Perturbation of Incenp function impedes anaphase chromatid movements and chromosomal passenger protein flux at centromeres.
ABSTRACT: Incenp is an essential mitotic protein that, together with Aurora B, Survivin, and Borealin, forms the core of the chromosomal passenger protein complex (CPC). The CPC regulates various mitotic processes and functions to maintain genomic stability. The proper subcellular localization of the CPC and its full catalytic activity require the presence of each core subunit in the complex. We have investigated the mitotic tasks of the CPC using a function blocking antibody against Incenp microinjected into cells at different mitotic phases. This method allowed temporal analysis of CPC functions without perturbation of complex assembly or activity prior to injection. We have also studied the dynamic properties of Incenp and Aurora B using fusion protein photobleaching. We found that in early mitotic cells, Incenp and Aurora B exhibit dynamic turnover at centromeres, which is prevented by the anti-Incenp antibody. In these cells, the loss of centromeric CPC turnover is accompanied by forced mitotic exit without the execution of cytokinesis. Introduction of anti-Incenp antibody into early anaphase cells causes abnormalities in sister chromatid separation through defects in anaphase spindle functions. In summary, our data uncovers new mitotic roles for the CPC in anaphase and proposes that CPC turnover at centromeres modulates spindle assembly checkpoint signaling.
Project description:A single inner centromere protein (INCENP) found throughout eukaryotes modulates Aurora B kinase activity and chromosomal passenger complex (CPC) localization, which is essential for timely mitotic progression. It has been proposed that INCENP might act as a rheostat to regulate Aurora B activity through mitosis, with successively higher activity threshold levels for chromosome alignment, the spindle checkpoint, anaphase spindle transfer and finally spindle elongation and cytokinesis. It remains mechanistically unclear how this would be achieved. Here, we reveal that the urochordate, <i>Oikopleura dioica</i>, possesses two INCENP paralogs, which display distinct localizations and subfunctionalization in order to complete M-phase. INCENPa was localized on chromosome arms and centromeres by prometaphase, and modulated Aurora B activity to mediate H3S10/S28 phosphorylation, chromosome condensation, spindle assembly and transfer of the CPC to the central spindle. Polo-like kinase (Plk1) recruitment to CDK1 phosphorylated INCENPa was crucial for INCENPa-Aurora B enrichment on centromeres. The second paralog, INCENPb was enriched on centromeres from prometaphase, and relocated to the central spindle at anaphase onset. In the absence of INCENPa, meiotic spindles failed to form, and homologous chromosomes did not segregate. INCENPb was not required for early to mid M-phase events but became essential for the activity and localization of Aurora B on the central spindle and midbody during cytokinesis in order to allow abscission to occur. Together, our results demonstrate that INCENP paralog switching on centromeres modulates Aurora B kinase localization, thus chronologically regulating CPC functions during fast embryonic divisions in the urochordate <i>O. dioica</i>. <b>Abbreviations:</b> CCAN: constitutive centromere-associated network; CENPs: centromere proteins; cmRNA: capped messenger RNA; CPC: chromosomal passenger complex; INCENP: inner centromere protein; Plk1: polo-like kinase 1; PP1: protein phosphatase 1; PP2A: protein phosphatase 2A; SAC: spindle assembly checkpoint; SAH: single ?-helix domain.
Project description:The mitotic checkpoint, also known as the spindle assembly checkpoint, delays anaphase onset until all chromosomes have reached bipolar tension on the mitotic spindle [1-3]. Once this is achieved, the protease separase is activated to cleave the chromosomal cohesin complex, thereby triggering anaphase. Cohesin cleavage releases tension between sister chromatids, but why the mitotic checkpoint now remains silent is poorly understood. Here, using budding yeast as a model, we show that loss of sister chromatid cohesion at anaphase onset would engage the mitotic checkpoint if this was not prevented by concomitant separase-dependent activation of the Cdc14 phosphatase. Cdc14, in turn, inactivates the mitotic checkpoint by dephosphorylating Sli15(INCENP), a subunit of the conserved Aurora B kinase complex that forms part of the proposed chromosomal tension sensor. Dephosphorylation-dependent relocation of Sli15(INCENP) from centromeres to the central spindle during anaphase is seen in organisms from yeast to human [4-8]. Our results suggest that Sli15(INCENP) dephosphorylation is part of an evolutionarily conserved mechanism that prevents the mitotic checkpoint from reengaging when tension between sister chromatids is lost at anaphase onset.
