Accurate cell division depends on proper attachment of chromosomes to the microtubule-based division apparatus. An impressive in vitro study shows how applied force plays a pivotal part in regulating such attachment. See Letter p.576
The main safety feature of seat belts is that if the vehicle jolts, an abrupt pull locks the belt, keeping the passenger in place. Cells also seem to carry a nanoscale version of seat belts: the kinetochores — macromolecular machines that consist of more than 50 different proteins and connect chromosomes to dynamic microtubules of the cell-division apparatus — keep the chromosomes from accidentally ending up in the wrong daughter cell (Akiyoshi et al.1, page 576 of this issue).
Stable propagation of genomes through mitotic cell division depends on the equal partitioning of replicated DNA, which is packaged into sister chromatids. Equal division depends on chromosome bi-orientation — that is, attachment of sister chromatids to microtubules that extend from opposite ends of the bipolar spindle (Fig. 1a). Failure of bi-orientation is common, but the improper attachments that emerge somehow get corrected2. Classic studies in grasshopper cells indicated3 that differences in physical forces acting on a chromosome could be crucial for distinguishing between correct and incorrect attachments. But how force-based regulation may work has remained largely mysterious. A major barrier to progress has been the biochemical complexity of the kinetochores4 and, therefore, the tremendous difficulty in isolating them in a functional form.
Enter Akiyoshi and co-workers1. The authors tagged different kinetochore proteins and developed conditions to isolate functional kinetochores from dividing yeast cells. Budding yeast is an ideal model system for such studies: not only can it be easily manipulated genetically, but also its kinetochore binds only one microtubule — unlike a human kinetochore, which can bind more than 20 microtubules. Nonetheless, the kinetochore architecture is essentially conserved across eukaryotes (organisms with nucleated cells), with microtubule-binding capacity increasing largely as a result of juxtaposing multiple copies of the core unit of the yeast kinetochore5.
A key feature of kinetochores in vivo is that they can remain attached to the ends of disassembling microtubules. The kinetochores Akiyoshi and colleagues isolate can also do this. What's more, although many of the typical structural proteins are present in the isolated kinetochores, key proteins — such as the enzyme Aurora kinase6 — that regulate chromosome attachment to the mitotic spindle are absent. These 'minimal' kinetochores therefore allow tests of how forces might regulate microtubule binding, independently of any potential regulation through protein phosphorylation.
Akiyoshi et al. attach the minimal kinetochores to a bead that they can manipulate with optical tweezers7 (Fig. 1b). A bead 'trapped' by optical tweezers behaves as if it is attached to a mechanical spring, such that a force restoring its position is proportional to the change in displacement. The authors examine interactions of the kinetochores with polymerizing and depolymerizing microtubules under different forces. This in vitro experiment recapitulates the pulling force that a kinetochore of a bi-oriented chromosome experiences within a cell.
It is reasonable to expect that the lifetime of the attachment between any two interacting partners, such as a ligand and its receptor, decreases as an applied force increases; this is because the mechanical work helps to overcome the detachment energy barrier8. Remarkably, however, Akiyoshi et al. reveal that force — in the range relevant to physiological forces that act on chromosomes — increases the lifetime of kinetochore–microtubule attachment twofold. The authors' further analysis reveals that the kinetochore–microtubule attachment behaves like a 'catch bond' — similar to a seat belt that locks in place when pulled abruptly9.
A catch bond can be modelled as a system with both a strongly bound state and a weakly bound state; force favours the strongly bound state. The minimal kinetochores are weakly bound to microtubules that are disassembling, and strongly bound to growing microtubules. Notably, applied force suppresses microtubule disassembly and can therefore favour the strongly bound state. On the basis of direct measurements and simple assumptions, Akiyoshi et al. develop a quantitative catch-bond model that accounts for the observed kinetochore–microtubule-attachment behaviour.
The catch-bond mechanism may be considered as a mechanical extension of biochemical allosteric regulation. Force can be considered to be the equivalent of a molecule binding a protein's regulatory site and inducing a conformational change that modulates activity. Evidence from other cellular components with catch-bond behaviour, such as the bacterial adhesion protein FimH, is consistent with this idea10. In the case of the kinetochore–microtubule interaction, it is possible that force directly induces a conformational change in microtubule tips11. The strongly bound state could involve kinetochore interactions with microtubule protofilaments that are relatively straight, as seen in growing microtubules in vitro12. The weakly bound state could have protofilaments splaying outwards, as seen in disassembling filaments12.
Examining the structure of the minimal kinetochores and how they bind different microtubule-tip structures are essential next steps. Combining these structural studies with mutagenesis analysis should allow the design of experiments to test the catch-bond mechanism in dividing cells. Aurora kinases, or other proteins that correct errors in chromosome–spindle attachments, could have a role in fine-tuning the catch-bond mechanism. Experiments with purified kinetochores will also no doubt be useful in dissecting the interplay between these chemical and mechanical regulatory mechanisms.
In vitro studies of isolated kinetochores might help to settle another outstanding question regarding the regulation of chromosome segregation. If chromosomes are improperly attached to the spindle, a signalling network called the spindle-assembly checkpoint blocks mitotic cell division before its anaphase step. It is unclear whether the spindle-assembly checkpoint directly responds to force (or tension)13. As the purified kinetochores contain proteins required for the spindle-assembly checkpoint, these kinetochores can be used to investigate whether the recruitment of checkpoint proteins — an early step in the signalling — is sensitive to force. Keep your seat belts fastened for the next phase of this exciting journey.
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Structural, morphological and nanomechanical characterisation of intermediate states in the ageing of erythrocytes
Journal of Molecular Recognition (2012)
Seminars in Cell & Developmental Biology (2011)