Specific detection of fission yeast primary septum reveals septum and cleavage furrow ingression during early anaphase independent of mitosis completion.

It is widely accepted in eukaryotes that the cleavage furrow only initiates after mitosis completion. In fission yeast, cytokinesis requires the synthesis of a septum tightly coupled to cleavage furrow ingression. The current cytokinesis model establishes that simultaneous septation and furrow ingression only initiate after spindle breakage and mitosis exit. Thus, this model considers that although Cdk1 is inactivated at early-anaphase, septation onset requires the long elapsed time until mitosis completion and full activation of the Hippo-like SIN pathway. Here, we studied the precise timing of septation onset regarding mitosis by exploiting both the septum-specific detection with the fluorochrome calcofluor and the high-resolution electron microscopy during anaphase and telophase. Contrarily to the existing model, we found that both septum and cleavage furrow start to ingress at early anaphase B, long before spindle breakage, with a slow ingression rate during anaphase B, and greatly increasing after telophase onset. This shows that mitosis and cleavage furrow ingression are not concatenated but simultaneous events in fission yeast. We found that the timing of septation during early anaphase correlates with the cell size and is regulated by the corresponding levels of SIN Etd1 and Rho1. Cdk1 inactivation was directly required for timely septation in early anaphase. Strikingly the reduced SIN activity present after Cdk1 loss was enough to trigger septation by immediately inducing the medial recruitment of the SIN kinase complex Sid2-Mob1. On the other hand, septation onset did not depend on the SIN asymmetry establishment, which is considered a hallmark for SIN activation. These results recalibrate the timing of key cytokinetic events in fission yeast; and unveil a size-dependent control mechanism that synchronizes simultaneous nuclei separation with septum and cleavage furrow ingression to safeguard the proper chromosome segregation during cell division.

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.

A DII Domain-Based Auxin Reporter Uncovers Low Auxin Signaling during Telophase and Early G1.

A sensitive and dynamically responsive auxin signaling reporter based on the DII domain of the INDOLE-3-ACETIC ACID28 (IAA28, DII) protein from Arabidopsis (Arabidopsis thaliana) was modified for use in maize (Zea mays). The DII domain was fused to a yellow fluorescent protein and a nuclear localization sequence to simplify quantitative nuclear fluorescence signal. DII degradation dynamics provide an estimate of input signal into the auxin signaling pathway that is influenced by both auxin accumulation and F-box coreceptor concentration. In maize, the DII-based marker responded rapidly and in a dose-dependent manner to exogenous auxin via proteasome-mediated degradation. Low levels of DII-specific fluorescence corresponding to high endogenous auxin signaling occurred near vasculature tissue and the outer layer and glume primordia of spikelet pair meristems and floral meristems, respectively. In addition, high DII levels were observed in cells during telophase and early G1, suggesting that low auxin signaling at these stages may be important for cell cycle progression.

The lysine demethylase LSD1 is required for nuclear envelope formation at the end of mitosis.

The metazoan nucleus breaks down and reassembles during each cell division. Upon mitotic exit, the successful reestablishment of an interphase nucleus requires the coordinated reorganization of chromatin and formation of a functional nuclear envelope. Here, we report that the histone demethylase LSD1 (also known as KDM1A) plays a crucial role in nuclear assembly at the end of mitosis. Downregulation of LSD1 in cells extends telophase and impairs nuclear pore complex assembly. In vitro, LSD1 demethylase activity is required for the recruitment of MEL28 (also known as ELYS and AHCTF1) and nuclear envelope precursor vesicles to chromatin, crucial steps in nuclear reassembly. Accordingly, the formation of a closed nuclear envelope and nuclear pore complex assembly are impaired upon depletion of LSD1 or inhibition of its activity. Our results identify histone demethylation by LSD1 as a new regulatory mechanism linking the chromatin state and nuclear envelope formation at the end of mitosis.

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.

The synaptonemal complex protein, Zip1, promotes the segregation of nonexchange chromosomes at meiosis I.

Crossing over establishes connections between homologous chromosomes that promote their proper segregation at the first meiotic division. However, there exists a backup system to ensure the correct segregation of those chromosome pairs that fail to cross over. We have found that, in budding yeast, a mutation eliminating the synaptonemal complex protein, Zip1, increases the meiosis I nondisjunction rate of nonexchange chromosomes (NECs). The centromeres of NECs become tethered during meiotic prophase, and this tethering is disrupted by the zip1 mutation. Furthermore, the Zip1 protein often colocalizes to the centromeres of the tethered chromosomes, suggesting that Zip1 plays a direct role in holding NECs together. Zip3, a protein involved in the initiation of synaptonemal complex formation, is also important for NEC segregation. In the absence of Zip3, both the tethering of NECs and the localization of Zip1 to centromeres are impaired. A mutation in the MAD3 gene, which encodes a component of the spindle checkpoint, also increases the nondisjunction of NECs. Together, the zip1 and mad3 mutations have an additive effect, suggesting that these proteins act in parallel pathways to promote NEC segregation. We propose that Mad3 promotes the segregation of NECs that are not tethered by Zip1 at their centromeres.

beta-Catenin asymmetries after all animal/vegetal- oriented cell divisions in Platynereis dumerilii embryos mediate binary cell-fate specification.

