The Chromosomal Passenger Complex Is Required for Chromatin-Induced Microtubule Stabilization and Spindle Assembly

AbstractIn cells lacking centrosomes, such as those found in female meiosis, chromosomes must nucleate and stabilize microtubules in order to form a bipolar spindle. Here we report the identification of Dasra A and Dasra B, two new components of the vertebrate chromosomal passenger complex containing Incenp, Survivin, and the kinase Aurora B, and demonstrate that this complex is required for chromatin-induced microtubule stabilization and spindle formation. The failure of microtubule stabilization caused by depletion of the chromosomal passenger complex was rescued by codepletion of the microtubule-depolymerizing kinesin MCAK, whose activity is negatively regulated by Aurora B. By contrast, we present evidence that the Ran-GTP pathway of chromatin-induced microtubule nucleation does not require the chromosomal passenger complex, indicating that the mechanisms of microtubule assembly by these two pathways are distinct. We propose that the chromosomal passenger complex regulates local MCAK activity to permit spindle formation via stabilization of chromatin-associated microtubules

The chromosomal passenger complex and the spindle assembly checkpoint: kinetochore-microtubule error correction and beyond

During mitosis, correct bipolar chromosome attachment to the mitotic spindle is an essential prerequisite for the equal segregation of chromosomes. The spindle assembly checkpoint can prevent chromosome segregation as long as not all chromosome pairs have obtained bipolar attachment to the spindle. The chromosomal passenger complex plays a crucial role during chromosome alignment by correcting faulty chromosome-spindle interactions (e.g. attachments that do not generate tension). In the process of doing so, the chromosomal passenger complex generates unattached chromosomes, a specific situation that is known to promote checkpoint activity. However, several studies have implicated an additional, more direct role for the chromosomal passenger complex in enforcing the mitotic arrest imposed by the spindle assembly checkpoint. In this review, we discuss the different roles played by the chromosomal passenger complex in ensuring proper mitotic checkpoint function. Additionally, we discuss the possibility that besides monitoring the presence of unattached kinetochores, the spindle assembly checkpoint may also be capable of responding to chromosome-microtubule interactions that do not generate tension and we propose experimental set-ups to study this

Kinesins relocalize the chromosomal passenger complex to the midzone for spindle disassembly.

Mitotic spindle disassembly after chromosome separation is as important as spindle assembly, yet the molecular mechanisms for spindle disassembly are unclear. In this study, we investigated how the chromosomal passenger complex (CPC), which contains the Aurora B kinase Ipl1, swiftly concentrates at the spindle midzone in late anaphase, and we researched the role of this dramatic relocalization during spindle disassembly. We showed that the kinesins Kip1 and Kip3 are essential for CPC relocalization. In cells lacking Kip1 and Kip3, spindle disassembly is severely delayed until after contraction of the cytokinetic ring. Purified Kip1 and Kip3 interact directly with the CPC and recruit it to microtubules in vitro, and single-molecule experiments showed that the CPC diffuses dynamically on microtubules but that diffusion stops when the CPC encounters a Kip1 molecule. We propose that Kip1 and Kip3 trap the CPC at the spindle midzone in late anaphase to ensure timely spindle disassembly

The inner centromere is a biomolecular condensate scaffolded by the chromosomal passenger complex.

The inner centromere is a region on every mitotic chromosome that enables specific biochemical reactions that underlie properties, such as the maintenance of cohesion, the regulation of kinetochores and the assembly of specialized chromatin, that can resist microtubule pulling forces. The chromosomal passenger complex (CPC) is abundantly localized to the inner centromeres and it is unclear whether it is involved in non-kinase activities that contribute to the generation of these unique chromatin properties. We find that the borealin subunit of the CPC drives phase separation of the CPC in vitro at concentrations that are below those found on the inner centromere. We also provide strong evidence that the CPC exists in a phase-separated state at the inner centromere. CPC phase separation is required for its inner-centromere localization and function during mitosis. We suggest that the CPC combines phase separation, kinase and histone code-reading activities to enable the formation of a chromatin body with unique biochemical activities at the inner centromere

Regulation of Chromosome Segregation by the Chromosomal Passenger Complex

Life depends on faithful DNA segregation. The molecular underpinnings controlling this segregation are not fully understood. Here I describe the role of the chromosomal passenger complex (CPC) in the regulation of chromosome segregation, using Xenopus egg extracts. The CPC contains the kinase Aurora B, which is well known for its phosphorylation of serine 10 on histone H3 (H3S10). While this phosphorylation is a hallmark of M-phase, its functional significance is enigmatic. In the first part of my thesis, I, with my collaborators, provide a molecular function for this phosphorylation in the chromosomal dissociation of HP1 (heterochromatin protein 1) in M-phase. The phosphorylation of H3S10 by Aurora B ejects HP1 from its stably methylated histone H3 lysine 9 (H3K9) chromatin binding site, clearly demonstrating the existence of a “methyl/phos switch.†While the significance of this chromosomal HP1 removal is unclear, I propose that it functions in proper M-phase chromosome compaction. In eukaryotes, chromosome segregation depends on spindle formation. Chromosome-induced spindle assembly requires the chromosomal recruitment and activation of Aurora B. How this chromosome-activated kinase spatially disseminates signals that lead to spindle formation remains unclear. In the second part of my thesis, I show that CPC must detect chromosomes and microtubules to support spindle assembly. While the CPC is enriched on chromosomes in metaphase, I establish that a fraction of the CPC is targeted to the metaphase spindle. I demonstrate that this understudied metaphase CPCmicrotubule interaction is required to target chromosomally activated Aurora B to emerging microtubules near chromosomes for spindle assembly. I propose that the dual detection of chromosomes and microtubules by the CPC ensures that spindle formation is spatially limited to the vicinity of chromosomes. The spindle assembly checkpoint is vital for faithful chromosome segregation, as the checkpoint monitors the proper attachment of chromosomes to the spindle. In the third part of my thesis, based on preliminary data, I discuss the possibility that the dual detection of chromosome and microtubules may also function in the signaling of this checkpoint. Together, my work demonstrates three different functions of the CPC that impinge on chromosome segregation and genomic integrity

