A temperature-sensitive mutation of the Schizosaccharomyces pombe gene nuc2+ that encodes a nuclear scaffold-like protein blocks spindle elongation in mitotic anaphase.

A temperature-sensitive mutant nuc2-663 of the fission yeast Schizosaccharomyces pombe specifically blocks mitotic spindle elongation at restrictive temperature so that nuclei in arrested cells contain a short uniform spindle (approximately 3-micron long), which runs through a metaphase plate-like structure consisting of three condensed chromosomes. In the wild-type or in the mutant cells at permissive temperature, the spindle is fully extended approximately 15-micron long in anaphase. The nuc2' gene was cloned in a 2.4-kb genomic DNA fragment by transformation, and its complete nucleotide sequence was determined. Its coding region predicts a 665-residues internally repeating protein (76.250 mol wt). By immunoblots using anti-sera raised against lacZ-nuc2+ fused proteins, a polypeptide (designated p67; 67,000 mol wt) encoded by nuc2+ is detected in the wild-type S. pombe extracts; the amount of p67 is greatly increased when multi-copy or high-expression plasmids carrying the nuc2+ gene are introduced into the S. pombe cells. Cellular fractionation and Percoll gradient centrifugation combined with immunoblotting show that p67 cofractionates with nuclei and is enriched in resistant structure that is insoluble in 2 M NaCl, 25 mM lithium 3,5'-diiodosalicylate, and 1% Triton but is soluble in 8 M urea. In nuc2 mutant cells, however, soluble p76, perhaps an unprocessed precursor, accumulates in addition to insoluble p67. The role of nuc2+ gene may be to interconnect nuclear and cytoskeletal functions in chromosome separation.

Temporal and spatial coordination of chromosome movement, spindle formation, and nuclear envelope breakdown during prometaphase in Drosophila melanogaster embryos.

The spatial and temporal dynamics of diploid chromosome organization, microtubule arrangement, and the state of the nuclear envelope have been analyzed in syncytial blastoderm embryos of Drosophila melanogaster during the transition from prophase to metaphase, by three-dimensional optical sectioning microscopy. Time-lapse, three-dimensional data recorded in living embryos revealed that congression of chromosomes (the process whereby chromosomes move to form the metaphase plate) at prometaphase occurs as a wave, starting at the top of the nucleus near the embryo surface and proceeding through the nucleus to the bottom. The time-lapse analysis was augmented by a high-resolution analysis of fixed embryos where it was possible to unambiguously trace the three-dimensional paths of individual chromosomes. In prophase, the centromeres were found to be clustered at the top of the nucleus while the telomeres were situated at the bottom of the nucleus or towards the embryo interior. This polarized centromere-telomere orientation, perpendicular to the embryo surface, was preserved during the process of prometaphase chromosome congression. Correspondingly, breakdown of the nuclear envelope started at the top of the nucleus with the mitotic spindle being formed at the positions of the partial breakdown of the nuclear envelope. Our observation provide an example in which nuclear structures are spatially organized and their functions are locally and coordinately controlled in three dimensions.

A cytoplasmic dynein heavy chain is required for oscillatory nuclear movement of meiotic prophase and efficient meiotic recombination in fission yeast.

Meiotic recombination requires pairing of homologous chromosomes, the mechanisms of which remain largely unknown. When pairing occurs during meiotic prophase in fission yeast, the nucleus oscillates between the cell poles driven by astral microtubules. During these oscillations, the telomeres are clustered at the spindle pole body (SPB), located at the leading edge of the moving nucleus and the rest of each chromosome dangles behind. Here, we show that the oscillatory nuclear movement of meiotic prophase is dependent on cytoplasmic dynein. We have cloned the gene encoding a cytoplasmic dynein heavy chain of fission yeast. Most of the cells disrupted for the gene show no gross defect during mitosis and complete meiosis to form four viable spores, but they lack the nuclear movements of meiotic prophase. Thus, the dynein heavy chain is required for these oscillatory movements. Consistent with its essential role in such nuclear movement, dynein heavy chain tagged with green fluorescent protein (GFP) is localized at astral microtubules and the SPB during the movements. In dynein-disrupted cells, meiotic recombination is significantly reduced, indicating that the dynein function is also required for efficient meiotic recombination. In accordance with the reduced recombination, which leads to reduced crossing over, chromosome missegregation is increased in the mutant. Moreover, both the formation of a single cluster of centromeres and the colocalization of homologous regions on a pair of homologous chromosomes are significantly inhibited in the mutant. These results strongly suggest that the dynein-driven nuclear movements of meiotic prophase are necessary for efficient pairing of homologous chromosomes in fission yeast, which in turn promotes efficient meiotic recombination.

