Cellular choke artist: Aurora B doesn’t perform well under pressure

From the Biggins Lab, Basic Sciences Division

The finger trap (more scientifically known as a biaxial braid) is a devious novelty device that has confounded and alarmed generations of unsuspecting victims. Slide your fingers into the trap and you’ll find that they can’t be easily removed. Panic and struggle to free yourself, and the trap only closes tighter around your defenseless digits. Only by taking a deep breath and examining the device will you realize that the key to escape is not to struggle but to relax, releasing the tension generated by your pulling and allowing your fingers to slip free. Our cells contain a piece of machinery that acts much like a finger trap, though this is no mere novelty. The process of cell division, so crucial to our lives, depends on the segregation of duplicated chromosomes to either end of a dividing cell. Segregation is carried out by the mitotic spindle, a collection of microtubules that extend out from either pole of the cell, grab hold of the chromosomes via a structure called the kinetochore, and drag them to their respective sides. When microtubules attach to the kinetochore, they want to start pulling, but their attachment is tenuous. Surprisingly, increased pulling does not, as one might expect, break this attachment and send the microtubule slinking away empty handed. Rather, like a finger stuck in a trap, the tension generated by pulling actually strengthens the attachment between microtubule and kinetochore, while the relaxation of tension is what causes it to falter. “[This process] requires the Aurora B protein kinase, which selectively phosphorylates the relaxed kinetochores to trigger their release from microtubules,” explains Dr. Sue Biggins, Professor and Director of Fred Hutch’s Basic Sciences Division. However, “how Aurora B distinguishes tense versus relaxed kinetochores is a long-standing mystery.” In a recent article in Nature Communications, Biggins group members Dr. Anna de Regt and Cordell Clark, in collaboration with the University of Washington’s Dr. Chip Asbury, utilized a new experimental strategy to address this elusive question.

In explaining why this mystery has remained unsolved for so long, Dr. Biggins points to “the extreme difficulty in manipulating kinetochore tension in vivo and unambiguously determining how it alters Aurora B activity.” To overcome this challenge, the group reconstituted this interaction in vitro by attaching microtubules to a coverslip, allowing them to bind purified kinetochores attached to beads, and using an optical trapping approach to pull on the bead, thus placing a defined amount of tension on the microtubules-kinetochore attachment. They could then add Aurora B to the mix and observe the release dynamics of this attachment. While in vitro reconstitution is an established approach in the Biggins lab, “this is the most difficult reconstitution we have achieved to date,” she says. Importantly, though, this experiment “allows direct observation of Aurora B-triggered detachments at different levels of tension.” First, the authors examined how Aurora B affects microtubule-kinetochore attachments in the absence of tension. Purified kinetochores bound to both stabilized and dynamically growing microtubules, and the addition of active Aurora B destabilized these interactions. Conversely, adding kinase-dead Aurora B, or adding active Aurora B to a mutant kinetochore that couldn’t be phosphorylated, failed to destabilize these interactions. These experiments indicated that phosphorylation of the kinetochore by Aurora B directly destabilizes these attachments. Next, they addressed the role of tension in this process by adding Aurora B to attachments placed under low or high tension using the optical trap. In these experiments, attachments under low tension were destabilized by Aurora B but attachments under high tension were not, revealing a direct role for tension in suppressing Aurora B-mediated detachment.

“This reconstitution avoids the complications that have confounded prior in vivo studies and provided the first direct experimental evidence that Aurora B-triggered detachment is directly suppressed when tension is applied to the kinetochore-microtubule interface,” explains Dr. Biggins. “The next question this paper raises is HOW tension can inhibit the outcome of the kinase.  We can think of two major possibilities.  One is that the substrate cannot thread into the active site [of Aurora B] when it is under tension due to a change in how it interacts with the microtubule when it is under tension…Alternatively, another interesting possibility is that the kinase still phosphorylates the substrate but that the phosphorylation is no longer able to result in detachment of the substrate from the microtubule.” Continuing work in this in vitro system is sure to reveal further insights into the nature of this complex and mysterious interaction.

aurora b model
(Top) Under low tension, Aurora B can access and phosphorylate sites on the kinetochore to destabilize microtubule-kinetochore attachments. (Bottom) Under high tension, Aurora B cannot access these sites, resulting in stabilized attachments. Image provided by Dr. Sue Biggins

This work was supported by the American Cancer Society, the David and Lucile Packard Foundation, the National Institutes of Health, and The Howard Hughes Medical Institute

Fred Hutch/UW Cancer Consortium members Dr. Sue Biggins and Dr. Chip Asbury contributed to this work.

de Regt AK, Clark CJ, Asbury CL, Biggins S. Tension can directly suppress Aurora B kinase-triggered release of kinetochore-microtubule attachments. Nat Commun. 2022 Apr 20;13(1):2152. doi: 10.1038/s41467-022-29542-8. PMID: 35443757; PMCID: PMC9021268.