Reconstitution of endoplasmic reticulum in rapidly dividing cells of early Xenopus embryos

The cytology of early blastomeres of Xenopus laevis embryos was examined. Particular attention was given to the organization of the nuclear envelope of karyomeres (chromosome vesicles) and the endoplasmic reticulum (ER) at different stages in early cleavage cycles of frog development. Nuclear envelope formation was observed to occur rapidly around individual chromosomes during early anaphase, and karyomeres fused subsequently to yield the final nucleus during telophase. Endoplasmic reticulum in the perinuclear cytoplasm was observed to be vesicular during metaphase and cisternal in form during telophase. Following microinjection of rat liver rough microsomes into early blastomeres, heterologous ER components were identified by electron microscope immunocytochemistry. The foreign ER was observed as large, reconstituted cisternae at stages in the cell cycle when the nuclear envelope was intact. Therefore, transplanted ER maintained the capacity to reconstitute in the cytoplasm of a rapidly dividing cell. In an attempt to better assess ER structure at the metaphase stage of the cell cycle, we next slowed down the division process by treating Xenopus embryos with anti-microtubule agents. Treatment with critical concentrations of colchicine, nocodazole, or vinblastine led to cleavage arrest but not to inhibition of the nuclear cycle. Following such treatment, homologous ER was observed in a vesicular form at all stages of the nuclear cycle. Heterologous ER, however, identified by immunocytochemistry in microinjected cells treated with nocodazole, displayed both vesicular and cisternal forms. We conclude that microinjected ER membranes exhibit cell-cycle-specific behavior, which is different from that of the host cell ER.     

An ultrastructural analysis of mitosis and cytokinesis in the zygote of the sea urchin, Arbacia punctulata

Post-fertilization events leading to the cleavage of the zygote of the sea-urchin, Arbacia punctulata were examined with the light and electron microscopes. Prior to prophase of the first cleavage division, endoplasmic reticulum and annulate lamellae become organized around the zygotic nucleus to produce a crescent-shaped structure which is defined as the streak (Harvey, '56). With the advent of prophase the streak undergoes morphogenic events which lead to the formation of the mitotic asters. During this transition there is a loss of annulate lamellae and a concomitant increase in endoplasmic reticulum. Annulate lamellae are not found as a part of the mitotic apparatus and are not again observed within the embryo until the two cell stage. During telophase, karyomeres are formed which consist of chromosomes delimited by a porous bilaminar envelope. Blastomere nuclei are produced following the fusion of the outer laminae, and subsequently by the fusion of the inner laminae of the envelopes encompassing the karyomeres.     

The chromosome periphery during mitosis

A complex structure, visible by electron microscopy, surrounds each chromosome during mitosis. The organization of this structure is distinct from that of the chromosomes and the cytoplasm. It forms a perichromosomal layer that can be isolated together with the chromosomes. This layer covers the chromosomes except in centromeric regions. The perichromosomal layer includes nuclear and nucleolar proteins as well as ribonucleoproteins (RNPs). The list of proteins and RNAs identified includes nuclear matrix proteins (perichromin, peripherin), nucleolar proteins (perichro-monucleolin, Ki-67 antigen, B23 protein, fibrillarin, p103, p52), ribosomal proteins (S1) and snRNAs (U3 RNAs). Only limited information is available about how and when the perichromosomal layer is formed. During early prophase, the proteins extend from the nucleoli towards the periphery of the nucleus. Thin cordon-like structures reach the nuclear envelope delimiting areas in which chromosomes condense. At telophase, the proteins are associated with the part of the chromosomes remaining condensed and accumulate in newly formed nucleoli in regions where chromatin is already decondensed. The perichromosomal layer contains several different classes of proteins and RNPs and it has been attributed various roles: (1) in chromosome organization, (2) as a barrier around the chromosomes, (3) involvement in compartmentation of the cells in prophase and telophase and (4) a binding site for chromosomal passenger proteins necessary to the early process of nuclear assembly.     