A novel fission yeast gene, tht1+, is required for the fusion of nuclear envelopes during karyogamy.

We have isolated a fission yeast karyogamy mutant, tht1, in which nuclear congression and the association of two spindle pole bodies occurs but the subsequent fusion of nuclear envelopes is blocked. The tht1 mutation does not prevent meiosis, so cells execute meiosis with two unfused nuclei, leading to the production of aberrant asci. The tht1(+) gene was cloned and sequenced. Predicted amino acid sequence has no significant homology to previously known proteins but strongly suggests that it is a type I membrane protein. The tht1(+) gene is dispensable for vegetative growth and expressed only in conjugating cells. Tht1p is a glycoprotein susceptible to endoglycosilase H digestion. Site- directed mutagenesis showed that the N-glycosylation site, as well as the COOH-terminal region of Tht1p, is essential for its function. A protease protection assay indicated that the COOH terminus is cytoplasmic. Immunocytological analysis using a HA-tagged Tht1p suggested that the protein is localized in nuclear envelopes and in the ER during karyogamy and that its levels are reduced in cells containing fused nuclei.

The onset of homologous chromosome pairing during Drosophila melanogaster embryogenesis.

We have determined the position within the nucleus of homologous sites of the histone gene cluster in Drosophila melanogaster using in situ hybridization and high-resolution, three-dimensional wide field fluorescence microscopy. A 4.8-kb biotinylated probe for the histone gene repeat, located approximately midway along the short arm of chromosome 2, was hybridized to whole-mount embryos in late syncytial and early cellular blastoderm stages. Our results show that the two homologous histone loci are distinct and separate through all stages of the cell cycle up to nuclear cycle 13. By dramatic contrast, the two homologous clusters were found to colocalize with high frequency during interphase of cycle 14. Concomitant with homolog pairing at cycle 14, both histone loci were also found to move from their position near the midline of the nucleus toward the apical side. This result suggests that coincident with the initiation of zygotic transcription, there is dramatic chromosome and nuclear reorganization between nuclear cycles 13 and 14.

The use of a charge-coupled device for quantitative optical microscopy of biological structures.

The properties of a charge-coupled device (CCD) and its application to the high-resolution analysis of biological structures by optical microscopy are described. The CCD, with its high resolution, high sensitivity, wide dynamic range, photometric accuracy, and geometric stability, can provide data of such high quality that quantitative analysis on two- and three-dimensional microscopic images is possible. For example, the three-dimensional imaging properties of an epifluorescence microscope have been quantitatively determined with the CCD. This description of the imaging properties of the microscope, and the high-quality image data provided by the CCD, allow sophisticated computational image processing methods to be used that greatly improve the effective resolution obtainable for biological structures. Image processing techniques revealed fine substructures in Drosophila embryonic diploid chromosomes in two and three dimensions. The same approach can be extended to structures as small as yeast chromosomes or to other problems in structural cell biology.

Telomere-led premeiotic chromosome movement in fission yeast.

The movement of chromosomes that precedes meiosis was observed in living cells of fission yeast by fluorescence microscopy. Further analysis by in situ hybridization revealed that the telomeres remain clustered at the leading end of premeiotic chromosome movement, unlike mitotic chromosome movement in which the centromere leads. Once meiotic chromosome segregation starts, however, centromeres resume the leading position in chromosome movement, as they do in mitosis. Although the movement of the telomere first has not been observed before, the clustering of telomeres is reminiscent of the bouquet structure of meiotic-prophase chromosomes observed in higher eukaryotes, which suggests that telomeres perform specific functions required for premeiotic chromosomal events generally in eukaryotes.

Telomere binding of the Rap1 protein is required for meiosis in fission yeast.

