Highway to hell-thy meiotic divisions: Chromosome passenger complex functions driven by microtubules

The chromosome passenger complex (CPC) localizes to chromosomes and microtubules, sometimes simultaneously. The CPC also has multiple domains for interacting with chromatin and microtubules. Interactions between the CPC and both the chromatin and microtubules is important for spindle assembly and error correction. Such dual chromatin-microtubule interactions may increase the concentration of the CPC necessary for efficient kinase activity while also making it responsive to specific conditions or structures in the cell. CPC-microtubule dependent functions are considered in the context of the first meiotic division. Acentrosomal spindle assembly is a process that depends on transfer of the CPC from the chromosomes to the microtubules. Furthermore, transfer to the microtubules is not only to position the CPC for a later role in cytokinesis; metaphase I error correction and subsequent bi-orientation of bivalents may depend on microtubule associated CPC interacting with the kinetochores.     

染色体移动复合物的结构、定位与功能

染色体移动复合物主要由蛋白激酶Aurora B、内层着丝粒蛋白、存活蛋白及蛋白Borealin组成。它在细胞分裂的不同阶段,能及时精确地定位到相关部位并作用于相应底物;具有调节染色质组蛋白磷酸化,控制姐妹染色单体的粘着、分离,参与分裂纺锤体组装及其对染色体的捕捉,纠正动粒与微管间不适当附着,将染色体精确分配到子细胞及促进胞浆分离等重要功能。文章简要介绍了染色体移动复合物的结构成分,在染色体臂部、内层着丝粒及纺锤体中区的定位过程,及其定位在不同部位的相应功能。     

RCC1 regulates inner centromeric composition in a Ran-independent fashion

RCC1 associates to chromatin dynamically within mitosis and catalyzes Ran-GTP production. Exogenous RCC1 disrupts kinetochore structure in Xenopus egg extracts (XEEs), but the molecular basis of this disruption remains unknown. We have investigated this question, utilizing replicated chromosomes that possess paired sister kinetochores. We find that exogenous RCC1 evicts a specific subset of inner KT proteins including Shugoshin-1 (Sgo1) and the chromosome passenger complex (CPC). We generated RCC1 mutants that separate its enzymatic activity and chromatin binding. Strikingly, Sgo1 and CPC eviction depended only on RCC1's chromatin affinity but not its capacity to produce Ran-GTP. RCC1 similarly released Sgo1 and CPC from synthetic kinetochores assembled on CENP-A nucleosome arrays. Together, our findings indicate RCC1 regulates kinetochores at the metaphase-anaphase transition through Ran-GTP-independent displacement of Sgo1 and CPC.     

A physical model for the condensation and decondensation of eukaryotic chromosomes

During the eukaryotic cell cycle, chromatin undergoes several conformational changes, which are believed to play key roles in gene expression regulation during interphase, and in genome replication and division during mitosis. In this paper, we propose a scenario for chromatin structural reorganization during mitosis, which bridges all the different scales involved in chromatin architecture, from nucleosomes to chromatin loops. We build a model for chromatin, based on available data, taking into account both physical and topological constraints DNA has to deal with. Our results suggest that the mitotic chromosome condensation/decondensation process is induced by a structural change at the level of the nucleosome itself.     

Condensin and Repo-Man-PP1 co-operate in the regulation of chromosome architecture during mitosis

The reversible condensation of chromosomes during cell division remains a classic problem in cell biology. Condensation requires the condensin complex in certain experimental systems, but not in many others. Anaphase chromosome segregation almost always fails in condensin-depleted cells, leading to the formation of prominent chromatin bridges and cytokinesis failure. Here, live-cell analysis of chicken DT40 cells bearing a conditional knockout of condensin subunit SMC2 revealed that condensin-depleted chromosomes abruptly lose their compact architecture during anaphase and form massive chromatin bridges. The compact chromosome structure can be preserved and anaphase chromosome segregation rescued by preventing the targeting subunit Repo-Man from recruiting protein phosphatase 1 (PP1) to chromatin at anaphase onset. This study identifies an activity critical for mitotic chromosome structure that is inactivated by Repo-Man-PP1 during anaphase. This activity, provisionally termed 'regulator of chromosome architecture' (RCA), cooperates with condensin to preserve the characteristic chromosome architecture during mitosis.     

