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Tension on kinetochore substrates is insufficient to prevent Aurora-triggered detachment

Anna K de Regt, Charles L Asbury, Sue Biggins

Preprint posted on September 13, 2018 https://www.biorxiv.org/content/early/2018/09/13/415992

What tension? Pulling forces in a proper bi-polar spindle are thought to turn off Aurora B-directed corrections. An in vitro study suggests that tension on kinetochores alone cannot deter this kinase

Selected by Angika Basant

Categories: cell biology

Background:

When proliferating, cells divide their genetic material by employing a highly complex and dynamic machine – the mitotic spindle. Microtubules emanating from opposite poles of the spindle contact duplicated chromosomes at specialised macromolecular structures called kinetochores. Microtubule-kinetochore interactions are a vital aspect of cell division and have incidentally been the focus of many preprints quite recently (1-3).

To faithfully pull apart sister chromatids, i.e. to provide every new cell a full complement of genetic material, each kinetochore in a pair must attach to a microtubule from opposite poles. Crucially, cells must be able determine whether this has been achieved before segregating chromosomes. This complex task is performed by Aurora B kinase. Proper attachments involve kinetochore pairs pulled on from opposite poles, thereby generating tension on kinetochores. Aurora B selectively phosphorylates its substrate proteins on kinetochores under low tension (4), where presumably both kinetochores attach to one pole. This phosphorylation reduces their affinity for microtubules, destabilising these erroneous attachments. How does Aurora B sense the level of tension on kinetochores, and is that the only cue underlying kinase-triggered detachment of microtubules?

Kinetochores are known to undergo dramatic structural changes when under tension (5-7). An appealing hypothesis is that this occludes key Aurora B substrates from being phosphorylated, thus retaining correct microtubule attachments. It is a considerable challenge to test such models in vivo. Aurora B performs several roles in cell division and isolating its function at kinetochores, particularly in the presence of opposing phosphatase activity, is not trivial.

Key findings:

The authors in this study distil this complex intracellular phenomenon into a reconstituted in vitro assay. They purify yeast kinetochores such that endogenous kinase and phosphatase activity has been quenched. Next, they design and purify an active recombinant Aurora B kinase (AurB*) that can phosphorylate key microtubule-interacting substrates (particularly the Ndc80 protein) on isolated kinetochores.They show that AurB* phosphorylation impairs the ability of these kinetochores to bind fluorescently labelled, stabilised microtubules in vitro. This effect is partially Ndc80 dependent.

What is the effect of AurB* on physiologically relevant microtubule attachments that are load-bearing, end-on and dynamic? The authors modified an optical trap assay (8) to generate tension on microtubule-attached kinetochores where AurB* can be flowed in. In such experiments, kinetochores linked to polystyrene beads are added to coverslips on which anchored microtubules are grown. The position and force experienced by these beads can be controlled via a tightly focused laser beam (optical trap). The kinetochores are then indirectly manipulated by the laser to attach to tips of dynamic microtubules and held at a desired force (to generate tension). Exogenous AurB* is added once the attachment is made.

While these manipulations are technically challenging, the authors were able to observe that phosphorylation by AurB* releases tips of microtubules from kinetochores under low tension (~1 piconewton (pN)), as would be predicted for an improper attachment in vivo. A kinase-dead mutant of AurB* is 3-fold less effective in performing this “correction”.

Importantly, the prevailing model also predicts that a properly attached kinetochore under high tension should not be detached from microtubules by Aurora B. However, quite surprisingly, the authors find that AurB* activity releases microtubule attachments even at high tension regimes (5 and 8 pN). This suggests that tension across kinetochores is not sufficient to block Aurora B phosphorylation on kinetochore substrates.

adapted from de Regt et al., 2018

 

What I like about this preprint:

It is very exciting to see a reconstituted system that draws on decades of work and has the potential to test several dogmas in the field of chromosome segregation. To quote the authors, “Rigorously testing such models requires independent control of tension, attachment, and enzyme activity”. This study (a) tackles all three of these challenges, (b) reveals unexpected complexities in the system and (c) sets the stage to further probe elusive mechanisms of how our genomes are faithfully divided.

