Chromosomal Passenger Research Articles

U3 snoRNA inter-regulates with DDX21 in the perichromosomal region to control mitosis

U3 snoRNA is essential for ribosome biogenesis during interphase. Upon mitotic onset, the nucleolus disassembles and U3 snoRNA relocates to the perichromosomal region (PR) to be considered as a chromosome passenger. Whether U3 controls mitosis remains unknown. Here, we demonstrate that U3 snoRNA is required for mitotic progression. We identified DDX21 as the predominant U3-binding protein during mitosis and confirmed that U3 snoRNA colocalizes with DDX21 in the PR. DDX21 knockdown induces mitotic catastrophe and similar mitotic defects caused by U3 snoRNA depletion. Interestingly, the uniform PR distribution of U3 snoRNA and DDX21 is interdependent. DDX21 functions in mitosis depending on its PR localization. Mechanistically, U3 snoRNA regulates DDX21 PR localization through maintaining its mobility. Moreover, Cy5-U3 snoRNA downsizes the fibrous condensates of His-DDX21 at proper molecular ratios in vitro. This work highlights the importance of the equilibrium between U3 snoRNA and DDX21 in PR formation and reveals the potential relationship between the PR assembly and mitotic regulation.

Dysregulation of mitotic machinery genes precedes genome instability during spontaneous pre-malignant transformation of mouse ovarian surface epithelial cells.

BackgroundBased in epidemiological evidence, repetitive ovulation has been proposed to play a role in the origin of ovarian cancer by inducing an aberrant wound rupture-repair process of the ovarian surface epithelium (OSE). Accordingly, long term cultures of isolated OSE cells undergo in vitro spontaneous transformation thus developing tumorigenic capacity upon extensive subcultivation. In this work, C57BL/6 mouse OSE (MOSE) cells were cultured up to passage 28 and their RNA and DNA copy number profiles obtained at passages 2, 5, 7, 10, 14, 18, 23, 25 and 28 by means of DNA microarrays. Gene ontology, pathway and network analyses were focused in passages earlier than 20, which is a hallmark of malignancy in this model.ResultsAt passage 14, 101 genes were up-regulated in absence of significant DNA copy number changes. Among these, the top-3 enriched functions (>30 fold, adj p < 0.05) comprised 7 genes coding for centralspindlin, chromosome passenger and minichromosome maintenance protein complexes. The genes Ccnb1 (Cyclin B1), Birc5 (Survivin), Nusap1 and Kif23 were the most recurrent in over a dozen GO terms related to the mitotic process. On the other hand, Pten plus the large non-coding RNAs Malat1 and Neat1 were among the 80 down-regulated genes with mRNA processing, nuclear bodies, ER-stress response and tumor suppression as relevant terms. Interestingly, the earliest discrete segmental aneuploidies arose by passage 18 in chromosomes 7, 10, 11, 13, 15, 17 and 19. By passage 23, when MOSE cells express the malignant phenotype, the dysregulated gene expression repertoire expanded, DNA imbalances enlarged in size and covered additional loci.ConclusionPrior to early aneuploidies, overexpression of genes coding for the mitotic apparatus in passage-14 pre-malignant MOSE cells indicate an increased proliferation rate suggestive of replicative stress. Concomitant down-regulation of nuclear bodies and RNA processing related genes suggests altered control of nuclear RNA maturation, features recently linked to impaired DNA damage response leading to genome instability. These results, combined with cytogenetic analysis by other authors in this model, suggest that transcriptional profile at passage 14 might induce cytokinesis failure by which tetraploid cells approach a near-tetraploid stage containing primary chromosome aberrations that initiate the tumorigenic drive.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3068-5) contains supplementary material, which is available to authorized users.

