Functional Coupling between DNA Replication and Sister Chromatid Cohesion Establishment.

Several lines of evidence suggest the existence in the eukaryotic cells of a tight, yet largely unexplored, connection between DNA replication and sister chromatid cohesion. Tethering of newly duplicated chromatids is mediated by cohesin, an evolutionarily conserved hetero-tetrameric protein complex that has a ring-like structure and is believed to encircle DNA. Cohesin is loaded onto chromatin in telophase/G1 and converted into a cohesive state during the subsequent S phase, a process known as cohesion establishment. Many studies have revealed that down-regulation of a number of DNA replication factors gives rise to chromosomal cohesion defects, suggesting that they play critical roles in cohesion establishment. Conversely, loss of cohesin subunits (and/or regulators) has been found to alter DNA replication fork dynamics. A critical step of the cohesion establishment process consists in cohesin acetylation, a modification accomplished by dedicated acetyltransferases that operate at the replication forks. Defects in cohesion establishment give rise to chromosome mis-segregation and aneuploidy, phenotypes frequently observed in pre-cancerous and cancerous cells. Herein, we will review our present knowledge of the molecular mechanisms underlying the functional link between DNA replication and cohesion establishment, a phenomenon that is unique to the eukaryotic organisms.

Telophase correction refines division orientation in stratified epithelia.

During organogenesis, precise control of spindle orientation balances proliferation and differentiation. In the developing murine epidermis, planar and perpendicular divisions yield symmetric and asymmetric fate outcomes, respectively. Classically, division axis specification involves centrosome migration and spindle rotation, events occurring early in mitosis. Here, we identify a novel orientation mechanism which corrects erroneous anaphase orientations during telophase. The directionality of reorientation correlates with the maintenance or loss of basal contact by the apical daughter. While the scaffolding protein LGN is known to determine initial spindle positioning, we show that LGN also functions during telophase to reorient oblique divisions toward perpendicular. The fidelity of telophase correction also relies on the tension-sensitive adherens junction proteins vinculin, α-E-catenin, and afadin. Failure of this corrective mechanism impacts tissue architecture, as persistent oblique divisions induce precocious, sustained differentiation. The division orientation plasticity provided by telophase correction may enable progenitors to adapt to local tissue needs.

ASXL1 interacts with the cohesin complex to maintain chromatid separation and gene expression for normal hematopoiesis.

ASXL1 is frequently mutated in a spectrum of myeloid malignancies with poor prognosis. Loss of Asxl1 leads to myelodysplastic syndrome-like disease in mice; however, the underlying molecular mechanisms remain unclear. We report that ASXL1 interacts with the cohesin complex, which has been shown to guide sister chromatid segregation and regulate gene expression. Loss of Asxl1 impairs the cohesin function, as reflected by an impaired telophase chromatid disjunction in hematopoietic cells. Chromatin immunoprecipitation followed by DNA sequencing data revealed that ASXL1, RAD21, and SMC1A share 93% of genomic binding sites at promoter regions in Lin-cKit+ (LK) cells. We have shown that loss of Asxl1 reduces the genome binding of RAD21 and SMC1A and alters the expression of ASXL1/cohesin target genes in LK cells. Our study underscores the ASXL1-cohesin interaction as a novel means to maintain normal sister chromatid separation and regulate gene expression in hematopoietic cells.

ICRF-193, an anticancer topoisomerase II inhibitor, induces arched telophase spindles that snap, leading to a ploidy increase in fission yeast.

ICRF-193 [meso-4,4-(2,3-butanediyl)-bis(2,6-piperazinedione)] is a complex-stabilizing inhibitor of DNA topoisomerase II (topo II) that is used as an effective anticancer drug. ICRF-193 inhibits topo II catalytic activity in vitro and blocks nuclear division in vivo. Here, we examined the effects of ICRF-193 treatment on chromatin behavior and spindle dynamics using detailed live mitotic cell analysis in the fission yeast, Schizosaccharomyces pombe. Time-lapse movie analysis showed that ICRF-193 treatment leads to an elongation of presumed chromatin fibers connected to kinetochores during mid-mitosis. Anaphase spindles begin to arch, and eventually spindle poles come together abruptly, as if the spindle snapped at the point of spindle microtubule overlap in telophase. Segregating chromosomes appeared as elastic clumps and subsequently pulled back and merged. The snapped spindle phenotype was abolished by microtubule destabilization after thiabendazole treatment, accompanied by unequal chromosome segregation or severe defects in spindle extension. Thus, we conclude that ICRF-193-treated, unseparated sister chromatids pulling toward opposite spindle poles produce the arched and snapped telophase spindle. ICRF-193 treatment increased DNA content, suggesting that the failure of sister chromatids to separate properly in anaphase, causes the spindle to break in telophase, resulting in polyploidization.

