Immunolocalization of the 100 kDa nucleolar protein during the mitotic cycle in CHO cells

The localization of a major nucleolar protein with a molecular weight of 100,000 has been followed during mitosis in Chinese hamster ovary CHO cells using specific antibodies to this protein and immunocytochemical techniques. The 100 kDa protein was visualized at discrete sites on metaphase chromosomes, corresponding to nucleolus organizer regions, and in large, immunostained nucleolar remnants that are discarded in the cytoplasm after nucleolar disintegration. After mitosis, the 100 kDa protein was shown to play an early role in nucleolar reformation. It was first detected in small deposits around the anaphase chromosomes. In telophase, the protein accumulated simultaneously in prenucleolar bodies and in the reforming nucleoli. The early presence of the 100 kDa protein in the telophase nucleus suggests that it is essential for the reestablishment of nucleolar function after mitosis. Thus this protein is present throughout the CHO cell cycle, an observation which supports the hypothesis that it plays a fundamental role in cell organization.     

The Three-dimensional Study of Chromosomes and Upstream Binding Factor-immunolabeled Nucleolar Organizer Regions Demonstrates Their Nonrandom Spatial Arrangement during Mitosis

The volumic rearrangement of both chromosomes and immunolabeled upstream binding factor in entire well-preserved mitotic cells was studied by confocal microscopy. By using high-quality three-dimensional visualization and tomography, it was possible to investigate interactively the volumic organization of chromosome sets and to focus on their internal characteristics. More particularly, this study demonstrates the nonrandom positioning of metaphase chromosomes bearing nucleolar organizer regions as revealed by their positive upstream binding factor immunolabeling. During the complex morphogenesis of the progeny nuclei from anaphase to late telophase, the equal partitioning of the nucleolar organizer regions is demonstrated by quantification, and their typical nonrandom central positioning within the chromosome sets is revealed.     

Mitotic Microtubule Development and Histone H3 Phosphorylation in the Holocentric Chromosomes of Rhynchospora Tenuis (Cyperaceae)

In the present work we report the phosphorylation pattern of histone H3 and the development of microtubular structures using immunostaining techniques, in mitosis of Rhynchospora tenuis (2n = 4), a Cyperaceae with holocentric chromosomes. The main features of the holocentric chromosomes of R. tenuis coincide with those of other species namely: the absence of primary constriction in prometaphase and metaphase, and the parallel separation of sister chromatids at anaphase. Additionaly, we observed a highly conserved chromosome positioning at anaphase and early telophase sister nuclei. Four microtubule arrangements were distinguished during the root tip cell cycle. Interphase cells showed a cortical microtubule arrangement that progressively forms the characteristic pre-prophase band. At prometaphase the microtubules were homogeneously distributed around the nuclear envelope. Metaphase cells displayed the spindle arrangement with kinetochore microtubules attached throughout the entire chromosome extension. At anaphase kinetochoric microtubules become progressively shorter, whereas bundles of interzonal microtubules became increasingly broader and denser. At late telophase the microtubules were observed equatorially extended beyond the sister nuclei and reaching the cell wall. Immunolabelling with an antibody against phosphorylated histone H3 revealed the four chromosomes labelled throughout their entire extension at metaphase and anaphase. Apparently, the holocentric chromosomes of R. tenuis function as an extended centromeric region both in terms of cohesion and H3 phosphorylation.     

Kinetochore and microtubules in two members of Chlorophyceae,Cladophora fracta and Spirogyra majuscula

Evidence is presented for the existence of a localised kinetochore with stratified fine structure in Cladophora and in Spirogyra. In the latter, there is the possibility of two kinetochores on the longer chromosomes. There is no evidence for a diffuse kinetochore. The nucleolus persists during mitosis in Cladophora on the nucleolar organising chromosomes, the granular material being lost from it very largely during metaphase and anaphase but the fibrillar material remaining. The persistent nucleolar material at metaphase and anaphase in Spirogyra is not attached to the nucleolar organising chromosomes but accumulates around all the chromosomes and chromatids, the microtubules of the spindle at anaphase passing through and possibly attaching to this nucleolar material and possibly assisting in the movement of the chromatids which are embedded within it.     