Project description:Cell division is orchestrated by a complex protein network that aims to maintenance of genomic stability. Visualisation of mitotic protein-protein associations in space and time has been limited due to the lack of proper biochemical and easy-to-use imaging tools. Here we report adaptation of the in situ proximity ligation assay (is-PLA) to study mitotic protein interactions with spatio-temporal resolution. We examined the composition of the Chromosomal Passenger Complex (CPC) at various mitotic phases and after chemical treatments using is-PLA with antibodies against the core CPC subunits Aurora B, INCENP, Survivin and Borealin. Our results support the notion that the core CPC functions as a single structural unit at centromeres in early mitosis and at central spindle after the onset of anaphase. Treatment of cells with the Aurora B inhibitor ZM447439 diminished the is-PLA signals at centromeres suggesting that Aurora B activity contributes to structural maintenance and/or proper subcellular localization of the core CPC. Is-PLA-based analysis of interaction between INCENP and Polo-like kinase 1 (Plk1) proposes that the kinase co-travels with CPC during late mitosis. The data illustrates both the strengths and limitations of the is-PLA in the analysis of mitotic macromolecule associations at sub-organelle level.
Project description:The function of the Aurora B kinase at centromeres and the central spindle is crucial for chromosome segregation and cytokinesis, respectively. Herein, we have investigated the regulation of human Aurora B by its complex partners inner centromere protein (INCENP) and survivin. We found that overexpression of a catalytically inactive, dominant-negative mutant of Aurora B impaired the localization of the entire Aurora B/INCENP/survivin complex to centromeres and the central spindle and severely disturbed mitotic progression. Similar results were also observed after depletion, by RNA interference, of either Aurora B, INCENP, or survivin. These data suggest that Aurora B kinase activity and the formation of the Aurora B/INCENP/survivin complex both contribute to its proper localization. Using recombinant proteins, we found that Aurora B kinase activity was stimulated by INCENP and that the C-terminal region of INCENP was sufficient for activation. Under identical assay conditions, survivin did not detectably influence kinase activity. Human INCENP was a substrate of Aurora B and mass spectrometry identified three consecutive residues (threonine 893, serine 894, and serine 895) containing at least two phosphorylation sites. A nonphosphorylatable mutant (TSS893-895AAA) was a poor activator of Aurora B, demonstrating that INCENP phosphorylation is important for kinase activation.
Project description:The chromosomal passenger complex (CPC) is a master regulator of mitosis. CPC consists of inner centromere protein (INCENP), Survivin, Borealin, and the kinase Aurora B and plays key roles in regulating kinetochore-microtubule attachments and spindle assembly checkpoint signaling. However, the role of CPC in sister chromatid cohesion, mediated by the cohesin complex, remains incompletely understood. Here, we show that Aurora B kinase activity contributes to centromeric cohesion protection partly through promoting kinetochore localization of the kinase Bub1. Interestingly, disrupting the interaction of INCENP with heterochromatin protein 1 (HP1) in HeLa cells selectively weakens cohesion at mitotic centromeres without detectably reducing the kinase activity of Aurora B. Thus, through this INCENP-HP1 interaction, the CPC also protects centromeric cohesion independently of Aurora B kinase activity. Moreover, the requirement for the INCENP-HP1 interaction in centromeric cohesion protection can be bypassed by tethering HP1 to centromeres or by depleting the cohesin release factor Wapl. We provide further evidence suggesting that the INCENP-HP1 interaction protects centromeric cohesion by promoting the centromere localization of Haspin, a protein kinase that antagonizes Wapl activity at centromeres. Taken together, this study identifies Aurora B kinase activity-dependent and -independent roles for the CPC in regulating centromeric cohesion during mitosis in human cells.
