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.
Highlights
The accurate segregation of chromosomes during anaphase requires that all sister kinetochores biorientate, an attachment state in which sisters form stable attachments to the plus-ends of microtubules that originate at opposite spindle poles
Bi-orientation begins immediately after nuclear envelope breakdown during prometaphase when scattered chromosomes engage the nascent mitotic spindle, and concludes with the formation of the metaphase plate – a state where all sister kinetochores are bi-orientated and aligned on the equator of a bipolar spindle (McIntosh et al, 2012)
By aligning profiles of the inter-sister distance – which reflects tension in the centromeric chromatin, 40 s before and 40 s after the first sister switching event (Figure 4A), we demonstrate clearly that lead initiated directional switch (LIDS) and trail initiated directional switch (TIDS) both have strong pre-event and postevent inter-sister distance signatures (Figure 4A; compare red [TIDS] and black [LIDS] traces)
Summary
The accurate segregation of chromosomes during anaphase requires that all sister kinetochores biorientate, an attachment state in which sisters form stable attachments to the plus-ends of microtubules that originate at opposite spindle poles. Directed motility is possible because one sister adopts a poleward (P) moving state (the lead sister) while the other is in an away-from-the-pole (AP) moving state (the trailing sister) These two movement states reflect the balance of microtubule polymerisation/depolymerisation within the kinetochore-fibre (K-fibre), which is typically 20–25 microtubules in human cells (Compton, 2000; Rieder, 2005; Wendell et al, 1993). The pairs of sister kinetochores repeatedly switch between moving backwards and forwards giving rise to oscillations in the positions of the sister chromatids It is not clear how the two sister kinetochores are able to communicate with each other so that they can co-ordinate their backwards and forwards movement. Using computational techniques to analyse kinetochore movements in living cells, the experiments reveal that the trailing sister kinetochore can sometimes change direction before the lead sister.
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