Anaphase-telophase Transition Research Articles

FBXW8-dependent degradation of MRFAP1 in anaphase controls mitotic cell death.

Mof4 family associated protein 1 (MRFAP1) is a 14 kDa nuclear protein, which involves in maintaining normal histone modification levels by negatively regulating recruitment of the NuA4 (nucleosome acetyltransferase of H4) histone acetyltransferase complex to chromatin. MRFAP1 has been identified as one of the most up-regulated proteins after NEDD8 (neural precursor cell expressed developmentally down-regulated 8) inhibition in multiple human cell lines. However, the biological function of MRFAP1 and the E3 ligase that targets MRFAP1 for destruction remain mysterious. Here we show, by using an immunoprecipitation-based proteomics screen, that MRFAP1 is an interactor of the F-box protein FBXW8. MRFAP1 is degraded by means of the ubiquitin ligase Cul7/FBXW8 during mitotic anaphase-telophase transition and accumulated in mitotic metaphase. Overexpression of FBXW8 increased the polyubiquitination and decreased the stability of MRFAP1, whereas knockdown of FBXW8 prolonged the half-life of MRFAP1. Moreover, forced expression of MRFAP1 in HeLa cells caused growth retardation and genomic instability, leading to severe mitotic cell death. Thus, Cul7/FBXW8-mediated destruction of MRFAP1 is a regulatory component monitoring the anaphase-telophase transition and preventing genomic instability.

Late mitotic functions of Aurora kinases

The coordination between late mitotic events such as poleward chromosome motion, spindle elongation, DNA decondensation, and nuclear envelope reformation (NER) is crucial for the completion of chromosome segregation at the anaphase-telophase transition. Mitotic exit is driven by a decrease of Cdk1 kinase activity and an increase of PP1/PP2A phosphatase activities. More recently, Aurora kinases have also emerged as master regulators of late mitotic events and cytokinesis. Aurora A is mainly associated with spindle poles throughout mitosis and midbody during telophase, whereas Aurora B re-localizes from centromeres in early mitosis to the spindle midzone and midbody as cells progress from anaphase to the completion of cytokinesis. Functional studies, together with the identification of a phosphorylation gradient during anaphase, established Aurora B as a major player in the organization of the spindle midzone and in the spatiotemporal coordination between chromosome segregation and NER. Aurora A has been less explored, but a cooperative role in spindle midzone stability has also been proposed, implying that both Aurora A and B contribute to accurate chromosome segregation during mitotic exit. Here, we review the roles of the Aurora kinases in the regulation of late mitotic events and discuss how they work together with other mitotic players to ensure an error-free mitosis.

BioEssays 3∕2015

BioEssaysVolume 37, Issue 3 Cover PictureFree Access BioEssays 3∕2015 First published: 18 February 2015 https://doi.org/10.1002/bies.201470031AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract A chromosome separation checkpoint. On pages 257–266, Maiato et al. discuss a “chromosome separation checkpoint” that regulates the anaphasetelophase transition. The cover depicts an LLC-PK cell at the anaphase-telophase transition. This transition is spatially regulated by a spindle midzone Aurora B activity gradient (light pink) revealed by the gradual dephosphorylation of Histone H3 on Ser10 (green) as chromosomes (magenta) migrate towards the poles where nuclear envelope (cyan blue) reformation begins. Microtubules are shown in white and actin filaments also in cyan blue define the cell borders. Interphase nuclei are shown in violet. Photo credits: Helder Maiato and Olga Afonso. Volume37, Issue3March 2015 RelatedInformation

A chromosome separation checkpoint: A midzone Aurora B gradient mediates a chromosome separation checkpoint that regulates the anaphase-telophase transition.