Australin: a chromosomal passenger protein required specifically for Drosophila melanogaster male meiosis

The chromosomal passenger complex (CPC), which is composed of conserved proteins aurora B, inner centromere protein (INCENP), survivin, and Borealin/DASRA, localizes to chromatin, kinetochores, microtubules, and the cell cortex in a cell cycle-dependent manner. The CPC is required for multiple aspects of cell division. Here we find that Drosophila melanogaster encodes two Borealin paralogues, Borealin-related (Borr) and Australin (Aust). Although Borr is a passenger in all mitotic tissues studied, it is specifically replaced by Aust for the two male meiotic divisions. We analyzed aust mutant spermatocytes to assess the effects of fully inactivating the Aust-dependent functions of the CPC. Our results indicate that Aust is required for sister chromatid cohesion, recruitment of the CPC to kinetochores, and chromosome alignment and segregation but not for meiotic histone phosphorylation or spindle formation. Furthermore, we show that the CPC is required earlier in cytokinesis than previously thought; cells lacking Aust do not initiate central spindle formation, accumulate anillin or actin at the cell equator, or undergo equatorial constriction.     

HP1α targets the chromosomal passenger complex for activation at heterochromatin before mitotic entry

The chromosomal passenger complex (CPC) is directed to centromeres during mitosis via binding to H3T3ph and Sgo1. Whether and how heterochromatin protein 1α (HP1α) influences CPC localisation and function during mitotic entry is less clear. Here, we alter HP1α dynamics by fusing it to a CENP‐B DNA‐binding domain. Tethered HP1 strongly recruits the CPC, destabilising kinetochore–microtubule interactions and activating the spindle assembly checkpoint. During mitotic exit, the tethered HP1 traps active CPC at centromeres. These HP1‐CPC clusters remain catalytically active throughout the subsequent cell cycle. We also detect interactions between endogenous HP1 and the CPC during G₂. HP1α and HP1γ cooperate to recruit the CPC to active foci in a CDK1‐independent process. Live cell tracking with Fab fragments reveals that H3S10ph appears well before H3T3 is phosphorylated by Haspin kinase. Our results suggest that HP1 may concentrate and activate the CPC at centromeric heterochromatin in G₂ before Aurora B‐mediated phosphorylation of H3S10 releases HP1 from chromatin and allows pathways dependent on H3T3ph and Sgo1 to redirect the CPC to mitotic centromeres.     

Delaying the final cut: A close encounter of checkpoint kinases at the midbody

How chromatin bridges are relayed to the chromosomal passenger complex (CPC) during mammalian cell division is unknown. In this issue, Petsalaki and Zachos (2020. J. Cell Biol. https://doi.org/10.1083/jcb.202008029) show that the DNA damage checkpoint kinases ATM and Chk2 signal to the CPC to associate with a pool of cytoskeletal regulators, MKLP2–Cep55, in the midbody center and to delay abscission.