Centrosome‐, chromosomal‐passenger‐ and cell‐cycle‐associated mRNAs are differentially regulated in the development of sporadic colorectal cancer

Dysregulation of the centrosome complex and chromosomal segregation has been associated with aneuploid cells and aggressive solid tumours, but the relevance of this mechanism to the adenoma-carcinoma sequence of sporadic colorectal cancer (sCRC), especially tumours showing chromosomal instability (CIN), is still unknown. In a series of matching normal epithelial cells (n = 41), dysplastic cells (n = 18), and invasive carcinoma cells (n = 41) from cases with sCRC, mRNA levels of the centrosomal kinase Aurora-A/STK15 and the chromosomal passenger- and cell cycle-associated molecules Incenp, Survivin, Mad-2, and Cyclin-D1 were therefore measured with specific reference to the type of genetic instability. Compared with normal epithelium, significant up-regulation of mRNAs was already present for Aurora-A/STK15 (p = 0.0313) in dysplastic cells and for all investigated markers in invasive carcinoma. Whereas Aurora-A/STK15 mRNA levels were similarly up-regulated in dysplastic and invasive carcinoma cells (p = 0.0797), Survivin (p = 0.0046) and Cyclin-D1 (p = 0.0017) mRNA levels increased from dysplastic to invasive carcinoma cells. In carcinomas, Incenp mRNA correlated with T category (p = 0.0149), and Survivin (p = 0.0382) and Cyclin-D1 (p = 0.0185) were associated with tumour differentiation. Importantly, a significantly higher (p = 0.0419) fold-change of Aurora-A/STK15 mRNA (p = 0.0419), but not Incenp, Survivin, Mad-2 or Cyclin-D1, was observed in sCRC cases with CIN (n = 29) when compared with tumours showing microsatellite instability (MIN, n = 10). The present data are the first to show an early increase of the centrosomal kinase Aurora-A/STK15 in the adenoma-carcinoma sequence of sCRC. The regulation of this kinase differs in CIN- and MIN-type sCRCs and the pattern of changes is different from those of the cell-cycle-associated markers Survivin, Mad-2, and Cyclin-D1. This reinforces the concept of preferential dysregulation of the centrosome complex in CIN-type (aneuploid), compared with MIN-type, sporadic colorectal cancers and may influence the response to and efficiency of novel therapeutics targeting Aurora kinases.

ASCR-007 ESPRG3: a novel chromosomal passenger taking part in ES self-renewal and spermatogenesis

ASCR-007 ESPRG3: a novel chromosomal passenger taking part in ES self-renewal and spermatogenesis

Phosphorylation of the Chromosomal Passenger Protein Bir1 Is Required for Localization of Ndc10 to the Spindle during Anaphase and Full Spindle Elongation

The Saccharomyces cerevisiae inhibitor of apoptosis (IAP) repeat protein Bir1 localizes as a chromosomal passenger. A deletion analysis of Bir1 identified two regions important for function. The C-terminal region is essential for growth, binds Sli15, and is necessary and sufficient for the localization of Bir1 as a chromosomal passenger. The middle region is not essential but is required to localize the inner kinetochore protein Ndc10 to the spindle during anaphase and to the midzone at telophase. In contrast, precise deletion of the highly conserved IAP repeats conferred no phenotype and did not alter the cell cycle delay caused by loss of cohesin. Bir1 is phosphorylated in a cell cycle-dependent manner. Mutation of all nine CDK consensus sites in the middle region of Bir1 significantly decreased the level of phosphorylation and blocked localization of Ndc10 to the spindle at anaphase. Moreover, immunoprecipitation of Ndc10 with Bir1 was dependent on phosphorylation. The loss of Ndc10 from the anaphase spindle prevented elongation of the spindle beyond 7 microm. We conclude that phosphorylation of the middle region of Bir1 is required to bring Ndc10 to the spindle at anaphase, which is required for full spindle elongation.