CELL DIVISION IN SPIROGYRA. I. MITOSIS

Dividing cells of Spirogyra sp. were examined with both the light and electron microscopes. By preprophase many of the typical transverse wall micro-tubules disappeared while others were seen in the thickened cytoplasmic strands. Microtubules appeared in the polar cytoplasm at prophase and by prometaphase they penetrated the nucleus. They were attached to chromosomes at metaphase and early anaphase, and formed a sheath surrounding the spindle during anaphase; they were seen in the interzonal strands and cytoplasmic strands at telophase. The interphase nucleolus, containing 2 distinct zones and chromatinlike material, fragmented at prophase; at metaphase and anaphase nucleolar material coated the chromosomes, obscuring them by late anaphase. The chromosomes condensed in the nucleoplasm at prophase, moving into the nucleolus at prometaphase. The nuclear envelope was finally disrupted at anaphase during spindle elongation; at telophase membrane profiles coated the reforming nuclei. During anaphase and early telophase the interzonal region contained vacuoles, a few micro-tubules, and sometimes eliminated n ucleolar material; most small organelles, including swollen endoplasmic reticulum and tubular membranes, were concentrated in the polar cytoplasm. Quantitative and qualitative cytological observations strongly suggest movement of intact wall rnicrotubules to the spindle at preprophase and then back again at telophase.     

Behaviour of nucleolus during mitosis

The aim of the present work was to study the distribution and the behaviour of the silver-staining nucleolar organizer region (Ag-NOR) proteins at the ultrastructural level during interphase and mitosis in five human and murine cancerous cell lines each characterized by a typical nucleolar morphology. During interphase the Ag-NOR proteins are restricted to the fibrillar centres (F.C.) and/or to the dense fibrillar component (D.F.C.). During prophase the silver-staining components come into close contact with some chromosomes and are arranged with a typical polarity: chromosome, F.C. and D.F.C. Then F.C. and D.F.C. together form roundish silver-stained structures and integrate in part within indentations at the periphery of the metaphase chromosomes. During anaphase and telophase large and small spherical silver-staining structures may be seen. They correspond respectively to the metaphase NORs and to numerous structures which appear de novo within ribonucleoprotein (RNP) material localized between the chromosomes. During late telophase the number of the small silver-staining structures decreases whereas the size of the larger ones increases. Then the interphase nucleoli recover their typical shape. These results suggest that when rRNA synthesis is impaired during mitosis the inactive NORs assume a structure and a localization which are not typical of the cell line. In contrast the F.C. and D.F.C. are probably two aspects of the NORs whose typical distribution, relative to the other nucleolar components, gives the interphasic nucleolus its characteristic morphology.     

A temperature-sensitive CHO-K1 cell mutant (tsTM13) defective in chromosome decondensation and spindle deconstruction in M phase

A temperature-sensitive CHO-K1 cell mutant, tsTM13, exhibited a delayed cell cycle progression from metaphase to telophase at a nonpermissive temperature and was finally arrested from anaphase to telophase. Metaphase chromosomes were overcondensed and chromosome disjunction in anaphase was uncoordinated. In telophase, sister chromatids were segregated and cytokinesis was completed, but chromosome structure remained in a condensed state and the spindle was not deconstructed. The level of phosphorylation of histones H1 and H3 remained high in the later stages of mitosis and the activity of histone H1 kinase was also maintained at a high level. These results strongly suggest that the pleiotropic defects of tsTM13 cells in mitosis are associated with a lack of inactivation of activated histone H1 kinase.     

“Cuckoo” Aegilops addition chromosome in wheat ensures its transmission by causing chromosome breaks in meiospores lacking it

In monosomic additions of Aegilops sharonensis to Chinese Spring wheat (2n=42 wheat chromosomes + 1 homoeologous group 4 Aegilops chromosome known as 4S¹), probably all functional gametes carried one Aegilops chromosome. Such preferential transmission is unusual in monosomic alien additions. Male and female meiosis seemed usually normal, but about 75% of embryo sacs and 28% of pollen grains were visibly abnormal near anthesis. Before the first gametophyte mitosis, up to about 13% of megaspores and pollen grains showed abnormalities usual in wheat aneuploids. However, in first mitosis, 50% of megaspores at metaphase and anaphase and 41% of pollen grains at anaphase and telophase contained acentric chromosome (or chromatid) segments of various sizes, up to about 32 in a cell, which were separated from the rest of the chromosome (or chromatid) by a gap or a thin Feulgen-positive thread. Such separated segments (SSs) are not normally seen in wheat and its aneuploids. The data indicat that alien and wheat chromosomes interact in meiocytes so that meiospores with the alien chromosome develop into normal gametophytes, but meiospores lacking the alien chromosome have SSs at first mitosis by whose loss or unequal distribution between daughter nuclei sterilizing deficiencies arise. Thus only gametophytes with the alien chromosome are competent.     