Zinc maintains prophase I arrest in mouse oocytes through regulation of the MOS-MAPK pathway.

Meiosis in mammalian females is marked by two arrest points, at prophase I and metaphase II, which must be tightly regulated in order to produce a haploid gamete at the time of fertilization. The transition metal zinc has emerged as a necessary and dynamic regulator of the establishment, maintenance, and exit from metaphase II arrest, but the roles of zinc during prophase I arrest are largely unknown. In this study, we investigate the mechanisms of zinc regulation during the first meiotic arrest. Disrupting zinc availability in the prophase I arrested oocyte by treatment with the heavy metal chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) causes meiotic resumption even in the presence of pharmacological inhibitors of meiosis. We further show that the MOS-MAPK pathway mediates zinc-dependent prophase I arrest, as the pathway prematurely activates during TPEN-induced meiotic resumption. Conversely, inhibition of the MOS-MAPK pathway maintains prophase I arrest. While prolonged zinc insufficiency ultimately results in telophase I arrest, early and transient exposure of oocytes to TPEN is sufficient to induce meiotic resumption and bypass the telophase I block, allowing the formation of developmentally competent eggs upon parthenogenetic activation. These results establish zinc as a crucial regulator of meiosis throughout the entirety of oocyte maturation, including the maintenance of and release from the first and second meiotic arrest points.

CGGBP1 is a nuclear and midbody protein regulating abscission.

Abscission marks the completion of cell division and its failure is associated with delayed cytokinesis and even tetraploidization. Aberrant abscission and consequential ploidy changes can underlie various diseases including cancer. Midbody, a transient structure formed in the intercellular bridge during telophase, contains several proteins including Aurora kinase B (AURKB), which participate in abscission. We report here an unexpected expression pattern and function of the transcription repressor protein CGG triplet repeat-binding protein 1 (CGGBP1), in normal human fibroblasts. We show that CGGBP1, a chromatin-associated protein, trans-localizes to spindle midzone and midbodies in a manner similar to that of AURKB. CGGBP1 depletion resulted in a cell cycle block at G2, characterized by failure of cells to undergo mitosis and also reduced entry into S phase. Consistent with its presence in the midbodies, live microscopy showed that CGGBP1 deficiency caused mitotic failure at abscission resulting in tetraploidy, which could be rescued by CGGBP1 overexpression. These results show that CGGBP1 is a bona fide midbody protein required for normal abscission and mitosis in general.

The active form of the metabolic sensor: AMP-activated protein kinase (AMPK) 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 alpha-catalytic AMPK subunit (P-AMPKalpha(Thr172))-but not its total form (AMPKalpha)-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-AMPKalpha(Thr172) associates with the two asters of microtubules that begin to nucleate from mature centrosomes. The overlapping localization of P-AMPKalpha(Thr172) 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-AMPKalpha(Thr172) cannot be detected following chromatid separation following anaphase-telophase transition. Rather, toward the end of anaphase and in telophase P-AMPKalpha(Thr172) 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-AMPKalpha(Thr172) at the central spindle and midbody persisted during the furrowing process and, at the completion of telophase, staining of P-AMPKalpha(Thr172) as doublet was apparent on either side of the midbody within the intercellular cytokinetic bridge. An identical mitotic geography of P-AMPKalpha(Thr172) was observed in cancer cells lacking the AMPK kinase LKB1, in non-cancerous human epithelial cells, and in mouse fibroblasts. The active form of AMPKalpha 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 alpha 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.

Aurora B kinases restrict chromosome decondensation to telophase of mitosis.

The Aurora kinases comprise a family of evolutionary conserved serine/threonine kinases that have important functions in centrosome duplication, mitotic spindle assembly, chromosome condensation, chromosome biorientation on the spindle and chromosome segregation. Vertebrates have three Aurora kinases, Aurora-A, -B and -C, while invertebrates have only Aurora-A and -B and yeasts have a single Aurora kinase, IpI1 in S. cerevisiae and Ark1 in S. pombe. Recently, the role of Aurora kinases in chromosome condensation has been defined; Aurora B plays a crucial role in the axial shortening of chromosomes during anaphase, presumably in order to prevent chromosome arms from becoming trapped within the cytokinetic plate.