Here we discuss a "chromosome separation checkpoint" that might regulate the anaphase-telophase transition. The concept of cell cycle checkpoints was originally proposed to account for extrinsic control mechanisms that ensure the order of cell cycle events. Several checkpoints have been shown to regulate major cell cycle transitions, namely at G1-S and G2-M. At the onset of mitosis, the prophase-prometaphase transition is controlled by several potential checkpoints, including the antephase checkpoint, while the spindle assembly checkpoint guards the metaphase-anaphase transition. Our hypothesis is based on the recently uncovered feedback control mechanism that delays chromosome decondensation and nuclear envelope reassembly until effective separation of sister chromatids during anaphase is achieved. A central player in this potential checkpoint is the establishment of a constitutive, midzone-based Aurora B phosphorylation gradient that monitors the position of chromosomes along the spindle axis. We propose that this surveillance mechanism represents an additional step towards ensuring mitotic fidelity.

Spatial control of the anaphase-telophase transition

The ultimate goal of mitosis is to physically separate the entire set of duplicated chromosomes in order to propagate the genetic information during cell division. The perpetuation of a faithful maintenance of the genome integrity can only be guaranteed by the existence of multiple cell cycle checkpoints.1 In particular, mitosis is a highly regulated process where checkpoints assume a major importance.2 The latest stages of mitosis have been extensively studied; however, the mechanisms underlying the coordination between chromosome separation during anaphase and mitotic exit remain to be unveiled. It has long been assumed that anaphase and telophase are events that occur passively after satisfaction of the spindle-assembly checkpoint (SAC).2 However, it seems counterintuitive to assume that these crucial moments of mitosis are not being tightly monitored. A basic assumption for successful chromosome segregation during anaphase is that the 2 sets of chromosomes effectively separate before nuclear envelope reformation (NER). Recently, we showed that an inverse correlation exists between chromosome separation velocity and anaphase duration in Drosophila and human cultured cells, suggesting the presence of an additional surveillance mechanism that spatially regulates the anaphase-telophase transition.3 Importantly, spatial regulation implies the presence of a sensory ruler capable of “measuring” the chromosome position along the division axis. In fact, we showed that the previously reported Aurora B phosphorylation gradient4 creates an “area of exclusion” that inhibits DNA decondensation and NER (Fig. 1). When global Aurora B activity is impaired, or its localization at the spindle midzone affected, this spatial regulation is lost and NER occurs independently of the chromosome position. The precocious assembly of the nuclear envelope around unseparated chromosomes may lead to aneuploidy and/or polyploidy that will ultimately perturb cell or tissue homeostasis. Figure 1. After SAC satisfaction, mitotic cells proceed into anaphase. Aurora B is transferred to the spindle midzone and establishes a phosphorylation gradient where its substrates are phosphorylated and chromosomes remain condensed. This phosphorylation gradient ... Although Aurora B activity dictates a spatial control of the anaphase-telophase transition, the prolonged extension of anaphase after the expression of non-degradable Cyclin B1 mutants indicates that a final step of degradation is required to determine true mitotic exit. Additionally, inhibition of Cdk1 activity during anaphase shortens anaphase duration, suggesting that Cyclin B1 is necessary to provide time for proper chromosome separation. Therefore, both Aurora B and Cdk1 kinase activities must be coordinated to assure the spatiotemporal control of the anaphase-telophase transition. An additional level of regulation is related to the phosphatases, which must be in place to counteract the kinase activities. Inhibition of PP1/PP2A at the anaphase onset caused an “endless anaphase” phenotype, with persistent condensed chromosomes and no evident NER. Our data suggests that the kinase activity is predominant in the spindle midzone and, as chromosomes move to opposite poles, phosphatase activity will prevail and account for the dephosphorylation of substrates and consequent mitotic exit (Fig. 1). Some outstanding questions still remain and in the future, it would be important to understand how AuroraB, Cdk1 and PP1/PP2A activities are mechanistically coordinated: is there a feedback loop combining these 3 players? Or is there a hierarchical series of phosphorylation/dephosphorylation events that promote the balanced activity that ultimately leads to accurate chromosome segregation and NER? In the context of a potential chromosome separation checkpoint it will be important to understand which are the substrates that mediate the anaphase-telophase transition in coordination with chromosome separation. In this context, the localization of the Drosophila Condensin I subunit Barren, whose loading onto chromosomes is dependent on Aurora B,5 was found to be sensitive to the midzone Aurora B phosphorylation gradient (Fig. 1). Additionally, Barren depletion caused a similar phenotype to the one found with Aurora B inhibition, further supporting that chromosome condensation could be a readout for chromosome position during anaphase. Overall, sister-chromatid separation during anaphase is actively monitored by the presence of a phosphorylation gradient of Aurora B established at the spindle midzone. This hub of kinase activity constitutively phosphorylates its substrates as long as they are under the influence of the gradient. The inhibitory action of Aurora B activity must therefore feedback to Cdk1 in order to dampen the kinase activity and allow the phosphatases to prevail. The proposed feedback control of the anaphase-telophase transition is just the tip of the iceberg - how it is molecularly regulated and whether this mechanism is conserved between different cells in different tissues and organisms will certainly be a direction of further research.