Telomeres are essential for chromosome integrity, protecting the ends of eukaryotic linear chromosomes during cell proliferation. Telomeres also function in meiosis; a characteristic clustering of telomeres beneath the nuclear membrane is observed during meiotic prophase in many organisms from yeasts to plants and humans, and the role of the telomeres in meiotic pairing and the recombination of homologous chromosomes has been demonstrated in the fission yeast Schizosaccharomyces pombe and in the budding yeast Saccharomyces cerevisiae. Here we report that S. pombe Rap1 is a telomeric protein essential for meiosis. While Rap1 is conserved in budding yeast and humans, schemes for telomere binding vary among species: human RAP1 binds to the telomere through interaction with the telomere binding protein TRF2; S. cerevisiae Rap1, however, binds telomeric DNA directly, and no orthologs of TRF proteins have been identified in this organism. In S. pombe, unlike in S. cerevisiae, an ortholog of human TRF has been identified. This ortholog, Taz1, binds directly to telomere repeats [18] and is necessary for telomere clustering in meiotic prophase. Our results demonstrate that S. pombe Rap1 binds to telomeres through interaction with Taz1, similar to human Rap1-TRF2, and that Taz1-mediated telomere localization of Rap1 is necessary for telomere clustering and for the successful completion of meiosis. Moreover, in taz1-disrupted cells, molecular fusion of Rap1 with the Taz1 DNA binding domain recovers telomere clustering and largely complements defects in meiosis, indicating that telomere localization of Rap1 is a key requirement for meiosis.

Identification of ribonucleotide reductase protein R1 as an activator of microtubule nucleation in Xenopus egg mitotic extracts.

Microtubule nucleation on the centrosome and the fungal equivalent, the spindle pole body (SPB), is activated at the onset of mitosis. We previously reported that mitotic extracts prepared from Xenopus unfertilized eggs convert the interphase SPB of fission yeast into a competent state for microtubule nucleation. In this study, we have purified an 85-kDa SPB activator from the extracts and identified it as the ribonucleotide reductase large subunit R1. We further confirmed that recombinant mouse R1 protein was also effective for SPB activation. On the other hand, another essential subunit of ribonucleotide reductase, R2 protein, was not required for SPB activation. SPB activation by R1 protein was suppressed in the presence of anti-R1 antibodies or a partial oligopeptide of R1; the oligopeptide also inhibited aster formation on Xenopus sperm centrosomes. In accordance, R1 was detected in animal centrosomes by immunofluorescence and immunoblotting with anti-R1 antibodies. In addition, recombinant mouse R1 protein bound to gamma- and alpha/beta-tubulin in vitro. These results suggest that R1 is a bifunctional protein that acts on both ribonucleotide reduction and centrosome/SPB activation.

Nucleolar accumulation of poly (A)+ RNA in heat-shocked yeast cells: implication of nucleolar involvement in mRNA transport.

Transport of mRNA from the nucleus to the cytoplasm plays an important role in gene expression in eukaryotic cells. In wild-type Schizosaccharomyces pombe cells poly(A)+ RNA is uniformly distributed throughout the nucleoplasm and cytoplasm. However, we found that a severe heat shock blocks mRNA transport in S. pombe, resulting in the accumulation of bulk poly(A)+ RNA, as well as a specific intron-less transcript, in the nucleoli. Pretreatment of cells with a mild heat shock, which induces heat shock proteins, before a severe heat shock protects the mRNA transport machinery and allows mRNA transport to proceed unimpeded. In heat-shocked S. pombe cells, the nucleolar region condensed into a few compact structures. Interestingly, poly(A)+ RNA accumulated predominantly in the condensed nucleolar regions of the heat-shocked cells. These data suggest that the yeast nucleolus may play a role in mRNA transport in addition to its roles in rRNA synthesis and preribosome assembly.

Nucleolar accumulation of poly (A)+ RNA in heat-shocked yeast cells: implication of nucleolar involvement in mRNA transport.

Transport of mRNA from the nucleus to the cytoplasm plays an important role in gene expression in eukaryotic cells. In wild-type Schizosaccharomyces pombe cells poly(A)+ RNA is uniformly distributed throughout the nucleoplasm and cytoplasm. However, we found that a severe heat shock blocks mRNA transport in S. pombe, resulting in the accumulation of bulk poly(A)+ RNA, as well as a specific intron-less transcript, in the nucleoli. Pretreatment of cells with a mild heat shock, which induces heat shock proteins, before a severe heat shock protects the mRNA transport machinery and allows mRNA transport to proceed unimpeded. In heat-shocked S. pombe cells, the nucleolar region condensed into a few compact structures. Interestingly, poly(A)+ RNA accumulated predominantly in the condensed nucleolar regions of the heat-shocked cells. These data suggest that the yeast nucleolus may play a role in mRNA transport in addition to its roles in rRNA synthesis and preribosome assembly.