Titin in insect spermatocyte spindle fibers associates with microtubules, actin, myosin and the matrix proteins skeletor, megator and chromator.

Titin, the giant elastic protein found in muscles, is present in spindles of crane-fly and locust spermatocytes as determined by immunofluorescence staining using three antibodies, each raised against a different, spatially separated fragment of Drosophila titin (D-titin). All three antibodies stained the Z-lines and other regions in insect myofibrils. In western blots of insect muscle extract the antibodies reacted with high molecular mass proteins, ranging between rat nebulin (600-900 kDa) and rat titin (3000-4000 kDa). Mass spectrometry of the high molecular mass band from the Coomassie-Blue-stained gel of insect muscle proteins indicates that the protein the antibodies bind to is titin. The pattern of staining in insect spermatocytes was slightly different in the two species, but in general all three anti-D-titin antibodies stained the same components: the chromosomes, prophase and telophase nuclear membranes, the spindle in general, along kinetochore and non-kinetochore microtubules, along apparent connections between partner half-bivalents during anaphase, and various cytoplasmic components, including the contractile ring. That the same cellular components are stained in close proximity by the three different antibodies, each against a different region of D-titin, is strong evidence that the three antibodies identify a titin-like protein in insect spindles, which we identified by mass spectrometry analysis as being titin. The spindle matrix proteins skeletor, megator and chromator are present in many of the same structures, in positions very close to (or the same as) D-titin. Myosin and actin also are present in spindles in close proximity to D-titin. The varying spatial arrangements of these proteins during the course of division suggest that they interact to form a spindle matrix with elastic properties provided by a titin-like protein.

Dose-dependent effects of stable cyclin B1 on progression through mitosis in human cells.

The disassembly of the mitotic spindle and exit from mitosis require the inactivation of Cdk1. Here, we show that expression of nondegradable cyclinB1 causes dose-dependent mitotic arrest phenotypes. By monitoring chromosomes in living cells, we determined that pronounced overexpression of stable cyclinB1 entailed metaphase arrest without detectable sister chromatid separation, while moderate overexpression arrested cells in a pseudometaphase state, in which separated sister chromatids were kept at the cellular equator by a bipolar 'metaphase-like' spindle. Chromosomes that left the pseudometaphase plate became pulled back and individual kinetochores were found to be merotelically attached to both spindle poles in stable cyclinB1 arrested cells. Inactivation of the chromokinesin hKid, by RNAi or antibody microinjection, prevented the formation of stable bipolar spindles and the 'metaphase-like' alignment of chromosomes in cells expressing stable cyclinB1. These experiments show that cyclinB1 is able to maintain a bipolar spindle even after sister chromatids had become separated and suggest an important role of hKid in this process. Cells expressing low levels of nondegradable cyclinB1 progressed further in mitosis and arrested in telophase.

Human LATS1 is a mitotic exit network kinase.

The kinase LATS/WARTS is a tumor suppressor protein conserved in evolution, but its function at the molecular level is not well understood. We report here that human LATS1 interacts with MOB1A, a protein whose homologue in budding yeast associates with kinases involved in mitotic exit. This suggested that LATS1 may be a component of the previously uncharacterized mitotic exit network in higher eukaryotes. Indeed, moderate overexpression of human LATS1 in cells exposed to microtubule poisons facilitated mitotic exit, and this activity required MOB1A. Reciprocally, small interfering RNA-mediated suppression of LATS1 or MOB1A prolonged telophase, but had no effect on the length of the earlier phases of mitosis. A role of LATS1 in mitotic exit may explain its previously described abilities to induce G2 arrest and promote cytokinesis.

Nuclear ploidy is contingent on the microtubular cycle responsible for plant cytokinesis.