The active form of the metabolic sensor AMP-activated protein kinase α (AMPKα) directly binds the mitotic apparatus and travels from centrosomes to the spindle midzone during mitosis and cytokinesis

The metabolic rheostat AMP-activated protein kinase (AMPK) is unexpectedly required for proper cell division and faithful chromosomal segregation during mitosis. Although it is conceptually attractive to assume that AMPK-interpreted microenvironmental bioenergetics may strictly engage cell’s energy status, cell grow, and cell division to avoid that energy stresses trigger cell death, the ultimate framework of AMPK activity towards chromosomal and cytoskeletal mitotic regulation is a question that remains unanswered. We herein reveal that the active form of the α-catalytic AMPK subunit (P-AMPKαThr172) -but not its total form (AMPKα)- transiently associates with several mitotic structures including centrosomes, spindle poles, the central spindle midzone and the midbody throughout all of the mitotic stages and cytokinesis in human cancer-derived epithelial cells. At prophase, P-AMPKαThr172 associates with the two asters of microtubules that begin to nucleate from mature centrosomes. The overlapping localization of P-AMPKαThr172 with the mitotic centrosomal Aurora-A kinase is also apparent on the microtubules near the spindle poles in metaphase and in early anaphase. This Aurora A-like centrosomal localization of P-AMPKαThr172 cannot be detected following chromatid separation following anaphase-telophase transition. Rather, toward the end of anaphase and in telophase P-AMPKαThr172 reactivity exhibited a similar but not identical localization to that occupied by the bona fide chromosomal passenger proteins INCENCP and Aurora-B. This localization of P-AMPKαThr172 at the central spindle and midbody persisted during the furrowing process and, at the completion of telophase, a prominent staining of P-AMPKαThr172 as doublet was apparent on either side of the midbody within the intercellular cytokinetic bridge. An identical mitotic geography of P-AMPKαThr172 was observed in cancer cells lacking the AMPK kinase LKB1, in non-cancerous human epithelial cells, and in mouse fibroblasts. The active form of AMPKα bound to the mitotic apparatus may physically tether the bioenergetic state of a cell to the four-dimensional regulation of the chromosomal and cytoskeletal mitotic events, thus suggesting a putative cytokinetic suppressor function. In this newly discovered scenario, we suggest a primordial mitotic role for the α catalytic AMPK subunit in the eukaryotic evolutionary process as it may ensure, at the cell level, an exquisite coordination between sensing of energy resources and the fundamental biological process of genome division.

Inducible nitric oxide synthase-derived nitric oxide regulates germinal vesicle breakdown and first polar body emission in the mouse oocyte