Dynamic behavior of microtubules during dynein-dependent nuclear migrations of meiotic prophase in fission yeast.

During meiotic prophase in fission yeast, the nucleus migrates back and forth between the two ends of the cell, led by the spindle pole body (SPB). This nuclear oscillation is dependent on astral microtubules radiating from the SPB and a microtubule motor, cytoplasmic dynein. Here we have examined the dynamic behavior of astral microtubules labeled with the green fluorescent protein during meiotic prophase with the use of optical sectioning microscopy. During nuclear migrations, the SPB mostly follows the microtubules that extend toward the cell cortex. SPB migrations start when these microtubules interact with the cortex and stop when they disappear, suggesting that these microtubules drive nuclear migrations. The microtubules that are followed by the SPB often slide along the cortex and are shortened by disassembly at their ends proximal to the cortex. In dynein-mutant cells, where nuclear oscillations are absent, the SPB never migrates by following microtubules, and microtubule assembly/disassembly dynamics is significantly altered. Based on these observations, together with the frequent accumulation of dynein at a cortical site where the directing microtubules interact, we propose a model in which dynein drives nuclear oscillation by mediating cortical microtubule interactions and regulating the dynamics of microtubule disassembly at the cortex.

Fission yeast genes nda1+ and nda4+, mutations of which lead to S-phase block, chromatin alteration and Ca2+ suppression, are members of the CDC46/MCM2 family.

Fission yeast cold-sensitive mutants nda1-376 and nda4-108 display a cell cycle block phenotype at the restrictive temperature (cell elongation with the single nucleus) accompanied by an alteration in the nuclear chromatin region. DNA content analysis shows that the onset of DNA synthesis is blocked or greatly delayed in both mutant cells, the block being reversible in nda4-108. Upon release to the permissive temperature, nda4-108 cells resumed replicating DNA, followed by mitosis and cytokinesis. The nda4 phenotype was partly rescued by the addition of Ca2+ to the medium; Ca2+ plays a positive role in the nda4+ function. The predicted protein sequences of nda1+ and nda4+ isolated by complementation are similar to each other and also, respectively, to those of the budding yeast, MCM2 and CDC46, both of which are members of the gene family required for the initiation of DNA replication. The central domains of these proteins are conserved, whereas the NH2- and COOH- domains are distinct. Results of the disruption of the nda1+ and nda4+ genes demonstrates that they are essential for viability.

Dynamics of centromeres during metaphase-anaphase transition in fission yeast: Dis1 is implicated in force balance in metaphase bipolar spindle.

In higher eukaryotic cells, the spindle forms along with chromosome condensation in mitotic prophase. In metaphase, chromosomes are aligned on the spindle with sister kinetochores facing toward the opposite poles. In anaphase A, sister chromatids separate from each other without spindle extension, whereas spindle elongation takes place during anaphase B. We have critically examined whether such mitotic stages also occur in a lower eukaryote, Schizosaccharomyces pombe. Using the green fluorescent protein tagging technique, early mitotic to late anaphase events were observed in living fission yeast cells. S. pombe has three phases in spindle dynamics, spindle formation (phase 1), constant spindle length (phase 2), and spindle extension (phase 3). Sister centromere separation (anaphase A) rapidly occurred at the end of phase 2. The centromere showed dynamic movements throughout phase 2 as it moved back and forth and was transiently split in two before its separation, suggesting that the centromere was positioned in a bioriented manner toward the poles at metaphase. Microtubule-associating Dis1 was required for the occurrence of constant spindle length and centromere movement in phase 2. Normal transition from phase 2 to 3 needed DNA topoisomerase II and Cut1 but not Cut14. The duration of each phase was highly dependent on temperature.

Pore configuration landscape of granular crystallization.