The C. elegans Tousled-like Kinase Contributes to Chromosome Segregation as a Substrate and Regulator of the Aurora B Kinase

The Aurora kinases control multiple aspects of mitosis, among them centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis. Aurora activity is regulated in part by a subset of Aurora substrates that, once phosphorylated, can enhance Aurora kinase activity. Aurora A substrate activators include TPX2 and Ajuba, whereas the only known Aurora B substrate activator is the chromosomal passenger INCENP. We report that the C. elegans Tousled kinase TLK-1 is a second substrate activator of the Aurora B kinase AIR-2. Tousled kinase (Tlk) expression and activity have been linked to ongoing DNA replication, and Tlk can phosphorylate the chromatin assembly factor Asf. Here, we show that TLK-1 is phosphorylated by AIR-2 during prophase/prometaphase and that phosphorylation increases TLK-1 kinase activity in vitro. Phosphorylated TLK-1 increases AIR-2 kinase activity in a manner that is independent of TLK-1 kinase activity but depends on the presence of ICP-1/INCENP. In vivo, TLK-1 and AIR-2 cooperate to ensure proper mitotic chromosome segregation. The C. elegans Tousled kinase TLK-1 is a substrate and activator of the Aurora B kinase AIR-2. These results suggest that Tousled kinases have a previously unrecognized role in mitosis and that Aurora B associates with discrete regulatory complexes that may impart distinct substrate specificities and functions to the Aurora B kinase.

Aurora-B Phosphorylation in Vitro Identifies a Residue of Survivin That Is Essential for Its Localization and Binding to Inner Centromere Protein (INCENP) in Vivo

The chromosomal passengers, aurora-B kinase, inner centromere protein (INCENP), and survivin, are essential proteins that have been implicated in the regulation of metaphase chromosome alignment, spindle checkpoint function, and cytokinesis. All three share a common pattern of localization, and it was recently demonstrated that aurora-B, INCENP, and survivin are present in a complex in Xenopus eggs and Saccharomyces cerevisiae. The presence of aurora-B kinase in the complex and its ability to bind the other components directly suggest that INCENP and survivin could potentially be aurora-B substrates. This hypothesis was recently proven for INCENP in vitro. Here we report that human survivin is specifically phosphorylated in vitro by aurora-B kinase at threonine 117 in its carboxyl alpha-helical coil. Mutation of threonine 117 to alanine prevents survivin phosphorylation by aurora-B in vitro but does not alter its localization in HeLa cells. By contrast, a phospho-mimic, in which threonine 117 was mutated to glutamic acid, was unable to localize correctly at any stage in mitosis. Mutation at threonine 117 also prevented immunoprecipitation of INCENP with survivin in vivo. These data suggest that phosphorylation of survivin at threonine 117 by aurora-B may regulate targeting of survivin, and possibly the entire passenger complex, in mammals.

Chromosomal passengers

What are they? Chromosomal passengers are proteins associated with chromosomes during the early stages of mitosis, then with the spindle midzone and equatorial cortex during anaphase and telophase, and finally with the midbody/spindle remnant at the exit from mitosis. It was this unusual dynamic behaviour that first brought INCENP to notice in 1985. Also known as… INCENP, Aurora-B, BIR1/survivin and TD-60. Aliases for INCENP include Sli15p in budding yeast and ICP-1 in C. elegans. Aurora-B is also known in mammals as AIM-1 and Aurora-1, in budding yeast as Ipl1p and as AIR-2 in C. elegans. Survivin is known as BIR1 in both yeasts and C. elegans, and as Deterin in Drosophila.Fig. 1 Most mysterious… TD-60 is a passenger protein, but next to nothing is known about its function. Its localization has been described as a ‘telophase disk’, a distribution seen for all passengers during anaphase/telophase. Whether the disk is a discrete structure or simply a manifestation of the spindle midzone remains a matter of some debate. Not to be confused with… CENP-E, chromokinesin, Polo kinase... A number of proteins are localised a bit like chromosomal passengers. However these proteins occupy slightly different locations on the chromosomes: the kinetochore for CENP-E and Polo; all along the chromosome arms for chromokinesin. Some of these proteins may interact functionally or physically with the passenger proteins. Strangest behaviour… Canonical passenger proteins accumulate in the inner centromeres during metaphase prior to transferring to the spindle midzone at anaphase. If chromosomal passengers do not first concentrate at centromeres, it seems they are not able to move on to the central spindle and midbody later in mitosis. No one knows what is so special about the inner centromere. Who needs them? Chromosomal passengers are essential in eukaryotes from yeast to mouse. INCENP and BIR1/survivin have been knocked out in mouse and budding yeast: result-dead mice and dead yeast. RNAi has shown that INCENP, Aurora-B and Survivin are essential in C. elegans and that the first two are also essential in Drosophila cultured cells. What do they do? The active ingredient in the passenger complex is probably Aurora-B, which apparently phosphorylates a number of important substrates required for chromosome structure, kinetochore function, chromosome bi-orientation at metaphase, and the completion of cytokinesis. INCENP is required for the correct targeting of Aurora-B at all times in mitosis and for Survivin movement to the centromere and all of its spindle and midbody locations late in mitosis. BIR1/Survivin targets Aurora-B during mitosis in C. elegans. Most controversial… BIR1/Survivin seems to love to confound researchers. BIR1/Survivin looks like an inhibitor of apoptosis protein, and dominant-negative mutants can kill cells like nobody's business. In fact, a number of clear and well controlled studies seem to suggest that Survivin may help prevent mitotic cells from apoptosis. However, both yeast and mouse knockouts show pretty convincingly that BIR1/Survivin mutants die, not because of programmed cell death, but because they make a hash of chromosome segregation or cytokinesis. The jury is still out on this one, but at the moment the apoptosis camp looks like they have the most to prove. Where can I find out more BIR1/Survivin-GFP (green) localised to the spindle midzone at anaphase. Image courtesy of Dr. Sally P. Wheatley.