Ultrastructure of mitosis and cytokinesis inZygnema sp. (Zygnematales,Chlorophyta)

Summary Mitosis and cytokinesis have been studied in the green algaZygnema C. A. Agardh using interference-contrast light and transmission electron microscopy. At prophase, the nucleolus disintegrates and numerous extranuclear microtubules near the nuclear periphery penetrate into the nucleoplasm. When aligned in the equatorial plane of the open metaphase spindle the chromosomes are coated with persistent nucleolar fragments. At anaphase, vacuoles intrude into the interzonal spindle region and seemingly contribute to the anaphase movement of the chromosomes. At telophase, the spindle is persistent and the reforming nuclei are separated by cytoplasmic strands containing microtubules, interspersed with vacuoles. Extensive bundles of microtubules, dictyosomes and parallel, slightly inflated ER-profiles extend from the poles of the telophase nucleus along the longitudinal side of the chloroplast. Conceivably, these microtubules guide the nucleus during its post-mitotic migration towards its central interphase position between the two halves of the dividing chloroplast. Throughout the mitotic cycle, ubiquitous dictyosomes, positioned near the chloroplast core, seem very active. Arrays of microtubules run towards these dictyosomes and may conduct the dictyosome-vesicles to the cleavage plane. At metaphase, septum growth becomes visible as an annular ingrowth of the plasmalemma. At late telophase or at entering interphase, an extensive clump of vesicles, associated with longitudinal bundles of microtubules, appears between the leading edges of the advanced furrow. Apparent fusion of these vesicles with the head of the centripetally-growing furrow results in its completion. The pattern of mitosis and cytokinesis inZygnema is compared with that of closely related green algae.     

禾谷镰孢菌Fusarium graminearum Schwabe分生孢子萌发过程中的核相变化及有丝分裂过程观察

通过Giemsa染色观察禾谷镰孢菌Fusarium graminearum分生孢子萌发过程中的核相变化及有丝分裂过程。观察表明,分生孢子细胞为单核,细胞核在分生孢子细胞内分裂后进入芽管,在芽管内进行多次分裂,使芽管内细胞核数目不断变化。禾谷镰孢菌有丝分裂过程可以分为4个时期,前期染色体逐渐浓缩变短,中期染色体清晰可见,后期染色单体发生分离并向相反的两极移动,末期形成新的子核。有丝分裂过程中染色体的分离同步或不同步,不同步分离中的滞后染色体形成后期桥的现象更为普遍。     

Microinjected centromere [corrected] kinetochore antibodies interfere with chromosome movement in meiotic and mitotic mouse oocytes [published erratum appears in J Cell Biol 1990 Dec;111(6 Pt 1):following 2800]

Kinetochores may perform several functions at mitosis and meiosis including: (a) directing anaphase chromosome separation, (b) regulating prometaphase alignment of the chromosomes at the spindle equator (congression), and/or (c) capturing and stabilizing microtubules. To explore these functions in vivo, autoimmune sera against the centromere/kinetochore complex are microinjected into mouse oocytes during specific phases of first or second meiosis, or first mitosis. Serum E.K. crossreacts with an 80-kD protein in mouse cells and detects the centromere/kinetochore complex in permeabilized cells or when microinjected into living oocytes. Chromosome separation at anaphase is not blocked when these antibodies are microinjected into unfertilized oocytes naturally arrested at second meiotic metaphase, into eggs at first mitotic metaphase, or into immature oocytes at first meiotic metaphase. Microtubule capture and spindle reformation occur normally in microinjected unfertilized oocytes recovering from cold or microtubule disrupting drugs; the chromosomes segregate correctly after parthenogenetic activation. Prometaphase congression is dramatically influenced when antikinetochore/centromere antibodies are introduced during interphase or in prometaphase-stage meiotic or mitotic eggs. At metaphase, these oocytes have unaligned chromosomes scattered throughout the spindle with several remaining at the poles; anaphase is aberrant and, after division, karyomeres are found in the polar body and oocyte or daughter blastomeres. Neither nonimmune sera, diffuse scleroderma sera, nor sham microinjections affect either meiosis or mitosis. These results suggest that antikinetochore/centromere antibodies produced by CREST patients interfere with chromosome congression at prometaphase in vivo.     