Division of the plant cell relies on the preprophase band of microtubules (PPB)-phragmoplast system. Cells of onion (Allium cepa L.) root meristems were rendered binucleate by preventing the consolidation of cell plate formation in telophase with 5 mM caffeine. These binucleates developed either a single PPB around one of their two nuclei or two PPBs, one per nucleus, in the prophase of the ensuing mitosis. Prophase cells developing one single PPB were shorter in length (42.3 +/- 4.1 microm) than those developing 2 PPBs (49.8 +/- 4.1 microm), and interphase duration was inversely related to cell length. Cells whose length was less than or equal to 42 microm, i.e., which had not even reached the mean size of the small binucleates in prophase, were followed throughout mitosis. In metaphase, they always assembled two mitotic spindles (one per nucleus). However, the cells that had assembled a single PPB also developed a single phragmoplast in telophase, leading to polyploidization. As these meristematic cells were not wide enough to accommodate the midzones of both mitotic spindles in any single plane transversal to the cell elongation axis, the spindles tilted until their midzones formed a continuum where the single common phragmoplast assembled. Its position was thereby uncoupled from that of the preceding PPB. Subsequently, the chromosomes from two different half-spindles were included, by a common nuclear envelope, in a single tetraploid nucleus. Finally, the cytokinetic plate segregated the two tetraploid nuclei formed at each side of the phragmoplast into two independent sister cells.

Dynamic organization of vacuolar and microtubule structures during cell cycle progression in synchronized tobacco BY-2 cells.

In higher plant cells, vacuoles show considerable diversity in their shapes and functions. The roles of vacuoles in the storage, osmoregulation, digestion and secretory pathway are well established; however, their functions in cell morphogenesis and cell division are still unclear. To observe the dynamic changes of vacuoles in living plant cells, we attempted to visualize the vacuolar membrane (VM) by pulse-labeling tobacco BY-2 cells with a styryl fluorescent dye, FM4-64. By time-sequence observations using confocal laser scanning microscopy (CLSM), we could follow the dynamics of vacuolar structures throughout the cell cycle in living higher plant cells. We also confirmed the dynamic changes of VM structures by the observation using transgenic BY-2 cells expressing GFP-AtVam3p fusion protein (BY-GV). Furthermore, by using transgenic BY-2 cells that stably express a GFP-tubulin fusion protein [BY-GT16, Kumagai et al. (2001) Plant Cell Physiol. 42: 723], we could study the relationship between the dynamics of vacuoles and microtubules. From these observations, we identified, for the first time, some remarkable events: (1) at the late G(2) phase, tubular structures of the vacuolar membrane developed in the central region of the cell, probably in the premitotic cytoplasmic band (phragmosome), surrounding the mitotic apparatus; (2) from anaphase to telophase, these tubular structures invaded the region of the phragmoplast within which the cell plate was being formed; (3) at the early G(1) phase, some of the tubular structures expanded rapidly between the cell plate and daughter nuclei, and subsequently developed into large vacuoles at interphase.

TMBP200, a microtubule bundling polypeptide isolated from telophase tobacco BY-2 cells is a MOR1 homologue.

Bundles of microtubules and cross-bridges between microtubules in the bundles have been observed in phragmoplasts, but proteins responsible for forming the cross-bridges have not been identified. We isolated TMBP200, a novel microtubule bundling polypeptide with an estimated relative molecular mass of about 200,000 from telophase tobacco BY-2 cells. Ultrastructural observation of microtubules bundled by purified TMBP200 in vitro revealed that TMBP200 forms cross-bridges between microtubules. The structure of the bundles and lengths of the cross-bridges were quite similar to those observed in phragmoplasts, suggesting that TMBP200 participates in the formation of microtubule bundles in phragmoplasts. The cDNA encoding TMBP200 was cloned and the deduced amino acid sequence showed homology to a class of microtubule-associated proteins including Xenopus XMAP215, human TOGp and Arabidopsis MOR1.

Effect of telophase enucleation on bovine somatic nuclear transfer.