Borealin: a novel chromosomal passenger required for stability of the bipolar mitotic spindle

The chromosomal passenger complex of Aurora B kinase, INCENP, and Survivin has essential regulatory roles at centromeres and the central spindle in mitosis. Here, we describe Borealin, a novel member of the complex. Approximately half of Aurora B in mitotic cells is complexed with INCENP, Borealin, and Survivin; and Borealin binds Survivin and INCENP in vitro. A second complex contains Aurora B and INCENP, but no Borealin or Survivin. Depletion of Borealin by RNA interference delays mitotic progression and results in kinetochore–spindle misattachments and an increase in bipolar spindles associated with ectopic asters. The extra poles, which apparently form after chromosomes achieve a bipolar orientation, severely disrupt the partitioning of chromosomes in anaphase. Borealin depletion has little effect on histone H3 serine10 phosphorylation. These results implicate the chromosomal passenger holocomplex in the maintenance of spindle integrity and suggest that histone H3 serine10 phosphorylation is performed by an Aurora B–INCENP subcomplex

The chromosomal passenger complex: guiding Aurora-B through mitosis

During mitosis, the chromosomal passenger complex (CPC) orchestrates highly different processes, such as chromosome alignment, histone modification, and cytokinesis. Proper and timely localization of this complex is the key to precise control over the enzymatic core of the CPC, the Aurora-B kinase. We discuss the molecular mechanisms by which the CPC members direct the dynamic localization of the complex throughout cell division. Also, we summarize posttranslational modifications that occur on the CPC and discuss their roles in regulating localization and function of this mitotic complex

The Chromosomal Passenger Complex Activates Polo Kinase at Centromeres

The coordinated activities at centromeres of two key cell cycle kinases, Polo and Aurora B, are critical for ensuring that the two sister kinetochores of each chromosome are attached to microtubules from opposite spindle poles prior to chromosome segregation at anaphase. Initial attachments of chromosomes to the spindle involve random interactions between kinetochores and dynamic microtubules, and errors occur frequently during early stages of the process. The balance between microtubule binding and error correction (e.g., release of bound microtubules) requires the activities of Polo and Aurora B kinases, with Polo promoting stable attachments and Aurora B promoting detachment. Our study concerns the coordination of the activities of these two kinases in vivo. We show that INCENP, a key scaffolding subunit of the chromosomal passenger complex (CPC), which consists of Aurora B kinase, INCENP, Survivin, and Borealin/Dasra B, also interacts with Polo kinase in Drosophila cells. It was known that Aurora A/Bora activates Polo at centrosomes during late G2. However, the kinase that activates Polo on chromosomes for its critical functions at kinetochores was not known. We show here that Aurora B kinase phosphorylates Polo on its activation loop at the centromere in early mitosis. This phosphorylation requires both INCENP and Aurora B activity (but not Aurora A activity) and is critical for Polo function at kinetochores. Our results demonstrate clearly that Polo kinase is regulated differently at centrosomes and centromeres and suggest that INCENP acts as a platform for kinase crosstalk at the centromere. This crosstalk may enable Polo and Aurora B to achieve a balance wherein microtubule mis-attachments are corrected, but proper attachments are stabilized allowing proper chromosome segregation

Analysis of mitotic phosphorylation of Borealin

BACKGROUND: The main role of the chromosomal passenger complex is to ensure that Aurora B kinase is properly localized and activated before and during mitosis. Borealin, a member of the chromosomal passenger complex, shows increased expression during G2/M phases and is involved in targeting the complex to the centromere and the spindle midzone, where it ensures proper chromosome segregation and cytokinesis. Borealin has a consensus CDK1 phosphorylation site, threonine 106 and can be phosphorylated by Aurora B Kinase at serine 165 in vitro. RESULTS: Here, we show that Borealin is phosphorylated during mitosis in human cells. Dephosphorylation of Borealin occurs as cells exit mitosis. The phosphorylated form of Borealin is found in an INCENP-containing complex in mitosis. INCENP-containing complexes from cells in S phase are enriched in the phosphorylated form suggesting that phosphorylation may encourage entry of Borealin into the chromosomal passenger complex. Although Aurora B Kinase is found in complexes that contain Borealin, it is not required for the mitotic phosphorylation of Borealin. Mutation of T106 or S165 of Borealin to alanine does not alter the electrophoretic mobility shift of Borealin. Experiments with cyclohexamide and the phosphatase inhibitor sodium fluoride suggest that Borealin is phosphorylated by a protein kinase that can be active in interphase and mitosis and that the phosphorylation may be regulated by a short-lived phosphatase that is active in interphase but not mitosis. CONCLUSION: Borealin is phosphorylated during mitosis. Neither residue S165, T106 nor phosphorylation of Borealin by Aurora B Kinase is required to generate the mitotic, shifted form of Borealin. Suppression of phosphorylation during interphase is ensured by a labile protein, possibly a cell cycle regulated phosphatase

The Chromosomal Passenger Complex Activates Polo Kinase at Centromeres

INCENP acts as a protein scaffold that integrates the functions of two crucial mitotic kinases, Aurora B and Polo, at centromeres of mitotic chromosomes