The present study investigated the subcellular localization of inducible nitric oxide synthase (iNOS) during mouse oocyte meiotic maturation and fertilization using confocal microscopy, and further studied the roles of iNOS-derived NO in oocyte maturation by using an iNOS-specific inhibitor aminoguanidine (AG) and iNOS antibody microinjection. In germinal vesicle-stage oocytes, iNOS immunoreactivity was mainly localized in the germinal vesicle. Shortly after germinal vesicle breakdown, the iNOS immunoreactivity accumulated around the condensed chromosomes. At metaphase I and metaphase II, with the organization of chromosomes to the equatorial plate, iNOS immunoreactivity was concentrated around the aligned chromosomes, putatively the position of the metaphase spindle. The accumulation of iNOS immunoreactivity could not be detected at anaphase I and anaphase II. However, at telophase I and telophase II, the staining of iNOS was concentrated in the region between the separating chromosomes/chromatids. Furthermore, the staining of iNOS also accumulated in the male and female pronuclei in fertilized eggs. Germinal vesicle breakdown and the first polar body emission of the oocytes were significantly blocked by the iNOS-specific inhibitor AG in a dose-dependent manner. The germinal vesicle breakdown in oocytes injected with iNOS antibody was also inhibited. We found that the phosphorylation of mitogen-activated protein kinase in oocytes after germinal vesicle breakdown was inhibited by AG treatment. The control oocytes extruded a normal first polar body, while the AG-treated oocytes exhibited an elongated protrusion or no elongated protrusion. The results of confocal microscopy showed that the AG-treated oocytes were arrested at anaphase I-telophase I. Our results suggest that the iNOS-derived NO pathway plays important roles in mouse oocyte meiotic maturation, especially in germinal vesicle breakdown and the anaphase-telophase transition.

The role of MEN (mitosis exit network) proteins in the cytokinesis of Saccharomyces cerevisiae.

At the latest stages of their cell cycle, cells carry out crucial processes for the correct segregation of their genetic and cytoplasmic material. In this work, we provide evidence demonstrating that the cell cycle arrest of some MEN (mitosis exit network) mutants in the anaphase-telophase transition is bypassed. In addition, the ability of cdc15 diploid mutant strains to develop non-septated chains of cells, supported by nuclear division, is shown. This phenotype is also displayed by haploid cdc15 mutant strains when cell lysis is prevented by osmotic protection, and shared by other MEN mutants. By contrast, anaphase-telophase arrest is strictly observed in double MEN-FEAR (fourteen early anaphase release) mutants. In this context, the overexpression of a FEAR component, SPO12, in a MEN mutant background enhances the ability of MEN mutants to bypass cell cycle arrest. Taken together, these data suggest a critical role of Cdc15 and other MEN proteins in cytokinesis, allowing a new model for their cellular function to be proposed.

Nuclear lamins A and B1: different pathways of assembly during nuclear envelope formation in living cells.

At the end of mitosis, the nuclear lamins assemble to form the nuclear lamina during nuclear envelope formation in daughter cells. We have fused A- and B-type nuclear lamins to the green fluorescent protein to study this process in living cells. The results reveal that the A- and B-type lamins exhibit different pathways of assembly. In the early stages of mitosis, both lamins are distributed throughout the cytoplasm in a diffusible (nonpolymerized) state, as demonstrated by fluorescence recovery after photobleaching (FRAP). During the anaphase-telophase transition, lamin B1 begins to become concentrated at the surface of the chromosomes. As the chromosomes reach the spindle poles, virtually all of the detectable lamin B1 has accumulated at their surfaces. Subsequently, this lamin rapidly encloses the entire perimeter of the region containing decondensing chromosomes in each daughter cell. By this time, lamin B1 has assembled into a relatively stable polymer, as indicated by FRAP analyses and insolubility in detergent/high ionic strength solutions. In contrast, the association of lamin A with the nucleus begins only after the major components of the nuclear envelope including pore complexes are assembled in daughter cells. Initially, lamin A is found in an unpolymerized state throughout the nucleoplasm of daughter cell nuclei in early G1 and only gradually becomes incorporated into the peripheral lamina during the first few hours of this stage of the cell cycle. In later stages of G1, FRAP analyses suggest that both green fluorescent protein lamins A and B1 form higher order polymers throughout interphase nuclei.

Colocalization of a high molecular mass phosphoprotein of the nuclear matrix (p255) with spliceosomes.

