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Drosophila kinesin-8 stabilises kinetochore-microtubule interaction

Tomoya Edzuka, Gohta Goshima

Preprint posted on October 25, 2018 https://www.biorxiv.org/content/early/2018/10/25/363150

Article now published in Journal of Cell Biology at http://dx.doi.org/10.1083/jcb.201807077

Sizing up the mitotic spindle: KLP67A regulates microtubule dynamics and stabilises kinetochore-microtubule attachments in Drosophila.

Selected by Ben Craske, Gaetan Dias Mirandela, Thibault Legal and Toni McHugh

Categories: biochemistry, cell biology

Context:

The collective efforts of microtubule motors during cell division facilitates the organisation of the bipolar spindle, the establishment of stable kinetochore – microtubule attachments and faithful segregation of genetic material. Kinesin-8 is a widely conserved processive motor which generally localises to the kinetochore and is required for proper chromosome alignment in budding yeast and mammalian cells. However, the proposed role of Kinesin-8 motors as active microtubule depolymerases is under debate. Here, the authors investigate the motility and depolymerase activity of the sole Drosophila Kinesin-8 motor KLP67A using in vitro reconstitutions with both GMPCPP stabilised and dynamic microtubules. Additionally, they characterise the importance of KLP67A in stabilising the kinetochore-microtubule attachment and regulating spindle length in mitotic S2 cells.

Key findings:
As is characteristic of Kinesin-8 motors, the authors demonstrate that full length Drosophila KLP67A is able to processively walk along microtubules and accumulate at the plus tips in vitro. However, no detectable depolymerisation phenotype was observed for KLP67A after application to GMPCPP stabilised microtubules, in contrast to what has been characterised for S. cerevisiae Kip3 or Drosophila Kinesin-13 depolymerase, KLP10A. Despite the absence of obvious depolymerase activity upon stabilised microtubules, the localisation of KLP67A to the tips of dynamic microtubules induced a significantly higher frequency of both catastrophe and subsequent rescue. This indicates that the sole Drosophila Kinesin-8 motor is able to induce microtubule depolymerisation, but also provide a subsequent stabilising effect at the plus tip to prevent severe shrinkage events and therefore operating by a distinct mechanism to both KLP10A and Kip3.

Figure adapted from Edzuka and Goshima, 2018

In S2 cells, RNAi knockdowns of KLP67A resulted in extensive elongation of the spindle microtubules, consistent with their conclusion that Drosophila Kinesin-8 can regulate the length of dynamic microtubules in vitro. In order to investigate KLP67A’s potential function in stabilising the kinetochore – microtubule attachment, S2 cells with monopolar spindles were generated and GFP-Rod was used as a marker of kinetochore attachment status following KLP67A knockdown. Under such conditions, the authors observed an increased frequency of detachment after initial establishment of an end-on bound state. This indicates that KLP67A is important for maintaining stable kinetochore-microtubule attachments and proper chromosome alignment during mitosis. In order to test whether detachment frequency was enhanced due to the loss of KLP67A’s microtubule length regulation, S2 cells were treated with colcemid to destabilise microtubules in the absence of KLP67A. However, artificial shortening of the spindle was unable to rescue the phenotype, suggesting that KLP67A’s stabilising function at the kinetochore-microtubule interface was independent of its depolymerase activity. As KLP67A was also able to stabilise microtubule plus ends after inducing catastrophe in vitro, the authors hypothesised that this function may be important for the stabilisation of end-on attachments and thus could be potentially rescued with another plus-end stabilising factor. Ectopic expression of GFP-Mast/Orbit (CLASP in humans) in the absence of KLP67A was able suppress microtubule detachment from the kinetochore, but had no impact on the other effects of KLP67A depletion e.g. spindle elongation and mitotic duration.

Finally, the authors carried out their own RNAi analysis of Kif18a depletion in GFP-Mad2 expressing HeLa cell lines. GFP-Mad2 recruitment to the kinetochore was used as a read-out for incorrect attachments. Although no clear differences were observed between the localisation of Mad2 to unaligned kinetochores, recruitment of GFP-Mad2 to aligned kinetochores was doubled in Kif18a depleted cells. This suggests that Kif18a is required to maintain stable kinetochore-microtubule attachments similarly to KLP67A in Drosophila.

All in all, this preprint highlights the importance of KLP67A in (i) regulating microtubule dynamics within the spindle, (ii) stabilising the kinetochore-microtubule attachment and (iii) highlighting functional similarity to the roles of human Kif18a during mitosis.

Open Questions:

What effect does the KLP67A tail domain have on microtubule dynamics?

Can similar depolymerase and stabilisation activity be reconstituted for human Kif18a in vitro on dynamic microtubules to clarify the controversy around its function?

Are stabilising factors at the human kinetochore-microtubule interface, such as the Ska and SKAP/Astrin complex, responsible for dampening the severity of Kif18a depletion in human cells?

Further reading:

Goshima, G., and R.D. Vale. 2005. Cell cycle-dependent dynamics and regulation of mitotic kinesins in Drosophila S2 cells. Mol Biol Cell. 16:3896-3907.

Stumpff, J., Y. Du, C.A. English, Z. Maliga, M. Wagenbach, C.L. Asbury, L. Wordeman, and R. Ohi. 2011. A tethering mechanism controls the processivity and kinetochore-microtubule plus-end enrichment of the kinesin-8 Kif18A. Mol Cell. 43:764-775.

McHugh, T., A.A. Gluszek, and J.P.I. Welburn. 2018. Microtubule end tethering of a processive kinesin-8 motor Kif18b is required for spindle positioning. J Cell Biol. 217: 2403-2416

Tags: drosophila, kif18a, kinesin-8, kinetochore, klp67a, mitosis

Posted on: 16th November 2018

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  • Author's response

    Tomoya Edzuka and Gohta Goshima shared

    What effect does the KLP67A tail domain have on microtubule dynamics?

    The motor domain alone can induce microtubule catastrophe but not slow down shrinkage or elevate the rescue frequency. We therefore speculate that KLP67A prevents rapid shrinkage or promotes rescue through free tubulin binding at the tail region; for example, KLP67A might increase the local concentration of free tubulin near microtubule plus ends. A corroborative data for this idea is the negative correlation between the amount of KLP67A on microtubule plus ends and the microtubule shrinkage rate (Fig. 3J). 

    Can similar depolymerase and stabilisation activity be reconstituted for human Kif18a in vitro on dynamic microtubules to clarify the controversy around its function?

    We have tried KIF18A (full-length) purification quite long time ago. It did not work well. The band was not clean and the protein was somehow lost during gel filtration. But in retrospect, we might have been able to get the protein of “okay” quality by using sucrose gradient centrifugation, like we later did for fly KLP67A. We guess we would then see the two different activities reported for KIF18A by Mayer and Ohi groups in an appropriate assay condition.

    Are stabilising factors at the human kinetochore-microtubule interface, such as the Ska and SKAP/Astrin complex, responsible for dampening the severity of Kif18a depletion in human cells?

    We agree that it is an interesting possibility, as fly does not possess those complexes. We have not tested the idea experimentally.

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