In response to Wnt signaling during animal development, beta-catenin accumulates in nuclei to mediate the transcriptional activation of target genes. Here, we show that a highly conserved beta-catenin in the annelid Platynereis dumerilii exhibits a reiterative, nearly universal embryonic pattern of nuclear accumulation remarkably similar to that observed in the nematode Caenorhabditis elegans. Platynereis exhibits beta-catenin sister-cell asymmetries after all cell divisions that occur along the animal/vegetal axis beginning early in embryogenesis, but not after two transverse divisions that establish bilateral symmetry in the trunk. Moreover, ectopic activation of nuclear beta-catenin accumulation in Platynereis causes animal-pole sister cells, which normally have low nuclear beta-catenin levels, to adopt the fate of their vegetal-pole sisters, which normally have high nuclear beta-catenin levels. The presence of reiterative and functionally important beta-catenin asymmetries in two distantly related animal phyla suggests an ancient metazoan origin of a beta-catenin-mediated binary cell-fate specification module.

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.

Zinc availability regulates exit from meiosis in maturing mammalian oocytes.

Cellular metal ion fluxes are known in alkali and alkaline earth metals but are not well documented in transition metals. Here we describe major changes in the zinc physiology of the mammalian oocyte as it matures and initiates embryonic development. Single-cell elemental analysis of mouse oocytes by synchrotron-based X-ray fluorescence microscopy (XFM) revealed a 50% increase in total zinc content within the 12-14-h period of meiotic maturation. Perturbation of zinc homeostasis with a cell-permeable small-molecule chelator blocked meiotic progression past telophase I. Zinc supplementation rescued this phenotype when administered before this meiotic block. However, after telophase arrest, zinc triggered parthenogenesis, suggesting that exit from this meiotic step is tightly regulated by the availability of a zinc-dependent signal. These results implicate the zinc bolus acquired during meiotic maturation as an important part of the maternal legacy to the embryo.

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.

Microtubule dynamics in mitosis in Aspergillus nidulans.

Mitosis in Aspergillus nidulans is very rapid, requiring less than 5 min at 37 °C in germlings (Bergen and Morris, 1983). In this time the cytoplasmic microtubules (MTs) must disassemble, the mitotic spindle assemble, function and disassemble, and cytoplasmic MTs reassemble. It follows that cytoplasmic MTs must be extremely dynamic in this period and we were interested, in particular, in examining the processes of MT disassembly in prophase and reassembly in anaphase and telophase. We observed a diploid strain that expressed GFP-α-tubulin. We used a spinning disk confocal microscope that allowed rapid image capture, which proved necessary because microtubule dynamics were extremely rapid. We found, for the first time, that microtubule severing occurs in prophase in a filamentous fungus and that catastrophe rather than nucleation limits astral microtubule growth.

The polarity and dynamics of microtubule assembly in the budding yeast Saccharomyces cerevisiae.

Microtubule assembly in Saccharomyces cerevisiae is initiated from sites within spindle pole bodies (SPBs) in the nuclear envelope. Microtubule plus ends are thought to be organized distal to the SPBs, while minus ends are proximal. Several hypotheses for the function of microtubule motor proteins in force generation and regulation of microtubule assembly propose that assembly and disassembly occur at minus ends as well as at plus ends. Here we analyse microtubule assembly relative to the SPBs in haploid yeast cells expressing green fluorescent protein fused to alpha-tubulin, a microtubule subunit. Throughout the cell cycle, analysis of fluorescent speckle marks on cytoplasmic astral microtubules reveals that there is no detectable assembly or disassembly at minus ends. After laser-photobleaching, metaphase spindles recover about 63% of the bleached fluorescence, with a half-life of about 1 minute. After anaphase onset, photobleached marks in the interpolar spindle are persistent and do not move relative to the SPBs. In late anaphase, the elongated spindles disassemble at the microtubule plus ends. These results show for astral and anaphase interpolar spindle microtubules, and possibly for metaphase spindle microtubules, that microtubule assembly and disassembly occur at plus, and not minus, ends.