A UNIQUE MITOTIC VARIATION IN THE MARINE DINOFLAGELLATE OXYRRHIS MARINA (PYRROPHYTA)1

Mitosis is described in the flagellate Oxyrrhis marina Dujardin and is compared in related genera. Dense plaques develop in the nuclear envelope at prophase and give rise to an intranuclear spindle. Some of the microtubules associate with the chromosomes while others extend across the nucleus. The basal bodies migrate toward the poles early in division and retain a position lateral to the nuclear poles throughout mitosis. Microtubules are not present between the nucleus and the basal bodies. The nucleolus is persistent and elongates throughout anaphase and telophase. Chromosomal separation is accomplished by sliding of non-chromosomal microtubules and by elongation of the nuclear envelope rather than by shortening of the spindle microtubules. The nuclear envelope begins to constrict in the center early in anaphase. Continued constriction of the envelope and elongation of the nucleus leads to the formation of a dumbbell-shaped nucleus by late telophase. Mitosis culminates by the constriction of the nucleus into two daughter nuclei. The taxonomic position of Oxyrrhis marina is discussed in light of these findings.     

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.     

Structural analysis of the mitotic cycle in pre-gastrula Xenopus embryos

The long-known phenomenon of karyomere (chromosome vesicle) formation at early telophase of the nuclear cycle during early embryogenesis of a wide range of organisms including amphibians (Rubaschkin 1905; for review, see Richards 1917) was investigated in the early cleavage cycles of Xenopus laevis embryos before the mid blastula transition. Embryos were fixed and Epon embedded at successive time intervals and consecutive thick (3 m) and ultrathin sections cut. Using conventional light microscopy at low magnification as well as phase and/or interference contrast video microscopy at high magnification, a substantial amount of information could be obtained from the analysis of optical sections in thick-sectioned material. In addition, details of the ultrastructural organization could be analysed from corresponding ultrathin sections by electron microscopy. The light microscopic analysis of serial thick sections allowed precise determination of the arrangement and sizes of telophase karyomere structures during the embryonic nuclear division cycle. It was found that small, widely spaced 1st order karyomeres fuse to larger (2nd order) karyomeres which then progressively exhibit lateral fusion of neighbouring karyomeres. The final coalescence of adjacent karyomeres marks the onset of the reorganization of the typical interphase nuclear structure. The data are discussed with regard to the occurrence of karyomeres during the embryonic nuclear cycle of arthropods, dipteran insects, and echinoderms as well as recent progress in the use of Xenopus egg extracts for in vitro assembly of nuclear structures around protein-free DNA.     

Interaction of antibodies against nuclear envelope-associated proteins from rat liver nuclei with rodent and human cells

Antibodies have been prepared against the three major polypeptides of the nuclear pore complex-lamina fraction from rat liver nuclei. The three antisera prepared in chickens give similar results in indirect immunofluorescence microscopy. In rat embryo fibroblasts we observe bright fluorescence at the level of the nuclear envelope, with no fluorescence of the nuclear interior and little or no fluorescence of the cytoplasm. The nuclear envelope regions of rat hepatoma cells, mouse A9 cells, HeLa cells and rat liver nuclei also fluoresce brightly. HeLa nucleoids, which are depleted of nuclear envelope components, still exhibit specific fluorescence when reacted with these antibodies. Distribution of the antigens changes during mitosis. Fluorescence in the cytoplasm is observed following the breakdown of the nuclear envelope at prometaphase. The antigens appear to progressively accumulate at the periphery of the chromosomes until telophase. In late telophase fluorescence occurs predominantly at the periphery of the chromosomes where the new nuclear envelope is formed.     

Ultrastructure of mitosis inAmoeba proteus

Summary The fine structure ofAmoeba proteus nuclei has been studied during interphase and mitosis. The interphase nucleus is discoidal, the nuclear envelope is provided with a honeycomb layer on the inside. There are numerous nucleoli at the periphery and many chromatin filaments and nuclear helices in the central part of nucleus.In prophase the nucleus becomes spherical, the numerous chromosomes are condensed, and the number of nucleoli decreases. The mitotic apparatus forms inside the nucleus in form of an acentric spindle. In metaphase the nuclear envelope loses its pore complexes and transforms into a system of rough endoplasmic reticulum cisternae (ERC) which separates the mitotic apparatus from the surrounding cytoplasm; the nucleoli and the honeycomb layer disappear completely. In anaphase the half-spindles become conical, and the system of ERC around the mitotic spindle persists. Electron dense material (possibly microtubule organizing centers—MTOCs) appears at the spindle pole regions during this stage. The spindle includes kinetochore microtubules attached to the chromosomes, and non-kinetochore ones which pierce the anaphase plate. In telophase the spindle disappears, the chromosomes decondense, and the nuclear envelope becomes reconstructed from the ERC. At this stage, nucleoli can already be revealed with the light microscope by silver staining; they are visible in ultrathin sections as numerous electron dense bodies at the periphery of the nucleus.The mitotic chromosomes consist of 10 nm fibers and have threelayered kinetochores. Single nuclear helices still occur at early stages of mitosis in the spindle region.     

