Congrats @krunovuk.bsky.social on this remarkable work! Thanks @erc.europa.eu, www.hrzz.hr, and www.srce.unizg.hr for supporting us!

We propose a kinetochore-centrosome feedback mechanism controlling chromosome movement: chromosomes near centrosomes rely on CENP-E because high Aurora A activity boosts Aurora B at kinetochores, restricting end-on attachments. CENP-E counteracts this by downregulating Aurora B.

Notably, the dependence of polar chromosomes on CENP-E can be bypassed by using constitutively dephosphorylated Hec1, emphasizing the role of Aurora kinase-mediated Hec1 phosphorylation in preventing congression when CENP-E is absent.

Does Aurora A at spindle poles directly promote Aurora B activity at kinetochores? Yes, our results show that acute inhibition of Aurora A reduces Aurora B activity at polar kinetochores, accompanied by a decrease in the number of polar chromosomes.

At poles that contain centrioles, high Aurora A kinase enhances Aurora B activity on kinetochores of polar chromosomes, increasing phosphorylation of microtubule-binding proteins and preventing stable microtubule attachments in the absence of CENP-E.

Centrosomes inhibit the initiation of congression in the absence of CENP-E, and depletion of centrioles allows chromosomes near acentriolar poles to initiate congression independently of CENP-E.

In our second paper, we asked why only polar chromosomes depend on CENP-E for congression. We inhibited Plk4 to generate spindles where one pole had centrioles and the other did not. Strikingly, the acentriolar pole (on the right in the movie) rarely had polar chromosomes.

In sum, the first paper shows that chromosome congression is coupled with biorientation, with CENP-E linking the two. A thread on the second paper is coming soon! Congrats @krunovuk.bsky.social on this amazing work! We appreciate support from @erc.europa.eu, www.hrzz.hr, and www.srce.unizg.hr.

We propose that congression operates as a negative feedback loop between the activity of Aurora kinases that oppose congression by destabilizing end-on attachments, but also activate CENP-E, which then promotes congression by opposing the activity of Aurora kinases.

In our model, stabilization of end-on attachments without CENP-E is delayed due to Aurora kinase-mediated phosphorylation of microtubule-binding proteins and expansion of the fibrous corona, which obstructs attachments. CENP-E counters this by reducing phosphorylation.

Why can’t kinetochores start their movement without CENP-E? Is dynein holding them? No. This is due to Aurora kinases that oppose the formation of end-on attachments near the spindle poles. When Aurora B is inhibited in the absence of CENP-E, kinetochores start to move!

To directly test if congression is coupled with biorientation, we performed STED super-resolution imaging. Indeed, reactivation of CENP-E led to a gradual transition from lateral to end-on attachments on polar chromosomes.

Chromosome congression involves progressive capture and stabilization of end-on microtubule attachments, based on recruitment of the attachment marker Astrin, together with a gradual reduction of SAC and fibrous corona proteins on kinetochores.

We found that without CENP-E kinetochores move in the same manner as normally, just not as frequently. Thus, CENP-E is not required for chromosome movement during congression, challenging the current view, but instead for the initiation of this movement.

@krunovuk.bsky.social pushed the limits of long-term live-cell imaging by simultaneously imaging hundreds of cells for 24 h using lattice light-sheet microscopy. By adding chemical inhibitors, he tested a time-resolved hierarchy of molecular networks working during congression.

Do you know how chromosomes travel to the spindle midplane in mitosis? The motor protein CENP-E is crucial for this. Without it, some chromosomes stay trapped at the poles. The current model says it glides kinetochores along microtubules... but does it really?

Wonderful, Pavel, congrats!!! 🍸🍸