The birth of two daughter cells from one mother cell is the outcome of the intricate process of cell division. During cell division, the duplicated chromosomes are first equally separated and segregated in mitosis, after which cytoplasmic division or cytokinesis begins to physically separate two new daughter cells with identical copies of the genome. Cytokinesis starts in anaphase with processive ingression of the cell membrane at the cell equator (aka furrow ingression) caused by constriction of an actomyosin-based ring structure. It eventually ends in late telophase with abscission, the process during which the two daughter cells are pinched off from each other at a structure called the midbody. The midbody is part of a narrow, microtubule-filled intercellular canal that connects the two daughter cells until abscission and serves as a landing platform for the abscission machinery (Fig. 1; 1). To avoid chromosome damage by the act of cytokinesis (2, 3), cytokinesis onset and progression has to be coordinated with prior chromosome segregation to ensure that all chromatin is cleared from the area where the furrow ingresses and the intercellular canal is eventually formed. In line with this notion, the presence of unsegregated chromatin in the division plane delays the final phase of cytokinesis, and this abscission delay is referred to as the “abscission checkpoint.” Midbody-localized Aurora B kinase, the enzymatic subunit of the chromosomal passenger complex (CPC), is a central and evolutionary-conserved component of this checkpoint (3, 4). Over the years, several downstream targets of Aurora B have been identified that explain how Aurora B activity can delay the final cut (5). Interestingly, many of these Aurora B targets accumulate in a narrow, ring-shaped structure in the center of the midbody, known as the Flemming body, suggesting this specific part of the midbody might act as a signaling hub for the abscission checkpoint (5). Yet, it has remained elusive how an active pool of Aurora B is recruited to and retained in the Flemming body, particularly in response to missegregated chromatin in the division plane. In this issue, Petsalaki and Zachos shed new light on this mechanism (6).Open in a separate windowFigure 1.Checkpoint kinase encounter at the midbody. Cartoon of a cell undergoing cytokinesis while a chromatin bridge links the two daughter nuclei. The future daughter cells are connected by an intercellular canal through which the chromatin bridge passes. An enlargement of the intercellular canal is drawn to highlight the central part of the canal where the midbody is positioned. The antiparallel-oriented microtubules that form the midbody arms overlap in the very center of the midbody (indicated as a gap between the microtubule arms). A large number of proteins is recruited to the midbody during cytokinesis. These proteins either localize on the midbody arms (not indicated), the Flemming body (such as Cep55, indicated in red), or dynamically exchange between these locations. An example of the latter is the CPC, which localizes on the midbody arms in early telophase but also becomes detectable in the Flemming body in late telophase (12). In the present study, Petsalaki and Zachos reveal the mechanism of this Flemming body localization of the CPC. They find that MRN, ATM, and Chk2 are required to drive the association between S91-phosphorylated INCENP and Cep55-bound MKLP2 in late telophase and demonstrate that CPC recruitment to the Flemming body is essential to delay abscission. ABK, Aurora B kinase; S91ph, phosphorylated S91; N, N-terminus; C, C-terminus. Created with Biorender.com.Petsalaki and Zachos identified the DNA double-strand break (DSB) signaling kinase ataxia-telangiectasia mutated (ATM) and its downstream target checkpoint kinase 2 (Chk2) as regulators of Aurora B recruitment to the Flemming body. The researchers found that active ATM and Chk2 colocalize with Aurora B and its CPC interaction partner, INCENP, at Flemming bodies in late telophase in cancer cell lines. Inhibition of these DNA damage checkpoint kinases accelerated the onset of abscission, both in cells without or with chromatin bridges. In case of the latter, ATM or Chk2 inhibition caused the chromatin bridge to break, resulting in DNA damage.Interestingly, Petsalaki and Zachos identified serine 91 (S91) in INCENP as a substrate of Chk2 and found that this phosphorylation promoted the direct association of INCENP with the kinesin-6 motor protein MKLP2. While a more central part of MKLP2 is known to interact with INCENP (7, 8, 9), Petsalaki and Zachos found that the very C-terminal part of MKLP2 directly bound to Cep55, an established Flemming body protein and recruiter of abscission regulators (5). Deletion of the C-terminal part of MKLP2 specifically disrupted Flemming body localization of the CPC. Similarly, inhibition of Chk2 only affected INCENP-S91 phosphorylation in late Flemming bodies but not in other parts of the late midbody, suggesting that ATM and Chk2 act locally to mediate the binding between the CPC and Cep55-associated MKLP2 (Fig. 1). To further substantiate this, the authors engineered an INCENP fusion protein that directed INCENP and Aurora B specifically toward the Flemming body and not to any other part of the midbody. Expression of this fusion protein restored the abscission delay observed in Chk2-inhibited cells, implying that by recruiting Aurora B to the Flemming body, ATM and Chk2 ensure that a pool of active Aurora B is in close proximity of critical executors of abscission, thereby most likely allowing efficient inhibition of the process.ATM activity is required at DNA DSB sites to delay cell cycle progression and to facilitate DNA damage repair. ATM recruitment and activation at DSB sites is mediated by the Mre11–Rad50–Nbs1 (MRN) complex, which recognizes and processes the DNA damage site (10). Interestingly, in dividing cells with chromatin bridges, Petsalaki and Zachos found that the MRN proteins localized at late Flemming bodies and were required for ATM and Chk2 activation in, and for CPC recruitment to, these sites (Fig. 1). Similar to ATM and Chk2 inhibition, knockdown of MRN proteins accelerated abscission, causing chromatin bridges to break. The here-described involvement of DNA damage signaling kinases in activation of the abscission checkpoint is not without precedent. DNA lesions resulting from prior replication stress have been described to delay abscission in an Aurora B–dependent manner (11). However, instead of ATM and Chk2, the DNA damage signaling kinases ATR (ataxia-telangiectasia and Rad3–related) and Chk1 relayed the presence of these lesions in the nucleus to Aurora B at the midbody (11). The DNA lesions giving rise to the chromatin bridges in the Petsalaki and Zachos study most likely have a different origin, fueling the idea that, similar to DNA damage sensing and signaling in, for instance, S or G2 phase of the cell cycle, different types of DNA lesions are also detected by different sensors and DNA damage signaling kinases in cells undergoing cytokinesis. In the latter cells, however, these signaling cascades culminate in the retention of active Aurora B in the Flemming body, which inhibits the abscission machinery.As such, the findings of Petsalaki and Zachos raise the more specific question of how chromatin bridges promote the recruitment of MRN proteins toward the midbody center. It is tempting to speculate that the ring-shaped Flemming body through which the chromatin bridge likely passes causes some sort of DNA stress that is recognized by the MRN complex, which in turn activates the abscission checkpoint and promotes chromatin bridge resolution. It will be interesting to learn what eventually happens to chromatin bridges that recruit the MRN complex in cells with active ATM and Chk2. Is the bridge resolved before abscission proceeds, and if so, what kind of DNA repair mechanism would facilitate chromatin bridge resolution? The discovered role for the MRN–ATM–Chk2 pathway in the abscission checkpoint will guide future efforts in addressing these important questions.     