Project description: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.
Project description:We have performed a biochemical and double-stranded RNA-mediated interference (RNAi) analysis of the role of two chromosomal passenger proteins, inner centromere protein (INCENP) and aurora B kinase, in cultured cells of Drosophila melanogaster. INCENP and aurora B function is tightly interlinked. The two proteins bind to each other in vitro, and DmINCENP is required for DmAurora B to localize properly in mitosis and function as a histone H3 kinase. DmAurora B is required for DmINCENP accumulation at centromeres and transfer to the spindle at anaphase. RNAi for either protein dramatically inhibited the ability of cells to achieve a normal metaphase chromosome alignment. Cells were not blocked in mitosis, however, and entered an aberrant anaphase characterized by defects in sister kinetochore disjunction and the presence of large amounts of amorphous lagging chromatin. Anaphase A chromosome movement appeared to be normal, however cytokinesis often failed. DmINCENP and DmAurora B are not required for the correct localization of the kinesin-like protein Pavarotti (ZEN-4/CHO1/MKLP1) to the midbody at telophase. These experiments reveal that INCENP is required for aurora B kinase function and confirm that the chromosomal passengers have essential roles in mitosis.
Project description:The chromosomal passenger complex (CPC), composed of inner centromere protein (INCENP), Survivin, Borealin, and the kinase Aurora B, contributes to the activation of the mitotic checkpoint. The regulation of CPC function remains unclear. Here, we reveal that in addition to Survivin and Borealin, the single ?-helix (SAH) domain of INCENP supports CPC localization to chromatin and the mitotic checkpoint. The INCENP SAH domain also mediates INCENP's microtubule binding, which is negatively regulated by Cyclin-dependent kinase-mediated phosphorylation of segments flanking the SAH domain. The microtubule-binding capacity of the SAH domain is important for mitotic arrest in conditions of suppressed microtubule dynamics, and the duration of mitotic arrest dictates the probability, but not the timing, of cell death. Although independent targeting of INCENP to microtubules or the kinetochore/centromere promotes the mitotic checkpoint, it is insufficient for a robust mitotic arrest. Altogether, our results demonstrate that dual recognition of chromatin and microtubules by CPC is important for checkpoint maintenance and determination of cell fate in mitosis.
Project description:The chromosomal passenger complex (CPC) is a critical regulator of chromosome segregation during mitosis by correcting nonbipolar microtubule-kinetochore interactions. By severing these interactions, the CPC is thought to create unattached kinetochores that are subsequently sensed by the spindle assembly checkpoint (SAC) to prevent premature mitotic exit. We now show that spindle checkpoint function of the CPC and its role in eliminating nonbipolar attachments can be uncoupled. Replacing the chromosomal passenger protein INCENP with a mutant allele that lacks its coiled-coil domain results in an overt defect in a SAC-mediated mitotic arrest in response to taxol treatment, indicating that this domain is critical for CPC function in spindle checkpoint control. Surprisingly, this mutant could restore alignment and cytokinesis during unperturbed cell divisions and was capable of resolving syntelic attachments. Also, Aurora-B kinase was localized and activated normally on centromeres in these cells, ruling out a role for the coiled-coil domain in general Aurora-B activation. Thus, mere microtubule destabilization of nonbipolar attachments by the CPC is insufficient to install a checkpoint-dependent mitotic arrest, and additional, microtubule destabilization-independent CPC signaling toward the spindle assembly checkpoint is required for this arrest, potentially through amplification of the unattached kinetochore-derived checkpoint signal.
Project description: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.