INCENP binds the Aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow.

Cytoskeletal rearrangements during mitosis must be co-ordinated with chromosome movements. The 'chromosomal passenger' proteins [1], which include the inner centromere protein (INCENP [2]), the Aurora-related serine-threonine protein kinase AIRK2 [3,4] and the unidentified human autoantigen TD-60 [5], have been suggested to integrate mitotic events. These proteins are chromosomal until metaphase but subsequently transfer to the midzone microtubule array and the equatorial cortex during anaphase. Disruption of INCENP function affects both chromosome segregation and completion of cytokinesis [6,7], whereas interference with AIRK2 function primarily affects cytokinesis [3,8]. Here, we report that INCENP is stockpiled in Xenopus eggs in a complex with Xenopus AIRK2 (XAIRK2), and that INCENP and AIRK2 kinase bind one another in vitro. This association was found to be evolutionarily conserved. Sli15p, the binding partner of yeast Aurora kinase Ipl1p, can be recognized as an INCENP family member because of the presence of a conserved carboxy-terminal sequence region, which we term the IN box. This interaction between INCENP and Aurora kinase was found to be biologically relevant. INCENP and AIRK2 colocalized exactly in human cells, and INCENP was required to target AIRK2 correctly to centromeres and the central spindle.

Autoantibodies to a novel cell cycle-regulated protein that accumulates in the nuclear matrix during S phase and is localized in the kinetochores and spindle midzone during mitosis

We have employed human autoantibodies to characterize a novel cell cycle-regulated nuclear protein, provisionally designated p330d (doublet polypeptide of 330 kDa). The expression and intracellular distribution of this protein was followed throughout the cell cycle using immunofluorescence microscopy, laser confocal microscopy, immunoelectron microscopy and flow cytometry. p330d was expressed only in proliferating cells and began accumulating in the nucleus during early S phase. The protein reached maximum expression levels during G2/M. In situ extractions with detergent, salt and nucleases failed to abolish the nuclear staining of interphase cells, suggesting a tight binding of p330d to the nuclear matrix during interphase. p330d was concentrated in the kinetochores during prophase but was relocated to the spindle midzone at the onset of anaphase. By late telophase, it was localized predominantly in the intercellular bridge regions flanking the midbody and disappeared gradually as the daughter cells separated. Immunoblotting analysis showed that the autoimmune sera recognized a doublet of 330 kDa, and affinity-purified antibodies from this doublet reproduced the fluorescence staining pattern of the whole serum. We propose that p330d is a novel member of the class of 'chromosomal passenger' proteins, which are associated transiently with centromeres during early mitosis and are then redistributed to other sites of the mitotic apparatus after the metaphase/anaphase transition. Possible in vivo functions for p330d and related proteins might include roles in centromere/kinetochore maturation and assembly, chromosome segregation, central spindle stabilization and cytokinesis.