Cell-cycle progression without an intact microtuble cytoskeleton.

For mammalian somatic cells, the importance of microtubule cytoskeleton integrity during interphase cell-cycle progression is uncertain. The loss, suppression, or stabilization of the microtubule cytoskeleton has been widely reported to cause a G1 arrest in a variable, and often high, proportion of cell populations, suggesting the existence of a "microtubule damage," "microtubule integrity," or "postmitotic" checkpoint in G1 or G2. We found that when normal human cells (hTERT RPE1 and primary fibroblasts) are continuously exposed to nocodazole, they remain in mitosis for 10-48 hr before they slip out of mitosis and arrest in G1; this finding is consistent with previous reports. To eliminate the persistent effects of prolonged mitosis, we isolated anaphase-telophase cells that were just finishing a mitosis of normal duration, then we rapidly and completely disassembled microtubules by chilling the preparations to 0 degrees C for 10 minutes in the continuous presence of nocodazole or colcemid treatment to ensure that the cells entered G1 without a microtubule cytoskeleton. Without microtubules, cells progressed from anaphase to a subsequent mitosis with essentially normal kinetics. Similar results were obtained for cells in which the microtubule cytoskeleton was partially diminished by lower nocodazole doses or augmented and stabilized with taxol. Thus, after a preceding mitosis of normal duration, the integrity of the microtubule cytoskeleton is not subject to checkpoint surveillance, nor is it required for the normal human cell to progress through G1 and the remainder of interphase.

SAF-A/hnRNP-U localization in interphase and metaphase.

SAF-A/hnRNP U is an abundant nuclear protein that interacts specifically with nuclear matrix attachment region DNA (MAR) and RNA as a component of hnRNPs. SAF-A/hnRNP U was also shown to specifically bind mouse major satellite DNA (satMa). Antibodies against SAF-A and GFP-fusion constructs were used in the current work in order to trace SAF-A localization. In accordance with its diverse nucleic acid binding specificity, SAF-A was found to be localized in three different domains: outside the chromosomes, on the surface of the chromosome arms (probably MARs), and in the centromere region where it apparently binds specifically to the satMa. GFP-fusion constructs with different SAF-A/hnRNP U domains confirms the functional significance of the protein's functional domains in interphase cells. In telophase cells, the anti-SAF-A antibody signal appeared as a kind of network covering unfolded chromosomes.

The drosophila protein asp is involved in microtubule organization during spindle formation and cytokinesis.

Abnormal spindle (Asp) is a 220-kD microtubule-associated protein from Drosophila that has been suggested to be involved in microtubule nucleation from the centrosome. Here, we show that Asp is enriched at the poles of meiotic and mitotic spindles and localizes to the minus ends of central spindle microtubules. Localization to these structures is independent of a functional centrosome. Moreover, colchicine treatment disrupts Asp localization to the centrosome, indicating that Asp is not an integral centrosomal protein. In both meiotic and mitotic divisions of asp mutants, microtubule nucleation occurs from the centrosome, and gamma-tubulin localizes correctly. However, spindle pole focusing and organization are severely affected. By examining cells that carry mutations both in asp and in asterless, a gene required for centrosome function, we have determined the role of Asp in the absence of centrosomes. Phenotypic analysis of these double mutants shows that Asp is required for the aggregation of microtubules into focused spindle poles, reinforcing the conclusion that its function at the spindle poles is independent of any putative role in microtubule nucleation. Our data also suggest that Asp has a role in the formation of the central spindle. The inability of asp mutants to correctly organize the central spindle leads to disruption of the contractile ring machinery and failure in cytokinesis.

The dynamics of postmitotic reassembly of the nucleolus.

Mammalian cell nucleoli disassemble at the onset of M-phase and reassemble during telophase. Recent studies showed that partially processed preribosomal RNA (pre-rRNA) is preserved in association with processing components in the perichromosomal regions (PRs) and in particles called nucleolus-derived foci (NDF) during mitosis. Here, the dynamics of nucleolar reassembly were examined for the first time in living cells expressing fusions of the processing-related proteins fibrillarin, nucleolin, or B23 with green fluorescent protein (GFP). During telophase the NDF disappeared with a concomitant appearance of material in the reforming nuclei. Prenucleolar bodies (PNBs) appeared in nuclei in early telophase and gradually disappeared as nucleoli formed, strongly suggesting the transfer of PNB components to newly forming nucleoli. Fluorescence recovery after photobleaching (FRAP) showed that fibrillarin-GFP reassociates with the NDF and PNBs at rapid and similar rates. The reentry of processing complexes into telophase nuclei is suggested by the presence of pre-rRNA sequences in PNBs. Entry of specific proteins into the nucleolus approximately correlated with the timing of processing events. The mitotically preserved processing complexes may be essential for regulating the distribution of components to reassembling daughter cell nucleoli.