Future directions and questions for the authors:

In the light of their data, the authors state that alternate models for error correction bear consideration. For example, we think of tension as primarily modifying the kinetochore but it may instead affect kinase activity or even that of a phosphatase. These would be very exciting new directions for the field. However, to delve further in the current experiments, I would love to ask the authors a few questions:

  1. How do detachment rates of non-phosphorylatable kinetochores (Ndc80-7A) compare with wild-type under low and high tension? In other words, could detachment under high tension be occurring via an unexpected mechanism?
  2. Could the response to kinetochore tension depend on strength of AurB activity? That is, if AurB* activity is too high, perhaps it phosphorylates its kinetochore substrates regardless of tension. In vivo AurB activity at kinetochores might be tightly regulated to respond to changes in tension. Were a range of AurB* concentrations tested, other than 0.5uM and 5uM, as even 0.5uM of exogenous, chimeric AurB* may exceed a threshold?
  3. It seems surprising that not only does AurB* function persist at high tension, but the detachment rates increase with increasing force. How would you explain this positive correlation?

References:

  1. Kuhn and Dumont, bioRxiv 2018; https://doi.org/10.1101/463471
  2. Roy et al., bioRxiv 2018; https://doi.org/10.1101/459594
  3. Doodhi et al., bioRxiv 2018; https://doi.org/10.1101/455873
  4. Kelly and Funabiki., Curr. Op. in Cell Biol 2009
  5. Wan et al., Cell 2009
  6. Joglekar et al., Curr. Biol 2009
  7. Tien et al., Genetics 2014
  8. Akiyoshi et al., Nature 2010

 

Posted on: 11th November 2018 , updated on: 12th November 2018

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  • Author's response

    Anna de Regt shared

    Hi Angika,

    Thanks for your excellent summary and great questions!

    To address your questions:

    We love the idea of using our assay to better understand what substrates and even what sites are most important for Aurora B error correction. However, we consider this a long-term goal at this time and beyond the scope of this work due to the work involved. Each “point” on our detachment-rate-versus-force graph has a huge amount of work behind it – remember, we can only measure a maximum of one event per slide because once AurB* is introduced, all the kinetochores will be phosphorylated and cannot be probed again. And, of course, because it’s single-particle biophysics, usually the best-case scenario doesn’t occur for every slide! So we have to set up and test many, many slides for each condition but certainly hope to pursue this question in the long-term.

    To more specifically address your question about Ndc80-7A, we did not measure detachment rates of kinetochores containing Ndc80-7A kinetochores for this work because we would not qualify these as “non-phosphorylateable kinetochores.” There is much evidence that phosphorylation sites on other kinetochore proteins (especially Dam1) also play an important role in Aurora-dependent error correction. We used the Ndc80-7A kinetochores to illustrate the point that AurB* is primarily phosphorylating known Aurora B sites but find it unlikely that these kinetochores would exhibit tension-dependent suppression of detachment rates.

    We did not test lower concentrations of AurB* because the detachment rate at low force with our two concentrations of AurB* is already quite low, and it is therefore unlikely that a lower concentration would yield a detachment rate that is distinguishable from the native (i.e., with kinase-dead AurB*) detachment rate. While we understand why it would be interesting to try as large a range of kinase concentrations as possible, we did not do this because we have likely found the lowest concentration of AurB* where it is still possible to measure kinase-triggered detachments at low force.

    The trend that you noticed where detachment rate increases with higher force is also true for the kinase-dead trials. This is actually typical behavior for protein-protein interactions: if you pull harder on them, they will come apart more readily. So it makes sense that this would also occur for the experiments that contained active AurB*. In fact, if we had seen the same detachment rates at low and high forces with AurB*, we think it would have been evidence in favor of our hypothesis, that tension across kinetochores suppresses Aurora B activity.

    Thanks again for your interest in our work and your questions! We hope that our assay will be a tool for probing some of these questions and more into the future.

    -Anna, Sue, and Chip

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