Trichomonas vaginalis: chromatin and mitotic spindle during mitosis

The mitotic phases and the changes that the chromatin and mitotic microtubules undergo during mitosis in the sexually transmitted parasite Trichomonas vaginalis are described. Parasites arrested in the gap 2 phase of the cell cycle by nutrient starvation were induced to mitosis by addition of fresh whole medium. [(3)H] Thymidine labeling of trichomonad parasites for 24 h showed that parasites have at least four synchronic duplications after mitosis induction. Fixed or live and acridine orange (AO)-stained trichomonads analyzed at different times during mitosis by epifluorescence microscopy showed that mitosis took about 45 min and is divided into five stages: prophase, metaphase, early and late anaphase, early and late telophase, and cytokinesis. The AO-stained nucleus of live trichomonads showed green (DNA) and orange (RNA) fluorescence, and the nucleic acid nature was confirmed by DNase and RNase treatment, respectively. The chromatin appeared partially condensed during interphase. At metaphase, it appeared as six condensed chromosomes, as recently reported, which decondensed at anaphase and migrated to the nuclear poles at telophase. In addition, small bundles of microtubules (as hemispindles) were detected only in metaphase with the polyclonal antibody anti-Entamoeba histolytica alpha-tubulin. This antibody showed that the hemispindle and an atractophore-like structure seem to duplicate and polarize during metaphase. In conclusion, T. vaginalis mitosis involves five mitotic phases in which the chromatin undergoes different degrees of condensation, from chromosomes to decondensed chromatin, and two hemispindles that are observed only in the metaphase stage.     

MITOSIS AND CYTOKINESIS IN THE CHLOROMONADOPHYCEAN ALGA GONYOSTOMUM SEMEN1

Mitosis and cytokinesis in Gonyostomum semen (Ehrenberg) Diesing have been investigated with the light microscope. During prophase nucleoli disappear and the chromatid structure of the chromosomes becomes apparent. Separation of chromatids at anaphase is accompanied by progressive fusion of the progeny chromosomes. This process continues into telophase by which stage the progeny nuclei consist of dense masses of chromatin with occasional chromosomes extending from their equatorial surfaces. By the end of telophase, nucleoli are reforming and the interphase nuclear morphology is reestablished. Mitosis is followed by cytokinesis, which is a relatively lengthy phase. In early cytokinesis the 2 interphase nuclei are present, and there is no indication of the forthcoming division of the cytoplasm. Later in cytokinesis a membrane is formed between the 2 nuclei. Final separation of the progeny individuals is accomplished by vigorous movements of swimming cells or, in the case of palmelloid cells, by the deposition of a mucilaginous layer.     

Cytogenetic Studies on Five Intermediate Forms of Progeny of Triticum aestivum Agropyron glaucum

A stuty has been made of the chromosome number of root-tip cells and the chro- mosome configuration of PMCs in five intermediate forms of progeny of Triticum aestivum × Agropyron glaucum. The somatic cells in each of these intermediate forms have 28 pairs of chromosomes (2n=56) and the PMCs at diakinesis or metaphase Ⅰ of meiosis have 28 bivalents. The process of meiosis in most PMCs is regular. Lagging chromosomes and fragments at anaphase Ⅰ or telophase Ⅰ were observed only in few PWCs. According to our results, all the five intermediate forms of progeny of T. aestivum × Ag. glaucum are allooctoploids. The application of these allooctoploids is also briefly discussed.     

Open spindle nuclear division in the amoebal phase of the acellular slime moldEchinostelium minutum with chromosomal movement related to the pronounced rearrangement of spindle microtubules

Summary Myxamoebae ofEchinostelium minutum exhibit extranuclear (open spindle) mitosis with centrioles present at the poles. Spindle microtubules are formed in association with a juxtanuclear MTOC which surrounds the cell's complement of centrioles. During late prophase or prometaphase the nuclear envelope breaks down and subsequently a metaphase plate is formed. Two anaphasic movements occur sequentially: firstly, the distance of the chromosomes to the poles shortens; secondly the distance between the spindle poles increases. The arrangement of spindle microtubules during anaphase is consistent with the hypothesis that chromosomal separation is due to lateral interaction (zippering) of microtubules. During telophase, reconstitution of the nuclear envelope usually takes place in the interzonal region prior to reformation in the polar region. Cytokinesis, which begins in anaphase or early telophase involves the participation of vesicles, microfilaments and microtubules.Based on the doctoral dissertation of the first author presented to the Department of Botany, University of Washington, Seattle, WA 98195, U.S.A.     