Dynamic Assembly of Brambleberry Mediates Nuclear Envelope Fusion during Early Development

To accommodate the large cells following zygote formation, early blastomeres employ modified cell divisions. Karyomeres are one such modification, mitotic intermediates wherein individual chromatin masses are surrounded by nuclear envelope; the karyomeres then fuse to form a single mononucleus. We identified brambleberry, a maternal-effect zebrafish mutant that disrupts karyomere fusion, resulting in formation of multiple micronuclei. As karyomeres form, Brambleberry protein localizes to the nuclear envelope, with prominent puncta evident near karyomere-karyomere interfaces corresponding to membrane fusion sites. brambleberry corresponds to an unannotated gene with similarity to Kar5p, a protein that participates in nuclear fusion in yeast. We also demonstrate that Brambleberry is required for pronuclear fusion following fertilization in zebrafish. Our studies provide insight into the machinery required for karyomere fusion and suggest that specialized proteins are necessary for proper nuclear division in large dividing blastomeres.     

MITOSIS IN THE AQUATIC FUNGUS RHIZOPHYDIUM SPHEROTHECA (CHYTRIDIALES)

The structure of centric, intranuclear mitosis and of organelles associated with nuclei are described in developing zoosporangia of the chytrid Rhizophydium spherotheca. Frequently dictyosomes partially encompass the sides of diplosomes (paired centrioles). A single, incomplete layer of endoplasmic reticulum with tubular connections to the nuclear envelope is found around dividing nuclei. The nuclear envelope remains intact during mitosis except for polar fenestrae which appear during spindle incursion. During prophase, when diplosomes first define the nuclear poles, secondary centrioles occur adjacent and at right angles to the sides of primary centrioles. By late metaphase the centrioles in a diplosome are positioned at a 40° angle to each other and are joined by an electron-dense band; by telophase the centrioles lie almost parallel to each other. Astral microtubules radiate into the cytoplasm from centrioles during interphase, but by metaphase few cytoplasmic microtubules are found. Cytoplasmic microtubules increase during late anaphase and telophase as spindle microtubules gradually disappear. The mitotic spindle, which contains chromosomal and interzonal microtubules, converges at the base of the primary centriole. Throughout mitosis the semipersistent nucleolus is adjacent to the nuclear envelope and remains in the interzonal region of the nucleus as chromosomes separate and the nucleus elongates. During telophase the nuclear envelope constricts around the chromosomal mass, and the daughter nuclei separate from each end of the interzonal region of the nucleus. The envelope of the interzonal region is relatively intact and encircles the nucleolus, but later the membranes of the interzonal region scatter and the nucleolus disperses. The structure of the mitotic apparatus is similar to that of the chytrid Phlyctochytrium irregulare.     

ELECTRON MICROSCOPIC STUDIES OF MITOSIS IN AMEBAE : II. The Giant Ameba Pelomyxa carolinensis

Dividing nuclei from the giant ameba Pelomyxa carolinensis were fixed in osmium tetroxide solutions buffered with veronal acetate to pH 8.0. If divalent cations (0.002 M calcium, magnesium, or strontium as chlorides) were added to the fixation solution, fibrils that are 14 mµ in diameter and have a dense cortex are observed in the spindle. If the divalent ions were omitted, oriented particles of smaller size are present and fibrils are not obvious. The stages of mitosis were observed and spindle components compared. Fibrils fixed in the presence of calcium ions are not so well defined in early metaphase as later, but otherwise have the same diameter in the late metaphase, anaphase, and early telophase. Fibrils are surrounded by clouds of fine material except in early telophase, when they are formed into tight bundles lying in the cytoplasm unattached to nuclei. Metaphase and anaphase fibrils fixed without calcium ions are less well defined and are not observably different from each other. The observations are consistent with the concept that spindle fibrils are composed of polymerized, oriented protein molecules that are in equilibrium with and bathed in non-oriented molecules of the same protein. Partially formed spindle fibrils and ribosome-like particles were observed in the mixoplasm when the nuclear envelope had only small discontinuities. Remnants of the envelope are visible throughout division and are probably incorporated into the new envelope in the telophase. Ribosome-like particles are numerous in the metaphase and anaphase spindle but are not seen in the telophase nucleus, once the envelope is reestablished, or in the interphase nucleus.     