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.

Differential interaction of splicing snRNPs with coiled bodies and interchromatin granules during mitosis and assembly of daughter cell nuclei.

In the interphase nucleus of mammalian cells the U1, U2, U4/U6, and U5 small nuclear ribonucleoproteins (snRNPs), which are subunits of spliceosomes, associate with specific subnuclear domains including interchromatin granules and coiled bodies. Here, we analyze the association of splicing snRNPs with these structures during mitosis and reassembly of daughter nuclei. At the onset of mitosis snRNPs are predominantly diffuse in the cytoplasm, although a subset remain associated with remnants of coiled bodies and clusters of mitotic interchromatin granules, respectively. The number and size of mitotic coiled bodies remain approximately unchanged from metaphase to early telophase while snRNP-containing clusters of mitotic interchromatin granules increase in size and number as cells progress from anaphase to telophase. During telophase snRNPs are transported into daughter nuclei while the clusters of mitotic interchromatin granules remain in the cytoplasm. The timing of nuclear import of splicing snRNPs closely correlates with the onset of transcriptional activity in daughter nuclei. When transcription restarts in telophase cells snRNPs have a diffuse nucleoplasmic distribution. As cells progress to G1 snRNP-containing clusters of interchromatin granules reappear in the nucleus. Coiled bodies appear later in G1, although the coiled body antigen, p80 coilin, enters early into telophase nuclei. After inhibition of transcription we still observe nuclear import of snRNPs and the subsequent appearance of snRNP-containing clusters of interchromatin granules, but not coiled body formation. These data demonstrate that snRNP associations with coiled bodies and interchromatin granules are differentially regulated during the cell division cycle and suggest that these structures play distinct roles connected with snRNP structure, transport, and/or function.

Delay of HeLa cell cleavage into interphase using dihydrocytochalasin B: retention of a postmitotic spindle and telophase disc correlates with synchronous cleavage recovery.

The molecular signals that determine the position and timing of the cleavage furrow during mammalian cell cytokinesis are presently unknown. We have studied in detail the effect of dihydrocytochalasin B (DCB), a drug that interferes with actin assembly, on specific late mitotic events in synchronous HeLa cells. When cleavage furrow formation is blocked at 10 microM DCB, cells return to interphase by the criteria of reformation of nuclei with lamin borders, degradation of the cyclin B component of p34cdc2 kinase, and loss of mitosis specific MPM-2 antigens. However, the machinery for cell cleavage is retained for up to one hour into G1 when cleavage cannot proceed. The components retained consist prominently of a "postmitotic" spindle and a telophase disc, a structure templated by the mitotic spindle in anaphase that may determine the position and timing of the cleavage furrow. Upon release from DCB block, G1 cells proceed through a rapid and synchronous cleavage. We conclude that the mitotic spindle is not inevitably destroyed at the end of mitosis, but persists as an integral structure with the telophase disc in the absence of cleavage. We also conclude that cell cleavage can occur in G1, and is therefore an event metabolically independent of mitosis. The retained telophase disc may indeed signal the position of furrow formation, as G1 cleavage occurs only in the position where the retained disc underlies the cell cortex. The protocol we describe should now enable development of a model system for the study of mammalian cell cleavage as a synchronous event independent of mitosis.

Golgi clusters and vesicles mediate mitotic inheritance independently of the endoplasmic reticulum.

We have examined the fate of Golgi membranes during mitotic inheritance in animal cells using four-dimensional fluorescence microscopy, serial section reconstruction of electron micrographs, and peroxidase cytochemistry to track the fate of a Golgi enzyme fused to horseradish peroxidase. All three approaches show that partitioning of Golgi membranes is mediated by Golgi clusters that persist throughout mitosis, together with shed vesicles that are often found associated with spindle microtubules. We have been unable to find evidence that Golgi membranes fuse during the later phases of mitosis with the endoplasmic reticulum (ER) as a strategy for Golgi partitioning (Zaal, K.J., C.L. Smith, R.S. Polishchuk, N. Altan, N.B. Cole, J. Ellenberg, K. Hirschberg, J.F. Presley, T.H. Roberts, E. Siggia, et al. 1999. Cell. 99:589-601) and suggest that these results, in part, are the consequence of slow or abortive folding of GFP-Golgi chimeras in the ER. Furthermore, we show that accurate partitioning is accomplished early in mitosis, by a process of cytoplasmic redistribution of Golgi fragments and vesicles yielding a balance of Golgi membranes on either side of the metaphase plate before cell division.