Telophase enucleation has been proven to be an efficient method for preparing recipient cytoplasts in bovine embryonic nuclear transfer (2, 11). This research was designed to study in vitro development of bovine oocytes containing transferred somatic cell nuclei, reconstructed by using enucleated in vitro-matured oocytes 32 h of age at telophase II stage as recipient cytoplasts, compared with those 24 h of age at metaphase II stage. Two protocols for donor cell injection were adopted, i.e., subzonal injection (SUZI) and intracytoplasmic injection (ICI). Bovine oviduct epithelial cells (BOECs) and bovine cumulus cells (BCCs) from an adult cow were used as nuclear donors for these experiments. In SUZI groups, the fusion rate of donor cells, both BOECs and BCCs, with MII enucleated oocytes were higher than those with TII enucleated oocytes (54% vs. 41% and 53% vs. 39%, respectively; P<0.05), but the development rates to morula plus blastocyst stage in MII groups were lower than those in TII groups (22% vs. 39% and 21% vs. 41%, respectively; P<0.05). In ICI groups, about 26% of enucleated MII oocytes injected with BOECs or BCCs cleaved and only small parts of them developed to blastocyst stage (4% and 3%, respectively; P>0.05). When BOECs or BCCs were intracytoplasmically injected into oocytes enucleated at TII stage, no blastocyst was formed in either donor cell group and no cleavage occurred in BOEC group. Our data demonstrated that telophase enucleation is beneficial to early embryo development when bovine somatic nuclei are transferred by subzonal injection. However, it is harmful when donor cells are directly injected into the cytoplast of the enucleated oocytes.

Isolation of polypeptides with microtubule-translocating activity from phragmoplasts of tobacco BY-2 cells.

As part of our efforts to understand the molecular basis of the microtubule-associated motility that is involved in cytokinesis in higher plant cells, an attempt was made to identify proteins with the ability to translocate microtubules in an extract from isolated phragmoplasts. Homogenization of isolated phragmoplasts in a solution that contained MgATP, MgGTP and a high concentration of NaCl resulted in the release from phragmoplasts of factors with ATPase and GTPase activity that were stimulated by microtubules. A protein fraction with microtubule-dependent ATPase and GTPase activity caused minus-end-headed gliding of microtubules in the presence of ATP or GTP. Polypeptides with microtubule-translocating activity cosedimented with microtubules that had been assembled in vitro from brain tubulin and were dissociated from sedimented microtubules by addition of ATP or GTP. After cosedimentation and dissociation procedures, a 125 kDa polypeptide and a 120 kDa polypeptide were recovered in a fraction that supported minus-end-headed gliding of microtubules. The rate of microtubule gliding that was caused by the fraction that contained the 125 kDa and 120 kDa polypeptides as main components was 1.28 microns/minute in the presence of ATP and 0.50 microns/minute in the presence of GTP. This fraction contained some microtubule-associated polypeptides in addition to the 125 kDa and 120 kDa polypeptides, but a fraction that contained only these additional polypeptides did not cause any translocation of microtubules. Thus, it appeared that the 125 kDa and 120 kDa polypeptides were responsible for translocation of microtubules. These polypeptides with plus-end-directed motor activity may play an important role in formation of the cell plate and in the organization of the phragmoplast.

The telophase disc: its possible role in mammalian cell cleavage.

The molecular signals that determine the position and timing of the furrow that forms during mammalian cell cytokinesis are presently unknown. It is apparent, however, that these signals are generated by the mitotic spindle after the onset of anaphase. Recently we have described a structure that bisects the cell during telophase at the position of the cytokinetic furrow. This structure, the telophase disc, appears to be templated by the mitotic spindle during anaphase, and precedes the formation of the cytokinetic furrow. The relationship of the telophase disc to the myosin and actin based furrowing mechanism is discussed here. We propose that the telophase disc may determine the position and timing of cleavage by recruitment and alignment of myosin.

Telophase disc: a new mammalian mitotic organelle that bisects telophase cells with a possible function in cytokinesis.

We have discovered a novel mitosis-specific human autoantigen that arises at the centromeres of prophase chromosomes, but ultimately participates in formation of an organelle that bisects the cell at late anaphase and during telophase. The organelle, discernible as a three-dimensional disc by confocal microscopy, encompasses the entire midzone diameter, and its distribution survives disassembly of interpolar microtubules by cold temperature treatment and detergent lysis of cells. Cytokinetic furrow contraction proceeds normally in dihydrocytochalasin B (DCB)-treated cells, and antigen distribution in the furrow is unaltered. In DCB, the furrow retracts in early interphase, coincident with loss of normal membrane association with the disc, resulting in the formation of binucleate cells. The midzone disc in both drug-treated and normal cells is present at the correct time and position to play a central role in cytokinesis. By immunocytochemistry, the disc appears to contain myosin but not actin. The position of the disc and the possible presence of myosin suggest that cytokinesis may involve the interaction of the disc organelle with actin in the cell cortex to produce cleavage in mammalian cells.