It was previously demonstrated that monoclonal antibody CC-3 binds to a phosphorylation dependent epitope present on a 255 kDa nuclear protein (p255). We show here that in interphase cells, p255 distributes to typical nuclear speckles that correspond to the localization of spliceosome components as revealed by antibodies to the m3G cap of snRNAs or to the non-snRNP splicing factor SC-35. Immunofluorescence and immunoblot studies indicated that p255 is resistant to extraction with non-ionic detergents, nucleases and high ionic strength buffers and may thus be defined biochemically as a nuclear matrix phosphoprotein. To determine the nature of the association of p255 with the nuclear structure, its distribution was studied at different stages of the cell cycle and after the cells were treated with nucleases or heat shocked. We found that the antigen diffused into the cytoplasm during metaphase but was reorganized into cytoplasmic speckles during anaphase-telophase transition, where it colocalized with SC-35. Nuclear matrix preparations that were digested with DNases and RNases showed that interphasic p255 still localized to nuclear speckles even though snRNA and snRNP antigens were removed. Heat-shocked cells labelled with monoclonal antibody CC-3 exhibited more rounded and less interconnected speckles, identical to those decorated by anti-SC-35 antibody under such conditions. These results indicate that p255 and SC-35 are present in the same nuclear structures, to which they are more tightly bound than the snRNP antigens. They further suggest that both proteins are implicated in spliceosome assembly or attachment.

Early changes in embryonic nuclei fused to chemically enucleated mouse oocytes.

Mouse oocytes were chemically enucleated by subjecting them to etoposide and cycloheximide treatment during the first meiotic division (Fulka, Jr. and Moor, Mol. Reprod. Dev. 34:427-430, 1993) and thereafter electrofused to karyoplasts prepared from: (i) two-cell stage embryos at the G2-phase; (ii) four-cell stage blastomeres (S- or G2-phase); or (iii) embryonic stem (ES) cells. In the first series of experiments we used fusion conditions which do not induce egg activation to define the series of nuclear changes that are initiated immediately following fusion. Although fusion is evident within 5-10 min of induction, nuclei remain visible for up to 20 min prior to chromatin condensation and the formation of metaphase plates (60-90 min post fusion). After activation, the anaphase-telophase transition is completed within 1-2 h, followed thereafter by cleavage of 75% of reconstituted eggs into two equal nucleated blastomeres, irrespective of the origin of the nuclei used for fusion. We conclude from the first study that a protocol involving fusion without activation, followed 90 min later by activation, is likely to be optimal for nuclear transplantation using MII-phase cytoplasts. In the second series of experiments the above optimized protocol was used to study the effects of different cell cycle combinations on chromosome organization in eggs reconstituted by nuclear transplantation. Both G1- and S-phase karyoplasts fused to MII-phase cytoplasts exhibited spindle abnormalities in all eggs studied. Characteristic abnormalities in these cell cycle combinations included chromatin fragmentation and joining or aggregations of chromatin.(ABSTRACT TRUNCATED AT 250 WORDS)

The fall of biological maturation promoting factor (MPF) and histone H1 kinase activity during anaphase and telophase in mouse oocytes.

Cell fusions have been used to determine the biological activity of the MPF complex in murine oocytes during their progression through anaphase and telophase to metaphase II. Oocytes (1) at metaphase I, (2) during the anaphase-telophase transition, or (3) at metaphase II were fused to germinal vesicle-staged (immature) oocytes. The hybrids were cultured for 1 h in the presence of db cAMP before fixation and nuclear evaluation. Metaphase I oocytes invariably induced germinal vesicle breakdown (GVBD) in the immature partner. By contrast, anaphase/telophase oocytes never induced GVBD in immature oocytes. The capacity to induce GVBD reappears after the formation of the second metaphase plate. In a second study, histone H1 kinase activity was measured during mouse oocyte maturation in single oocytes. H1 kinase activity was low in GV oocytes, increased sharply at MI, declined during anaphase and telophase and increased again at MII. After egg activation, H1 kinase activity was reduced to basal levels. These results provide direct evidence that a drop in activity of MPF in murine oocytes occurs concomitantly with the exit from metaphase I; MPF activity remains low until the cell re-enters metaphase.