MITOSIS IN THE ALGA VACUOLARIA VIRESCENS

Details of mitosis in the chloromonadophycean alga Vacuolaria virescens Cienk. have been studied with the light microscope. The chromosomes are relatively large (up to μ in length at metaphase) and so mitotic stages are readily distinguishable. Chromosomes can be recognized in interphase nuclei as fine strands of chromatin. Contraction of these chromosomes marks the beginning of mitosis and continues progressively until the transition from metaphase to anaphase. Disintegration of nucleoli is complete by late prophase and nucleolar reformation begins in telophase. Some chromosomes exhibit less densely stained regions; centromeres are also present as indicated by their differential staining and by the behavior of chromosomes at metaphase and anaphase. At anaphase progeny chromosomes move apart parallel to the division axis of the nucleus. As anaphase progresses the chromosomes fuse at the polar surface of the progeny chromosome groups. This process continues in telophase and the chromosome groups become more spherical. By the end of telophase nucleolar reformation has begun and the chromosomes have relaxed to their interphase condition.     

FURTHER OBSERVATIONS ON THE CLUSTERS OF GRANULAR MATERIAL AROUND THE CENTRIOLE IN THE SEA URCHIN EGG: CHANGES IN DISTRIBUTION DURING MITOSIS*

Changes in the distribution of pericentriolar material, which was called “clusters of granular material”, in a previous paper were observed during mitosis of the sea urchin egg by electron microscopy using thick sections. At prophase, small clusters in an early stage of formation were observed near the nucleus. At prometaphase, the clusters appeared to aggregate loosely at the poles of the spindle. They formed large masses at metaphase, while at late anaphase they became reduced in size and formed an array at right angles to the spindle axis. Some clusters still remained near the karyomeres at telophase and then became closely associated with the daughter nucleus. The clusters were closely associated with the astral microtubules and spindle microtubules at prophase and prometaphase, respectively. The granular material is suggested to be a nucleating site of microtubule assembly during mitosis.     

Ultrastructure of Chilomonas paramecium and the phylogeny of the cryptoprotists

The ultrastructure of the cryptoprotist Chilomonas paramecium is reviewed and compared to earlier accounts. Distinctive features include a complex cytoskeleton which defines the cell organization and interconnects cell components; trichocysts which resemble those in other cryptoprotists; and two non-photosynthetic plastids. During mitosis there is partial dispersal of the nuclear envelope early in prophase but some remains at the nuclear periphery throughout mitosis. At metaphase chromosomes are arranged on the longitudinal axis of an elongated elliptical nucleus. During telophase the chromosomes decondense and the nuclear envelope reforms. Cell structure is compared with that in other cryptoprotists, and origin of this taxon of algae is discussed.     

Cyclins A and B associate with chromatin and the polar regions of spindles, respectively, and do not undergo complete degradation at anaphase in syncytial Drosophila embryos

Maternally contributed cyclin A and B proteins are initially distributed uniformly throughout the syncytial Drosophila embryo. As dividing nuclei migrate to the cortex of the embryo, the A and B cyclins become concentrated in surface layers extending to depths of approximately 30-40 microns and 5-10 microns, respectively. The initiation of nuclear envelope breakdown, spindle formation, and the initial congression of the centromeric regions of the chromosomes onto the metaphase plate all take place within the surface layer occupied by cyclin B on the apical side of the blastoderm nuclei. Cyclin B is seen mainly, but not exclusively, in the vicinity of microtubules throughout the mitotic cycle. It is most conspicuous around the centrosomes. Cyclin A is present at its highest concentrations throughout the cytoplasm during the interphase periods of the blastoderm cycles, although weak punctate staining can also be detected in the nucleus. It associates with the condensing chromosomes during prophase, segregates into daughter nuclei in association with chromosomes during anaphase, to redistribute into the cytoplasm after telophase. In contrast to the cycles following cellularization, neither cyclin is completely degraded upon the metaphase-anaphase transition.     

The myc proteins are not associated with chromatin in mitotic cells.

The protein products of cellular and viral myc oncogenes are detected in nuclei by immunofluorescence. No myc fluorescence is found in nucleoli. In mitotic cells the myc antigens are not found associated with metaphase chromosomes, but are diffusely distributed throughout the cytoplasm. Cytoplasmic myc fluorescence is first observed when chromatin begins to condense in early prophase. Granular nuclear myc fluorescence is again discerned in telophase cells, when the nuclear envelope is formed and becomes more prominent upon cytokinesis; concomitantly the diffuse cytoplasmic myc staining is lost. These results suggest that myc proteins not only bind to DNA or chromatin, but are also associated with other structural systems in the nuclei.     