Progressive telomere dysfunction causes cytokinesis failure and leads to the accumulation of polyploid cells

Most cancer cells accumulate genomic abnormalities at a remarkably rapid rate, as they are unable to maintain their chromosome structure and number. Excessively short telomeres, a known source of chromosome instability, are observed in early human-cancer lesions. Besides telomere dysfunction, it has been suggested that a transient phase of polyploidization, in most cases tetraploidization, has a causative role in cancer. Proliferation of tetraploids can gradually generate subtetraploid lineages of unstable cells that might fire the carcinogenic process by promoting further aneuploidy and genomic instability. Given the significance of telomere dysfunction and tetraploidy in the early stages of carcinogenesis, we investigated whether there is a connection between these two important promoters of chromosomal instability. We report that human mammary epithelial cells exhibiting progressive telomere dysfunction, in a pRb deficient and wild-type p53 background, fail to complete the cytoplasmatic cell division due to the persistence of chromatin bridges in the midzone. Flow cytometry together with fluorescence in situ hybridization demonstrated an accumulation of binucleated polyploid cells upon serial passaging cells. Restoration of telomere function through hTERT transduction, which lessens the formation of anaphase bridges by recapping the chromosome ends, rescued the polyploid phenotype. Live-cell imaging revealed that these polyploid cells emerged after abortive cytokinesis due to the persistence of anaphase bridges with large intervening chromatin in the cleavage plane. In agreement with a primary role of anaphase bridge intermediates in the polyploidization process, treatment of HMEC-hTERT cells with bleomycin, which produces chromatin bridges through illegimitate repair, resulted in tetraploid binucleated cells. Taken together, we demonstrate that human epithelial cells exhibiting physiological telomere dysfunction engender tetraploid cells through interference of anaphase bridges with the completion of cytokinesis. These observations shed light on the mechanisms operating during the initial stages of human carcinogenesis, as they provide a link between progressive telomere dysfunction and tetraploidy.     

