Limits...
Super-resolution kinetochore tracking reveals the mechanisms of human sister kinetochore directional switching.

Burroughs NJ, Harry EF, McAinsh AD - Elife (2015)

Bottom Line: Here, we combine super-resolution tracking of kinetochores with automated switching-point detection to analyse sister switching dynamics over thousands of events.We discover that switching is initiated by both the leading (microtubules depolymerising) or trailing (microtubules polymerising) kinetochore.Surprisingly, trail-driven switching generates an overstretch of the chromatin that relaxes over the following half-period.

View Article: PubMed Central - PubMed

Affiliation: Warwick Systems Biology Centre, Warwick Mathematics Institute, University of Warwick, Coventry, United Kingdom.

ABSTRACT
The congression of chromosomes to the spindle equator involves the directed motility of bi-orientated sister kinetochores. Sister kinetochores bind bundles of dynamic microtubules and are physically connected through centromeric chromatin. A crucial question is to understand how sister kinetochores are coordinated to generate motility and directional switches. Here, we combine super-resolution tracking of kinetochores with automated switching-point detection to analyse sister switching dynamics over thousands of events. We discover that switching is initiated by both the leading (microtubules depolymerising) or trailing (microtubules polymerising) kinetochore. Surprisingly, trail-driven switching generates an overstretch of the chromatin that relaxes over the following half-period. This rules out the involvement of a tension sensor, the central premise of the long-standing tension-model. Instead, our data support a model in which clocks set the intrinsic-switching time of the two kinetochore-attached microtubule fibres, with the centromeric spring tension operating as a feedback to slow or accelerate the clocks.

No MeSH data available.


Related in: MedlinePlus

Standard model for kinetochore directional switching.Schematic outlining the prevailing model for how sister kinetochores coordinate directional switches. As the leading, poleward-moving, kinetochore (P; black) moves to the right, the centromeric chromatin (blue) – which functions as a compliant linkage between sisters – becomes progressively more stretched (steps i, ii). Stretching occurs because the trailing, away-from-the-pole, kinetochore (AP; red) is moving more slowly than the lead. Once sisters are at maximum stretch, the tension in the chromatin is thought to trigger the lead sister kinetochore to switch into an AP state. This results in a rapid loss of tension as both sisters are then in an AP state and moving towards each other (step iii). This relaxation is thought to trigger switching of the initially trailing sister into a P-moving state (step iv). Adapted from Wan et al., 2012.DOI:http://dx.doi.org/10.7554/eLife.09500.003
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4764575&req=5

fig1: Standard model for kinetochore directional switching.Schematic outlining the prevailing model for how sister kinetochores coordinate directional switches. As the leading, poleward-moving, kinetochore (P; black) moves to the right, the centromeric chromatin (blue) – which functions as a compliant linkage between sisters – becomes progressively more stretched (steps i, ii). Stretching occurs because the trailing, away-from-the-pole, kinetochore (AP; red) is moving more slowly than the lead. Once sisters are at maximum stretch, the tension in the chromatin is thought to trigger the lead sister kinetochore to switch into an AP state. This results in a rapid loss of tension as both sisters are then in an AP state and moving towards each other (step iii). This relaxation is thought to trigger switching of the initially trailing sister into a P-moving state (step iv). Adapted from Wan et al., 2012.DOI:http://dx.doi.org/10.7554/eLife.09500.003

Mentions: Initial investigations into the control of chromosome movement utilised time-lapse imaging in newt lung cells using video-enhanced differential interference contrast microscopy (Skibbens et al., 1993). Kinetochores were shown to undergo periods of relatively constant velocity separated by abrupt changes in direction – a behaviour termed ‘directional instability’. Subsequent experiments demonstrated that weakening the centromeric chromatin which links the sisters (with a laser) uncoupled the normally coordinated motility of sister kinetochores (Skibbens et al., 1995). These experiments led to a model (also see Rieder and Salmon, 1994) in which tension in the centromeric chromatin triggers a lead sister switch (P-to-AP) at a certain threshold, the loss of tension then triggering a directional switch in the second sister (AP-to-P). More recent kinetochore-tracking experiments in PtK1 cells are consistent with this model and show that switching initiates at maximum inter-kinetochore stretch (Wan et al., 2012), schematic shown in Figure 1. The polar ejection force, which increases with proximity to the pole, pushes the chromosomes towards the metaphase plate. When chromosomes stray far from the equator, this anti-poleward force increases the load on the leading (P) kinetochore and promotes switching – an idea supported by experiments in newt and human cells (Ke et al., 2009; Stumpff et al., 2012). The standard tension model thus predicts a fixed sequence of sister kinetochore-switching events during a directional reversal – lead switch first, followed by trail. However, the timeframe for these events is short (several seconds), requiring a sampling rate that avoids temporal aliasing. Existing kinetochore-tracking assays have a frame rate in the 7.5–15 s range (Dumont et al., 2012; Jaqaman et al., 2010; Wan et al., 2012), meaning that detailed analysis of the switching mechanism has not been possible to date.10.7554/eLife.09500.003Figure 1.Standard model for kinetochore directional switching.


