an unaligned non-biorientated sister pair that cannot move to the metaphase plate in a CENP-E-depleted cell (supplementary material Movie 6). t = 0, first frame of the movie. The dashed lines indicate the metaphase plate periphery.

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).


Chromosome congression is promoted by CENP-Q- and CENP-E-dependent pathways.

Bancroft J, Auckland P, Samora CP, McAinsh AD - J. Cell. Sci. (2014)

Fates of unaligned kinetochore pairs. (A) Schematic representing the orientation of unaligned kinetochore pairs within the mitotic spindle. The black dotted line represents the pole-to-pole axis, dotted green lines on chromosomes 4 and 5 represent the kinetochore sister–sister axis, the ∼90° angle between the sister–sister axis of chromosome 4 and the spindle pole-to-pole axis indicates non-biorientation. The reduced angle (≤45°) of the sister–sister axis relative to spindle axis of chromosome 5 indicates biorientation. Chromosomes 1, 2 and 3 are behind the pole and are therefore non-biorientated. Chromosome 6 is mono-orientated by the pole proximal kinetochore and laterally attached to an adjacent K-fibre by the pole distal kinetochore. Discriminating this chromosome from biorientated chromosomes (e.g. number 5) was not possible in our assay. (B) The orientation state of unaligned kinetochore pairs in CENP-Q- (n = 217 kinetochores) and CENP-E-siRNA- (n = 211 kinetochores) treated HeLa K cells stably expressing eGFP–CENP-A and eGFP–centrin1. Pink represents non-biorientated kinetochores (classes 1, 2, 3 and 4), and orange orientated kinetochores (classes 5 and 6). n≥3 independent experiments. (C) The fates of non-biorientated kinetochore pairs over 5 min in live HeLa K cells (stably expressing eGFP–CENP-A and eGFP–centrin1) after 48 h of treatment with CENP-Q or CENP-E siRNA. (D) The fates of orientated kinetochore pairs over 5 min in live HeLa K cells (stably expressing eGFP–CENP-A and eGFP–centrin1) after 48 h of treatment with CENP-Q or CENP-E siRNA. (E) Example frames from movies of kinetochore fates in HeLa K cells (stably expressing eGFP–CENP-A and eGFP–centrin1) after 48 h of treatment with CENP-Q or CENP-E siRNA. Orange chevrons indicate pairs that are orientated with the spindle axis, blue chevrons indicate pairs that are non-biorientated and white chevrons indicate where the designation of state is unclear. Yellow stars indicate the spindle pole when visible. The first column shows an example of a biorientated kinetochore pair in a CENP-Q-depleted cell, the pair does not congress but switches to a non-biorientated state (supplementary material Movie 3). The second column shows an example of an orientated kinetochore pair that fails to congress in CENP-Q-depleted cells (supplementary material Movie 4). In contrast, the third column shows an example of an orientated sister pair in CENP-E-depleted cells that is still able to congress to the metaphase plate (supplementary material Movie 5). The forth column shows an unaligned non-biorientated sister pair that cannot move to the metaphase plate in a CENP-E-depleted cell (supplementary material Movie 6). t = 0, first frame of the movie. The dashed lines indicate the metaphase plate periphery.
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f03: Fates of unaligned kinetochore pairs. (A) Schematic representing the orientation of unaligned kinetochore pairs within the mitotic spindle. The black dotted line represents the pole-to-pole axis, dotted green lines on chromosomes 4 and 5 represent the kinetochore sister–sister axis, the ∼90° angle between the sister–sister axis of chromosome 4 and the spindle pole-to-pole axis indicates non-biorientation. The reduced angle (≤45°) of the sister–sister axis relative to spindle axis of chromosome 5 indicates biorientation. Chromosomes 1, 2 and 3 are behind the pole and are therefore non-biorientated. Chromosome 6 is mono-orientated by the pole proximal kinetochore and laterally attached to an adjacent K-fibre by the pole distal kinetochore. Discriminating this chromosome from biorientated chromosomes (e.g. number 5) was not possible in our assay. (B) The orientation state of unaligned kinetochore pairs in CENP-Q- (n = 217 kinetochores) and CENP-E-siRNA- (n = 211 kinetochores) treated HeLa K cells stably expressing eGFP–CENP-A and eGFP–centrin1. Pink represents non-biorientated kinetochores (classes 1, 2, 3 and 4), and orange orientated kinetochores (classes 5 and 6). n≥3 independent experiments. (C) The fates of non-biorientated kinetochore pairs over 5 min in live HeLa K cells (stably expressing eGFP–CENP-A and eGFP–centrin1) after 48 h of treatment with CENP-Q or CENP-E siRNA. (D) The fates of orientated kinetochore pairs over 5 min in live HeLa K cells (stably expressing eGFP–CENP-A and eGFP–centrin1) after 48 h of treatment with CENP-Q or CENP-E siRNA. (E) Example frames from movies of kinetochore fates in HeLa K cells (stably expressing eGFP–CENP-A and eGFP–centrin1) after 48 h of treatment with CENP-Q or CENP-E siRNA. Orange chevrons indicate pairs that are orientated with the spindle axis, blue chevrons indicate pairs that are non-biorientated and white chevrons indicate where the designation of state is unclear. Yellow stars indicate the spindle pole when visible. The first column shows an example of a biorientated kinetochore pair in a CENP-Q-depleted cell, the pair does not congress but switches to a non-biorientated state (supplementary material Movie 3). The second column shows an example of an orientated kinetochore pair that fails to congress in CENP-Q-depleted cells (supplementary material Movie 4). In contrast, the third column shows an example of an orientated sister pair in CENP-E-depleted cells that is still able to congress to the metaphase plate (supplementary material Movie 5). The forth column shows an unaligned non-biorientated sister pair that cannot move to the metaphase plate in a CENP-E-depleted cell (supplementary material Movie 6). t = 0, first frame of the movie. The dashed lines indicate the metaphase plate periphery.
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Mechanochemical Cell Biology Building, Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK.

Show MeSH
Related in: MedlinePlus