Microinjected fluorescent phalloidin in vivo reveals the F-actin dynamics and assembly in higher plant mitotic cells.

Endosperm mitotic cells microinjected with fluorescent phalloidin enabled us to follow the in vivo dynamics of the F-actin cytoskeleton. The fluorescent probe immediately bound to plant microfilaments. First, we investigated the active rearrangement of F-actin during chromosome migration, which appeared to be slowed down in the presence of phalloidin. These findings were compared with the actin patterns observed in mitotic cells fixed at different stages. Our second aim was to determine the origin of the actin filaments that appear at the equator during anaphase-telophase transition. It is not clear whether this F-actin is newly assembled at the end of mitosis and could control plant cytokinesis or whether it corresponds to a passive redistribution of broken polymers in response to microtubule dynamics. We microinjected the same cells twice, first in metaphase with rhodamine-phalloidin and then in late anaphase with fluorescein isothiocyanate-phalloidin. This technique enabled us to visualize two F-actin populations that are not co-localized, suggesting that actin is newly assembled during cell plate development. These in vivo data shed new light on the role of actin in plant mitosis and cytokinesis.

Large-scale chromatin structural domains within mitotic and interphase chromosomes in vivo and in vitro

Higher-order chromatin structural domains approximately 130 nm in width are observed as prominent components of both Drosophila melanogaster and human mitotic chromosomes using buffer conditions which preserve chromosome morphology as determined by light microscopic comparison with chromosomes within living cells. Spatially discrete chromatin structural domains of similar size also exist as prominent components within interphase nuclei prepared under equivalent conditions. Examination of chromosomes during the anaphase-telophase transition suggests that chromosomes decondense largely through the progressive straightening or uncoiling of these large-scale chromatin domains. A quantitative analysis of the size distribution of these higher-order domains in telophase nuclei indicated a mean width of 126 +/- 36 nm. Three-dimensional views using stereopairs of chromosomes and interphase nuclei from 0.5 micron thick sections suggest that these large-scale chromatin domains consist of 30 nm fibers packed by tight folding into larger, linear, fiber-like elements. Reduction in vitro of either polyamine or divalent cation concentrations within two different buffer systems results in a loss of these large-scale domains, with no higher-order chromatin organization evident above the 20-30 nm fiber. Under these conditions the DNA distribution within mitotic chromosomes and interphase nuclei appears significantly diffuse relative to the appearance by light microscopy within living cells, or, by electron microscopy, within cells fixed directly without permeabilization in buffer. These results suggest that these large-scale chromatin structural domains are fundamental elements of chromosome architecture in vivo.

Plant actin filament and microtubule interactions during anaphase-telophase transition: effects of antagonist drugs

F-actin and microtubule co-distribution and interaction were studied during anaphase-telophase. Rapid and drastic changes in the cytoskeleton during these particular stages were studied in isolated plant endosperm cells of the blood lily. These wall-free cells can be considered as natural dividing protoplasts. As identified previously, an F-actin cytoskeletal network characterized the plant cortex and formed an elastic cage around the spindle, remaining throughout interphase, mitosis and cytokinesis. Actin was specifically labeled by fluorescent phalloidin and/or monoclonal antibodies. Gold-labeled secondary antibodies were used for ultrastructural observations and silver-enhancement was applied for video-enhanced microscopy. Microtubule and microfilament dynamics and interaction were studied using drug antagonists to actin (cytochalasins B, D) and to tubulin (colchicine). This permitted precise correlations to be made between chromosome movement inhibition and alteration in the actin/tubulin cytoskeleton. During anaphase chromosome migration, the cortical actin network was stretched along the microtubular spindle, while it remained homogeneous when anaphase was inhibited by colchicine. Cytochalasins did not inhibit chromosome movement but altered actin distribution. A new population of actin filaments appeared at the equator in late anaphase before the microtubular phragmoplast was formed and contributed to cell plate formation. Our conclusion is that F-actin-microtubule interaction may contribute to the regulatory mechanism of plant cytokinesis.