Mapping the mammalian intercellular bridge

Mapping the mammalian intercellular bridge

Analysis of the distribution of the INCENPs throughout mitosis reveals the existence of a pathway of structural changes in the chromosomes during metaphase and early events in cleavage furrow formation

The INCENPs are two polypeptides of 135 x 10(3) and 150 x 10(3) Mr that enter mitosis as tightly bound chromosomal proteins, but subsequently leave the chromosomes altogether and become associated with the central spindle and cell cortex at the contractile ring. In the experiments reported here we have used confocal microscopy and immunoelectron microscopy to provide a detailed picture of the intracellular location of these proteins during mitosis. The experiments have not only revealed a number of new details concerning the properties of the INCENPs in mitosis, but have revealed a number of novel aspects of the mitotic process itself. The first of these is the existence of a sequential pathway of structural changes in the chromosomes that occurs during metaphase. This pathway is revealed by the existence of four distinct INCENP staining patterns in mitotic cells. In 'early' and 'early/mid' metaphase, the INCENPs gradually become concentrated at the centromeres, forming a ring at the center of the metaphase plate. During 'mid/late' metaphase they exit from the chromosomes, so that by late metaphase they are found solely in streaks that traverse the plate parallel to the spindle axis. The streaks probably correspond to INCENPs closely associated with microtubule bundles, perhaps as part of the stem body material. Examination of transverse optical sections of the spindle interzone during early anaphase reveals an unexpectedly high degree of order. The INCENP antigens are localized on fibers that are organized into a hollow ring 8 microns in diameter and approximately 4 microns beneath the cell cortex. Measurement of cellular dimensions in the confocal microscope reveals that the maximum diameter of early anaphase cells lies across the spindle equator, so that when the cleavage furrow forms, it does so around the maximum circumference of the cell. During anaphase, a subpopulation of the INCENP antigen becomes localized to the cortex where the furrow will subsequently form. This occurs prior to any other evidence of furrowing. Thus, binding of the INCENPs to this region may represent an early step in furrow formation. Together, these results suggest that the INCENPs may represent a new class of 'chromosomal passenger' proteins that are carried to the spindle equator by the chromosomes and subsequently perform a cytoskeletal role following their release from the chromosomes at the metaphase:anaphase transition.

Chromosomal passengers: Toward an integrated view of mitosis

The major events of mitosis have traditionally been considered to represent two distinct pathways and have been studied by two separate groups of workers. The chromosomal events (chromosome condensation and sister chromatid disjunction) have been the principal focus for one group, while the cytoskeletal events (nuclear envelope breakdown, chromosomal movements, cytokinesis) have been the focus for the other. This historical division is epitomized by the view of many cell biologists, which was aptly caught by Mazia's comparison of the role of the chromosome arms in mitosis to that of "the corpse at the funeral" which "provide a reason for the proceedings but do not take an active part in them" (Mazia 1961). More recent studies have demonstrated that the role of the chromosomes in mitotic movements is somewhat more active than this. That the kinetochore may play an important role in chromosome movements has long been suspected (see early references in Mazia 1961) but was only proven rather recently (Brinkley et al. 1988; Gorbsky et al. 1987; Nicklas 1989). This has led to a burst of recent interest in all aspects of kinetochore structure and function. Our studies have led us to ask whether chromosomes may play an even more extensive role in the events of mitosis. We suggest here that in addition to their active role in movements, the chromosome may make important structural contributions to the anaphase spindle and cleavage furrow, which are normally thought of as "cytoskeletal" functions. These structural contributions may be made by members of a new class of "chromosomal passenger" proteins that use the chromosomes as a means of conveyance so that they are correctly positioned at the metaphase plate to carry out their nonchromosomal functions during anaphase and the subsequent mitotic events.