Super-resolution kinetochore tracking reveals the mechanisms of human sister kinetochore directional switching.

Burroughs NJ, Harry EF, McAinsh AD - Elife (2015)

Standard model for kinetochore directional switching.Schematic outlining the prevailing model for how sister kinetochores coordinate directional switches. As the leading, poleward-moving, kinetochore (P; black) moves to the right, the centromeric chromatin (blue) – which functions as a compliant linkage between sisters – becomes progressively more stretched (steps i, ii). Stretching occurs because the trailing, away-from-the-pole, kinetochore (AP; red) is moving more slowly than the lead. Once sisters are at maximum stretch, the tension in the chromatin is thought to trigger the lead sister kinetochore to switch into an AP state. This results in a rapid loss of tension as both sisters are then in an AP state and moving towards each other (step iii). This relaxation is thought to trigger switching of the initially trailing sister into a P-moving state (step iv). Adapted from Wan et al., 2012.DOI:http://dx.doi.org/10.7554/eLife.09500.003
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4764575&req=5

fig1: Standard model for kinetochore directional switching.Schematic outlining the prevailing model for how sister kinetochores coordinate directional switches. As the leading, poleward-moving, kinetochore (P; black) moves to the right, the centromeric chromatin (blue) – which functions as a compliant linkage between sisters – becomes progressively more stretched (steps i, ii). Stretching occurs because the trailing, away-from-the-pole, kinetochore (AP; red) is moving more slowly than the lead. Once sisters are at maximum stretch, the tension in the chromatin is thought to trigger the lead sister kinetochore to switch into an AP state. This results in a rapid loss of tension as both sisters are then in an AP state and moving towards each other (step iii). This relaxation is thought to trigger switching of the initially trailing sister into a P-moving state (step iv). Adapted from Wan et al., 2012.DOI:http://dx.doi.org/10.7554/eLife.09500.003
Mentions: Initial investigations into the control of chromosome movement utilised time-lapse imaging in newt lung cells using video-enhanced differential interference contrast microscopy (Skibbens et al., 1993). Kinetochores were shown to undergo periods of relatively constant velocity separated by abrupt changes in direction – a behaviour termed ‘directional instability’. Subsequent experiments demonstrated that weakening the centromeric chromatin which links the sisters (with a laser) uncoupled the normally coordinated motility of sister kinetochores (Skibbens et al., 1995). These experiments led to a model (also see Rieder and Salmon, 1994) in which tension in the centromeric chromatin triggers a lead sister switch (P-to-AP) at a certain threshold, the loss of tension then triggering a directional switch in the second sister (AP-to-P). More recent kinetochore-tracking experiments in PtK1 cells are consistent with this model and show that switching initiates at maximum inter-kinetochore stretch (Wan et al., 2012), schematic shown in Figure 1. The polar ejection force, which increases with proximity to the pole, pushes the chromosomes towards the metaphase plate. When chromosomes stray far from the equator, this anti-poleward force increases the load on the leading (P) kinetochore and promotes switching – an idea supported by experiments in newt and human cells (Ke et al., 2009; Stumpff et al., 2012). The standard tension model thus predicts a fixed sequence of sister kinetochore-switching events during a directional reversal – lead switch first, followed by trail. However, the timeframe for these events is short (several seconds), requiring a sampling rate that avoids temporal aliasing. Existing kinetochore-tracking assays have a frame rate in the 7.5–15 s range (Dumont et al., 2012; Jaqaman et al., 2010; Wan et al., 2012), meaning that detailed analysis of the switching mechanism has not been possible to date.10.7554/eLife.09500.003Figure 1.Standard model for kinetochore directional switching.

Bottom Line: Here, we combine super-resolution tracking of kinetochores with automated switching-point detection to analyse sister switching dynamics over thousands of events.We discover that switching is initiated by both the leading (microtubules depolymerising) or trailing (microtubules polymerising) kinetochore.Surprisingly, trail-driven switching generates an overstretch of the chromatin that relaxes over the following half-period.

View Article: PubMed Central - PubMed

Affiliation: Warwick Systems Biology Centre, Warwick Mathematics Institute, University of Warwick, Coventry, United Kingdom.

ABSTRACT
The congression of chromosomes to the spindle equator involves the directed motility of bi-orientated sister kinetochores. Sister kinetochores bind bundles of dynamic microtubules and are physically connected through centromeric chromatin. A crucial question is to understand how sister kinetochores are coordinated to generate motility and directional switches. Here, we combine super-resolution tracking of kinetochores with automated switching-point detection to analyse sister switching dynamics over thousands of events. We discover that switching is initiated by both the leading (microtubules depolymerising) or trailing (microtubules polymerising) kinetochore. Surprisingly, trail-driven switching generates an overstretch of the chromatin that relaxes over the following half-period. This rules out the involvement of a tension sensor, the central premise of the long-standing tension-model. Instead, our data support a model in which clocks set the intrinsic-switching time of the two kinetochore-attached microtubule fibres, with the centromeric spring tension operating as a feedback to slow or accelerate the clocks.

No MeSH data available.


Related in: MedlinePlus