Uncovering grain-scale mechanisms that underlie the disorder-order transition in assemblies of dissipative, athermal particles is a fundamental problem with technological relevance. To date, the study of granular crystallization has mainly focussed on the symmetry of crystalline patterns while their emergence and growth from irregular clusters of grains remains largely unexplored. Here crystallization of three-dimensional packings of frictional spheres is studied at the grain-scale using X-ray tomography and persistent homology. The latter produces a map of the topological configurations of grains within static partially crystallized packings. Using numerical simulations, we show that similar maps are measured dynamically during the melting of a perfect crystal. This map encodes new information on the formation process of tetrahedral and octahedral pores, the building blocks of perfect crystals. Four key formation mechanisms of these pores reproduce the main changes of the map during crystallization and provide continuous deformation pathways representative of the crystallization dynamics.

Cloning and expression of a human/mouse Polycomb group gene, ENX-2/Enx-2.

The Drosophila Polycomb group (Pc-G) genes encode transcriptional factors involved in development. Little is known about members of the vertebrate Pc-G genes. In this study, we have isolated a cDNA encoding a human Pc-G protein and the mouse equivalent. The human and mouse genes, which were named ENX-2 and Enx-2, encode 702 and 750 amino acids, respectively. ENX-2/Enx-2 protein exhibits a high homology (53-55% identity) to Drosophila Enhancer of zeste [E(z)] protein belonging to the Pc-G. The expression of Enx-2 was observed in mouse kidney, adrenal gland, testis and brain at high levels by Northern blot analysis. A cell line of mouse neuroblastoma, Neuro-2a, also expresses Enx-2 mRNA and its level is elevated by induction of neuronal differentiation of the cell.

Cloning and characterization of Xenopus laevis xSox7 cDNA.

A family of SRY-related genes has been termed SOX. We have isolated and sequenced a cDNA encoding a novel Sox protein from Xenopus laevis ovary. This cDNA contains an open reading frame (ORF) coding for 362 amino acids, which encompasses an HMG box and exhibits a strong (90%) identity to that of mouse Sox7; the cDNA was named xSox7 in this study. Northern analysis revealed that the xSox7 mRNA was 2.0 kb in length. Various adult frog tissues were tested by reverse transcription/polymerase chain reaction for xSox7 mRNA, and the results showed that xSox7 is expressed in a wide range of tissues. Furthermore, electrophoretic mobility shif assay indicated that recombinant xSox7 is capable of binding to AACAAT sequence.

Isolation and characterization of a mouse SRY-related cDNA, mSox7.

SOX is a family of SRY-related genes, which encode transcriptional factors involved in development. In this study, we newly isolated and sequenced mouse cDNA clones for mSox7. The mSox7 gene encodes 380 amino acids containing an SRY-type HMG box. Genomic Southern analysis suggested that the mSox7 gene was a single-copy gene. Tissue specific expression of mSox7 was investigated by Northern analysis. The expression was restricted to the ovary and heart, and the size of the transcripts was estimated to be 3.6 knt. Electrophoretic mobility shift assay indicated that recombinant mSox7 polypeptide was capable of binding to a nucleotide sequence, AACAAT. Immunohistochemical study revealed that mSox7 protein was localized in oocytes in the mouse ovary.

Molecular cloning and expression of Xenopus laevis requiem cDNA1.

Requiem is an apoptosis-associated gene, which was originally identified in mouse (T.G. Gabig et al., J. Biol. Chem. 269 (1994) 29515-29519). In this study, we isolated five independent cDNA clones for frog Requiem (xReq) from Xenopus laevis ovary. Sequence analysis of the multiple cDNAs has suggested that Xenopus genome contains at least two non-allelic copies of the xReq gene. The amino acid sequence deduced from the cDNAs contained a single Krüppel-type zinc-finger motif and two PHD-finger motifs. Northern analysis revealed that the ovary expressed xReq transcripts with different sizes.

Cloning and expression of Xenopus laevis xSox12 cDNA.

A family of SRY-related genes has been termed SOX. We have isolated and sequenced a cDNA encoding xSox12 from Xenopus laevis ovary. The cDNA contained an open reading frame (ORF) coding for 470 amino acids encompassing an HMG box characteristic of the SOX family, a leucine zipper motif and glutamine-rich segments. The size of the xSox12 mRNA was determined to be 3.0 knt by Northern analysis. The ovary was the most prominent in the expression of the Sox mRNA among the various tissues of adult frog as far as examined.