Chromatin Protein HP1α Interacts with the Mitotic Regulator Borealin Protein and Specifies the Centromere Localization of the Chromosomal Passenger Complex

Accurate mitosis requires the chromosomal passenger protein complex (CPC) containing Aurora B kinase, borealin, INCENP, and survivin, which orchestrates chromosome dynamics. However, the chromatin factors that specify the CPC to the centromere remain elusive. Here we show that borealin interacts directly with heterochromatin protein 1α (HP1α) and that this interaction is mediated by an evolutionarily conserved PXVXL motif in the C-terminal borealin with the chromo shadow domain of HP1α. This borealin-HP1α interaction recruits the CPC to the centromere and governs an activation of Aurora B kinase judged by phosphorylation of Ser-7 in CENP-A, a substrate of Aurora B. Consistently, modulation of the motif PXVXL leads to defects in CPC centromere targeting and aberrant Aurora B activity. On the other hand, the localization of the CPC in the midzone is independent of the borealin-HP1α interaction, demonstrating the spatial requirement of HP1α in CPC localization to the centromere. These findings reveal a previously unrecognized but direct link between HP1α and CPC localization in the centromere and illustrate the critical role of borealin-HP1α interaction in orchestrating an accurate cell division.     

The Chromosomal Passenger Complex Is Required for Meiotic Acentrosomal Spindle Assembly and Chromosome Biorientation

DURING meiosis in the females of many species, spindle assembly occurs in the absence of the microtubule-organizing centers called centrosomes. In the absence of centrosomes, the nature of the chromosome-based signal that recruits microtubules to promote spindle assembly as well as how spindle bipolarity is established and the chromosomes orient correctly toward the poles is not known. To address these questions, we focused on the chromosomal passenger complex (CPC). We have found that the CPC localizes in a ring around the meiotic chromosomes that is aligned with the axis of the spindle at all stages. Using new methods that dramatically increase the effectiveness of RNA interference in the germline, we show that the CPC interacts with Drosophila oocyte chromosomes and is required for the assembly of spindle microtubules. Furthermore, chromosome biorientation and the localization of the central spindle kinesin-6 protein Subito, which is required for spindle bipolarity, depend on the CPC components Aurora B and Incenp. Based on these data we propose that the ring of CPC around the chromosomes regulates multiple aspects of meiotic cell division including spindle assembly, the establishment of bipolarity, the recruitment of important spindle organization factors, and the biorientation of homologous chromosomes.     

CENP-V is required for centromere organization, chromosome alignment and cytokinesis

The mechanism of mitotic chromosome condensation is poorly understood, but even less is known about the mechanism of formation of the primary constriction, or centromere. A proteomic analysis of mitotic chromosome scaffolds led to the identification of CENP-V, a novel kinetochore protein related to a bacterial enzyme that detoxifies formaldehyde, a by-product of histone demethylation in eukaryotic cells. Overexpression of CENP-V leads to hypercondensation of pericentromeric heterochromatin, a phenotype that is abolished by mutations in the putative catalytic site. CENP-V depletion in HeLa cells leads to abnormal expansion of the primary constriction of mitotic chromosomes, mislocalization and destabilization of the chromosomal passenger complex (CPC) and alterations in the distribution of H3K9me3 in interphase nucleoplasm. CENP-V-depleted cells suffer defects in chromosome alignment in metaphase, lagging chromosomes in anaphase, failure of cytokinesis and rapid cell death. CENP-V provides a novel link between centromeric chromatin, the primary constriction and the CPC.     

Cell division control by the Chromosomal Passenger Complex

The Chromosomal Passenger Complex (CPC) consisting of Aurora B kinase, INCENP, Survivin and Borealin, is essential for genomic stability by controlling multiple processes during both nuclear and cytoplasmic division. In mitosis it ensures accurate segregation of the duplicated chromosomes by regulating the mitotic checkpoint, destabilizing incorrectly attached spindle microtubules and by promoting the axial shortening of chromosomal arms in anaphase. During cytokinesis the CPC most likely prevents chromosome damage by imposing an abscission delay when a chromosome bridge connects the two daughter cells. Moreover, by controlling proper cytoplasmic division, the CPC averts tetraploidization. This review describes recent insights on how the CPC is capable of conducting its various functions in the dividing cell to ensure chromosomal stability.     