Dynamic interaction between BAF and emerin revealed by FRAP, FLIP, and FRET analyses in living HeLa cells

Barrier-to-autointegration factor (BAF) is a conserved 10 kDa DNA-binding protein. BAF interacts with LEM-domain proteins including emerin, LAP2 beta, and MAN1 in the inner nuclear membrane. Using fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP), we compared the mobility of BAF to its partners emerin, LAP2 beta, and MAN1 in living HeLa cells. Like endogenous BAF, GFP-BAF was enriched at the nuclear envelope, and found inside the nucleus and in the cytoplasm during interphase. At every location, FRAP and FLIP analysis showed that GFP-BAF diffused rapidly; the halftimes for recovery in a 0.8 microm square area were 260 ms at the nuclear envelope, and even faster inside the nucleus and in the cytoplasm. GFP-fused emerin, LAP2 beta, and MAN1 were all relatively immobile, with recovery halftimes of about 1 min, for a 2 microm square area. Thus, BAF is dynamic and mobile during interphase, in stark contrast to its nuclear envelope partners. FLIP results further showed that rapidly diffusing cytoplasmic and nuclear pools of GFP-BAF were distinctly regulated, with nuclear GFP-BAF unable to replenish cytoplasmic BAF. Fluorescence resonance energy transfer (FRET) results showed that CFP-BAF binds directly to YFP-emerin at the inner nuclear membrane of living cells. We propose a "touch-and-go" model in which BAF binds emerin frequently but transiently during interphase. These findings contrast with the slow mobility of both GFP-BAF and GFP-emerin during telophase, when they colocalized at the 'core' region of telophase chromosomes at early stages of nuclear assembly.     

Electron microscopic study of the nuclear membrane of SPEV cells during mitosis. II. Reconstruction of the nuclear membrane (anaphase and telophase)]

Reformation of the nuclear envelope of the PKLV cells starts in the early anaphase when numerous contacts between membranous elements and chromosomes become visible. The nuclear envelope reforms, first, on the periphery of chromosomes and in chromosome's centromeric regions, and later - in the telomeric regions. The latest reconstruction of nuclear envelope occurs in spaces where microtubules come up close to the "nuclei", The appearance of normal pore complexes in the late telophase is preceded by the appearance, in the early and middle telophase, of pores with unusual structure.     

Nuclear responses to MPF activation and inactivation in Xenopus oocytes and early embryos

The cell cycle of most organisms is highlighted by characteristic changes in the appearance and activity of the nucleus. Structural changes in the nucleus are particularly evident when a cell begins to divide. At this time, the nuclear envelope is disassembled, the chromatin condenses into metaphase chromosomes, and the chromosomes associate with a newly formed spindle. Upon completion of cell division the nuclear envelope reassembles around the chromosomes as they form telophase nuclei, and subsequently interphase nuclei, in the daughter cells. The cytoplasmic control of nuclear behavior has been the theme of Yoshio Masui's research for much of his career. His pioneering demonstration that the cytoplasm of maturing amphibian oocytes causes the resumption of the meiotic cell cycle when it is injected into an immature oocyte provided unequivocal evidence that a cytoplasmic factor could initiate the transition from interphase to metaphase (M-phase) in intact cells. As described in several reviews in this and the previous issue of Biology of the Cell (see Beckhelling and Ford; Duesbery and Vande Woude; Maller), Masui initially called this activity maturation promoting factor (MPF), but when it was realized that it was a ubiquitous regulator of both mitotic and meiotic cell cycles, MPF came to stand for M-phase promoting factor. Biochemical evidence indicates that MPF activity is composed of a mitotic B-type cyclins and cyclin-dependent kinase 1. The increase in the protein kinase activity of cdk1 initiates the changes in the nucleus associated with oocyte maturation and with the entry into mitosis. This article will attempt to provide a brief summary of the responses of the nucleus to the activation of MPF. In addition, the effect of MPF inactivation on nuclear envelope assembly at the end of mitosis will be discussed. This article is written as a tribute to Yoshio Masui on his retirement from the University of Toronto, and as an expression of gratitude for his guidance while I was a student in his laboratory. I have felt very privileged to have known him as a mentor and a friend.