Sensing Chromosome Bi-Orientation by Spatial Separation of Aurora B Kinase from Kinetochore Substrates

Abstract

Successful cell division requires that chromosomes attach to opposite poles of the mitotic spindle (bi-orientation). Aurora B kinase regulates chromosome-spindle attachments by phosphorylating kinetochore substrates that bind microtubules. Centromere tension stabilizes bi-oriented attachments, but how physical forces are translated into signaling at individual centromeres is unknown. Using FRET-based biosensors to measure localized phosphorylation dynamics in living cells, we found that phosphorylation of an Aurora B substrate at the kinetochore depended on its distance from the kinase at the inner centromere. Furthermore, repositioning Aurora B closer to the kinetochore prevented stabilization of bi-oriented attachments and activated the spindle checkpoint. Thus, centromere tension can be sensed by increased spatial separation of Aurora B from kinetochore substrates, which reduces phosphorylation and stabilizes kinetochore microtubules.Accurate chromosome segregation during cell division is essential to maintain genome integrity. Prior to segregation, kinetochores of sister chromatids attach to microtubules from opposite spindle poles (bi-orientation). This configuration is achieved through a trial-and-error process in which correct attachments exert tension across the centromere, which stabilizes kinetochore-microtubule interactions. Incorrect attachments, for example if both sister chromatids attach to a single spindle pole, exert less tension and are destabilized, providing a new opportunity to bi-orient (1,2). How tension is coupled to kinetochore-microtubule stability is not known.The mitotic kinase Aurora B (Ipl1 in budding yeast) localizes to the inner centromere, between sister kinetochores, and destabilizes microtubule attachments by phosphorylating kinetochore substrates, including Dam1 and the Ndc80 complex (3-10). An appealing model is that Aurora B substrates are selectively phosphorylated at incorrect attachments. To test this model we first examined phosphorylation of CENP-A Ser-7, a known kinetochore substrate (11). We used an assay in which Aurora B inhibition leads to a high frequency of syntelic attachment errors, with sister chromatids connected to a single spindle pole (6) (Fig. S1A). We compared phospho-CENP-A staining at correct and incorrect attachments 10 min after removing the reversible Aurora B kinase inhibitor ZM447439 (12), which re-activates Aurora B. Phospho-** Publisher's Disclaimer: This manuscript has been accepted for publication in Science. This version has not undergone final editing.Please refer to the complete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the

Temporal changes in Hec1 phosphorylation control kinetochore–microtubule attachment stability during mitosis

“…Aurora B localizes prominently to the centromere region of mitotic chromosomes and it has been suggested that a gradient of Aurora B kinase originating from the centromere can only 'reach' outer kinetochore substrates such as Hec1 when sister kinetochores are under low tension (Tanaka et al, 2002;Andrews et al, 2004;Pinsky et al, 2006). In support of this, a recent study found that phosphorylation of an engineered Aurora B substrate at the kinetochore was dependent on its distance from the centromere and, furthermore, that experimentally positioning Aurora B closer to the outer kinetochore prevented stabilization of bi-oriented kinetochore-MT attachments (Liu et al, 2009). In regard to Aurora B phosphorylation of Hec1 specifically, our results are not fully consistent with a spatial positioning model for two reasons.…”

“…Under this experimental condition, active Aurora B kinase remains at the outer kinetochore, indicating that although the kinase is spatially positioned to rephosphorylate Hec1, it does not. Because it is probable that other outer kinetochore targets are regulated by spatial positioning of the kinase (Liu et al, 2009;Liu et al, 2010;Welburn et al, 2010), the mechanism controlling Aurora-Bdependent phosphorylation cycles might differ between kinetochore targets.…”

“…It has been proposed that phosphorylation of outer kinetochore targets by Aurora B is regulated by spatial positioning of the kinase, which is enriched at the centromere of mitotic chromosomes (Tanaka et al, 2002;Andrews et al, 2004;Liu et al, 2009). In such a model, under conditions of low centromere tension (prior to chromosome bi-orientation), outer kinetochore targets of Aurora B are proximal to centromeric Aurora B kinase, allowing for interaction and phosphorylation.…”

Dynein prevents erroneous kinetochore-microtubule attachments in mitosis

“…[36][37][38] Once chromosomes become bi-oriented and tension is applied between sister kinetochores, Ndc80 moves away from Aurora B, resulting in kinetochoremicrotubule attachment stabilization. 39,40 Here we elaborate on recent studies that demonstrated a role of Dynein, 5 chromokinesins 5,41 and Aurora A kinase 5,41,42 in the prevention and correction of erroneous kinetochore-microtubule attachments during chromosome congression.…”

Cyclin B3 controls anaphase onset independent of spindle assembly checkpoint in meiotic oocytes

“…Aurora B kinase and Mps1 are at the top of this pathway and they are thought to regulate each other, [20][21][22][23] the downstream components Bub1, BubR1, Bub3, Mad1 and Mad2 form 3 complexes: Bub1-Bub3, BubR1-Bub3 and Mad1-Mad2. 24 They are recruited to kinetochores in a KMN-dependent manner, [25][26][27] and when the kinetochore is unattached, the checkpoint is activated.…”