Cine-micrographic studies on dicentric chromosomes

Summary The behavior during mitosis of SR (sister reunion) bridges and other types of dicentric anaphase bridges was analysed. The endosperm of Haemanthus katharinae was used as material, and 16 mm time-lapse cine-film was analysed frame by frame. Most observations concern anaphase and telophase. The bridges break or are stretched and their variation in behavior depends on the length of the bridge and the stage when it is stretched. Breakage in anaphase is due to the pulling action of the kinetochores, while breakage in telophase is due to phragmoplast activity and a formation of a cell plate. The kinetochore movements of the dicentrics are usually, but not always, slowed down when the bridges stretch up to when the bridges have broken. Whether the movement of the kinetochores is slowed down or not depends mostly on the metaphase position of the kinetochore relative to the equator. A retarded movement of one kinetochore influences the movements of others in its close neighborhood while kinetochores situated further away are not influenced at all. In cells with many short bridges whole groups of chromosomes are retarded and the formation of a restitution nucleus follows. The formation of a restitution nucleus may also result from the existence of numerous bridges causing failure of phragmoplast formation. The contraction of the bridges during telophase pulls the two parts of the restitution nucleus together and contributes to maintain their connection.Some conclusions concerning the mechanism responsible for the movements of the chromosomes are also given.     

Nuclear division in the red algae Chondria nidifica and Chondria tenuissima

Cytological investigations are reported for two Chondria species, the Pacific species Chondria nidifica Harvey and Chondria tenuissima (Goodenough et Woodward) C. A. Agardh from the shore of the Marmara Sea in Istanbul. Nuclear division during mitosis and meiosis has been followed in somatic cells and in tetrasporangial mother cells respectively of diploid tetrasporic plants. The spherical interphase nucleus stains densely, showing many chromatin granules. Mitotic nuclei in the apical groove show a large number of chromosomes at metaphase; the chromosome number has been estimated at diakinesis to be 40 in both C. nidifica and C. tenuissima. The meiotic nuclei of tetraspore mother cells in prophase contain several relatively large nucleolar-derivatives in both species. The nucleolar derivatives disappear completely before the chromosomes begin to differentiate. In meiotic prophase the tetraspore mother cell enlarges from its original diameter. The period of the second meiotic anaphase seems to be extremely short in comparison with other nuclear phases. When the chromosomes reach the poles, they spread and subsequently form a relatively compact mass at telophase. The spindle has not been observed in C. tenuissima. Photographs are presented of nucleoli and nucleolar-derivatives in mitotic and meiotic divisions.     

A class of nonribosomal nucleolar components is located in chromosome periphery and in nucleolus-derived foci during anaphase and telophase

The subcellular location of several nonribosomal nucleolar proteins was examined at various stages of mitosis in synchronized mammalian cell lines including HeLa, 3T3, COS-7 and HIV-1 Rev-expressing CMT3 cells. Nucleolar proteins B23, fibrillarin, nucleolin and p52 as well as U3 snoRNA were located partially in the peripheral regions of chromosomes from prometaphase to early telophase. However, these proteins were also found in large cytoplasmic particles, 1–2 μm in diameter, termed nucleolus-derived foci (NDF). The NDF reached maximum numbers (as many as 100 per cell) during mid- to late anaphase, after which their number declined to a few or none during late telophase. The decline in the number of NDF approximately coincided with the appearance of prenucleolar bodies and reforming nucleoli. The HIV-1 Rev protein and a mutant Rev protein defective in its nuclear export signal were also found in the NDF. The mutant Rev protein precisely followed the pattern of localization of the above nucleolar proteins, whereas the wild-type Rev did not enter nuclei until G1 phase. The nucleolar shuttling phosphoprotein Nopp 140 did not follow the above pattern of localization during mitosis: it dispersed in the cytoplasm from prometaphase through early telophase and was not found in the NDF. Although the NDF and mitotic coiled bodies disappeared from the cytoplasm at approximately the same time during mitosis, protein B23 was not found in mitotic coiled bodies, nor was p80 coilin present in the NDF. These results suggest that a class of proteins involved in preribosomal RNA processing associate with chromosome periphery and with NDF as part of a system to conserve and deliver preexisting components to reforming nucleoli during mitosis. Edited by: S. A. Gerbi     

Kid-mediated chromosome compaction ensures proper nuclear envelope formation

Toward the end of mitosis, neighboring chromosomes gather closely to form a compact cluster. This is important for reassembling the nuclear envelope around the entire chromosome mass but not individual chromosomes. By analyzing mice and cultured cells lacking the expression of chromokinesin Kid/kinesin-10, we show that Kid localizes to the boundaries of anaphase and telophase chromosomes and contributes to the shortening of the anaphase chromosome mass along the spindle axis. Loss of Kid-mediated anaphase chromosome compaction often causes the formation of multinucleated cells, specifically at oocyte meiosis II and the first couple of mitoses leading to embryonic death. In contrast, neither male meiosis nor somatic mitosis after the morula-stage is affected by Kid deficiency. These data suggest that Kid-mediated anaphase/telophase chromosome compaction prevents formation of multinucleated cells. This protection is especially important during the very early stages of development, when the embryonic cells are rich in ooplasm.