Chromosomal enrichment and activation of the aurora B pathway are coupled to spatially regulate spindle assembly

Chromatin-induced spindle assembly depends on regulation of microtubule-depolymerizing proteins by the chromosomal passenger complex (CPC), consisting of Incenp, Survivin, Dasra (Borealin), and the kinase Aurora B, but the mechanism and significance of the spatial regulation of Aurora B activity remain unclear. Here, we show that the Aurora B pathway is suppressed in the cytoplasm of Xenopus egg extract by phosphatases, but that it becomes activated by chromatin via a Ran-independent mechanism. While spindle microtubule assembly normally requires Dasra-dependent chromatin binding of the CPC, this function of Dasra can be bypassed by clustering Aurora B-Incenp by using anti-Incenp antibodies, which stimulate autoactivation among bound complexes. However, such chromatin-independent Aurora B pathway activation promotes centrosomal microtubule assembly and produces aberrant achromosomal spindle-like structures. We propose that chromosomal enrichment of the CPC results in local kinase autoactivation, a mechanism that contributes to the spatial regulation of spindle assembly and possibly to other mitotic processes.     

Unequal cell division and chromatin differentiation in pollen grain cells

Pinus pollen grains, normally developing, were subjected to centrifugal force, low temperature and caffeine solution. In the former two treatments, daughter cells with some abnormal directions of division, abnormal volume and chromatin dispersion were induced in pollen grains treated. Regardless of the direction of division, of the two daughter cells produced by the unequal division, the larger one contained strongly dispersed chromatin and the smaller one weakly dispersed chromatin. In the two daughter cells produced by approximately equal division, the chromatin was dispersed strongly to a similar degree, and by halfway unequal division, chromatin in the larger cell was dispersed strongly and in the smaller one intermediately. Chromatin in bi-nucleate cells induced by caffeine treatment was dispersed strongly to an identical degree. It is suggested that for the occurrence of heteronomous chromatin configuration in natural pollen grains the unequal cell division was indispensable, although the axis of division didn't directly contribute. After both the treatments of centrifugation and low temperature during microspore and embryonal cell divisions, the affected daughter cells divided in terms of the certain fixed axis of division and chromatin dispersion, instead of exhibiting abnormal development.     

TopBP1/Dpb11 binds DNA anaphase bridges to prevent genome instability

DNA anaphase bridges are a potential source of genome instability that may lead to chromosome breakage or nondisjunction during mitosis. Two classes of anaphase bridges can be distinguished: DAPI-positive chromatin bridges and DAPI-negative ultrafine DNA bridges (UFBs). Here, we establish budding yeast Saccharomyces cerevisiae and the avian DT40 cell line as model systems for studying DNA anaphase bridges and show that TopBP1/Dpb11 plays an evolutionarily conserved role in their metabolism. Together with the single-stranded DNA binding protein RPA, TopBP1/Dpb11 binds to UFBs, and depletion of TopBP1/Dpb11 led to an accumulation of chromatin bridges. Importantly, the NoCut checkpoint that delays progression from anaphase to abscission in yeast was activated by both UFBs and chromatin bridges independently of Dpb11, and disruption of the NoCut checkpoint in Dpb11-depleted cells led to genome instability. In conclusion, we propose that TopBP1/Dpb11 prevents accumulation of anaphase bridges via stimulation of the Mec1/ATR kinase and suppression of homologous recombination.     

Spindle-to-cortex communication in cleaving,polyspermic Xenopus eggs

Mitotic spindles specify cleavage planes in early embryos by communicating their position and orientation to the cell cortex using microtubule asters that grow out from the spindle poles during anaphase. Chromatin also plays a poorly understood role. Polyspermic fertilization provides a natural experiment in which aster pairs from the same spindle (sister asters) have chromatin between them, whereas asters pairs from different spindles (nonsisters) do not. In frogs, only sister aster pairs induce furrows. We found that only sister asters recruited two conserved furrow-inducing signaling complexes, chromosome passenger complex (CPC) and Centralspindlin, to a plane between them. This explains why only sister pairs induce furrows. We then investigated factors that influenced CPC recruitment to microtubule bundles in intact eggs and a cytokinesis extract system. We found that microtubule stabilization, optimal starting distance between asters, and proximity to chromatin all favored CPC recruitment. We propose a model in which proximity to chromatin biases initial CPC recruitment to microtubule bundles between asters from the same spindle. Next a positive feedback between CPC recruitment and microtubule stabilization promotes lateral growth of a plane of CPC-positive microtubule bundles out to the cortex to position the furrow.